CN101490559A - Homogeneous double receptor agglutination assay for immunosuppressant drugs - Google Patents

Abstract

A homogeneous, non-competitive, double receptor agglutination assay for measuring immunosuppressant drugs is described. The assay employs at least two receptors wherein each receptor is specific for a separate binding site on the drug and wherein each receptor is bound to a detection particle. The immunosuppressant drug binds to the receptors and causes particle agglutination, which can be measured and correlated with the presence or amount of immunosuppressant drug in a sample.

Description

The homogeneous double receptor agglutination assay of immunosuppressive drug

Invention field

The present invention relates to medicine monitoring field, especially relate to the existence of immunosuppressant drug in the working sample or the immunoassay of its content.More particularly, the present invention relates to be used to measure the noncompetitive dual receptor determination method of homogeneity (homogeneous) of immunosuppressive drug.

Background technology

Several important immunosuppressive drugs that act on immunophilin are arranged, as cyclosporin, tacrolimus, rapamycin and everolimus.Immunophilin is the protein chaperone with peptidyl prolyl isomerase activity, and it belongs to cyclophilin (CyPs) in conjunction with cyclosporin and FK506 in conjunction with one of them of two extended familys of albumen (FKBPs).

Cyclosporin (being also referred to as cyclosporine and Ciclosporin A) is the natural metabolic product of soil fungi, by the peptide that 11 amino acid are formed, with tacrolimus, is the calcineurin inhibitor.Cyclosporin is considered to combine with the cytosol albumen cyclophilin (a kind of immunophilin) of immunocompetent lymphocytes (particularly T lymphocyte).The compound of this cyclosporin and cyclophilin can be suppressed at the calcineurin of inducing interleukin 2 to transcribe under the normal environment.This medicine also suppresses the generation of lymphokine and the release of interleukins, thereby causes the hypofunction of effector T cell.

Tacrolimus (FK506) also is epiphyte product (Streptomyces tsukubaensis).It is macrolide and works by suppressing calcineurin.This medicine especially is used in the transplanting of liver, kidney, heart and lung.It combines with immunophilin, and then compound combines and suppress the activity of its phosphatase with calcineurin.By this way, it has stoped the G0 phase to be transferred to the G1 phase.Tacrolimus render a service stronger than cyclosporin and remarkable spinoff still less.

Rapamycin (sirolimus) is very potent immunosuppressive drug.It forms compound closely with FKBP 12 (FK504 of 12kDa combines albumen), and rapamycin-FKBP12 compound combines with the rapamycin target (yTOR) of mammiferous rapamycin target (mTOR) or yeast more successively.The form that two kinds of yTOR are arranged, yTOR1 and yTOR2.As a member in the relevant kinases family of new phosphatidyl inositol kinase, the kinase whose activity of mTORs is that its signal conduction function is necessary.In mTOR, the FKBP12-rapamycin is differentiated the fragment that is to be positioned at the 11kDa on the amino terminal of kinases territory in conjunction with territory (FRBD).

Although cyclosporin is different with the molecular structure of tacrolimus (FK506), their immunosuppressive properties is similar with molecular mechanism.Both all with in the cytosol albumen that enriches is the immunophilin born of the same parents combine.As for tacrolimus, main it seems in conjunction with albumen is FKBP12, and cyclosporin combines with the immunophilin that gang is called cyclophilin.This then medicine-immunophilin compound can form the pentamer compound with calcineurin, calmodulin and calcium, causes the noncompetitive inhibition of calcineurin-phosphatase activity.

Everolimus is immunosuppressant macrolide, and it has stable 2-hydroxyethyl chain in the structural C-40 of sirolimus position.Everolimus has bigger polarity than sirolimus, is to develop in the work of the PK (pharmacokinetic) profile that improves sirolimus.

