CN102186590A

CN102186590A - Device, kit and method for pulsing biological samples with an agent and stabilising the sample so pulsed

Info

Publication number: CN102186590A
Application number: CN2008801316095A
Authority: CN
Inventors: 帕特里克·斯托德尔; 米歇尔·高曼; 马里奥斯·特因德
Original assignee: Universite Libre de Bruxelles ULB
Current assignee: Universite Libre de Bruxelles ULB
Priority date: 2008-10-17
Filing date: 2008-10-17
Publication date: 2011-09-14
Also published as: JP2012505639A; CA2740150A1; CA2740150C; WO2010043271A1; IL212082A0; EP2346606A1

Abstract

The present invention is related to a device and kitfor pulsing a biological sample with a pulsing agent, subsequently stabilizing the biological sample so pulsed, which device or kit provides a control reaction. The invention has application in the fields of medical diagnostics, particularly relating to immunity.

Description

Be used for making biological sample pulsation and stable so device, kit and the method for the sample of pulsation with reagent

Invention field

The present invention relates to be used for device, method and the kit of diagnostic assay, and be applied in the field of immunology.

Introduce

The monitoring nucleic acid level, for example, the mRNA level is directly determining that a kind of reagent is valuable to biosystem as the time spent.For example, if a kind of reagent is introduced into the time that continues specific length in the biosystem, then the reaction of this system and this reagent can be determined by the level of measuring mRNA.This can be effective to monitor immunity, and wherein, for example, this reagent is that antigen and monitored mRNA are cytokines mRNA, for example, and interleukin.

By blood drawing from individuality and will later time point in time add effect that reagent tests this reagent blood leave circulation and with this reagent stimulation between introduced variable delay.At this timing period, blood may experience slowly or chemical modification fast, for example, depends on the temperature that keeps it.In addition, delay is that the comparative study between the sample of continuous drawing is invalid in variable meaning.

When test during nucleic acid, main challenge, especially exists when detecting the requiring of low-level RNA or unstable RNA in external unstability owing to RNA.Even only the degraded of sub-fraction RNA also may change the explanation to rna level.Some transcripts are known to be present in the cell with low copy; Other transcripts have " rich AU " sequence in its 3 ' end, promote it to be degraded fast by endogenous RNA enzyme.Interior RNA of a few hours significantly degraded rapidly after research had been presented at the sample collection.In addition, in case after sample was gathered, the RNA of some kind increased by gene induced process.RNA degraded and outer-gene induce both all can cause underestimating or over-evaluating of vivo gene transcript number.

Therefore, when measuring the do time spent of a kind of reagent to the blood extracted out, the challenge that exists in this area is the process of management mRNA degraded, and this process has just begun after blood is drawn out of at once, and restarts after introducing antigen.Because " before " degraded can exert an influence to the degraded of " afterwards ", so error is all relevant with two processes; Therefore, the bigger and error of the possibility of error is difficult to illustrate more.

Another problem in this area is to carry out must using several equipment when biological sample is exposed to reagent and carries out its foranalysis of nucleic acids subsequently.Typically, need reagent bottle, accurate pipette, refrigerating equipment to carry out quantitative measurment at least.If under the condition that lacks suitable laboratory facility, obtain sample, for example, in the family of individuality or in the operating room of basic equipment, then be not suitable for or be convenient to carry out accurate substrate adding, and may not have cold storage establishment to use in addition.

In case sample has been exposed to reagent, exist many methods to separate and measure wherein nucleic acid, for example, mRNA.Low-level transcript is determined in certain methods even permission in the transcript mixture.Yet, do not have a kind of possibility that the level of one or more transcripts of existing in the biological sample is determined the time in sampling that is provided in these methods.Even under refrigerated condition, the preservation of biological sample causes incorrect mRNA level.In fact, in practice, because the place of sampling is positioned at different places with the place that RNA analyzes, it is infeasible therefore analyzing fresh sample.

In recent years, PreAnalytiX (joint venture of Becton Dickinson and Qiagen) has produced PAXgene ^TMThe blood rna system.This PAXgene ^TMBlood rna system (being also referred to as the Qiagen method) is a kind of integrated standardization system that is used to gather with stabilisation whole blood sample and isolated cell RNA.According to PreAnalytiX, at PAXgene ^TMIn the blood rna system, blood directly gathers PAXgene ^TMIn the blood rna pipe, and use PAXgene subsequently ^TMBlood rna kit isolation of RNA.Use this system, can from whole blood, obtain complete cell RNA.

PAXgene ^TMThe blood rna pipe is a kind of plastics vacuum tube (evacuated tube) that is used to gather whole blood and stabilized cell RNA spectrum (profile).This pipe is equipped with the additive (a kind of have Patent right agent blend) of stabilized cell RNA, can eliminate the in-vitro inducing of genetic transcription thing and the acute variation of the cell RNA express spectra that prevents from normally to be taken place under conditions in vitro.Use by PAXgene then ^TMThe pellosil technology isolation of RNA that provides in the blood rna kit.According to PreAnalytiX, the RNA that obtains has accurately represented the express spectra in the body and has been suitable in the downstream application of certain limit.According to supplier's saying, use accurately quantitate gene transcript of this system.This PAXgene ^TMThe major defect of blood rna system is each PAXgene ^TMThe blood rna pipe needs and PAXgene ^TMThe blood rna kit is combined (referring to PAXgene ^TMThe specification guide of blood rna pipe).Yet, this mandatory combination restriction the further improvement of this system.

More recent, developed Tempus ^TMBlood rna pipe (applying biological system (Applied Biosystems)), it is a kind of plastics vacuum tube similarly, is used to gather whole blood and stabilized cells RNA spectrum.Each pipe is equipped with the additive (a kind of have Patent right agent blend) of stabilized cell RNA, can eliminate the in-vitro inducing of genetic transcription thing and the acute variation of the cell RNA express spectra that prevents from normally to be taken place under conditions in vitro.

Goal of the invention

An object of the present invention is to provide and be used for making biological sample to be exposed to device, kit and the method for the nucleic acid of reagent and the stable sample that so exposes.

An object of the present invention is to provide and be used to make biological sample to be exposed to reagent, device, kit and the method for the nucleic acid in the stable sample that so exposes, described device, kit and method also provide control reaction.

Another object of the present invention provides the time that obtains biological sample and described sample is exposed between the reagent is shortened, and keeps constant or keeps almost constant device, kit and method.

Another object of the present invention provides the time shortening that will make sample be exposed to reagent and stablize the nucleic acid in the described sample, keeps constant or keeps almost constant device, kit and method.

Another object of the present invention provides under the condition of the amount that need not to measure sample and/or reagent and makes reagent be exposed to device, kit and the method for sample.

Another object of the present invention provides and makes reagent and contrast be exposed to sample, and makes described sample and contrast be exposed to reagent and time of stablizing the nucleic acid in the described sample keeps constant or keeps almost keeping constant single assembly.

Another object of the present invention provides under the condition of the amount that need not to measure stabilizing agent and makes reagent be exposed to device, kit and the method for sample.

Another object of the present invention provides and is used to make biological sample to be exposed to reagent, and the nucleic acid in the stable sample that so exposes also therefrom extracts nucleic acid and is used for further device, kit and the method for analyzing.

Another object of the present invention provides and is used to make biological sample to be exposed to reagent, the device of the nucleic acid in the stable sample that so exposes, and described device also provides control reaction and helps the robot automation.

Another object of the present invention provides device, kit and the method for the combination that solves one or more above-mentioned purposes.

Summary of the invention

One embodiment of the invention are a kind of kits that are used to measure liquid biological sample, and it comprises:

Be suitable for the receiving fluids biological sample, make described sample be exposed to first material and be exposed to the vessel of nucleic acid stability agent subsequently, described vessel comprise:

A) be present in first material of described vessel inside,

B) wherein have the container of described stabilizing agent,

C) connection between described vessel inside and the described internal tank,

D) block the physical barriers of described connection temporarily;

With

Be suitable for the receiving fluids biological sample, make described sample be exposed to the contrast material and be exposed to the contrast vessel of nucleic acid stability agent subsequently, described contrast vessel comprise:

A) be present in the contrast material of described contrast vessel inside,

B) wherein have the control container of described stabilizing agent,

C) connection between described contrast vessel inside and the described control container inside,

D) block the physical barriers of described connection temporarily.

Another embodiment of the invention is aforesaid kit, and wherein said first material is fixed on part or all inner surface of described vessel.

Another embodiment of the invention is aforesaid kit, and wherein said first material is fixed on the solid phase carrier.

Another embodiment of the invention is aforesaid kit, and wherein said first material is a liquid.

Another embodiment of the invention is aforesaid kit, and wherein said first material is a solid.

Another embodiment of the invention is aforesaid kit, and wherein said vessel and/or contrast vessel comprise one or more zones that syringe needle penetrates that are adapted to pass through.

Another embodiment of the invention is aforesaid kit, and wherein said zone is reclosable barrier film.

Another embodiment of the invention is aforesaid kit, and wherein said vessel and/or contrast vessel comprise the accessory that is suitable for admitting syringe and content is wherein transferred to the inside of described vessel or contrast vessel.

Another embodiment of the invention is aforesaid kit, and wherein said vessel and/or contrast vessel comprise the accessory that is suitable for admitting syringe needle.

Another embodiment of the invention is aforesaid kit, and wherein said vessel and/or contrast vessel comprise can minimizing from the gas stream/liquid of vessel stream and permission liquid biological sample and flow to valve in the described vessel.

Another embodiment of the invention is aforesaid kit, and wherein said vessel and/or contrast vessel comprise a kind of equipment, can discharge the gas of being replaced by described equipment.

Another embodiment of the invention is aforesaid kit, and wherein said vessel and/or contrast vessel remain under the negative pressure.

Another embodiment of the invention is aforesaid kit, wherein by applying physical force and make a d to described vessel or contrast vessel) physical barriers open.

Another embodiment of the invention is aforesaid kit, and wherein said power is sent to described physical barriers with opening device.

Another embodiment of the invention is aforesaid kit, and wherein said power is irreversibly opened described physical barriers.

Another embodiment of the invention is aforesaid kit, and wherein said vessel and/or contrast vessel are included in the indication of the stabilizing agent distribution that wherein distributes known volume.

Another embodiment of the invention is aforesaid kit, and wherein said first material comprises one or more immune system antigen.

Another embodiment of the invention is aforesaid kit, and wherein said immune system antigen is vaccine composition.

Another embodiment of the invention is aforesaid kit, and wherein said immune system antigen is the antigen that excites super allergen reaction (hyperallergenic response).

Another embodiment of the invention is aforesaid kit, and wherein said immune system antigen is to be selected among histocompatibility antigen, bacterium LPS, tetanus toxoid (tetanous toxoid), cancer immunotherapy antigen, MAGE-3, cat allergen, Feld1, the antigen presenting cell from organ donor, autoantigen, the GAD65 one or more.

Another embodiment of the invention is aforesaid kit, and wherein said stabilizing agent is cell RNA degraded and/or gene induced inhibitor.

Another embodiment of the invention is aforesaid kit, and wherein said cell RNA degraded and/or gene induced inhibitor are to be present in PAXgene ^TMOr Tempus ^TMIn the blood rna pipe those.

Another embodiment of the invention is aforesaid kit, and the external engagement of wherein said vessel and contrast vessel is together to form single entity.

Another embodiment of the invention is the determinator that is used for liquid biological sample, and it helps to make described sample to be exposed to first material and contrast material respectively, and is exposed to the nucleic acid stability agent subsequently, and described device comprises:

Wherein there is described first material in-the first Room,

Wherein there is described contrast material in-the second Room,

-Di three Room, wherein exist described stabilizing agent and

-be used for the support of biological sample pipe.

Another embodiment of the invention is aforesaid determinator, one or more sealed in the wherein said chamber, and comprise one or more zones that hollow needle pierces through that are adapted to pass through.

Another embodiment of the invention is aforesaid determinator, and wherein said zone is reclosable barrier film.

Another embodiment of the invention is aforesaid determinator, also comprise and be used for the support that is connected with transfer tube dismounting ground, described transfer tube is included in hollow flexible or the rigid pipe that arbitrary end disposes hollow needle, and it is suitable at fluid transfer between any two chambers or between a chamber and described biological sample pipe.

Another embodiment of the invention is aforesaid determinator, also comprises as top defined transfer tube.

Another embodiment of the invention is aforesaid determinator, a pin of wherein said transfer tube is connected in the parallel alignment mode with the hollow needle of forcing pipe, described forcing pipe comprises that an end disposes the flexible hollow pipe of described pin, thereby and is suitable for applying vacuum or pressure to chamber or biological sample pipe and promotes liquid and pass through transfer tube.

Another embodiment of the invention is aforesaid determinator, and wherein said first material is fixed on part or all inner surface of described first vessel.

Another embodiment of the invention is aforesaid determinator, and wherein one or more described chambers comprise passage.

Another embodiment of the invention is aforesaid determinator, and wherein said first and/or second Room remains under the negative pressure.

Another embodiment of the invention is aforesaid determinator, and wherein said first and/or second Room is included in the indication of the stabilizing agent distribution that wherein distributes known volume.

Another embodiment of the invention is aforesaid determinator, and wherein said first material is as top definition.

Another embodiment of the invention is aforesaid determinator, and wherein said stabilizing agent is as top definition.

Detailed Description Of The Invention

One aspect of the present invention relates to the vessel that are suitable for holding biological sample, and described vessel hold first material of scheduled volume, and described first material can be pulsation agent (pulsing agent).

As used herein " pulsation agent " comprise that biological sample can be to any material of its exposure.The example of material comprises peptide, nucleic acid, antigen.The pulsation agent can comprise other compositions that described material is outer, such as stabilizing agent, and indicator, bridging agent (linker), matrix etc.

Term " biological sample " be meant the sample that comprises nucleic acid/biological agent such as clinical (for example, cell fraction, whole blood, blood plasma, serum, urine, tissue, cell etc.), the agricultural, environment (for example, soil, mud, mineral, water, air), food (any foodstuff), court or other possible samples." whole blood " be meant blood such as it by the venous blood collection collection, that is, contain leucocyte and red blood cell, blood platelet, blood plasma and infectious agent at last; Infectious agent can be viral, bacillary or parasitics.Clinical sample can be originated from the human or animal.The character of the sample of analyzing can be solid or liquid.Obviously, when using solid material, they at first are dissolved in the suitable solution, and described solution can be the RNAIater reagent of being sold by Qiagen.According to the present invention, this solution does not always have real " buffer solution " of the composition of at least two kinds of abundant balances.It can be strong hypotonic solution such as independent NaCl or extract solution such as containing alcohol.

Vessel (vessel) are also referred to as reaction vessels in this article, can hold the pulsation agent in many ways.According to an aspect of the present invention, the pulsation agent can be fixed on the inwall of vessel.The inwall of vessel can serve as a contrast so that the suitable coating that described pulsation agent can be adhered to.Alternatively, described pulsation agent can be attached directly on part or all inwall of vessel.Being suitable for such suitable coating that adheres to, method and vessel material is well known in the art.According to another aspect of the present invention, described pulsation agent exists as solid.Described solid can be bead, gel, the paste of powder, freeze-drying.Suitable solid is formed and their preparation method is well known in the art.According to another aspect of the present invention, described pulsation agent is fixed on the solid phase carrier.Solid phase carrier can be attached to the inside of vessel.Alternatively, solid phase carrier can break away from the inside in vessel.The example of solid phase carrier includes but not limited to, chromatography matrix, magnetic bead.According to another aspect of the present invention, the pulsation agent exists as liquid.Suitable liquid is formed and their preparation method is well known in the art.

The measurement device that carries out analytical pulsation test requirements document calibration outside the scope of laboratory condition is such as pipette.Because the error that causes of unregulated measurement mechanism can cause constant error, and divide the human error of timing also can cause error between the different samples, this makes comparative analysis invalid.Provide the vessel of the pulsation agent that is supplied with scheduled volume to eliminate needs, and got rid of artificial measure error other equipment.In addition, provide independently to contrast the vessel permission, help to detect error result standardization.The use of contrast is important, because RNA is in case be unsettled and quick degraded after taking out from circulation.Inconsistent delay in making whole blood pulsation will cause the result that can't contrast.And pulsation agent incubation period between, adopt different (biology) approach according to reagent.The present invention allows to measure this biological process by the mRNA of quantitative biomarker, described biomarker such as cell factor, chemotactic factor (CF), transcription factor or represent other gene outcomes or the surrogate markers thing of particular procedure.Do not having under the condition of reference value, quantitatively these biomarkers are almost meaningless in mensuration; Use comprehensive contrast to allow to carry out apace control reaction.By comparative measurements and contrast, the meaning of the amount of biomarker and pulsation agent are to the influence of biosystem.As an example, generally acknowledge that the Mx1 gene is under the control of I type IFN.We have utilized this knowledge, and use this gene outcome as an alternative mark measure the curative effect of I type IFN.With whole blood quantitative Mx1 gene outcome behind I type IFN incubation.The amount of Mx1 gene outcome can be expressed as the mRNA copy number of comparing with the mRNA copy number of standardization gene outcome (that is, its transcriptional activity is not subjected to the gene of the function influence of IFN).But do not having under the condition of control reaction, this is quantitatively with meaningless, and described control reaction is a quantitative surrogate markers thing under unprovoked collating condition (that is the same treatment that, does not contain I type IFN).Identical reason goes for any immune response.In order to have strict contrast, it is very important that control sample is handled in an identical manner, that is, control sample is from once extracting identical incubation time and temperature and parallel processing together; The combination of measuring and contrast the chamber in single assembly will make this become feasible.

Type according to vessel of the present invention can be any kind that is suitable for preserving biological sample.According to an aspect of the present invention, will hold the vessel sealing of the agent of pulsing.According to an aspect of the present invention, the vessel that hold the agent of pulsing have water-tight equipment such as nut again, and pusher cap (push-on cap) is renovated (flip-cap).Referring to, for example, Fig. 5.According to an aspect of the present invention, biological sample or other fluids can be introduced in the vessel by using syringe needle to pierce through to vessel wall.The wall of vessel can be in that to pierce through the back reclosable, or the wall of vessel can be not reclosable after piercing through, or the wall of vessel can be equipped with reclosable zone such as barrier film.Referring to, for example, Fig. 4.

According to an aspect of the present invention, biological sample or other liquid thereby help transfer tube and introduce, and described transfer tube is included in the hollow pipe (for example, trochar) that arbitrary end disposes pin.Pin is set and is used to pierce through the sample cell that holds sample or the barrier film of other containers, and another root pin is set the barrier film that is used to pierce through vessel; Use pin biological sample can be transferred to vessel from sample cell.Described pipe can be flexible or rigidity.The shape of transfer tube can be a U-shaped.Referring to, for example, Figure 22.

According to an aspect of the present invention, one or more accessories of the biological sample container that can admit syringe or other to be equipped with connecting device by means of being used for of being connected with vessel and be introduced into vessel.For example, vessel can be equipped with Rule (Luer) accessory, and described Rule accessory can be admitted needleless injector.Referring to for example, Fig. 3.In another embodiment, vessel can be equipped with non-Rule (non-Luer) accessory, and the container that described accessory can connect design with (reciprocating) the non-Rule with complementation closely cooperates.