Calcineurin (CaN) is the heterodimer protein phosphatase, and it is regulated subunit B (CaNB) by 61kDa catalytic subunit A (CaNA) and 19kDa and is formed.Known, tacrolimus (FK506) mainly mediates by FKBP12 for the inhibiting effect of CaN enzymatic activity.In fact the FK506/FKBP12 compound is under the situation that calcium ion exists, the combining of high-affinity with CaN, thus may develop amboceptor " sandwich " determination method of tacrolimus in the detection by quantitative blood sample.

Because the interaction when CaN A that crystal structure analysis shows and CaN B all help to combine with the FK506-FKBP12 compound between the interface, desirable fusion should be striden this two subunits.Recently, reported that the 97kDa strand merges calcineurin A/B-calmodulin (scCN) polypeptide expression and purifying (Y.Qin et al, Biochimica et Biophysica Acta, 1747,171-178,2005).Although this has hinted that the CaN-CaM of strand may show the activity of phosphatase, whether have the ability that combines with the FK506-FKBP12 compound and still be not identified.In addition, the expression of this 97kDa scCN is very low.Under for the effort that produces the less and easy-to-operate protein of volume, Schreiber and his colleague disclose by the generation of the CaN fusions of brachymemma.In this fusions, for the CAB (fCAB) of energy expressive function, the amino acid/11 2-394 of CaNA and the amino terminal of whole C aNB merge.Similar, also made up minimum CAB by name (mCAB) than short run this, it contains amino acid 340-394 and the whole C aNB of CaNA.The activity that these fCAB and mCAB combine with the FK506-FKBP12 compound is to measure (P.demons et al, the Chemistry shown in the indication by the report of transcribing of the Jurkat T cell of transient transfection; Biology, 9,49-61,2002).Yet so far just more not deep expression and purifying about fCAB and mCAB is disclosed with Study of Interaction FK506-FKBP12 external directly determining.

In order suitably to manage the transplant patient who accepts the immunosuppressive drug treatment, the optimum blood medicine concentration that obtains medicine is even more important.The use because immunosuppressive drug often mutually combines, the selectivity of parent drug are crucial.

The paratope that the similar conjugate of competitive immunometric assay method Chinese traditional medicine of known commercially available immunosuppressive drug and free drug are competed limited quantity jointly.Although these immunoassays have good selectivity for parent drug, they show significant cross reactivity to one or more metabolic products usually, therefore compared to contrast method, can cause unwanted overgauge (bias) in the immunoassay.The determination method of current immunosuppressive drug also comprises high performance liquid chromatography connexus Zymography (LC/MS/MS).These methodologies need special equipment and technology.

Competitive immunometric assay for example another problem of rapamycin is need utilize the like derivatives of rapamycin, and its character to be also unstable.Another problem of competitive immunometric assay method is that the assay sensitivity that will reach enough when measuring low-down active drug concentration range (for example 5-20ng/ml) is very difficult.

Form in the uniceptor immunosuppressive drug determination method that is used for detecting rapamycin, verified hop protein and the direct covalent coupling of particle of finishing, see, the pending trial and the US60/714 of transfer simultaneously jointly, 712 (proposing) and US 11/468 on September 7th, 2005,940 (proposing on August 31st, 2006), the content of these two applications are by reference and in this paper.The use of nano particle is also open therein.

Armstrong etc., in Clinical Chemistry 44:12,2516-2523 (1998) have described its active metabolite of measuring tacrolimus and extracting in whole blood pentamer forms determination method.They have described and have utilized protein-bonded determination method based on microtiter plate.

Sagi-Eisenberg has taught and has utilized immobilized FKBP12 to detect the heterogeneous determination method of rapamycin on July 14th, 2005 disclosed WO 2005/062708.

Huster has described the immunoassay of measuring sirolimus on May 11st, 2006 disclosed US 2006/0099654, the sample that suspection is contained sirolimus combines with two kinds of latex globule reagent and biotinylated analyte receptor.A kind of globule pack is by streptavidin and comprised the photonasty dyestuff.Second kind of bead pack is by the antibody produced with the sirolimus molecule fragment and comprise the chemiluminescence dyestuff.KFBP can be used as analyte receptor.