According to an aspect of the present invention, biological sample can be by means of with the matched trochar of described vessel or hypodermic syringe needle and be introduced in the vessel, and described trochar or hypodermic syringe needle are suitable for directly extracting biological sample from individuality.Referring to, for example, Fig. 6.

According to an aspect of the present invention, biological sample can water-tight equipment be introduced in the vessel by opening again.Referring to, for example, Fig. 5.

Known as those of skill in the art, sample is introduced into sealing will causes therefrom displacing isopyknic air or gas in the vessel, or the pressure of accumulation therein.Therefore, vessel can be equipped with suitable device to discharge from described vessel with the gas that allows to be replaced, or the accumulation of pressure is provided.Described equipment is well known in the art, and comprises valve, anti-weeper (non-drip holes), and passage has obducent passage (clothed-vents), the use of negative pressure in the distensible vessel wall, described vessel.Referring to, for example, the arrow 31 on Figure 11.

In one aspect of the invention, sealing vessel pressure inside is a negative pressure.The pressure that accumulates in the time of can utilizing described negative pressure to be released in biological sample is introduced into described sealing vessel.Alternatively, or additionally, negative pressure can be in predeterminated level and can be used for allowing to introduce the biological sample of fixed volume.

According to another aspect of the present invention, vessel are equipped with aforesaid sealable barrier film, be suitable for admitting first pin (trochar) and second pin, can enter in the vessel, can apply malleation or negative pressure to vessel inside by second pin by the first pin liquid biological sample.When applying negative pressure (vacuum) by second pin, biological sample can be drawn in the vessel by first pin.Preferably, first pin is the part of foregoing transfer tube.Second pin can be the part of forcing pipe described below, and described forcing pipe is connected with air (extracting out or pressurization) pump.First pin and second pin can engage in the mode of parallel alignment.Referring to, for example, Figure 24.

The vessel that wherein have been supplied with the pulsation agent of scheduled volume allow individuality is carried out diagnostic test, need not to use the equipment that is used to measure described antigen.In addition, when carrying out diagnostic test outside the scope at laboratory condition, the problem of pollution and distribution accuracy may cause the error result in the quantitative assay.Vessel as described herein have overcome these problems.

Another aspect of the present invention is the contrast vessel, and it can be aforesaid vessel, and difference is that it does not hold beyond the biological pulsation agent, and holds contrast pulsation agent (contrast material).For the purpose of describing this present invention and local mentioned as other, vessel as described herein are used for holding the pulsation agent, and are also referred to as " reaction vessels " sometimes, and contrast vessel as described herein are used for holding contrast pulsation agent.Vessel and contrast vessel are operated independently; They receive sample and stabilizing agent independently, and inside is not connected.Yet can there be the part as kit together in vessel and contrast vessel.Alternatively, the outer surface of vessel and contrast vessel can mechanically be connected the single entity that comprises vessel and contrast vessel with formation.According to an aspect of the present invention, it is sealed the contrast vessel of contrast pulsation agent to be housed.The contrast vessel can have again water-tight equipment such as, for example, nut, pusher cap is renovated.The contrast vessel can have easily broken strip of paper used for sealing such as peel-open viscosity strip of paper used for sealing (peel-back adhesive seal), break formula strip of paper used for sealing (snap-off seal).According to an aspect of the present invention, the wall of contrast vessel can be equipped with reclosable zone such as barrier film, can material be introduced in the contrast vessel by using syringe needle to pierce through by this zone.The barrier film of contrast vessel can be reclosable after piercing through.According to an aspect of the present invention, biological sample or other liquid thereby help above-mentioned transfer tube and introduce, and arbitrary end of described transfer tube disposes pin (for example, trochar).Pin is set and is used for piercing through the pipe that holds sample cell or the barrier film of other containers, and another root pin is set the barrier film that is used for piercing through the contrast vessel; Use described pin liquid biological sample can be transferred to the contrast vessel from sample cell.Described pipe can be flexible or rigidity.The shape of transfer tube can be a U-shaped.

In one aspect of the invention, the contrast vessel can have the equipment that extracts liquid biological sample when being connected with the biological sample pipe fluid from the biological sample pipe; Example includes but not limited to the syringe type plunger, or applies any equipment of negative pressure.

It being understood that the contrast vessel receive a part that is introduced into the identical biological sample in the reaction vessels.Biological sample can separated into two parts, and portion is used to contrast vessel, and another part is used for reaction vessels.Alternatively, the biological sample of a five equilibrium sample can be introduced in the reaction vessels, and second equates that aliquot can be introduced in the contrast vessel.

According to another aspect of the present invention, the contrast vessel are equipped with aforesaid sealable barrier film, be suitable for admitting first pin (trochar) and second pin, can enter in the contrast vessel by the first pin liquid biological sample, can apply malleation or negative pressure to the inside of contrast vessel by second pin.When applying negative pressure (vacuum) by second pin, biological sample can be drawn in the contrast vessel by first pin.Preferably, first pin is the part of foregoing transfer tube.Second pin can be the part of forcing pipe described below, and described forcing pipe is connected with air (extracting out or pressurization) pump.First pin and second pin can engage in the mode of parallel alignment.Referring to, for example, Figure 24.

Parameter when contrast pulsation agent will be depended on pulsation agent and research is as those of skill in the art fully understand.For example, when the pulsation agent is a peptide, one group of peptide, or during peptide mixer, contrast pulsation agent can comprise one or more peptides with equal length, and difference is the fragment that the sequence of described one or more peptides was randomized and/or was different from existing albumen or peptide.This contrast pulsation agent will with the whole blood identical and the reagent of same amount, the blood of equal volume one identical period of incubation together and under identical temperature with the pulsation agent.In another embodiment, the pulsation agent can be the protein for treatment agent, such as the IFN-β that is dissolved to desired concn in excipient such as water, PBS or salt solution.Then contrast pulsation agent can be this excipient.In a further embodiment, the pulsation agent can be an antigen, such as the Feld1 that is dissolved to desired concn in excipient such as water, PBS or salt solution; Then contrast pulsation agent can be the excipient of this antigen.Alternatively, the contrast vessel can not have dummy or empty, in the case, for example, the immune response of inducing, or the gene expression of inducing is compared with untreated sample.

Another aspect of the present invention relates to vessel as described herein, and described vessel further comprise the container that wherein has stabilizing agent; Described stabilizing agent temporarily is prevented from contacting with the biological sample of pulsing agent or be exposed to described reagent.

In one aspect of the invention, stabilizing agent comprises nucleic acid stability agent and/or cell RNA degradation inhibitor and/or gene induced inhibitor, and/or described stabilizing agent is as being present in PAXgene ^TMIn the blood rna pipe those and/or stabilizing agent are as being present in Tempus ^TMIn the blood rna pipe those.Reagent and its combination are as known in the art, or can be derived out by those of skill in the art.

PAXgene ^TMAnd Tempus ^TMThe blood rna pipe to be comprising the solution supply of additive, described additive stabilized cell RNA and can eliminate the in-vitro inducing of genetic transcription.Do not provide detailed information to describe the character of this additive.For this purpose, with PAXgene ^TMThe brochure referenced patents US 5,906,744 that pipe provides together.Yet the pipe described in this patent allows those skilled in the art from the blood plasma but not prepare nucleic acid from whole blood as carrying out among the present invention.Particularly, US 5,906, and 744 device preferably includes plastics or glass tube, be used for suppressing the device of blood clotting and be used for from the whole blood separated plasma device (US 5,906,744 the 2nd hurdles, I.42-43).Therefore, according to the present invention, as US 5,906, content described in 744 and PAXgene ^TMThe true content of blood rna pipe is uncorrelated, because it relates to different purposes.

According to the present invention, be contained in PAXgene ^TMSolution in the blood rna pipe can contain quaternary amine surfactants.Therefore, according to the present invention, quaternary amine surfactants can be used used as stabilizers.US 5,010 in the past, put down in writing in 183 and used quaternary amine surfactants to come nucleic acid in the stabilizing biological sample.This patent provides the method for from biomaterial mixture purify DNA or RNA.Said method comprising the steps of: a certain amount of cationic detergent is added in the mixture that contains RNA or DNA, described amount is enough to dissolved cell, makes any contaminating protein and the lipid dissolving in the mixture and forms insoluble hydrophobic compound between nucleic acid and detergent.Therefore the compound that comprises RNA or DNA and detergent separates with the pollutant of dissolving.In nearer patent, identical inventor mentions the use surfactant, as US 5,010, described in 183 and the deposition efficiency dissolving low and haemocyte that causes RNA of other surfactants that are purchased incomplete.Owing to there is demand for this purpose to improved cationic surfactant, US 5,010,183 inventor searches for the new method that comprises isolation of RNA the blood from biological sample, this method comprises the waterborne cation surfactant solution that uses the quaternary amine that comprises selection, and (US 5,985,572).The novel aqueous quaternary amine surfactants that can stablize from the RNA of biological sample also is documented among WO94/18156 and the WO02/00599.Can be according to the synthetic possible different surfaces activating agent that can be used for any method of the present invention of the rules of announcing in the above-cited or relevant patent.An example that can be used for the quaternary amine of method of the present invention is myristyl trimethyl ammonium oxalate (US 5,985,572).Alternatively, described cationic detergent can be the Catrimox-14TM (US5,010,183) as shown in embodiments of the invention 1.Further about the stabilisation of described biological sample, described application has been described and has been used conventional isolation technics such as the column chromatography isolating nucleic acid.Because forcibly with PAXgene ^TMBlood rna pipe and PAXgene ^TMBlood rna kit (also using column chromatography) is combined, and supplier hints PAXgene ^TMThe compound that exists in the blood rna pipe may be only compatible with described chromatography.

In one aspect of the invention, stabilizing agent is contained in the described container until such as when biological sample mixes with the pulsation agent, and/or the user is when need introduce stabilizing agent.

Container can be integrated in the vessel, means that this container can mechanically be bonded together with these vessel, thereby forms the integral type parts.According to an aspect of the present invention, the inside of described container is connected with the inside of described vessel, and has the physical barriers of the described connection of blocking-up.Carve in due course, the power of applying is opened described physical barriers, allows stabilizing agent to mix with the biological sample of so pulsation.According to an aspect of the present invention, according to the physical force that applies, physical barriers is opening and closing reversibly.The power that applies can be passed to physical barriers itself, or is passed to physical barriers through stabilizing agent.Physical barriers can be any mechanical barrier.The example of such physical barriers comprises revolving valve, hole valve (aperture valve), slit valve (slit valve), diaphragm valve (diaphragm valve), ball valve (ball valve), clack valve (flap valve).According to another aspect of the present invention, can irreversibly open physical barriers by applying power.Physical force can be any mechanical force.The power that applies can be passed to physical barriers itself and (referring to for example, Fig. 7), or be passed to physical barriers (referring to for example, Fig. 8) through stabilizing agent.Another example of such physical barriers comprise the connector that is extruded the appropriate location (referring to, for example, Fig. 1), broken barrier after applying power (referring to, for example, Fig. 7).

According to another aspect of the present invention, the inside of described container is connected with the inside of described vessel, and stops stabilizing agent to flow to vessel from container by the surface tension of stabilizing agent with the combination of the described pore-size that is connected.According to this aspect of the invention, carve in due course, apply and be passed to the moving stabilizing agent of trying hard to recommend of stabilizing agent and enter vessel from container.This power can for example apply by pushing, putting upside down continuously and stir.

Another aspect of the present invention relates to contrast vessel as described herein, and described contrast vessel further comprise the control container that wherein has stabilizing agent; Described stabilizing agent temporarily is prevented from contacting with the biological sample that contrasts the pulsation agent or be exposed to described contrast pulsation agent.Vessel with container of integration as herein described, and can there be the part as kit together in the contrast vessel with control container of integration.Alternative, the outside of the vessel of the container with integration as herein described, and the outside of contrast vessel with control container of integration can use bridging element to engage or connect, to form single assembly (Figure 19 and 20).

Control container can be integrated in the contrast vessel, means that control container can mechanically engage to form the integral type parts with the contrast vessel.According to another aspect of the present invention, the inside of described control container is connected with the inside of described contrast vessel, and has the physical barriers of the described connection of blocking-up.Carve in due course, the power of applying is opened described physical barriers, allows stabilizing agent to mix with the biological sample of so pulsation.According to an aspect of the present invention, according to the physical force that applies, physical barriers is opening and closing reversibly.The power that applies can be passed to physical barriers itself, or is passed to physical barriers through stabilizing agent.The example of such physical barriers comprises revolving valve, hole valve, slit valve, diaphragm valve, ball valve, clack valve.According to another aspect of the present invention, can irreversibly open physical barriers by applying power.The power that applies can be passed to physical barriers itself and (referring to for example, Figure 17), or be passed to physical barriers (referring to for example, Figure 18) through stabilizing agent.Another example of such physical barriers comprise the connector that is extruded the appropriate location (referring to, for example, Figure 12), broken barrier after applying power (referring to, for example, Figure 17).

According to another aspect of the present invention, the inside of described control container is connected with the inside of described contrast vessel, and stops stabilizing agent to flow to the contrast vessel from control container by the surface tension of stabilizing agent with the combination of the described pore-size that is connected.According to this aspect of the invention, carve in due course, apply and be passed to the moving stabilizing agent of trying hard to recommend of stabilizing agent and enter the contrast vessel from control container.This power can for example apply by pushing, putting upside down continuously and stir.Vessel as described herein or contrast vessel, it comprises container or the control container that is used to distribute stabilizing agent respectively, allow deconditioned technical staff to use blood that pulsation agent or contrast make blood pulses and stable so pulsation to analyze by those of skill in the art in the stage after a while being used for.Therefore, gather at needs under the situation of many samples, can save cost, make blood pulses and stablize described blood because can employ unskilled operator as vessel disclosed herein or contrast vessel.In addition, vessel or contrast vessel are allowed repeatability, because the pulsation agent of known quantity and stabilizing agent can be supplied in the described vessel in advance, make like this and the error minimize of inhaling the phase shift pass.In addition, greatly reduced to extract biological sample and made described sample be exposed to time between pulsation antigen or the contrast, because sample can directly be evacuated in the described pipe, or for example via syringe.In addition, owing to realize stabilizing agent is introduced in the sample by applying power simply, can set the time that makes between biological sample pulsation and the stabilizing biological sample exactly; Therefore do not exist because suction moves the delay that stabilizing agent causes.

Another embodiment of the present invention is to be suitable for making the biological sample pulsation with the pulsation agent, to wherein introducing so kit of the RNA composition in the biological sample of pulsation of stabilizing agent and test, it comprises one or more vessel as disclosed above and one or more container that wherein has described stabilizing agent subsequently.This kit also further comprises the contrast vessel that wherein have biological contrast pulsation agent.

In an embodiment of kit, the container unconformity that wherein has a described stabilizing agent is to the vessel that wherein have the pulsation agent, and/or the control container unconformity that wherein has a described stabilizing agent is to the contrast vessel that wherein have the contrast material.

Therefore container or control container can be separated, and can be any containers of this area, and it is suitable for holding the stabilizing agent in the kit.According to an aspect of the present invention, it is sealed the container or the control container of stabilizing agent to be housed.Container or control container can have water-tight equipment such as for example again, nut, and pusher cap is renovated.Container or control container can have easily broken strip of paper used for sealing such as peel-open viscosity strip of paper used for sealing, break the formula strip of paper used for sealing.According to an aspect of the present invention, the wall of container or control container can be equipped with reclosable zone such as barrier film; Stabilizing agent can be extracted out by using syringe needle to pierce through from container or control container.The barrier film of container or control container can be reclosable after piercing through.According to specific embodiment, container or control container and be used in combination at the transfer tube that arbitrary end disposes pin (for example, trochar).Respectively, a pin is set the barrier film that is used to pierce through container or control container, and another root pin is set the barrier film (hereinafter described) that is used to pierce through vessel or contrast vessel; Use transfer tube respectively stabilizing agent to be transferred to vessel or contrast vessel from container or control container.This pipe can be flexible or rigidity.The shape of transfer tube can be a U-shaped.Container or control container can comprise one or more accessories, and described accessory is suitable for connecting and describedly is equipped with the vessel of complementary connecting device and stabilizing agent is transferred to described vessel or contrast vessel.For example, container or control container can be equipped with Rule accessory, this Rule accessory can admit aforesaid be connected to described vessel (referring to, for example, Fig. 9,10 and 11), or be connected to Rule accessory of the complementation of described contrast vessel.In another embodiment, container or control container can be equipped with non-Rule accessory, and vessel or contrast vessel that this non-Rule accessory can connect design with the non-Rule with complementation closely cooperate.Stabilizing agent can be transferred to vessel or contrast vessel by the water-tight equipment again of opening vessel; Stabilizing agent can leave container or control container via any above-mentioned accessory or opening.Container or control container can randomly have the equipment that allows air to enter when stabilizing agent leaves.In one aspect of the invention, container or control container can have the equipment of promotion from the stabilizing agent of described container or control container; Example includes but not limited to the syringe type plunger, but the extruded wall of container or control container or apply any equipment of malleation.Container or control container randomly have the measurement device of the volume that is used for determining the stabilizing agent that is assigned with, for example, and meter.In one aspect of the invention, container or control container are held the volume of the stabilizing agent that is enough to the single use.In another aspect of the present invention, container or control container are held the volume of the stabilizing agent of the test that is enough to repeatedly to pulse.

According to another aspect of the present invention, container or control container are equipped with aforesaid salable barrier film, it is suitable for admitting first pin (trochar) and second pin, can leave vessel by the first pin stabilizing agent, can apply malleation or negative pressure to vessel inside by second pin.When applying malleation by second pin, stabilizing agent can be pushed out by first pin from container or control container.Preferred first pin is the part of foregoing transfer tube.Second pin can be the part of forcing pipe described below, and described forcing pipe is connected with air (extracting out or pressurization) pump.First pin and second pin can engage in the mode of parallel alignment.

In another embodiment of kit, wherein have the container of stabilizing agent and be connected with the vessel that wherein have the pulsation agent; The embodiment of vessel as mentioned above.In another embodiment of kit, wherein have the control container of described stabilizing agent and be connected with the contrast vessel that wherein have the contrast material; The embodiment of contrast vessel as mentioned above.

Randomly, kit of the present invention can comprise the specification handbook, and described handbook comprises the description to the method that is used to make the biological sample pulsation.

Another aspect of the present invention relates to as vessel disclosed herein and the kit of vessel as described in comprising, and wherein said pulsation agent comprises antigen.According to an aspect of the present invention, described antigen is bacterium LPS.According to another aspect of the present invention, described antigen is the immune response recall antigen.According to another aspect of the present invention, described antigen is tetanus toxoid.According to another aspect of the present invention, described antigen is cancer immunotherapy antigen.According to another aspect of the present invention, described antigen is MAGE-3.According to another aspect of the present invention, described antigen is the cat allergen.According to another aspect of the present invention, described antigen is Feld1.According to another aspect of the present invention, described antigen is the antigen presenting cell from organ donor.According to another aspect of the present invention, described antigen is autoantigen.According to another aspect of the present invention, described antigen is GAD65.

Parameter when contrast pulsation agent will be depended on pulsation agent and research is as those of skill in the art fully understand.For example, when the pulsation agent is a peptide, one group of peptide, or during peptide mixer, contrast pulsation agent can comprise one or more peptides with equal length, and difference is the fragment that the sequence of described one or more peptides was randomized and/or was different from existing albumen or peptide.This contrast pulsation agent will with the whole blood identical and the reagent of same amount, the blood of equal volume identical time period of incubation together and under identical temperature with the pulsation agent.In another embodiment, the pulsation agent can be the protein for treatment agent, such as the IFN-β that is dissolved to desired concn in excipient such as water, PBS or salt solution.Then contrast pulsation agent can be this excipient.In a further embodiment, the pulsation agent can be an antigen, such as the Feld1 that is dissolved to desired concn in excipient such as water, PBS or salt solution; Then contrast pulsation agent can be the excipient of this antigen.Alternatively, the contrast vessel can not have dummy or empty, in the case, for example, the immune response of inducing, or the gene expression of inducing is compared with untreated sample.

Another aspect of the present invention relates to antigen pulses biological sample, and stable subsequently nucleic acid is wherein also tested the method for the RNA composition in the stabilizing biological sample of so pulsing.This method comprise use randomly gather, pulse as vessel disclosed herein, contrast vessel and/or kit and stablize as described in sample.

One embodiment of the invention are the devices that are used to accept liquid biological sample, and it helps to make described sample to be exposed to first material and contrast material respectively, and is exposed to the nucleic acid stability agent subsequently, and described device comprises:

Wherein there is first material in-the first Room,

Wherein there is the contrast material in-the second Room,

-Di three Room, wherein exist stabilizing agent and

-be used for the support of biological sample pipe.

The example of device is presented among Figure 21 a and the 21b.According to an aspect of the present invention, at least one (for example, 1,2,3, all), preferred institute has family sealed.One or more (for example, 1,2,3, all) chamber can have water-tight equipment such as for example again, nut, and pusher cap is renovated.One or more (for example, 1,2,3, all) chamber can have easily broken strip of paper used for sealing such as peel-open viscosity strip of paper used for sealing, breaks the formula strip of paper used for sealing.According to an aspect of the present invention, one or more (for example, 1,2,3, all) chamber is equipped with reclosable zone such as barrier film, can material be introduced in the chamber by using syringe needle to pierce through by this barrier film.The barrier film of chamber can be reclosable after piercing through.

According to specific embodiment, one or more (for example, 1,2,3, all) (disposing hollow needle (for example, the trochar of hypodermic syringe needle) hollow pipe, as shown in for example Figure 22) at arbitrary end is used in combination for chamber and biological sample pipe and transfer tube.Pin is set and is used for piercing through a chamber (for example, first Room) barrier film, and another root pin is set the barrier film that is used for piercing through another chamber (for example, the 3rd Room).Alternatively, pin is set and is used for piercing through a chamber (for example, first Room) barrier film, and another root pin is set and is used for from biological sample pipe (by stent support) withdrawn fluid; In this way, two chambers or chamber and biological sample pipe can through described pipe provisionally fluid be connected.Fluid connects permission use transfer tube will be for example, and stabilizing agent is transferred to first Room and/or second Room from the 3rd Room.Described pipe can be flexible or rigidity.Transfer tube can be a U-shaped.

Biological sample pipe according to the present invention can be any pipe that is suitable for preserving biological sample.According to an aspect of the present invention, the biological sample pipe is sealed.According to an aspect of the present invention, the biological sample pipe has water-tight equipment such as, nut again, and pusher cap is renovated.According to an aspect of the present invention, biological sample can be introduced in the pipe by using syringe needle to pierce through to tube wall.Tube wall can be in that to pierce through the back reclosable, or tube wall can be not reclosable after piercing through, or the wall of sample cell can be equipped with reclosable zone such as barrier film.According to an aspect of the present invention, biological sample can be extracted out from the pipe that is introduced into by transfer tube, and described transfer tube is included in arbitrary end and disposes pin () hollow pipe for example, trochar is as described in this paper elsewhere.A pin is set the barrier film that is used for piercing through the sample cell that holds described sample or other liquid, and another root pin is set the barrier film that is used for piercing through the chamber; Use described pipe liquid biological sample can be transferred to the chamber from sample cell.Described pipe can be flexible or rigidity.The shape of transfer tube can be a U-shaped.According to an aspect of the present invention, biological sample can be introduced in the sample cell by means of one or more accessories, and described accessory is connected with sample cell, is suitable for syringe or other are equipped with the container of connecting device.For example, pipe can be equipped with Rule accessory, and described Rule accessory can be admitted needleless injector.In another embodiment, pipe can be equipped with non-Rule accessory, and the container that described non-Rule accessory can connect design with the non-Rule with complementation closely cooperates.According to an aspect of the present invention, biological sample can be by means of trochar or hypodermic syringe needle and is introduced in the described pipe, and described trochar or hypodermic syringe needle and described pipe fit are suitable for directly extracting biological sample from individuality.According to an aspect of the present invention, biological sample can water-tight equipment be introduced in the sample cell by opening again.

In one aspect of the invention, first Room can have and is used for liquid biological sample is evacuated to equipment first Room from the biological sample pipe when being connected with the biological sample pipe fluid.Second Room can have similarly and is used for liquid biological sample is evacuated to equipment second Room from the biological sample pipe when for example using transfer tube to be connected with the biological sample pipe fluid.The example of such equipment includes but not limited to use the syringe type plunger, or applies negative pressure to receiving chamber, or applies any equipment of malleation to supply room.According to the preferred embodiments of the invention, use the pressure in the following insertable forcing pipe control room.First Room and/or second Room can partly remain under the vacuum.

In one aspect of the invention, first Room can have and is used for stabilizing agent is evacuated to equipment first Room from the 3rd Room when being connected with the 3rd Room fluid.Second Room can have similarly and is used for stabilizing agent is evacuated to equipment second Room from the 3rd Room when for example using transfer tube to be connected with the 3rd Room fluid.The example of such equipment includes but not limited to use the syringe type plunger, or applies negative pressure to receiving chamber, or applies any equipment of malleation to supply room.According to the preferred embodiments of the invention, use the pressure in the following insertable forcing pipe control room.First Room and/or second Room can partly remain under the vacuum.

In another aspect of the present invention, the 3rd Room can have and is used for that when for example using transfer tube to be connected with first Room and/or the second Room fluid stabilizing agent is advanced into first Room from the 3rd Room and/or is advanced into equipment second Room.The example of such equipment includes but not limited to the syringe type plunger, or applies any equipment of malleation to the 3rd Room.According to the preferred embodiments of the invention, use the pressure in the following insertable forcing pipe control room.The 3rd Room can remain on direct draught.

According to an aspect of the present invention, first Room is aseptic.According to another aspect of the present invention, second Room is aseptic.According to another aspect of the present invention, the 3rd Room is aseptic.According to another aspect of the present invention, it is aseptic having family.

According to one embodiment of the invention, each chamber is equipped with aforesaid sealable barrier film, is suitable for admitting first pin and second pin, and liquid can pass through first pin, and can apply malleation or negative pressure to the inside of chamber by second pin.When applying malleation by second pin, liquid can be pushed out from the chamber by first pin.Alternatively, when applying negative pressure by second pin, liquid can be drawn in the chamber by first pin.Preferably, first pin is the part of foregoing transfer tube.Second pin can be the part of forcing pipe.First pin and second pin can engage in the mode of parallel alignment.Referring to, for example, Figure 24.

When needs, one or more (for example, 1,2,3, all) chamber can randomly dispose passage, and described passage allows indoor pressure to equate with environmental pressure.Passage can comprise check valve (for example, revolving valve, hole valve, slit valve, diaphragm valve, ball valve, clack valve), and described check valve is set and is used to provide gas passing through on direction only, and for example, it can allow to discharge the gas of discharge.

According to another aspect of the present invention, described device further is equipped with the support that is used for the biological sample pipe, is also referred to as the sample cell support in this article.The sample cell support can be any suitable structure, structures machinery or other modes, and it keeps sample cell to be in stand up position.In preferred embodiments, the sample cell support is the hole that wall is arranged, and it has and the shape of the base regions complementation (reciprocal) of sample cell at least.

According to another aspect of the present invention, described device further is equipped with the support that is used for transfer tube, is also referred to as pipe holder in this article.Pipe holder can be any suitable structure, structures machinery or other modes, and it is suitable for transfer tube removably is connected to described device.Pipe holder allows to preserve transfer tube in transportation and non-use period.It also provides clean environment for the pin end of transfer tube.In preferred embodiments, the transfer tube support comprises two grooves, and each groove has the shape complementary with each end of transfer tube.

Described device can take to satisfy any form to the requirement of chamber and support component.Should be understood that it should preferably be optimized for size, weight, enabling capabilities, durability and reuse.In a preferred embodiment of the invention, chamber mechanical connection each other.Preferably, the sample cell support also with the chamber mechanical connection.Preferably, pipe holder also with the chamber mechanical connection.Under the condition that so connects, this device is a single entity, and it has the convenience as autonomous unit (autonomous unit).Mechanical connection can use any suitable configuration as known in the art to realize, for example, adopt the chamber that forms by monolithic bio-inert material such as Merlon, polypropylene, cyclic olefine copolymer (COC) or glass that is connected with rack mechanical that identical or different material is made.

The preferred embodiments of the invention are presented among Figure 21 a and with cross section with 3-D view and are presented among Figure 21 b, it has been described by solid block material (solid block) 44 devices that form 30 such as the material of cyclic olefine copolymer (COC), it is equipped with first Room 32 that wherein has first material 2, wherein have second Room 34 that contrasts material 35 and the 3rd Room 36 that wherein has stabilizing agent 5.Chamber 32,34,36 are arranged on the cylinder open in the solid block material 44, and each chamber seals with lid 46,48,50, and described lid also can be made by COC at least in part.Alternatively, the chamber 32,34,36 can each cylindrical bottle that freely is fixed in the cylinder open that is arranged in the solid block material 44 form, and described cylindrical bottle is made by suitable material (for example, glass, polypropylene, Merlon), each personal lid 46,48,50 sealing of each bottle.Each lid 46,48,50 disposes reclosable barrier film 38,40,42, provides path for using pin inlet chamber inside.Sample cell 52 supports are arranged on the device.Described sample cell support 52 is cylinder open, and it is set the base regions that is used to support sample cell.This device further disposes pipe holder 54,56, and described pipe holder comprises two grooves, and each groove has the shape complementary with each end of transfer tube.Use the U-shaped transfer tube, the content of sample cell can be transferred in each of chamber, first (pulsation agent) chamber 32 and second (contrast) 34, and described chamber is under the negative pressure.Can use identical pin that stabilizing agent is transferred to each of first Room 32 and second Room 34 from the 3rd Room 36.

As described in the elsewhere, be included in arbitrary end and (for example dispose pin, the transfer tube of hollow pipe trochar) is used for liquid is transferred to another chamber or vessel from a chamber or vessel, by releasing described liquid to providing chamber (or container or control container) to apply malleation, or by in receiving chamber (or vessel or contrast vessel), applying the described liquid of negative pressure extracting.

The transmission bobbin is fit to pierce through reclosable (for example, rubber or siloxanes) barrier film makes fluid or air only can pass through in the pin inlet chamber (vessel or contrast vessel).The tip of pin is preferably in the slope, and its both sides are sharp, and can be (non-coring) that does not core.Described pin can be 22G, 23G, 24G or 25G, or the value in the scope between any two of aforementioned numerical value, preferred 23G.Every pin can be with discerptible waterproof lid protection to prevent entering of microorganism before use.Each pin lid can be arranged in pipe holder and be connected with pipe holder, so that when the U-shaped pin was mentioned from device, lid separated with every pin, and lid is still stayed in the pipe holder.

The length (L) (being the length of the straight line portion of pointer) of transmission bobbin can be identical or different in the transfer tube.Their length will depend on the degree of depth of chamber and depend on the volume of the liquid that will be transferred.As general principle, the length (L1) of a transmission bobbin can be for 25,26,28,30,32,33,34,35,36,38,40,42,44,46,48,50,55mm, or the value in the scope between any two of above mentioned numerical value, preferred 25-35mm, more preferably 30-35mm.The length (L2) of another root transmission bobbin can be for 25,26,28,30,32,33,34,35,36,38,40,42,44,46,48,50,55mm, or the value in the scope between any two of above mentioned numerical value, preferred 40-50mm, more preferably 42-46mm.The height of pipe (L3) will depend on available clearance space.As general principle, the length of a transfer tube (L3) can be 5,7,8,9,10,11,12,13,14,15,16 or 17mm, or the value in the scope between any two of above mentioned numerical value, preferred 10-15mm, more preferably 12-14mm.Hollow pipe can be rigidity or flexible.Preferably it is a rigidity.According to a preferred aspect of the present invention, transfer tube is formed by the minute hand with the U-shaped bending, forms transfer tube of the present invention thereby make second pin be connected to non-tip, and it has the rigidity hollow pipe.Distance (D) when adopting rigid pipe between two pins will depend on the spacing between each chamber.According to a preferred aspect of the present invention, the distance between the distance (D) between two pins and two adjacent chamber is basic identical; This can realize during with square 2 * 2 arrayed at chamber and sample holder usually.As rule, the distance between the pin can be 20,22,24,26,28,30 or 32mm, or the value in the scope between any two of above mentioned numerical value, preferred 24-28mm, more preferably 26mm.

Transfer tube can be suitable for connecting robot system (for example, manipulator or platform) thereby help the automation sample treatment.When adopting the rigidity hollow pipe, robot system is connected with transfer tube through described rigidity hollow pipe.The use of robot system allows and uses several devices of the present invention to carry out automatic assay, and it lacks potentially under the condition of other stimulants and carry out also in gnotobasis.In addition, this device can promote the transfer of liquid reagent by the effectiveness of single transfer tube.

Pressure to chamber, vessel or container can provide (Figure 23) by the hollow forcing pipe that comprises flexible pipe, described flexible pipe at one end disposes the pin of the barrier film that is used for being inserted into chamber, vessel or container, its other end is suitable for being connected with air pump, and described air pump can provide required controlled pressure to described chamber, vessel or container.This pressurization bobbin can be mechanically connected to a pin (referring to Figure 24) of transfer tube by joint (joint) such as welding point, adhint or clamp.The pressurization bobbin is suitable for piercing through reclosable (for example, rubber or siloxanes) barrier film makes air or gas only to enter or to leave chamber, vessel or container by described pin.The tip of pin is preferably in the slope, and its both sides are sharp, and can not core.Described pin can be 22G, 23G, 24G or 25G, or the value in the scope between any two of aforementioned numerical value, preferred 23G.

Described device disposes other closed chamber and comprises within the scope of the invention.Described chamber can be along arranged in a straight line or arrange in the mode (for example, 2x2,2x4 etc.) of array.Preferably, described chamber and sample holder are with 2x2 square of arrayed, and adjacent chamber and the distance between the sample holder equate.

In one embodiment of the invention, the method for biological sample pulsation is comprised the steps:

I) biological sample is introduced in the described vessel,

Ii) randomly stir described vessel,

Iii) after the preset time section, stabilizing agent is introduced in the described vessel and

Iv) test the level of its amplifying nucleic acid.

Another embodiment of the invention is the method for test individuality at the immune response of antigen, described individuality has carried out preimmunization at described antigen, described method comprises uses vessel as disclosed herein, and the agent of wherein pulsing is the antigen that is studied, and comprises the steps:

A) will be introduced into from the blood sample that described individuality obtains the vessel,

B) randomly stir described vessel,

C) after the preset time section, described nucleic acid stability agent is introduced in the described vessel,

D) level of test cell factor mRNA.

According to an aspect of the present invention, described cell factor is IL-2, IL-4, IL-13, one or more among the IFN-γ.

Another embodiment of the invention is the individual method of answering originality (hyperallergenicity) at the hypermutation of antigen of test, described method comprises uses vessel as disclosed herein, the agent of wherein pulsing is the antigen that is studied, and comprises the steps:

E) will be introduced into from the blood sample that described individuality obtains the vessel,

F) randomly stir described vessel,

G) after the preset time section, the nucleic acid stability agent is introduced in the described vessel,

H) level of test I L-4 mRNA.

Another embodiment of the invention is the individual method of repelling organ graft of test, and described method comprises uses vessel as disclosed herein, and the agent of wherein pulsing is the histocompatibility antigen of donor, and comprises the steps:

I) will be introduced into from the blood sample that described individuality obtains the vessel,

J) randomly stir described vessel,

K) after the preset time section, the nucleic acid stability agent is introduced in the described vessel,

L) level of test I L-2 mRNA.

Above-mentioned method can be suitable for using with device of the present invention, for example, the method for the liquid biological sample pulsation that one embodiment of the invention are to use device of the present invention to make to be present in the biological sample pipe, described method comprises the steps:

I) use the U-shaped pin that liquid biological sample is transferred to second Room that wherein has dummy from the biological sample pipe,

Ii) use the U-shaped pin that liquid biological sample is transferred to first Room that wherein has the pulsation agent from the biological sample pipe,

Iii) use the U-shaped pin that stabilizing agent is transferred to the biological sample pipe from the 3rd Room,

Iv) use the U-shaped pin that stabilizing agent is transferred to second Room that wherein has dummy and liquid biological sample from the 3rd Room,

V) use the U-shaped pin that stabilizing agent is transferred to first Room that wherein has pulsation agent and liquid biological sample from the 3rd Room,

Make the liquid biological sample pulsation with pulsation agent and dummy thus, and stable so sample of pulsation.

Above-mentioned additive method, promptly the test individuality is at the method for the immune response of antigen, and described individuality has carried out preimmunization at described antigen; The individual method of answering originality at the hypermutation of antigen of test; The individual method of repelling organ graft of test can be suitable for using with device of the present invention similarly.

Another aspect of the present invention relates to antigen makes the biological sample pulsation, and the method for stablizing nucleic acid wherein subsequently and testing the RNA composition in the stabilizing biological sample of so pulsing said method comprising the steps of:

A) use aforesaid kit, device and/or method, make described biological sample pulsation and add inhibition RNA degraded and/or gene induced compound to it with the pulsation agent,

B) form the sediment that comprises nucleic acid,

C) the described sediment with step (B) separates with supernatant,

D) the described sediment of use buffer solution dissolving step (C) forms suspension,

E) use automation equipment isolating nucleic acid from the described suspension of step (D),

F) distribution/distribution of use automation equipment is used for the reagent mixture of RT-PCR,

G) nucleic acid that uses automation equipment to separate in step (E) distributes/is distributed in the reagent mixture that has distributed of step (F), and

H) with the automation setting, the nucleic acid/RT-PCR reagent mixture of use step (G) is determined the interior level of the body of transcript.

Said method can randomly adopt contrast as herein described, and described biological sample is exposed to described contrast.Contrast does not contain reagent or contains contrast pulsation agent.This method is carried out when being exposed to the pulsation agent basically, and carries out in the mode identical with the method that adopts vessel.The result of contrast vessel can be used for the result of standardization available from vessel.