Summary of the invention

The present invention comprises the existence of immunosuppressive drug in the working sample or the method for amount, may further comprise the steps: (a) provide and suspect the sample that contains immunosuppressive drug, (b) in sample, add at least two kinds medicine had specific acceptor, wherein every kind of acceptor has specificity for the binding site that separates on the medicine, and wherein every kind of acceptor combines with the detection particle, and its Chinese traditional medicine and these receptors bind and produce particle agglutination reaction further, (c) measure the amount of particle agglutination, and (d) amount of particle agglutination is associated with existence or its amount of immunosuppressive drug in the sample.According to an embodiment, have only a kind of acceptor to combine, and the antibody that combines with receptor protein specifically rather than combine with particle is used for agglutinating particle with detecting particle.In another embodiment, to target immunosuppressive drug specific antibody surrogate has been arranged a kind of or whole two kinds acceptor, wherein this antibody with detect particle and combine.

The accompanying drawing summary

Fig. 1 is according to the description of the present invention in embodiment 8, with the rapamycin calibration curve of yTOR1 FRBD.Rapamycin concentrations is indicated on the x axle and absorbance is indicated on the y axle in (spiked) whole blood sample of admixture (contrast).

Fig. 2 is according to the description of the present invention in embodiment 8, with the rapamycin calibration curve of yTOR2 FRBD.Rapamycin concentrations is indicated on the x axle and absorbance is indicated on the y axle in the whole blood sample of admixture (contrast).

Fig. 3 has compared the chart of the measured value (y axle) of rapamycin concentrations with respect to the rapamycin concentrations (x axle) of admixture according to the description in the embodiment of the invention 8.The concentration of rapamycin is indicated on the x axle and the measured value of rapamycin concentrations is indicated on the y axle in the whole blood sample of admixture (contrast).

Fig. 4 is according to the description in the embodiment of the invention 15, with the rapamycin calibration curve of mTOR FRBD.Rapamycin concentrations has been indicated on the x axle and absorbance has been indicated on the y axle in the whole blood sample of admixture (contrast).

Fig. 5 has showed the interactional curve map of the calcineurin A/B fusions of the FKBP12 of FK506 combination and brachymemma.

Detailed Description Of The Invention

Term " detection particle " refers to any common to about 1000nm diameter of about 10nm herein Spheric granules, and can in the homogeneity determination method, be resolved out state of aggregation and non-state of aggregation. Inspection Surveying particle can be nano particle or particulate, but for convenience, with these all herein particles Be called " particulate ". The surface of detection particle may functionalised and chemical compound lot can be attached to its table On the face, comprise for example fluorogen, chemical luminophor, antibody, biotin or streptavidin.

Epi-position is the zone that can stimulate specific immune response on antigen molecule surface, and described replying is directed at this zone.Epitope tagging is a recombinant DNA method, by the known antibody of this method clone gene encoded protein confrontation immune response is arranged.The developing into simplify to purify and utilize affinity substrate and specific antibody to detect recombinant protein of fusion tag system provides huge dirigibility.The length range of epitope tag is 10 to 15 amino acid, and is designed to create molecule handle (handle) into protein.Thereby they are placed on amino or the c-terminus any potential destruction of as far as possible reducing the function of the tertiary structure of protein and corresponding proteins matter usually.Because label is all very little, they also can not disturb the 26S Proteasome Structure and Function of recombinant protein.Therefore, epitope tag does not need to be removed usually before the experiment of carrying out subsequently.Many different labels have been used, for example His ₆(sequence of 6 histidines is to containing metallic ion such as Ni ^{+ 2}Or Co ^{+ 2}Matrix very strong affinity is arranged), HA (being derived from the peptide sequence of human influenza virus hemagglutinin), and AviTag utilize colibacillary biotin protein ligase (BirA) the biotinylated sequence of enzyme process list on portion's lysine residue within it.This kind of enzyme BirA can join and express potpourri also in position by biotinylation.