When biological sampling, suppress the RNA degraded and/or gene induced be vital, its objective is and reclaim the RNA mixing pit that described RNA mixing pit can be used for determining transcript level in the body.Cell RNA can use PAXgene ^TMThe blood rna system is with its complete form purifying, yet the present invention proves the not interior level (referring to embodiment 2) of the real body of energy measurement of this " same " system of use.

Level only can be used and be suppressed extracellular and/or intracellular rna degraded and/or gene induced compound when the RNA mixing pit by the biological sample preparation that is stabilized begins in the body of show nucleic acid transcript of the present invention, measures/determine/quantitative; Use automation equipment to carry out the separation of nucleic acid thus, use the automation equipment branch to be used in the reagent mixture and the nucleic acid that separates of RT-PCR reaction thus, and carry out the mensuration of transcript level thus with the automation setting.According to the present invention, only this mode allows with RNA in the quantitative body of reproducible mode.The number of steps of carrying out in described method is reduced to minimum, so that avoid error." error " can be inhale move, in the processing, program and/or any error of calculating the error that causes or can causing by those skilled in the art.Thus, the present invention's suggestion is carried out RT and PCR reaction with single step.Method of the present invention when merging more intermediate steps should in addition more accurate.For example, in the method for the invention, can combining step (A) and (B).

In another aspect of the present invention, the distribution of nucleic acid (step (G)) can distribute the needed reagent mixture of RT-PCR (step (F)) to carry out afterwards, before or simultaneously.

The method according to this invention does not need to carry out OD and measures, and has eliminated the error that causes when calculating nucleic acid concentration.On the contrary, use complete PAXgene ^TMThe blood rna kit need carry out OD and measure.This has illustrated the method according to this invention once more, and to compare be more reliable and accurate method with a kind of system in back.For example understand this accuracy preferably of the present invention by the repeatability research that provides in the table 1.

In another aspect of the present invention, when in the step (D) in the method according to this invention during lysigenous sediment, the use that can combine with full automatic RNA extraction method and analytic approach of the suspension of acquisition.Only this combination allows the accurate optimization and the repeatability of the method carried out, and allows accurately and reproducibly to determine after pulsation rna level.Because PAXgene ^TMThe brochure of blood rna system is described corresponding pipe and cannot be used in combination with other separation methods, and does not have detailed information to describe the difference composition of this kit, therefore uses this PAXgene to those skilled in the art ^TMThe part of blood rna system and by its exploitation new method be not conspicuous.

Only there is a small amount of full automation ground isolation of RNA commercial system that allows.The example of this type of automatic nucleic acid extractor is: MagNA Pure LC instrument (Luo Shi diagnoses (Roche Diagnostics)), AutoGenprep 960 (Autogen), ABI PrismTM 6700 automatic nucleic acid work stations (applying biological system (Applied Biosystems)) have optional WAVE

The WAVE of fragment gatherer (Fragment Collector) FCW 200

Foranalysis of nucleic acids system (Transgenomic) and BioRobot 8000 (Qiagen).

The present invention is directed to such fact: be to use such material to begin for all these systems are requisite, described material is fresh as far as possible, or stabilized so that allow to determine the transcript level after pulsation, and wherein the RNA degraded is minimized.Problem about all these systems is that biological sample gathers and take to the laboratory in such pipe, and described pipe does not contain or only contains conventional additives, makes mRNA still can degrade apace.Therefore, use these methods to carry out the transcript that mRNA exists in quantitatively undoubtedly can quantitative described pipe, but this transcript level that exists in cell/biological agent when quantitatively not representing sampling.This experimental evidence is provided among Figure 32 .2 of embodiments of the invention 1.

The implication of term " quantitatively " is accurately and reproducibly to determine the RNA copy number; But not too importantly also can use RNA to carry out qualitative or sxemiquantitative research for those skilled in the art via method separation as described in the present invention.

Definition " transcript " is not limited to mRNA (mRNA), but also relates to the RNA molecule of the other types of the known existence of those skilled in the art.The method according to this invention can be extracted mRNA and total RNA.This allows the interior nRNA of estimated body correctly, thereby the strong instrument of estimating genetic transcription is provided.

Term " nucleic acid " is meant strand or double-strandednucleic acid sequence, and it maybe can be the cDNA of amplification or the genomic DNA of amplification that described nucleic acid can be made up of deoxyribonucleotide (DNA) or ribonucleotide (RNA), RNA/DNA crossbred, or its combination.The nucleotides that also can comprise any modification as known in the art according to nucleotide sequence of the present invention.

According to the present invention, in the biological sample amplifying nucleic acid may reside in extracellular or cell.

In the step (C) of the inventive method, sediment and supernatant " are separated " and can carry out via centrifugal, filtration, absorption or other means well known by persons skilled in the art.Described sediment can comprise cell, cell/fragment, nucleic acid or its combination.The basis of this notion is to stop containing nucleic acid reagent (biological agent) to contact with external source/pulse/signal.This can contain nucleic acid reagent by fixing, dissolve and/or decomposing, or is undertaken by any other means well known by persons skilled in the art.

The buffer solution that uses in the step of method of the present invention (D) can be the sedimentary buffer solution that obtains in the step (C) that is used for dissolving described method.This buffer solution can have additional effect such as dissolving or further dissolving contains nucleic acid reagent.

" automation equipment " that uses can be that moving device or the another kind of automation equipment that is suitable for carrying out required movement well known by persons skilled in the art are inhaled in automation.

" reagent mixture that is used for RT-PCR " is meant and carries out required all reagent (not comprising oligonucleotides when clearly mentioning) of RT and PCR reaction simultaneously.According to the present invention, " oligonucleotides " can comprise as being present in the short section nucleic acid in primer for example or the probe.According to the present invention, the method can be measured with microarray or RNA enzyme protection and be used in combination.

As noted earlier, preserve the incorrect mensuration that biological sample such as blood causes the mRNA level.In fact, in practice, the analysis of fresh sample is infeasible, because the place of sampling is positioned at different positions with the place that RNA analyzes.The method according to this invention can make biological sample be transported to suitable laboratory in the place from afar without any under the influence condition by transcript content in to its body.The transportation of biological sample can be in the method for the invention step (A) or step (B) after carry out.

Usually, when using blood sample, the preferential red blood cell of removing before isolating nucleic acid.Red blood cell is rich in hemoglobin, and their existence causes producing the lysate of high viscosity.Therefore, these removing can come isolating nucleic acid in the mode of improvement more.Yet, in the method for the invention, cancelled this step, comprise the insoluble precipitate of nucleic acid because form immediately, thereby these every other compositions with biological sample are separated.This explanation, except other advantages, it is better method that method of the present invention is compared with the most prior art method.

According to the present invention, the described buffer solution that uses in the step (D) of method of the present invention can be the buffer solution that contains guanidine-rhodanate (thiocyanate).

In an embodiment of the present invention, at PAXgene ^TMThe sediment that forms in the blood rna pipe is dissolved in the dissolving buffer solution that is provided by MagNA Pure LC mRNA separating kit I (Luo Shi diagnoses (Roche Diagnostics), molecular biochemistry product (Molecular Biochemicals)).Therefore, one of possible buffer solution that suggestion in the present invention can be used for method of the present invention is the buffer solution that contains guanidine-rhodanate that is provided by MagNA Pure LC mRNA separating kit I (Luo Shi diagnoses (Roche Diagnostics), molecular biochemistry product (Molecular Biochemicals)).

MagNA Pure LC mRNA separating kit Kit I (Luo Shi diagnoses (Roche Diagnostics), molecular biochemistry product (Molecular Biochemicals)) specialized designs is used on the MagNA Pure LC instrument to guarantee separating high-quality and undegradable RNA from whole blood, leucocyte and PBLC.According to its description of product, the RNA of acquisition is suitable for high sensitivity and quantitatively LightCycler RT-PCR reaction, and is suitable for standard module circulation instrument (block cycler) RT-PCR reaction, and Northern trace and other standard rnas are used.However, the present invention proves that " former state ground " uses the method to can not determine correct transcript level.The present invention shows the demand (referring to embodiment 1) of stablizing RNA before RNA separates that exists.The invention describes and use the compound stablize RNA to separate with automation/unique combination of routine analyzer.

According to the present invention, in case the sediment of step (D) is dissolved in the dissolving buffer solution, in the buffer solution that is provided by MagNA Pure LC mRNA separating kit I, method of the present invention can be abideed by about the described program of MagNA Pure LC mRNA separating kit I.After existing chaotropic salt to come sample dissolution by dissolving in the buffer solution, the magnetic particle of Streptavidin bag quilt with biotin labeled oligomeric-dT adds, and mRNA is incorporated into the surface of particulate.And then carry out the DNase digestion step.Use then magnet and several times washing step with mRNA and unconjugated separating substances.At last, the mRNA of wash-out purifying.This separating kit can separate pure mRNA as " from people (walk away) " system automation.It can separate and be suitable for about the high-quality of all main downstream application of gene expression analysis and the mRNA of integrality.Provide different testing programs according to the specimen material that uses.Sample can directly be placed on the MagNA pure LC instrument platform.When using whole blood, the cell that is present in the sample is preferentially manually dissolved.Can delay to carry out the mRNA separation then or directly go on foot to handle at this instrument enterprising.

The present invention proves the step (E) in the method according to this invention in an embodiment of the present invention, (Luo Shi diagnoses (Roche Diagnostics) to use MagNA Pure LC instrument in step (F) and/or the step (G), molecular biochemistry product (Molecular Biochemicals)) cause producing the RNA mixing pit as automation equipment, this RNA mixing pit can be used for determining being exposed to the accurate level of transcript after the pulsation agent.The pearl of capturing RNA such as via Streptavidin-biotin system or equivalent system turnkey by magnetic bead with oligomeric-dT, can be applied in the method for the present invention so as from cell fragment separating mRNA.

Alternatively, can use other automation equipments such as ABI PrismTM 6700 automatic nucleic acid work stations (applying biological system (Applied Biosystems)) maybe can be used for any other automation equipment of this purpose according to the present invention.

In the brochure of MagNA pure LC mRNA separating kit I (catalog number 3 004 015), do not mention the composition of the buffer solution that uses in this kit in detail.Therefore, to those skilled in the art, supposing that buffer solution that this kit provides can dissolve passes through PAXgene ^TMThe sediment that method obtained of blood rna pipe is not conspicuous.In addition, those skilled in the art can be based on by PAXgene ^TMThe information that blood rna pipe brochure provides and two kinds of methods are combined, this brochure claim these pipes only can with corresponding PAXgene ^TMThe blood rna kit combine (the 3rd page, referring to the restriction of system; The 6th page, referring to ordering information).

As noted above, when using blood sample, step in the method for the invention (A) back optimum solvation red blood cell.(Luo Shi diagnoses (Roche Diagnostics) at MagNA Pure LC mRNA separating kit I, molecular biochemistry product (Molecular Biochemicals)) in the design, before separating mRNA from leucocyte, dissolving is arranged and remove erythrocytic possibility.Yet because this step, sample can not processedly get enough soon to avoid the mRNA degraded.The inventor determines to use and is included in PAXgene ^TMStabilizing agent in the blood rna pipe combines with MagNA Pure mRNA separating kit on the MagNA Pure instrument.Use PAXgene ^TMThe blood rna pipe provides the sediment of nucleic acid, infers that this sediment can not be dissolved in the dissolving buffer solution of MagNA Pure mRNA separating kit.Even now, the inventor finds that this is actually possible.Abide by this observation, the inventor is with PAXgene ^TMThe use of the use of the stabilizing agent in the blood rna pipe and automation RNA piece-rate system combines.The inventor finds that unexpectedly this combination is possible, and this combination provides a kind of and is used for from the effective technology of biological sample accurate quantification mRNA.

The RNA that uses the method according to this invention to separate is ready for large-scale downstream application, comprises for example nucleic acid amplification technologies, such as RT-PCR and NASBA Express array and expression chip analysis, quantitative RT-PCR comprises TaqMan Technology, cDNA is synthetic, RNase and the protection of S1 nuclease, Northern, Dot blot and slot blot analysis and primer extend.

The inventor shows to use in embodiments of the invention 1 and embodiment 2 and suppresses RNA degraded and/or gene induced compound and separate with automation RNA with the combination of automated analysis method such as PCR in real time and can determine level in the body of transcript.Yet, according to the present invention, can use the analytical method except that PCR in real time, as long as they provide with the automation setting.

The major advantage of the method according to this invention is such fact, promptly by using the method can analyze little sample volume.This is a primary importance in the time only obtaining small size, for example, and when analysis neonate's blood sample or under the situation of massive blood loss (high blood loss).According to the present invention, it is quantitative to use little biological sample to 100 μ l to carry out RNA.Use Qiagen kit (PAXgenTM blood rna system) analysis come from childhood to the RNA of the sample of 100 μ l be impossible, this kit needs the blood (is 2.5ml according to this kit handbook) of larger volume.

As mentioned above, one aspect of the present invention is to be suitable for making biological sample pulsation with the pulsation agent, the kit of the nucleic acid of the stable subsequently biological sample from pulsation like this.In another aspect of the present invention, described kit comprises the annexing ingredient that is used for separating from biological sample stable, pulsation RNA that can be quantitative.According to an aspect of the present invention, described kit can comprise such as following annexing ingredient:

-be used for the reagent that automation RNA separates,

-being used for carrying out simultaneously independently compound of the reagent mixture of RT and real-time PCR reactions or its, it allows automation to distribute described mixture,

-randomly, be used for carrying out described RT-PCT reaction specific oligonucleotide and

-randomly, and the specification handbook, the method that its explanation automation RNA separates is for the automation of RT-PCR in real time distributes the method for reagent mixture and the nucleic acid that separates and the method that automation RNA analyzes.

In embodiments of the present invention, the inventor has used from Luo Shi diagnosis (Roche Diagnostics), and single step RT-PCR reaction is carried out in " Lightcycler mRNA hybridization probe kit " (catalogue #3 018 954) of molecular biochemistry product (Molecular Biochemicals).All regent pack that need are contained in this kit, except oligonucleotides (synthetic by Biosource).Yet PCR in real time also can be at other instruments, such as carrying out on applying biological system (Applied Biosystems) instrument as described in the present invention.This kit can additionally comprise buffer solution such as the buffer solution that contains guanidine-rhodanate, and it can be used for the step (b) of the method according to this invention.

The method according to this invention also can be used for quantitatively/DNA (two strands or strand) of detection of biological sample.Therefore, the invention still further relates to the method that is used for quantitatively from the DNA of biological sample, wherein use as, wherein saved the RT reaction, and wherein the compound of step (a) protects also DNA not to be degraded the method that quantitative RNA carried out according to the present invention.Because these nucleic acid are more stable than RNA, so it is stable more important than not too with the stable phase of RNA.

In addition, the present invention relates to the kit that is used for separating DNA that can be quantitative according to of the present invention, wherein lack the reagent mixture/compound that is used to carry out described RT reaction from biological sample.Situation that need to determine accurate dna level in the biological sample can be used for determining " existence " of one or more infection/one or more pollutions of being caused by unexpected gene, pathogen or parasite in the biological sample; And/or be used for determining " level " of described infection/pollution.For example, this method can be used for determining the percentage of cereal batch of material (cereal batch) transfer genetic stew.

The invention still further relates to use according to device of the present invention, kit and method, change with the nucleic acid in vivo of monitoring/detection, thereby diagnose specific disease with biomarker in a kind of reagent pulsation artifact sample.

The invention still further relates to use according to device of the present invention, kit and method, change with the nucleic acid in vivo of monitoring/detection with biomarker in a kind of reagent pulsation artifact sample, thus screening compounds, and described compound is used to prepare the medicine for the treatment of disease.

Therefore, the invention still further relates to and to pass through the method according to this invention compounds identified.

Device disclosed herein, kit and method can be used for treating and/or diagnosing the illness.An example of the disease that will be treated or diagnose is an immune correlated disease.According to the present invention, the example of immune correlated disease can be autoimmunity (autoimmunity), rheumatoid arthritis (rheumatoid arthritis), multiple sclerosis (multiple sclerosis), type 1 diabetes, cancer (for example, in cancer immunotherapy), immune deficiency (immunodeficiencies) (for example, in AIDS), allergy, graft rejection (graft rejection) or graft versus host disease(GVH disease) (Graft versus Host Disease, GVHD) (for example, in transplanting).Disclosed embodiment at length for example understands described application among the present invention.Therefore, immune regulative compound or reagent can influence one of described disease; Ia transcript or epitope specificity CTL the variation relevant or transcript that the auxiliary lymphocyte of T is relevant can be indicated the existence and/or the state of one of described disease; And the amynologic state that the state of one of described disease can be described.

Can use device of the present invention, kit and method can be for example to encode so that study the nucleic acid of described immune correlated disease quantitatively, the member of the cytokine receptor superfamily that chemotactic factor (CF), cell factor, growth factor, cytotoxicity mark, transcription factor, TNF are correlated with and their part, apoptosis mark, immunoglobulin (Ig), TXi Baoshouti are with the nucleic acid of any mark relevant with immune activation or inhibition known or to be found.

According to the present invention, described nucleic acid can the coding maker thing such as IL-1ra, IL-1 β, IL-2, IL-4, IL-5, IL-9, IL-10, IL-12p35, IL-12p40, IL-13, TNF-α, IFN-γ, IFN-α, TGF-β and participate in or do not participate in any interleukin or the cell factor of immune response.House-keeping gene such as beta-actin or GAPDH (GAPD) can be used as the inner mark thing.

According to the present invention, the nucleic acid of the albumen of signal transduction path in described epitope specificity CTL the member relevant or cytokine receptor superfamily that the relevant transcript of the auxiliary lymphocyte of T is the Codocyte factor, cytokine receptor, cytotoxin, inflammatory or anti-inflammatory medium, TNF is relevant and their part, G-G-protein linked receptor and their part, tyrosine kinase receptor and their part, transcription factor and the participation cell.

According to the present invention, the described nucleic acid granzyme of can encoding, any mark among perforin (perforine), prostaglandin, leukotriene, immunoglobulin (Ig) and immunoglobulin superfamily acceptor, Fas and Fas part, TXi Baoshouti, chemotactic factor (CF) and chemokine receptors, albumen-EGFR-TK C, albumen-SRCA, signal transducer and transcriptional activator (STAT), NF-kB, T-bet, GATA-3, the Oct-2.

The present invention has also described according to the application in detection/monitoring/screening compounds of device of the present invention, method or kit, wherein said compound is an immune regulative compound, and it can be selected from the group of being made up of following: eukaryotic, prokaryotic, virus, bacteriophage, parasite, medicine (natural extract, organic molecule, peptide, albumen, nucleic acid), therapeutic treatment, vaccine and graft.The application of described method is not limited to detection/monitoring/screening unification compound.Also can study the synergy of one group of material.

The invention still further relates to any application in detection/monitoring epi-position specific CTL or Ia transcript of aforesaid device, kit and method.

Also can be used for after immunological response in the monitoring body according to device of the present invention, kit and method with the medicine/therapy/vaccine therapy patient of the immune state that is easy to change the patient.According to the present invention, use patient or the detection of participating in patient's the whole blood of clinical testing of employing immunological regulation agent medicine or therapy or employing (therapeutic or preventative) vaccine pair cell factor mRNA (can extend to chemotactic factor (CF), growth factor, cytotoxicity mark, apoptosis mark or known or to be found with immune activation relevant any mark) effect, security and/or the final side effect that can be used for estimate treatment of described method in being in treatment.

The invention still further relates to and be used for device, kit and the method for amynologic state in the detection bodies with diagnosis/prognosis of being used to influence immune disease (cancer, autoimmune disease, allergy, graft rejection, GVHD etc.).

According to the present invention, use described method to detect cytokines mRNA (can extend to chemotactic factor (CF), growth factor, cytotoxicity mark, apoptosis mark or any mark relevant with immune activation known or to be found) in suffering from the direct or indirect whole blood that influences its immune disease patient, purpose is to obtain diagnosis or prognosis.

The present invention has also described by analyzing the epi-position specific CTL and has identified the compositions and methods of the amynologic state that can change the experimenter, said method comprising the steps of:

(a) one or more immunomodulators are applied to the experimenter,

(b) from the described experimenter whole blood of taking a sample,

(c) use aforesaid device, use with step (a) in identical/similar and/or different immunomodulator of using the haemocyte that exists in the whole blood sample of step (b) is pulsed,

(d) haemocyte of the pulsation of step (c) or the non-fluctuating haemocyte of step (b) are collected in the pipe, described pipe comprises and suppresses RNA degraded and/or gene induced compound, or with described compound be added to pulsation/non-fluctuating cell,

(e) form the sediment that comprises nucleic acid,

(f) the described sediment with step (e) separates with supernatant,

(g) the described sediment of use buffer solution dissolving step (f) forms suspension,

(h) use automation equipment isolating nucleic acid from the described suspension of step (g),

(i) distribution/distribution of use automation equipment is used for the reagent mixture of RT-PCR,

(j) nucleic acid that uses automation equipment to separate in step (h) distributes/is distributed in the reagent mixture that has distributed of step (i),

(k) with automation be provided with in the body of the transcript that epitope specificity CTL is relevant in the solution that has distributed of detection/monitoring/analytical procedure (j) level and

(l) evaluation can change the reagent of described experimenter's amynologic state,

Thus, Already under the situation among the experimenter, omit step (a) at the reagent of step (a).

The invention still further relates to the kit that comprises the component that to carry out top at least step (c).This kit can comprise other reagent and the specification that can carry out one or more other steps.The disclosure of this paper makes up the necessary component of required kit for the technical staff has instructed.

According to the present invention, under the situation of disease, or exist among the described experimenter under the situation of graft and can have one or more immunomodulators.In the present invention, the member of the cytokine receptor superfamily that " epitope specificity CTL relevant transcript " can be the Codocyte factor, cytokine receptor, cytotoxin (as granzyme, perforin etc.), TNF is relevant and the transcript of their part (for example, Fas and Fas part) or other cell receptors.

The present invention has also described the compositions and methods of identifying the amynologic state that can change the experimenter:

(a) one or more immunomodulators are applied to the experimenter,

(b) from the described experimenter whole blood of taking a sample,

(c) use aforesaid device or kit, use with step (a) in identical/similar and/or different immunomodulator of using the haemocyte that exists in the whole blood sample of step (b) is pulsed,

(e) form the sediment that comprises nucleic acid,

(f) the described sediment with step (e) separates with supernatant,

(k) with automation be provided with in the body of Ia transcript in the solution that has distributed of detection/monitoring/analytical procedure (j) level and

(l) identify the reagent of the amynologic state can change described experimenter, thus, Already under the situation among the experimenter, omit step (a) at the reagent of step (a).

In the present invention, " Ia transcript " can be for example to encode, member and their part or any mark relevant with immune activation known or to be found of the cytokine receptor superfamily that one or more cell factors, one or more chemotactic factor (CF)s, growth factor, cytotoxicity mark, transcription factor, TNF are correlated with.According to the present invention, under the situation of disease, or exist among the described experimenter under the situation of graft and can have one or more immunomodulators.According to experimenter of the present invention can be that the human or animal originates both.

The present invention also provide be used to diagnose/prognosis/monitoring influences the method for experimenter's immune clinical state, this method comprises the steps:

(a) from the described experimenter whole blood of taking a sample,

(b) use aforesaid device or kit, use be present in described experimenter in identical/similar and/or different immunomodulator the haemocyte that exists in the whole blood sample of step (a) is pulsed,

(c) haemocyte with the pulsation of step (b) is collected in the pipe, and described pipe comprises and suppresses RNA degraded and/or gene induced compound, or described compound is added to the cell of pulsation,

(d) form the sediment that comprises nucleic acid,

(e) the described sediment with step (d) separates with supernatant,

(f) the described sediment of use buffer solution dissolving step (e) forms suspension,

(g) use automation equipment isolating nucleic acid from the described suspension of step (f),

(h) distribution/distribution of use automation equipment is used for the reagent mixture of RT-PCR,

(i) nucleic acid that uses automation equipment to separate in step (g) distributes/is distributed in the reagent mixture that has distributed of step (h),

(j) with automation be provided with in the body of Ia transcript in the solution that has distributed of detection/monitoring/analytical procedure (i) level and

(k) in the body of the Ia transcript of detection/monitoring the variation of level and

(l) diagnosis/prognosis/immune disease of monitoring influence.

In the present invention, " clinical state " is any variation of experimenter's health, such as different diseases or the existence of graft.

Unless other regulation, all technology used herein and scientific terminology have the general identical meanings of understanding with those skilled in the art institute.Exemplary method and material are as described below, although implementing or test can be used and those similar or of equal value method and materials as herein described when of the present invention.All publications that this paper mentions and the full content of other lists of references merge by reference.Having under the situation of conflict, be suitable for this specification, comprising definition.Material, method and embodiment only are illustrative, and to be not intended to be restrictive.From following accompanying drawing, detailed description and claim, other features and advantages of the present invention will be conspicuous.

Accompanying drawing

Fig. 1 a-1d has described the embodiment according to vessel of the present invention and method.Fig. 1 a shows vessel 1, wherein has antigen particulate 2.Described vessel are equipped with the resealable equipment 3 of the inlet that is used for syringe needle.Container 4 also is the part of vessel 1; Stabilizing agent 5 is present in this container; And the connection between the inside of the inside of vessel 1 and container 4 is blocked by physical barriers provisionally, and in this example, physical barriers is a connector 25.Transmitting physical force provides with the equipment of the removing connector form with plunger 28.In this embodiment, plunger is covered by cap 23.In Fig. 1 b, biological sample 24 is introduced in the vessel by the resealable equipment 3 of syringe needle 6 by inlet, and allows that biological sample 24 is exposed to antigen 2.In Fig. 1 c, piston cap 23 is removed 26.In Fig. 1 d, introduce axostylus axostyle 7 and exert pressure 27 to it, therefore force plunger 28 to promote connector 25 away from container 4.When taking out connector 25, stabilizing agent 5 is released in the vessel 1 and allows and mixes with biological sample 24 and antigen 2.

Fig. 2 has described the embodiment that wherein there is the vessel 1 of antigen 2 according to of the present invention.These vessel can be open-topped 7, as shown in scheming to go up, maybe can be equipped with the closure or the equipment that are used to introduce sample or stabilizing agent, and the example is presented among Fig. 3-6.The body of vessel 1 also can comprise the container that wherein has stabilizing agent, as shown in Fig. 7 and 8.

Fig. 3 has described to be suitable for the embodiment of accessory of the type of the vessel shown in Fig. 2.The top 7 of vessel is equipped with Rule type accessory 8, and this accessory can be admitted Rule type accessory of the complementation on the syringe 11.This syringe can be equipped with according to biological sample of embodiment of the present invention or stabilizing agent.

Fig. 4 has described to be suitable for the embodiment of accessory of the type of the vessel shown in Fig. 2.The top 7 of vessel is equipped with reclosable barrier film 9, and described barrier film can be admitted syringe needle.This syringe can be equipped with according to biological sample of embodiment of the present invention or stabilizing agent.

Fig. 5 describes to be suitable for the embodiment of accessory of the type of the vessel shown in Fig. 2.The top 7 of vessel is equipped with the equipment that uses nut 10 to admit.

Fig. 6 describes to be suitable for the embodiment of accessory of the type of the vessel shown in Fig. 2.The top 7 of vessel is equipped with hypodermic syringe needle 19.These vessel can directly be used for from individual draw samples.

Fig. 7 has described the embodiment of the body of vessel 1, its can be for example with Fig. 2-6 in the vessel and the accessory that show be used in combination.The vessel 1 that wherein have antigen 2 comprise the container 12 that wherein has stabilizing agent 5.The all or part of material 15 by fragmentation when applying certain power of wall of a container is made.Thereby this container is equipped with and is used for sending part or all the broken equipment that makes container from user's power, comprises with sharp point (sharp point) but 14 area presseds that are connected 13.Depressing at regional 13 o'clock, but sharp point 14 contact crushing materials 15 make its fragmentation, thereby the physical barriers between removal container and the vessel allows in the moment of being determined by the user stabilizing agent to be flowed to vessel 1.

Fig. 8 has described the embodiment of the body of vessel 1, its can be for example with Fig. 2-6 in the vessel and the accessory that show be used in combination.The vessel 1 that wherein have antigen 2 comprise the container 16 that wherein has stabilizing agent 5.Connection between the inside of container and vessel 17 is physically blocked by barrier film 18, and barrier film 18 can break by applying power.When the wall of squeeze receptacle 16, pressure is sent to barrier film, causes membrane ruptures, thereby allows stabilizing agent 5 to enter in the vessel 1.

Fig. 9 has described the embodiment of container 20 of the present invention, and it is not connected with vessel.Stabilizing agent 5 is present in the container 20, and container 20 is equipped with and is suitable for Rule type accessory 22 (for example, as shown in Fig. 3 and Figure 11) of connecting with the vessel with complementary accessory.The wall of container 20 can be squeezable, can stabilizing agent be left.

Figure 10 has described the embodiment of container 29 of the present invention, and it is not connected with vessel.Stabilizing agent 5 is present in the container 29, and container 29 is equipped with and is used for Rule type accessory 22 (for example, as shown in Fig. 3 and Figure 11) of connecting with the vessel with complementary accessory.Described vessel further are equipped with plunger 30, and the power that applies thereon can make stabilizing agent 5 leave.

Figure 11 has described the embodiment according to kit of the present invention, and it comprises the vessel 1 that are equipped with Rule type accessory 8, and in this example, comprises that air that permission is replaced leaves the valve 31 of vessel.This kit also comprises container 29, is similar to and describes ground among Figure 10, has stabilizing agent 5 in the container 29.Accessory on the vessel 8 can connect with the accessory on the container 22.

Figure 12 a-12d has described the embodiment according to contrast vessel of the present invention and method.Figure 12 a shows the contrast vessel 37 that wherein have contrast material 35.The contrast vessel be equipped with the resealable equipment 3 of the inlet that is used for syringe needle '.Control container 4 ' also be the part of contrast vessel 37; Stabilizing agent 5 ' be present in the control container; And contrast inside of vessel 37 and control container 4 ' inside between connection blocked by physical barriers provisionally, in this example, physical barriers be connector 25 '.Transmit physical force with the equipment of removing connector with plunger 28 ' form provide.In this embodiment, plunger is by cap 23 ' covering.In Figure 12 b, biological sample 24 by syringe needle 6 ', via the resealable equipment 3 of inlet ' be introduced in the contrast vessel, and allow that biological sample 24 is exposed to contrast material 35.In Figure 12 c, piston cap 23 ' be removed 26 '.In Figure 12 d, introduce axostylus axostyle 7 ' and to its exert pressure 27 ', therefore force plunger 28 ' promotion connector 25 ' away from control container 4 '.Taking out 25 ' time of connector, stabilizing agent 5 ' be released in the contrast vessel 37 and allow with biological sample 24 and contrast material 35 and mix.

Figure 13 has described the embodiment according to the contrast vessel 37 of wherein existence contrast material 35 of the present invention.These contrast vessel 37 can be open-topped 7 ', as shown in scheming to go up, maybe can be equipped with the closure or the equipment that are used to introduce sample or stabilizing agent, the example is presented among Figure 14-16.The body of contrast vessel 37 also can comprise the control container 12 that wherein has stabilizing agent ', 16 ', as shown in Figure 17 and 18.

Figure 14 has described to be suitable for the embodiment of accessory of the type of the contrast vessel shown in Figure 13.Top 7 ' be equipped with Rule type accessory 8 of contrast vessel 35 ', this accessory can admit syringe 11 ' on Rule type accessory of complementation.This syringe can be equipped with according to biological sample of embodiment of the present invention or stabilizing agent.

Figure 15 has described to be suitable for the embodiment of accessory of the type of the contrast vessel shown in Figure 13.The reclosable barrier film 9 in top 7 ' be equipped with of contrast vessel 37 ', described barrier film can be admitted syringe needle.This syringe can be equipped with according to biological sample of embodiment of the present invention or stabilizing agent.

Figure 16 has described to be suitable for the embodiment of accessory of the type of the contrast vessel 37 shown in Figure 37.The equipment of nut 10 ' admittance is used at the top 7 ' be equipped with of contrast vessel 37.

Figure 17 has described the embodiment of the body of contrast vessel 37, its can be for example with Figure 14-16 in the contrast vessel and the accessory that show be used in combination.Wherein have the contrast material 35 contrast vessel 37 comprise wherein have stabilizing agent 5 ' control container 12 '.The all or part of material 15 of the wall of control container ' make by fragmentation when applying certain power.Thereby this control container is equipped with and is used for sending part or all the broken equipment that makes control container from user's power, but comprise with the area pressed 13 of sharp point 14 ' be connected '.Depressing regional 13 ' time, but sharp point 14 ' contact crushing material 15 ', make its fragmentation, thereby remove the physical barriers between control container and the contrast vessel, allow stabilizing agent to be flowed to contrasting in the vessel 37 in the moment of determining by the user.

Figure 18 has described the embodiment of the body of contrast vessel 37, its can be for example with Figure 14-16 in the contrast vessel and the accessory that show be used in combination.The contrast vessel 37 that wherein have tester 35 comprise the control container 16 that wherein has stabilizing agent 5 '.Connection between the inside of control container and contrast vessel 37 is by barrier film 18 ' physically blocking-up, barrier film 18 ' can break by applying power.Extruding control container 16 ' wall the time, pressure is sent to barrier film, causes membrane ruptures, thereby allows stabilizing agent 5 to enter in the contrast vessel 37.

Figure 19 has described the embodiment of device of the present invention, and described device comprises contrast vessel 37 as shown in Figure 17, and the outside of described contrast vessel 37 is connected with vessel 1 as shown in Figure 7 by bridging element 70.This device was allowed biological sample before stabilizing agent 5, was delivered to first material 2 and contrast material 35 respectively.Because response sample and control sample (side-by-side) concurrently keep, therefore avoided the error that occurs along with mixing control sample and response sample.

Figure 20 has described the embodiment of device of the present invention, and described device comprises contrast vessel 37 as shown in Figure 18, and the outside of described contrast vessel 37 mechanically is connected with vessel 1 as shown in Figure 8 by bridging element 70.This device was allowed biological sample before stabilizing agent 5, was delivered to first sample 2 and contrast material 35 respectively.Because response sample and control sample keep concurrently, therefore avoided the error that occurs along with mixing control sample and response sample.

Figure 21 a has shown the schematic diagram of the embodiment of the device 30 that is used to accept liquid biological sample of the present invention, and this device helps to make described sample to be exposed to first material and contrast material respectively, and is exposed to the nucleic acid stability agent subsequently.This device comprises first Room 32 that wherein has first material 2, wherein has second Room 34 of contrast material 35, wherein has the 3rd Room 36 of stabilizing agent 5, is used for the support 52 of biological sample pipe, and described support is a cylinder open.Each chamber forms the opening in the piece material of solid material 44.Each chamber seals with the

lid

46,48,50 that disposes

reclosable barrier film

38,40,42, and described barrier film can be broken with hollow needle such as subcutaneous trochar or acupuncture.This device further disposes the support that is used for transfer tube, and described support is taked the form of two

grooves

54,56, and described groove is suitable for admitting the pin end of transfer tube.

Figure 21 b has described the cross section that A-A ' along the line passes device of the present invention, and it has at length shown the content of second (contrast) chamber 34 and the 3rd (stabilizing agent) chamber 36.

Figure 22 has shown the viewgraph of cross-section of transfer tube 60 of the present invention, and described transfer tube comprises hollow thin long tube 64, and each end of described pipe disposes hollow needle (for example, trochar or hypodermic needle) 62,66 thus.Transfer tube 60 is configured to liquid can flow to another root pin 66 along pipe 64 by a pin 62.In embodiments shown,

62,66 has unequal length.

Figure 23 has shown the viewgraph of cross-section of forcing pipe 88 of the present invention, described forcing pipe comprises the flexible elongate tube 82 of hollow, an end of pipe and hollow needle are (for example thus, trochar or hypodermic syringe needle) 80 connect and the other end 85 is suitable for connecting the discharge coupling 87 of (pressurization or extract out) gas medium (for example, air) pump 86.Forcing pipe 88 is configured to pierce through the barrier film of vessel or chamber, and sets up pressure reduction therein by extracting or introduce gas medium (for example air) out.

Figure 24 shows the viewgraph of cross-section of composite set group, and described composite set group comprises forcing pipe 88 of the present invention and transfer tube 60.The one or more joints 84 of a pin 62 uses of transfer tube 60 are connected to the pin 80 of forcing pipe 88, so that two

62,80 parallel alignment.Therefore, barrier film is pierced through simultaneously by two

pins

62,80.

Figure 25 shows the viewgraph of cross-section of the transfer tube of the present invention 60 of the size with indication.D represents two minimum ranges between the pin 62,66.L1 is the length of a pin, it specifically is straight part, L2 is the length of another root pin, specifically be straight part, L3 is the distance on first imaginary line 65, and it is from

62,66 straight line portion extends towards pipe 64, its distance L 3 is limited by the end 67 of the straight line portion of pin, and to put 69 be second imaginary line 63 and first imaginary line, 65 intersections, second imaginary line 63 and the roof intersection of managing 64.

Figure 26-29 shows as the embodiment in 30 uses of device shown among Figure 21 a.

In Figure 26, the biological sample pipe 58 that comprises liquid biological sample (for example, blood, urine) is inserted in the pipe holder 52.One end of transfer tube 60 is inserted in the biological sample pipe 58 by barrier film 61, and the other end is inserted in second (contrast) chamber 34 by barrier film 40.60 directions at arrow flow liquid biological sample from biological sample pipe 58 along transfer tube, and enter in second Room 34, contrast material 35 in this its contact.Liquid biological sample can promote by using forcing pipe 88 (not shown)s, and the pin of forcing pipe can puncture the barrier film 38 of second Room 34 and set up vacuum, or punctures the barrier film 61 and the increase of pressure wherein of biological sample pipe 58.

In Figure 27, transfer tube has been removed and has inserted again, makes the end of transfer tube 60 in second Room 34 be arranged in first Room 32 by barrier film 38, and the end of transfer tube 60 in biological sample pipe 58 is still in the original place simultaneously.60 directions at arrow flow liquid biological sample from biological sample pipe 58 along transfer tube, and enter in first Room 32, contact first material 2 (pulsation agent) this its.Liquid biological sample can promote by using forcing pipe 88 (not shown)s, and the pin of forcing pipe can puncture the barrier film 40 of first Room 32 and set up vacuum, or punctures the barrier film 61 and the increase of pressure wherein of biological sample pipe 58.

Figure 28 shows the flushing of transfer tube 60 behind the transfer liquid biological sample, described flushing is inserted in the 3rd (stabilizing agent) chamber 36 by barrier film 42 by taking out with the end that inserts the transfer tube 60 feasible transfer tubes 60 that contact with biological sample pipe 58 in the past again, and the other end of transfer tube 60 is inserted in the biological sample pipe 58 by barrier film 61.Liquid stabilisers 5 flows from the 3rd Room 36 along transfer tube 60 in the direction of arrow, and enters in the biological sample pipe 58, preserves or is used for further processing as waste liquid this its.Liquid stabilisers 5 can promote by using forcing pipe 88 (not shown)s, and barrier film 42 and pressure wherein that the pin of forcing pipe can puncture the 3rd Room 36 increase, or puncture the barrier film 61 of biological sample pipe 58 and set up vacuum.After noting flushing, allow the sample incubation.Incubation time and temperature will depend on that Several Factors comprises the pulsation agent, the volume of sample and reagent and concentration.For rule, incubation can carry out under 37 ℃ 30 minutes-16 hours, the most typically 2-4 hour.

In Figure 29, transfer tube 60 is removed and inserts again, make an end be inserted in the 3rd (stabilizing agent) chamber 36 by barrier film 42, and the other end is inserted in second (contrast) chamber 34 by barrier film 40.Direction at arrow flows liquid stabilisers 5 from the 3rd Room 36 along transfer tube 60, and enters in second Room 34, contacts the mixture of biological sample and contrast material 2 this its.Liquid stabilisers 5 can promote by using forcing pipe 88 (not shown)s, and barrier film 42 and pressure wherein that the pin of forcing pipe can puncture the 3rd Room 36 increase, or puncture the barrier film 40 of second Room 34 and set up vacuum.

In Figure 30, transfer tube 60 is removed and inserts again, makes an end be inserted in the 3rd (stabilizing agent) chamber 36 by barrier film 42, and the other end is inserted in first (pulsation reaction) chamber 32 by barrier film 38.Direction at arrow flows liquid stabilisers from the 3rd Room 36 along transfer tube 60, and enters in first Room 32, contacts the mixture of the biological sample and first material 35 this its.Liquid stabilisers 5 can promote by using forcing pipe 88 (not shown)s, and barrier film 42 and pressure wherein that the pin of forcing pipe can puncture the 3rd Room 36 increase, or puncture the barrier film 38 of first Room 32 and set up vacuum.

When EP (end of program), first Room and second Room have all received biological sample and stabilizing agent.Move and to drive by robot by what transfer tube 60 and forcing pipe 88 caused in use, for example, have the manipulator of some one-movement-freedom-degrees or drive by X-Y robot platform (robotic table) by use.When adopting robot to drive, transfer tube will typically comprise rigid pipe 64, and this rigid pipe allows accurately clamping and location transfer tube 60.

The strategy of taking among the given embodiment of Figure 31 .1-31.4.

Figure 31 .1 is at the stripped monitoring of the immune response of tetanus toxoid.

The strategy of taking among Figure 31 .2 embodiment 3.

The strategy of taking among Figure 31 .3 embodiment 4.

The strategy of taking among Figure 31 .4 embodiment 5.

Figure 32 .1: at the RT-PCR of IFN-γ in the peripheral blood and IL-10 mRNA natural product.As described like that from from extracting total RNA 6 different healthy volunteers' (post 1-6) whole blood and the PBMC. Whole blood:In 1 minute after collected specimens with 0.6ml whole blood and 6ml Catrimox-14 ^TMMix.Sample is with the centrifugal 5min of 12000g then.The nucleic acid sediment that obtains washes with water carefully, and is dissolved in 1ml Tripure ^TMIn.Then according to Tripure ^TMManufacturer's specification carries out RNA and extracts.PBMC: from 15ml heparinize venous blood, prepare cell according to standardization program, and cytolysis is at 1ml Tripure ^TMIn be used to extract RNA.Begin to carry out about IFN-γ for all samples by the total RNA of 1 μ g, the RT-PCR of IL-10 and house-keeping gene HPRT as (Stordeur etc., (1995), Pradier etc., (1996)) are described.

Figure 32 .2: at the PCR in real time of IFN-γ in the whole blood and IL-10mRNA stability.From healthy donors, gather the sample of Citrated (citrated) venous blood.From then in the sample, in back 1 minute of blood sampling and in 5 hours per hour after, with 100 μ l aliquots and 900 μ l Catrimox-14 ^TMMix, simply blood sample is kept at room temperature getting between the aliquot at every turn.The nucleic acid sediment that obtains (referring to the legend of Figure 13 .1) is dissolved in the dissolving buffer solution of 300 μ l from " MagNA Pure LC mRNA separating kit I " (Luo Shi diagnoses (Roche Diagnostics), molecular biochemistry product (Molecular Biochemicals)).Use MagNA Pure LC instrument (Luo Shi diagnoses (Roche Diagnostics), molecular biochemistry product (Molecular Biochemicals)) to extract mRNA (final elution volume: 100 μ l) according to manufacturer's specification.(Luo Shi diagnoses (Roche Diagnostics) according to " Lightcycler-RNA Master hybridization probe kit ", molecular biochemistry product (Molecular Biochemicals)) standardization program described in, since 5 μ l mRNA preparations, adopt one-step method to carry out reverse transcription and PCR in real time.Primer and probe sequence and PCR condition such as Stordeur etc., J Immunol Methods (immunological method magazine), 259 (1-2): 55-64,2002) described in.

Figure 33: extract the method for RNA and the schematic comparison of comparing by the method that the present invention proposes from whole blood by what PreAnalytiX proposed.

Figure 34. the in-vitro inducing of tetanus toxoid pair cell factor blood mRNA.(10ug/ml Aventis) is added to the 500 μ l whole bloods of collection from the healthy volunteer to tetanus toxoid, and described volunteer carried out vaccine inoculation at lockjaw before 7 years.At 37 ℃ at 5%CO ₂Through after the different time periods, add the reagent that comprises in the 1.4ml PAXgene pipe in the atmosphere.The lysate that 300 μ l obtain is used for separating total mRNA on MagNA Pure instrument, and carries out RT-PCR described in the present invention.

Figure 35. with IL-1 β and IL-1RA mRNA dynamics after the LPS stimulation of whole.200 μ l heparinize blood and 10ng/ml LPS incubation 0 when beginning (cultivate), 0.5,1,2 and 6 hour.When cultivating end, add 500 μ l PAXgene ^TMThe reagent of pipe is used for whole cytolysises and nucleic acid precipitation.Described in the present invention, carry out RT and PCR in real time then at IL-1 β, IL-1RA and beta-actin mRNA with single step.The result represents with the mRNA copy number of per 1,000,000 beta-actin mRNA copy.Demonstration is about the average and the standard error of the average of 5 independent experiments.

Figure 36. linear regression: about the mRNA copy number of initial blood volume.Various blood volume in the presence of 10ng/ml LPS (scope from 20 to 200 μ l, X-axle) were cultivated 6 hours.Cultivate when finishing, described in the present invention, carry out RT and PCR in real time at IL-1 β and beta-actin mRNA.The Y-axle is represented original copy number.This line is used for linear regression.A test that shows is represented 6 times.

Figure 37. with the mRNA cell factor dynamics after the tetanus toxoid stimulation of whole.Taking heparin blood from 5 healthy volunteers, described volunteer carried out vaccine inoculation at lockjaw before at least 5 years.For each donor, 200 μ l whole blood aliquots and 10 μ g/ml tetanus toxoid incubations 0 when beginning (cultivate), 4,8,16,24 and 48 hours.When cultivating end, add 500 μ l PAXgene ^TMThe reagent that comprises in the pipe, and use the quantitative different transcripts of method of the present invention.The result represents with the mRNA copy number of per 1,000,000 beta-actin mRNA copy.Demonstration is about the average and the standard error of the average of 5 independent experiments.

Figure 38. regulate in the body of blood cell factor mRNA behind the intravenous injection LPS.5 healthy volunteers injection is with the 4ng/kg LPS of single dose.Behind preceding 10 minutes of lps injection and the lps injection 0.5,1,1.5,2,3 and 6 hour, PAXgene ^TMGet the 2.5ml blood sample in the pipe.The method according to this invention is carried out the quantitative of cytokines mRNA.The result represents with the mRNA copy number of per 1,000,000 beta-actin mRNA copy.Expression is about the average and the standard error of the average of each time point.

Figure 39. following up a case by regular visits to of anti-tetanus vaccine reaction.Selecting 6 healthy volunteers to accept anti-tetanus recalls.From with (filled circles) or do not cultivate 20 hours the whole blood quantitatively IL-2mRNA level with (open circles) 10 μ g/ml tetanus toxoids, and when recalling (the 0th day), before 14 days and after 3,7,14,21 and 90 days (X-axle) carry out.The result represents with the mRNA copy number (Y-axle) of per 1,000,000 beta-actin mRNA copy.Each of 6 chart boards (numbering 1-6) expression is from the individual data items (1 donor of each chart board) of 6 different donors.

Figure 40. the general introduction of the program of taking at the

embodiment

7,8,9,10 and 11 that is used for the analyzing blood mRNA expression of cytokines.

Figure 41. extract mRNA and preparation reagent mixture in automation on the MagNAPure: the directly related property between the amount of initial biomaterial and the copy number of existence.The Y-axle is represented original copy number.This line is used for linear regression.

Figure 42. extract mRNA and preparation reagent mixture in automation on the MagN APure: the directly related property between the amount of initial biomaterial and the copy number of existence.The Y-axle is represented original copy number.This line is used for linear regression.

Figure 43. participate in the patient's of cancer immunotherapy summary case report.Diagnose out melanoma in July, 1999.In August calendar year 2001, prove that many places shift, and this patient participates in immediately and accepts cancer vaccine after carrying out orchiectomy (orchydectomy) in April, 2002.This vaccine is present in some injections of MAGE-3 purifying protein (by the specific expressed antigen of melanoma cells) and adjuvant combination.

Figure 44. vaccine inoculation scheme and the diagram by PCR in real time monitoring immune response.The stimulated in vitro whole blood is with the immune response of assessment to MAGE-3.The patient accepts 3 vaccine injections, simultaneously blood sampling weekly in the time in 9 weeks.200 μ l aliquots of every patient's whole blood sample at 10 μ g/ml MAGE-3 albumen or as the 10 μ g/ml TRAP (plasmodium falciparum (plasmodium falciparum) antigen) of cloudy type contrast in the presence of incubation.When cultivate finishing, can to carry out IL-2 mRNA as described in example 6 above quantitative thereby add the reagent that comprises in the PAXgene pipe.The result provides in Figure 45.

Figure 45. the IL-2 mRNA in the whole blood after the MAGE-3 vaccine inoculation in patient #3.Behind MAGE-3 vaccine booster immunization, in the whole blood that MAGE-3 stimulates, observe higher IL-2 mRNA level.The Y-axle is represented the IL-2 mRNA copy number of per 1,000,000 beta-actin mRNA copy, and all number of X-axle when being blood sampling.Vaccine injection liquid was used when the 0th, 2 and 6 weeks.The whole blood of incubation when solid post is used to have MAGE-3, the whole blood of incubation when the shade terminal is used to have TRAP.

Figure 46. the stimulated in vitro of whole blood: estimate to allergenic immune response.Test, it is quantitative to carry out IL-4 mRNA behind whole blood and allergen incubation.From cat being had an allergic experimenter and blood sampling from two health volunteers.Whole blood different incubation time section of incubation under the condition that does not have or exist cat allergen (being Feld1) then, when incubation finished, can to carry out IL-4 mRNA as described in example 6 above quantitative thereby add the reagent that comprises in the PAXgene pipe.The result provides in Figure 47.

Figure 47. the stimulated in vitro of whole blood: estimate immune response to Feld1.The Feld1 allergen is significantly induced from the IL-4mRNA level in the whole blood that cat is had allergic experimenter and is higher than anergic experimenter.The Y-axle is represented the IL-4 mRNA copy number of per 1,000,000 beta-actin mRNA copy, and the X-axle is the different incubation time.The IL-4mRNA level that exists in the normal whole blood of green post representative and allergen incubation, the IL-4 mRNA level that exists in allergia experimenter's the whole blood is by red post (at the blood that has incubation under the condition of Feld1) and yellow post (blood of incubation under the condition that does not have Feld1) expression.

Figure 48. in this whole blood system, be specific relevant with dosage to replying of Feld1.Whole blood 1 from the allergenicity experimenter) in the presence of the Feld1 that increases concentration (diagonal line hatches post); 2) in the presence of the beta lactoglobulin (BLG) of another kind of allergen 10 μ g/ml (horizontal dotted line shade post); 3) incubation 2 hours in the presence of crosslinked IgE (spot post).The Y-axle is represented the IL-4 mRNA copy number of per 1,000,000 beta-actin mRNA copy.

Figure 49. be higher than normal healthy controls with the IL-4 mRNA level after the Feld1 stimulation of whole cat being had among allergic patient.The blood sample that cat is had allergic patient (ALL post) from 10 health volunteers (CTR post) and 10 is repeated the above test of lantern slide 9-11.Whole blood sample was incubation in the presence of the 10 μ g Feld1 or in the presence of as the crosslinked IgE of positive control 2 hours.Show that average peace standard error of the mean is poor.

Figure 50. the stimulated in vitro of whole blood: assessment is to the t cell response of GAD65.Be shown in the test of quantitatively carrying out for IL-2 mRNA behind the GAD65 albumen incubation of whole blood and purifying.Blood sample is taken from 6 type 1 diabetes patients and 5 health volunteers.Whole blood incubation 18 hours under the condition that does not have or exist 10 μ g/ml GAD65 stops cultivating by adding the reagent that comprises in the PAXgene pipe then then.Quantitative as described in example 6 above then IL-2 mRNA level.The result provides in Figure 51.

Figure 51. the stimulated in vitro of whole blood: assessment is to the t cell response of GAD65.Compare with the health volunteer, after GAD65 stimulates, demonstrate higher IL-2 mRNA level from type 1 diabetes patient's whole blood.The result is expressed as after proofreading and correct at beta-actin, with respect to the IL-2 mRNA copy number of the copy number calculating that exists in the whole blood of cultivating when not having GAD65.Use logarithmic scale.Show that average peace standard error of the mean is poor.Healthy donors: CTR post; Autoimmune diabetes patient: PAT post.

Figure 52. monitoring alloreactivity (alloreactive) immune response: IL-2 mRNA's is quantitative in whole blood+dendritic cells system.Behind whole blood and incoherent dendritic cells (DC) incubation, quantitatively test with assessment alloreactivity t cell response for IL-2 mRNA.In the presence of IL-4 and GM-CSF, from two incoherent healthy volunteers' (MT and MA) dendritic cells in external generation.From the whole blood sample of every donor (1) in the presence of the dendritic cells group of another donor or in the presence of they self dendritic cells (2) cultivate.To mix (3) from the whole blood sample of two donors, and mix two dendritic cells preparations (4).Behind the incubation 12 hours, stop cultivating by adding the reagent that comprises in the PAXgene pipe.Quantitative as described in example 6 above then IL-2 mRNA level.The result is presented among Figure 53.

Figure 53. monitoring alloreactivity immune response: IL-2 mRNA's is quantitative in whole blood+dendritic cells system.By IL-2 mRNA qualitative assessment alloreactivity t cell response in the whole blood.The IL-2 mRNA copy number that shows per 1,000,000 beta-actin mRNA copy.Condition from left to right is: the independent whole blood from donor MA, from the whole blood of donor MA+from the DC of donor MA, from the whole blood of donor MA+from the DC of donor MT, independent whole blood from donor MT, from the whole blood of donor MT+from the DC of donor MT, from the whole blood of donor MT+, from the whole blood of donor MT+, from the DC+ of donor MT DC from donor MA from the whole blood of donor MA from the DC of donor MA.

Embodiment

Embodiment 1: the analysis of spontaneous cytokines mRNA in the peripheral blood

By quantitatively should the estimating of the synthetic cytokines mRNA of PBC " periphery immune state ".Yet, only can from fresh whole blood sample, carry out accurately quantitatively, mRNA is protected not by nuclease digestion in fresh whole blood, and wherein genetic transcription is suppressed.As just discussing in this respect, by using surfactant reagent such as this possibility that become of myristyl trimethyl ammonium oxalate.Carry out IL-10 and the IFN-γ mRNA of RT-PCR with quantitatively spontaneous generation in peripheral blood.Result's demonstration is compared with the PMBC (PBMC) from same individual, and IFN-γ transcript level is higher in the whole blood, and IL-10 mRNA is not observed significant difference.In blood the IFN-γ mRNA of observed higher amount can be at least owing to the degraded of mRNA.Use real time pcr, can prove blood/IF N-γ mRNA practically in external degraded apace,

At room temperature be equivalent to about 1 hour.

Deng having analyzed the influence (Hartel etc., 2001) that the cell purification program generates spontaneous cytokines mRNA in the peripheral blood recently.The PMBC (PBMC) that their show fresh separated with compare from the whole blood of the fresh collection of same individual, express higher levels of IL-2, IL-4 and TNF-α mRNA, and IFN-γ mRNA level is not observed difference.In 6 different individualities, carry out the comparison of IFN-γ, and found different results.Observe the strongly expressed of IFN-γ mRNA in the whole blood of all donors, it is (Figure 32 .1) that obviously reduces in PBMC.The result who obtains and

Deng the result between this difference may be relevant with the program that is used for from whole blood, separating total RNA, used quantitative real time pcr although the fact is the latter. Deng having used heparinized blood, its in 2 hours by waiting ammonium chloride that oozes to handle haemolysis.Used myristyl trimethyl ammonium oxalate in the method for the invention, a kind of Catrimox-14 that is called ^TMCationic surfactant reagent (Qiagen, Westburg, Leusden, Holland), it directly mixes with blood, has avoided use anti-coagulants (Dahle and Macfarlane, (1993); Schmidt etc., (1995)).In addition, induce nucleic acid precipitation and nuclease inhibition in this reagent 1 minute after collected specimens.This provides total RNA preparation, and it may be near mRNA state in the body.This is a particular importance for cytokines mRNA, makes it to endogenous nuclease sensitivity owing to be arranged in the sequence that is rich in AU of its 3 ' non-translational region.Use real time pcr, in fact observe the spontaneous and quick degraded of peripheral blood IFN-γ mRNA, 1 hour this level has reduced roughly 50% after gathering blood.Yet this phenomenon is for all cells factor and not all correct, because find IL-10 mRNA level stable at least 5 hours (Figure 32 .2) after blood sampling.In addition, the IL-10 mRNA level of whole blood is compared with the IL-10 mRNA level of PBMC and is not found significant difference (Figure 32 .1).

At Catrimox-14 ^TMThe nucleic acid sediment (referring to the legend of Figure 32 .1) that dissolving back obtains can be dissolved in by Chomczynski and the described guanidine/thiocyanate salt solution of Sacchi (1987), with and the form that is purchased such as Tripure ^TMIn (Luo Shi diagnoses (Roche Diagnostics), molecular biochemistry product (Molecular Biochemicals), Brussels, Belgium), make that the application of this surfactant is easy especially.This means, except using Catrimox-14 ^TMFirst step beyond, the RNA separable programming be identical for whole blood and cell.Alternatively, PAXgene ^TMBlood rna pipe (Qiagen, Westburg, Leusden, Holland) can be used for replaced C atrimox-14 ^TMIn the case, the sediment that obtains can be dissolved in the dissolving buffer solution of " MagNA Pure LC mRNA separating kit I ", as in the legend of Figure 32 .2 for Catrimox-14 ^TMDescribed such.The spontaneous IL-10 mRNA that characterizes people's monokaryon haemocyte produces (Stordeur etc., (1995)), with induce (Pradier etc., (1996)) of monitoring OKT3 monoclonal antibody, two examples that wherein successfully use Catrimox-14 have been represented to in-vivo tissue factor mRNA.After adding ionophore A23187+ phorbol myristic acid acetic acid esters (phorbol myristate acetate), also observe strong IL-2 mRNA and induce (not shown), point out it to the application in the in vitro study of whole blood to whole blood.

The importance of carrying out RT-PCR as quickly as possible from the whole blood of dissolving has been given prominence in the observation that obtains in the present embodiment, thus accurately quantitative PBC factor mRNA.For this purpose, use reagent such as Catrimox-14 or PAXgene ^TMThe additive that comprises in the blood rna pipe, and real-time RT-PCR may have been represented the program of present the best.By doing like this, might under the condition of not using external strong stimulation thing such as ionomycin (ionomycin) or lectins, study the native state of PBC.

Embodiment 2:PAXgene ^TMComparison between the method for blood rna system and proposal according to the present invention

" PAXgene ^TMThe blood rna system " be meant PAXgene ^TMThe blood rna pipe " and " PAXgene ^TMThe blood rna kit " combination." Qiagen method " is meant " PAXgene ^TMBlood rna kit ".

Based on Stordeur etc., J Immunol Methods (immunological method magazine), 259 (1-2): 55-64, experimental evidence described in 2002, the present invention proposes a kind of new program and comes separating mRNA from whole blood, and it can use a kind of easy and reproducible method to measure transcript level in the body.PAXgene ^TMBlood rna system and the method according to this invention have carried out schematically comparing in Figure 33.

Material and method

From as by PAXgene ^TMSuch PAXgene that directly is captured in that blood rna system (Qiagen) is recommended ^TMPeripheric venous blood in the blood rna pipe carries out all tests (that is, 2.5ml blood vacuum is captured in vitro, and this test tube contains the unknown reagent of 6.9ml).After dissolving finished, the content of test tube is transferred in two other test tubes: 4.7ml was used for PAXgene blood rna kit, and 0.4ml is used for MagNA Pure and extracts.Discard remaining lysate.These two test tubes are with 2, the centrifugal 10min of 000g, and abandoning supernatant.The nucleic acid sediment is then:

A) PAXgene ^TMBlood rna pipe+PAXgene ^TMThe blood rna kit-... in water, wash, be dissolved in then and be used for total RNA in the BR1 buffer solution and extract, as recommending in the corresponding specification handbook.PAXgene ^TMThe program of blood rna system is as follows: blood sample (2.5ml) is captured in the PAXgene blood rna pipe, and at room temperature preserves if desired or transportation.The RNA separation begins with centrifugation step, and nucleic acid is deposited in the PAXgene blood rna pipe.Washing precipitate, and add Proteinase K to carry out proteopepsis.Add alcohol regulating in conjunction with condition, and with sample application in by PAXgene ^TMThe centrifugal adsorption column that the blood rna kit provides (spin column).Brief centrifugal during, pollutant by the time RNA optionally be incorporated into by PAXgene ^TMThe pellosil that the blood rna kit provides.Behind washing step, RNA is wash-out in the buffer solution of optimizing.As (" Cytokine mRNA Quantification by Real Time PCR (by the quantitative cytokines mRNA of PCR in real time) " J Immunol Methods (immunological method magazine) such as Stordeur, 259 (1-2): 55-64,2002) described reverse transcription and the PCR in real time that carries out at IFN-γ and beta-actin mRNA.

-b) PAXgene ^TM The blood rna pipe ++ MagNA Pure LC mRNA separating kit I-... be dissolved in the dissolving buffer solution of 300 μ l from MagNA Pure mRNA separating kit.On MagNA Pure LC instrument,, carry out extraction and the purifying of mRNA with the final elution volume of 100 μ l then according to specification from Luo Shi diagnosis (Roche Diagnostics), molecular biochemistry product (Molecular Biochemical).(Luo Shi diagnoses (Roche Diagnostics) according to " Lightcycler-RNA Master hybridization probe kit ", molecular biochemistry product (Molecular Biochemicals)) standardization program described in, since 5 μ l mRNA preparations, adopt one-step method to carry out reverse transcription and PCR in real time.

The result:

Extracting method and PAXgene by the Qiagen recommendation ^TMBlood rna pipe (PAXgene ^TMThe blood rna system) combination and also with PAXgene ^TMThe MagNA Pure LC instrument of blood rna pipe jointing is mentioned method and is compared.In two methods, PAXgene ^TMThe use of blood rna pipe can be stablized the RNA from haemocyte.The result is set forth in table 1.1 and 1.2.The result of this test shows that MagNA Pure LC technology has repeatability (for the variation coefficient of the IFN-γ mRNA copy number of proofreading and correct at beta-actin, Qiagen compares with MagNA Pure LC and is respectively 26% and 16%) preferably.

Notice enjoyably and carry out the employed volume of initial blood volume ratio Qiagen method little (for MagNA Pure is 0.11ml, and comparing for Qiagen is 1.25ml) that MagNA Pure extracts.If use so little volume to carry out the Qiagen method, then impossible measure R NA concentration even may not carried out reverse transcription.This has just given prominence to another advantage of the technology described in the present invention: with the possibility of very little blood volume (about 100 μ l) quantification of mrna.

Conclusion:

Embodiment 2 for example understands use PAXgene ^TMThe possibility of blood rna pipe jointing MagNA Pure LC mRNA separating kit I, or more accurately, will be from PAXgene ^TMThe sediment of blood rna pipe is dissolved in the possibility in the dissolving buffer solution that comprises in this kit, and this dissolving buffer solution must use with other components of this kit.

In this embodiment, proved opposite with other combinations, only the combination described in the present invention cause correctly/real body in transcript quantitative.

Embodiment 3: the immune response of monitoring at tetanus toxoid exsomatizes.

In embodiment 3, blood exsomatizes to stimulate with antigen (that is, tetanus toxoid), and blood donors should be at this antigen immune (because carrying out vaccine inoculation before 7 years).Carry out RT-PCR (Figure 31 .1) according to this method.Cytokines mRNA is measured as the reading of volunteer's immune system at this antigen reactive ability.IL-2, IL-4, IL-13 and IFN-γ mRNA preferentially analyze, but all potential proteins C reactives can quantitatively analyzing by their corresponding mRNA.The result of embodiment 3 is presented among Figure 34.The cardinal principle strategy of abideing by among this embodiment can schematically show as shown in Figure 31 .2.

Can applicable embodiment: cancer immunotherapy

For some years, develop for the elementary tactics of cancer immunotherapy mode with vaccine inoculation.In fact, can identify many growth tumour antigens in the progress aspect science of heredity and the immunology, they are expressed to surface of tumor cells.These antigens are presented to surface of tumor cells with the form of the peptide relevant with main histocompatibility complex (HLA).The example of antigen that can be considered to tumour antigen is described by Fong and Engleman (Annu.Rev.Immunol. (immunology annual review) 2000.18:245-273).The principle of anti-cancer vaccine inoculation is to abide by the strongest immunogenic mode these antigens is presented systemic immunity to the patient.This presents described peptide then from injections of antigens in the presence of additive or corresponding peptide on autoantigen presentation cell (for example, dendritic cells).Make tumor regression although the final purpose of anti-cancer vaccine inoculation remains, it is difficult that the effect of definite anti-cancer vaccine inoculation remains, and especially is under the situation of terminal stage of a disease the patient, and it only can utilize limited treatment window.Here it is, and why the anti-cancer vaccine inoculation is made us interested especially reason as complementary therapy or in the framework of prevention.What therefore exploitation was sensitive comes the immunology effect of evaluation experimental anti-cancer vaccine inoculation so that stipulate the application process of these vaccines and find that the biological mechanism that implies is very important with accurate monitoring technology, and this will help to determine better following therapeutic scheme.The difficulty of measuring the immunology effect of these vaccines mainly is to lack and is enough to the mensuration of detection bodies inner cell immune response delicately.Up to now, the technology of use be included in antigen existence down and the in vitro culture patient's who in the presence of the costimulatory molecules of the Elementary Function characteristic changing that is easy to induction of lymphocyte, concentrates for a long time PBMC.Therefore, be exceedingly difficult to lymphocyte precursor at the anergy state of tumour antigen or the analysis of tolerance status, this is in view of the reversible nature of their their functional statuses behind external incubation of the long period in the presence of the antigen.On the other hand, the technology based on the tetramer of MHC-peptide complexes that is used to detect low-frequency epitope specificity CTL precursor lacks usually and detects the lymphocytic sensitivity of tumour-specific.These technology do not provide any information about these lymphocytic functional responses in addition.

Can be for example thereby only enough sensitive, very the short time stimulates the back to detect lymphocyte could allow real evaluation anti-cancer vaccine vaccination regimen to the reactive technology of the original function of given antigen effect at external use antigen.

Show (Kammula in recent years, U.S., Marincola, F.M., and Rosenberg, S.A. (2000) Real-time quantitative polymerase chain reaction assessment of immune reactivity in melanoma patients aft

Embodiment 4: detect the immune activation of acceptor that the histocompatibility antigen by donor causes.

In embodiment 4, migrate in the acceptor from the organ (for example, liver, kidney, marrow etc.) of donor.The whole blood of acceptor is captured in the test tube that comprises according to inhibition according to the present invention RNA degraded and/or gene induced compound.Carry out RT-PCR according to this method.Cytokines mRNA is measured as the reading (Figure 31 .3) of the immune activation of the acceptor that the histocompatibility antigen by donor causes.

Embodiment 5: detect the reactivity of the immune system of acceptor to the histocompatibility antigen of donor.

In embodiment 5, migrate in the acceptor from the organ (for example, liver, kidney, marrow etc.) of donor.The whole blood of acceptor is captured in the test tube, and with the histocompatibility antigen of the donor incubation that exsomatizes.Be added in the blood according to inhibition RNA degraded of the present invention and/or gene induced compound.Carry out RT-PCR according to this method.Cytokines mRNA is measured as the immune reading of replying (Figure 31 .4) of the acceptor that the histocompatibility antigen by donor causes.

Application Example: the repulsion after the monitoring organ transplant

The repulsion of monitoring graft is basically based on the detection of (at blood urea nitrogen (BUN)-BIN-under the situation of kidney transplant thing or kreatinin) or the mark that measures when the biopsy of analyzing transplanted organ in patient's urine or blood.Yet these indexs only are detected when repulsion mechanism is fully made progress.In fact, graft rejection is the result prior to the amynologic mechanism of transplant organ destruction.Before transplant organ is impaired, detect the loss that these amynologic mechanisms can reduce transplant organ by regulating immunosuppressive therapy earlier considerablely.On the other hand, also recognize the subclinical incident (not bringing out clinical sign) that repulsion takes place continually after transplanting.The reason that these subclinical repulsion incidents may be chronic rejections.Several authors after deliberation the early immune that repels of sense organ learn mark and particularly, detect the alloreactivity T lymphocyte in the acceptor circulation at the alloantigen of donor.Method consists essentially of mixed culture and lymphopoiesis that passes through distinct methods (ELISA, ELISPOT, flow cytometry etc.) continuous measurement receptor or measurement production of cytokines is combined.Recently, other authors have paid close attention to the lymphocyte activator mark pattern that characterizes, and it is easy to emphasize in early days cause repulsion mechanism.The mRNA that quantitative PCR method by sensitivity detects the gene (granzyme B, perforin, different cell factors) that the cytotoxicity activated T lymphocytes T by activation expresses shows it is to measure to cause the excellent instrument that repels.For this purpose, according to the present invention, can study the courier of the different types of cell factor of coding, preferred target can be IL-2, IFN-γ, IL-4, IL-5, granzyme, perforin and FasFas-part.

Embodiment 6: the immunologic surveillance in the whole blood of use PCR in real time.

In embodiment 6, the whole blood method has been described, it can be in synthetic the inducing of mRNA horizontal survey cell factor.The intention of the method is the PAXgene that contains the mRNA stabilizing agent that is used to take a blood sample ^TMPipe is as the MagNA Pure that is used to extract mRNA and prepares the automated system of RT-PCR reagent mixture ^TMInstrument and about be used for accurate and reproducible quantitative transcript level at Lightcycler ^TMThe combination learned of real-time PCR method.This embodiment proves that at first the method is enough to measure inducing at IL (interleukin)-1 β when whole blood adds bacteria lipopolysaccharide (LPS) and IL-1 receptor antagonist (IL-1RA) mRNA.This embodiment proves that further this mode also is suitable for detecting the generation of the mRNA of the cell factor in encode T cell source in the whole blood (as the external immune response model to recall antigen) with the tetanus toxoid incubation.At last, this embodiment proves that the method can successfully be used for estimating inflammatory response and the t cell response in the body, because it can detect among the healthy volunteer inducing of IL-1 β and IL-1RA behind the injection LPS, can also detect inducing of IL-2 when using tetanus vaccine to recall immunity.

Material and method

The blood collection that is used for research in the body.For accurate quantitatively peripheral blood mRNA level, get the 2.5-ml blood sample in PAXgene ^TMBe used for cytolysis and nucleic acid precipitation in the pipe immediately.MRNA is stable 5 days at the most in this blood lysate, and this is guaranteed to be held under the room temperature and extracts until mRNA.

External whole blood is cultivated.To 200 μ l heparinize whole bloods, and after blood sampling, begin to carry out that external whole blood LPS stimulates or tetanus toxoid is attacked again 4 hours the latest the time.By adding 500 μ l PAXgene ^TMThe reagent of pipe stops cultivating, and it induces whole cytolysises and mRNA to stablize.Can use identical mRNA extraction scheme to be used for studying both in the external and body like this.

MRNA extracts.At PAXgene ^TMIn the pipe or the blood lysate that when whole blood cultivate to finish, obtains mix tout court, shift 300 μ l aliquots then and be used for to the 1.5-ml eppendorf pipe with maximum velocity centrifugation 5 minutes (12,000-16,000g depends on device).Abandoning supernatant, nucleic acid sediment fully are dissolved in MagNA Pure by vortex ^TMMRNA extracts in the 300 μ l dissolving buffer solution that comprises in the kit (Luo Shi applied science (Roche Applied Science)).Then at MagNA Pure ^TMInstrument (Luo Shi applied science (Roche Applied Science)) is gone up specification (" mRNA I cell " the Luo Shi experimental program (Roche ' s protocol) of abideing by the manufacturer, final elution volume 100 μ l), use this kit from 300 these solution of μ l, to extract mRNA.The quality of the mRNA that extracts proved by Northern engram analysis (Luo Shi applied science (Roche Applied Science), the data of not delivering) in the past.

PCR in real time and reagent mixture preparation.According to the standardization program described in " Lightcycler-RNA Master hybridization probe " kit (Luo Shi applied science (Roche Applied Science)), adopt one-step method to carry out reverse transcription and PCR in real time.More accurately, in containing 20 following μ l final volume, carry out RT-PCR reaction: the 1) H of 20 μ l at the most ₂O; 2) 7.5 μ l RNA Master hybridization probe 2.7xconc (RNA Master hybridization probe kits-Luo Shi applied science (Roche Applied Science); 3) 1.3 μ l 50mM Mn (OAc) ₂4) (final concentration 300,600 or 900nM depend on the mRNA target for 6 picomoles of 1,2 or 3 μ l/microlitre forward and reverse primer; Specific condition for every kind of mRNA target is documented in Stordeur etc. fully, J Immunol Methods (immunological method magazine), and 259 (1-2): 55-64, in 2002, except IL-2 and IL-4, they are set forth in the table 2); 5) 4 picomoles of 1 μ l/microlitre TaqMan probe (final concentration 200nM); 6) mRNA of 5 μ l purifying or standard dilution.Made the mRNA reverse transcription in 20 minutes and then after 95 ℃ of 30s carry out preliminary denaturing step at 61 ℃ of incubations, the beginning temperature cycles.Each circulation is read fluorescence (F1/F2 passage, achromatization compensation) by forming 95 ℃ 0 (zero) second and 60 ℃ of 20s when this second step finishes.Carry out 45 circulations altogether.All primers are selected as crossing over intron sequences, and making can not amplifying genom DNA.

By MagNA Pure ^TMInstrument is being used for Lightcycler ^TMOn capillary in directly fully preparation contain the RT-PCR reactant mixture of all reagent, oligonucleotides and sample.With these top capillaceous closures, centrifugal, be introduced into Lightcycler then ^TMIn be used for an one step RT-PCR.Therefore the sampling full automation of all RT-PCR components has been avoided the error of manual sampling.

The result is expressed as at the standardized copy number of beta-actin mRNA (the mRNA copy number of the cytokines mRNA of per 1,000,000 beta-actin mRNA copy).For each sample, the mRNA copy number uses Ct value (" the arithmetic match point is analyzed (Arithmetic Fit point analysis) ") to calculate from calibration curve by instrument software.This latter is at taking turns the PCR mapping from the serial dilution of purify DNA every, as Stordeur etc., and J Immunol Methods (immunological method magazine), 259 (1-2): 55-64 are described in 2002.

Experimental endotoxemia.5 healthy male volunteers (21-28 year) accept the intravenous injection single dose of LPS (from Escherichia coli (E.coli), lot G; American Pharmacopeia (United States Pharmacopeial Convention), Rockville, MD; The 4ng/kg body weight), do not take any medicine before these volunteers test at least in 10 days.Behind preceding 10 minutes of lps injection and lps injection 0.5,1,1.5,2,3 and 6 hours, get the 2.5ml blood sample in PAXgene ^TMIn the pipe.For in vitro study, take from 200 μ l heparinize whole bloods of healthy individual and 10ng/ml LPS (from e. coli serotype 0128:B12, Sigma-Aldrich, Bornem, Belgium) under 37 ℃ at 5%CO ₂Incubation 0 in the atmosphere (cultivating beginning), 0.5,1,2 and 6 hours.

Anti-tetanus is recalled vaccine inoculation.Healthy volunteer's (2 male sex, 4 women, 27-53 year) accepts the muscle intradermal vaccine and recalls inoculation (Tevax, Smith Kline Beecham Biologicals, Rixensart, Belgium), these volunteers' last tetanus toxoid vaccine inoculation is at least before 5 years.3,7,14,21 and 90 days taking heparin blood tube before the same day, 14 days of administration and after the administration.200 μ l blood under 37 ℃ at 5%CO ₂In the atmosphere, there was or do not exist under the condition of 10 μ g/ml tetanus toxoids (by Dr.E.Trannoy, Aventis Pasteur, Lyon, France is so kind as to give) incubation 20 hours.

The result

When in whole blood, adding bacterium LPS, measure IL-1 β and IL-1 RA mRNA.Such as among Figure 35 proof, in whole blood, add the rapid induction that LPS (10ng/ml) causes IL-1 β and IL-1 RA mRNA.This induces after adding LPS 30-60 minute clearly, causes the mRNA level of IL-1 β and IL-1 RA to increase to 47-respectively doubly and 22-times after 6 hours.The pattern of curve shows the quick of these two kinds of cytokines mRNA amounts and continues to increase.In order to estimate this system accuracy quantitative to mRNA, the whole blood that stimulates by the LPS of different volumes (20-200 μ l), it is quantitative to carry out mRNA at beta-actin and IL-1 β.As shown in Figure 36, the mRNA copy number of beta-actin and IL-1 β is in fact directly related with the blood volume of beginning.

Vitro responses at tetanus toxoid.In order to determine whether the method can be suitable for analyzing t cell response, whole blood cells in culture factor mRNA level after the quantitative adding tetanus toxoid, tetanus toxoid is the recall antigen that a kind of quilt is fully determined, because all individualities all carry out vaccine inoculation in boyhood.Find whole blood IFN-γ behind the antigen incubation therewith, IL-2, IL-4 and IL-13 mRNA fast and moment induce (Figure 37).As if when the amplitude of replying of every kind of cell factor was compared, inducing of IL-2 mRNA was the most outstanding.In fact, about 5 independent experiments that show among Figure 37, in the presence of this toxoid incubation after 16 hours IL-2 mRNA copy totally increase to about 220 times, and the maximum increase of IL-4 and IFN-γ mRNA is no more than 5 times in identical experiment.So the sensitiveest parameter in the whole blood system of quantitative this assessment t cell response seemingly of IL-2 mRNA.The data indication that provides in the table 3 is quite variable to the amplitude of replying of tetanus toxoid in this test, and it may depend on the time that last vaccine is recalled.After adding tetanus toxoid, do not observe inducing of IL-2mRNA effectively, illustrate that only the T cell and the non-inmature T cell of previous sensitization can be replied (table 3) in this measures to umbilical cord blood.

In whole blood, induce IL-1 RA and IL-1 β mRNA behind the intravenous injection LPS.As first application that is used for the method that the detection bodies inner cell factor induces, analyze the healthy volunteer's that low dosage (4ng/kg) bacteria lipopolysaccharide of use by oneself injects serial blood sample.Observe obviously the inducing of IL-1RA and IL-1 β mRNA (Figure 38).Inducing of IL-1 β mRNA is fast, and it is detected because use back 30-60 minute at endotoxin, and it is instantaneous, because IL-1 β mRNA level returns to numerical value before the injection after 6 hours.IL-1 RA mRNA is also induced, and compares the dynamics that it has delay with IL-1 β mRNA.

The detection of anti-tetanus toxoid immune response after the memory vaccine inoculation.Because the sensitive parameter that experiment in vitro prompting IL-2mRNA is a monitoring anti-tetanus toxoid to be replied, so select this parameter to analyze to recall in the body after the vaccine inoculation in the whole blood T cell to the variation of replying of tetanus toxoid.For this purpose, lacking or existing under the condition of tetanus toxoid, before using vaccine and afterwards several time points carry out the whole blood incubation.As shown in Figure 39, in all vaccinated individualities, the generation that is exposed to IL-2 mRNA in the whole blood of antigen all significantly increases.7 days IL-2 mRNA have induced clearly after vaccine inoculation, reach maximum horizontal when the 14th day or the 21st day.Variability between the individuality may relevant with the base state difference of anti-tetanus immunological (also referring to table 3).It is special that the IL-2 that measures in the whole blood after vaccine inoculation replys immunizing antigen, because lacking the not significantly change (table 3) of IL-2 mRNA level of measuring under the external condition that stimulates again.

Discuss

Why being called PCR in real time is because use the fluorescence molecule in conjunction with the PCR product can directly monitor the amplicon accumulation during the PCR process.This causes generating the fluorescence curve of each sample, by this curve, by comparing with the fluorescence curve that the standard items that use calibration obtain, might determine (c) DNA copy number of sample.In order to strengthen specificity, fluorescence molecule can be and the oligonucleotides of the PCR product sequence complementation between two primers.Described in the application, new methodology provides a kind of sensitivity and accurate way quantitatively to use nucleic acid in the biological sample that art methods can not be quantitative.The application by situation in representing body purifying cells or tissue quantitatively cytokines mRNA illustrate this point.

Using whole blood to be used for RT-PCR, to analyze one of difficulty of being run into be the cytolysis of carrying out before RNA extracts.Because a large amount of albumen are present in blood plasma and the red blood cell, the most methods of isolation of RNA relates to the potential cell source of the analyzed mRNA of purifying or removes red blood cell from whole blood, carries out RNA then and extracts.These intermediate steps can be degraded with mRNA and/or be gene induced relevant, and therefore relevant with the variation of mRNA level.In addition, get this true degraded that just can cause some mRNA of blood merely.Especially also be like this for cytokines mRNA, cytokines mRNA by the sequence that is rich in AU that is arranged in its 3 ' non-translational region to endogenous nuclease sensitivity.Shown that peripheral blood IFN-γ mRNA level in fact reduced roughly 50% (Stordeur etc., (2002) J.Immunol Meth. (immunological method magazine) 261:195) in 1 hour after gathering blood.This can use quaternary amine surfactants to be avoided such as myristyl trimethyl ammonium oxalate, and the latter is a kind of Catrimox-14 of being called ^TMThe cationic surfactant of (Qiagen, Westburg, Leusden, Holland), it induces full cytolysis, and induces the nucleic acid precipitation simultaneously.Present embodiment is observed and is used PAXgene ^TMThe nucleic acid sediment that pipe obtains can unexpectedly be dissolved in guanidine/thiocyanate salt solution.An example of described solution is to be used for mRNA isolated M agNA Pure ^TMThe dissolving buffer solution that LC kit (Luo Shi applied science (Roche Applied Science)) provides.This impels us with PAXgene ^TMThe use of pipe and MagNA Pure ^TMInstrument combines, and has utilized the high repeatability and the accuracy of a back device, and it is owing to the automated preparation of all components of PCR reactant mixture.

Interesting is that the application's method successfully is applied to the cytokine gene that endotoxin is attacked in the whole blood of back in the detection bodies and induces, and has proved that it can be used to monitor the whole body inflammatory response.Attack the instantaneous nature that back IL-1 replys in the body, with external after blood adds LPS continuing to increase of IL-1 mRNA form contrast.This may with the quick removing of LPS in the body and body in to produce the distribution again of cell of cell factor relevant, its rise with adhesion molecule and chemokine receptors is relevant.It is the t cell response of monitoring after the vaccine inoculation that another of this whole blood method may be used, as by carry out in the individuality of tetanus toxoid vaccine inoculation external attack as suggested in obviously the inducing of back viewed IL-2 mRNA again.This may be interested especially for extensive vaccine inoculation research, may be difficult to carry out cell separation in good condition undertissue in described research, especially in developing country, is in the evaluation at these several new vaccines of country.In order further to study the applicability of the method in vaccine test, it will be used as soon at the reading of t cell response behind the hepatitis B primary vaccination and test.

Directly related property (Figure 36) prompting between initial blood volume and the mRNA copy number uses the method not need utterly to measure mRNA concentration with ecbatic.Yet, because even little sample volume change and all may cause quantitative error, therefore preferred by measuring the copy number that house-keeping gene such as beta-actin comes calibration measurement simultaneously.This may still not be best, because the expression of house-keeping gene may change under some incentive condition.Therefore, can before extracting, mRNA in sample, add external perimysium reference.When the cell source of cell factor is fully determined, such as being under the situation of T cell for IL-2, can suitably proofread and correct the copy number of cytokine gene, such as the CD3 in one embodiment of back by the copy number that is coded in the gene of specifically expressing in the corresponding cell type.Equally, thus should develop the data that the international standard that is used for the caliberator by the quantitative cytokines mRNA of PCR in real time helps comparison different experiments chamber to generate.The measurement of cytokines mRNA is effective to monitoring and evaluation novel vaccine and the needed innate immune responses of immunotherapy and acquired immunity and replys in the whole blood.

Automation mRNA on the embodiment 7:MagNA Pure extracts and the reagent mixture preparation: the directly related property between the amount of initial biomaterial and the copy number of existence.

The method of abideing by among this embodiment is summarised among Figure 40.For the accuracy of this system is described, calculate of the linear regression (Figure 41) of mRNA copy number to the initial cell number.Extract mRNA from the PMBC (PBMC) of various numbers (100,000-600,000 cell, X-axle), and described in " material and method " chapters and sections of the embodiment of the invention 6, carry out a step RT-PCR in real time at beta-actin mRNA.This test by PBMC at beta-actin and TNF-α mRNA (Figure 42, chart board B and D) with by whole blood (Figure 42, chart board A) and CD4 ⁺The T cell of purifying (Figure 42, chart board C) repeats at beta-actin mRNA.

Embodiment 8: cancer immunotherapy

The program of abideing by among this embodiment is summarized among Figure 40.This methodology is applied to monitor the immune response of being induced by cancer vaccine.Figure 43,40 and 41 illustrate the result who melanoma patient is obtained in this field.

Embodiment 9: allergy

The program of abideing by among this embodiment is summarized among Figure 40.This methodology is applied to allergy.Whole blood and replying that relevant allergenic external incubation is induced by the allergia experimenter by using the quantitative IL-4mRNA of PCR in real time to analyze.Figure 46,47,48 and 49 illustrate the result who obtains in this field.

Embodiment 10: LADA

The program of abideing by among this embodiment is summarized among Figure 40.This methodology is applied to LADA.The IL-2 mRNA quantitative Application of using this whole blood system is in the t cell response of assessment to glutamate decarboxylase 65 (GAD65), and GAD65 is a kind of autoantigen, and it is the target of autoreactive T cell in the 1 type autoimmune diabetes.Figure 40 and 41 illustrates the result who obtains in this field.

Embodiment 11: transplant

The program of abideing by among this embodiment is summarized among Figure 40.This methodology is applied to transplant.Behind whole blood and alloreactivity non-T cell incubation, provide a kind of replacement scheme of classical mixed lymphocyte reaction (MLP) (MLR), with monitoring alloreactivity t cell response by the quantitative IL-2 mRNA of PCR in real time.Figure 52 and 53 illustrates the result who obtains in this field.

Although should be understood that in conjunction with detailed description of the present invention the present invention, the description of front is intended to explanation but does not limit the scope of the invention, and scope of the present invention is limited by the scope of accompanying Claim.Other aspects, advantage and change are included in the scope of following claim.

The comparison of table 1.Qiagen and MagNA Pure LC extraction method.

1.1.Qiagen mRNA extraction method.Blood mRNA from same blood sample extracts 9 times.

1.2.MagNA Pure LC (kit+instrument) mRNA extraction method.Blood mRNA by same blood sample preparation extracts 9 times.

Table 2. is used for (in real time) PCR ¹Oligonucleotides

1. for sufficient description, referring to Stordeur etc., J Immunol Methods (immunological method magazine), 259 (1-2): 55-64,2002.

2.F R and P represent forward and reverse primer and probe respectively; Numeral is from about the Genebank registration number X01586 of IL-2 with about the sequence location of the Genebank registration number NM_000589 of IL-4.

3. the final concentration of forward primer (F) and reverse primer (R).

4. the serial dilution by the PCR product for preparing by " classics " PCR generates calibration curve, and is as follows for the actual conditions of this PCR: at 95 ℃ of sex change 20s, extend 45s at 58 ℃ of renaturation 20s with at 72 ℃, amount to and carry out 35 circulations.MgCl ₂Final concentration is 1.5mM.

Table 3

Tetanus toxoid	Cord blood	Adult's whole blood (vaccine is recalled preceding)
			--	109±51	1,154±1,194
+	159±91	7,715±8,513

Claims

1. determinator (30) that is used for liquid biological sample, it helps to make described sample to be exposed to first material (2) and contrast material (35) respectively and is exposed to nucleic acid stability agent (5) subsequently, and described device comprises:

Wherein there is described first material (2) in-the first Room (32),

Wherein there is described contrast material (35) in-the second Room (34),

-Di three Room (36), wherein exist described stabilizing agent (5) and

-be used for the support (52) of biological sample pipe.

2. device according to claim 1, one or more sealed in wherein said chamber (32,34,36), and comprise one or more zones of being pierced through of being suitable for by hollow needle.

3. device according to claim 2, wherein said zone are reclosable barrier film (40,42,46).

4. according to each described device among the claim 1-3, also comprise and be used for the support (54 that removably is connected with transfer tube (60), 56), described transfer tube is included in arbitrary end and disposes hollow needle (62,66) hollow flexible or rigid pipe (64) are suitable at fluid transfer between any two chambers or between a chamber and described biological sample pipe.

5. device according to claim 4 also comprises transfer tube (60) as defined in claim 4.

6. according to claim 4 or 5 described devices, a pin (62) of wherein said transfer tube is connected with the mode of the hollow needle (80) of forcing pipe (88) with parallel alignment, described forcing pipe comprises the flexible hollow pipe (82) that at one end disposes described pin (80), thereby and is suitable for applying vacuum or pressure to chamber or biological sample pipe and promotes liquid and pass through transfer tube.

7. according to each described device among the claim 1-6, wherein said first material (2) is fixed on part or all inner surface of described first Room (32).

8. according to each described device among the claim 1-7, wherein one or more described chambers (32,34,36) comprise passage.

9. according to each described device among the claim 1-8, wherein said first Room (32) and/or second Room (34) remain under the negative pressure.

10. according to each described device among the claim 1-9, wherein said first Room (32) and/or second Room (34) are included in the indication of the stabilizing agent that wherein distributes known volume.

11. according to each described device among the claim 1-10, wherein said first material (5) comprises one or more immune system antigen.

12. device according to claim 11, wherein said immune system antigen is vaccine composition.

13. device according to claim 11, wherein said immune system antigen are the antigen that excites super allergen reaction.

14. device according to claim 11, wherein said immune system antigen are to be selected among histocompatibility antigen, bacterium LPS, tetanus toxoid, cancer immunotherapy antigen, MAGE-3, cat allergen, Feld1, the antigen presenting cell from organ donor, autoantigen, the GAD65 one or more.

15. according to each described device among the claim 1-14, wherein said stabilizing agent (35) is cell RNA degraded and/or gene induced inhibitor.

16. device according to claim 15, wherein said cell RNA degraded and/or gene induced inhibitor are to be present in PAXgene ^TMOr Tempus ^TMIn the blood rna pipe those.

17. a kit that is used to measure liquid biological sample comprises:

Vessel (1), it is suitable for accepting liquid biological sample, makes described sample be exposed to first material (2), and is exposed to the nucleic acid stability agent subsequently, and described vessel comprise:

A) be present in inner first material (1) of described vessel (2),

B) wherein have the container (4,12,16) of described stabilizing agent (35),

C) connection between described vessel (2) inside and described container (4,12, the 16) inside,

D) block the physical barriers (25,15,18) of described connection provisionally;

With

Contrast vessel (37), it is suitable for accepting liquid biological sample, makes described sample be exposed to the contrast material, and is exposed to the nucleic acid stability agent subsequently, and described contrast vessel comprise:

A) be present in the inner contrast material (35) of described contrast vessel (37),

B) wherein have described stabilizing agent (35 ') control container (4 ', 12 ', 16 '),

C) the inner and described control container of described contrast vessel (37) (4 ', 12 ', 16 ') connection between the inside,

D) block provisionally described connection physical barriers (15 ', 18 ', 25 ').

18. kit according to claim 17, wherein said first material (2) is fixed on part or all inner surface of described vessel (1).

19. kit according to claim 17, wherein said first material (2) is fixed on the solid phase carrier.

20. according to each described kit among the claim 17-19, wherein said first material (1) is a liquid.

21. according to each described kit among the claim 17-19, wherein said first material (1) is a solid.

22. according to each described kit among the claim 17-21, wherein said vessel (1) and/or contrast vessel (37) comprise one or more zones of being pierced through by syringe needle of being suitable for.

23. kit according to claim 22, wherein said zone are reclosable barrier films.

24. according to each described kit among the claim 17-23, wherein said vessel (1) and/or contrast vessel (37) comprise the accessory that is suitable for admitting syringe and content is wherein transferred to the inside of described vessel (1) or contrast vessel (37).

25. according to each described kit among the claim 17-24, wherein said vessel (1) and/or contrast vessel (37) comprise the accessory that is suitable for admitting syringe needle.

26. according to each described kit among the claim 17-25, wherein said vessel (1) and/or contrast vessel (37) comprise that the gas stream/liquid stream that can make from vessel minimizes and allow liquid biological sample to flow to valve in the described vessel.

27. according to each described kit among the claim 17-26, wherein said vessel (1) and/or contrast vessel (37) comprise a kind of equipment, can discharge the gas of being replaced by described equipment.

28. according to each described kit among the claim 17-27, wherein said vessel (1) and/or contrast vessel (37) remain under the negative pressure.

29. according to each described kit among the claim 17-28, wherein by applying physical force and make a d to described vessel or contrast vessel) physical barriers (15,18,25,15 ', 18 ', 25 ') open.

30. kit according to claim 29, wherein said power with opening device be passed to described physical barriers (15,18,25,15 ', 18 ', 25 ').

31. kit according to claim 29, wherein said power irreversibly open described physical barriers (15,18,25,15 ', 18 ', 25 ').

32. according to each described kit among the claim 17-31, wherein said vessel (1) and/or contrast vessel (37) are included in the indication of the stabilizing agent that wherein distributes known volume.

33. according to each described kit among the claim 17-32, wherein said first material (2) comprises one or more immune system antigen.

34. kit according to claim 32, wherein said immune system antigen is vaccine composition.

35. kit according to claim 32, wherein said immune system antigen are the antigen that excites super allergen reaction.

36. kit according to claim 32, wherein said immune system antigen are to be selected among histocompatibility antigen, bacterium LPS, tetanus toxoid, cancer immunotherapy antigen, MAGE-3, cat allergen, Feld1, the antigen presenting cell from organ donor, autoantigen, the GAD65 one or more.

37. according to each described kit among the claim 17-36, wherein said stabilizing agent is cell RNA degraded and/or gene induced inhibitor.

38. according to each described kit among the claim 17-37, wherein said cell RNA degraded and/or gene induced inhibitor are to be present in PAXgene ^TMOr Tempus ^TMIn the blood rna pipe those.

39. according to each described kit among the claim 17-38, the external engagement of wherein said vessel (1) and contrast vessel (37) is to form single entity.