Term " FK506 is in conjunction with albumen " or " FKBP " refer to the receptor protein that combines and form compound with rapamycin in this article, this compound energy specificity is incorporated into and is selected from following albumen: mTOR, yTOR1, yTOR2, and the FRBDs of mTOR, yTOR1 and yTOR2.

Term " specificity combination " refers to high affinity (avidity) and/or high-affinity (affinity) combination between two paired kinds (species), for example this paired kind such as part/target part have been comprised, enzyme/substrate, acceptor/activator, antibody/antigen, and agglutinin/carbohydrates.Binding interactions may be mediated by interaction covalently or non-covalently, or the combination of covalency and noncovalent interaction.Especially, one of them the paired member that is characterized as of specificity combination combines with specific kind, but not with the corresponding member of binding members under compound family in other kinds combine.Therefore, for example, antibody preferably combines with single epitope rather than with other epi-positions in the protein families.

Term " acceptor " or " bound fraction " refer to the protein or the glycoprotein that can combine with target compound specifically in this article.The example of acceptor is the immunophilin that combines with immunosuppressive drug.

Term " nanoshell (nanoshell) " refers to the particulate of being made up of the electrolyte nuclear (for example, silicon dioxide or calcium phosphate) of super thin metal layer (for example, gold) bag quilt in this article.Be applicable to that nanoshell diameter of the present invention is selected from the scope of 50nm to 250nm.The surface of particulate can functionalised, and comprises that the chemical compound lot as antibody or streptavidin can be attached to its surface.

Term " particulate " refers to the solia particle that diameter is about 10nm to 1000nm in this article.Generally be used for mean particle dia of the present invention and be about 100nm to 600nm.Particulate can be formed and comprised polystyrene and emulsion particle by multiple material, and its surface all randomly is functionalized, and chemical compound lot can be attached to its surface, comprises as antibody or streptavidin.

Term " antibody " refers to one or one group polypeptide that comprises at least one in conjunction with the territory herein, wherein antibody binding domain is that variable domain by antibody molecule is folded to form and is three-dimensionally formed in conjunction with the space, the feature complementary of the antigenic determinant of the shape of its inside surface and CHARGE DISTRIBUTION and antigen so just can produce immune response with antigen.Except as otherwise noted, in general described antibody is contained polyclone and monoclonal antibody.Term " antibody " also comprises the recombinant protein that comprises antibody binding domain and antibody fragment, and wherein antibody fragment comprises Fab, Fab ', F (ab) ₂And F (ab ') ₂Fragment.

" joint " is can be in conjunction with key, molecule or the group of the molecule of two corpus separatums herein.Thereby joint can provide optimized spacing or can further provide the unsettled key that connects that two entities are separated from each other for two entities.Unsettled connect that key comprises can be by the group of photodestruciton, the part of acid labile, alkali-sensitive part and the group that can be destroyed by enzyme.

Term " homogeneity determination method " refers to and determines the existence of compound or qualitatively or quantitatively determining of concentration in the sample herein, wherein, in order to detect or the measurement target compound concentrations, unreacted detectable does not need to separate from the reagent that has reacted with target compound.

Term " sandwich assay " refers to the homogeneity determination method that detects target compound herein, and this has wherein utilized two kinds of bound fractions, and each part combines with target compound specifically, but is combined on two independent position.

The present invention relates to immunosuppressive drug in the detection of biological liquid homogeneity, based on the sandwich assay of particle.More particularly, the present invention relates to utilize the bound fraction that can combine with immunosuppressive drug specifically homogeneity, based on the determination method of particle.Determination method comprises at least two kinds of bound fractions, and wherein the bound fraction of every kind of separation is combined on the diverse location of target immunosuppressive drug, and every kind of bound fraction combines with one or more detection particles.Therefore, the doubtful sample that contains interested immunosuppressive drug will contact with the composition that comprises two kinds of bound fractions in the mensuration, and the degree of reagent aggegation (promptly forming the quantity of sandwich complex between two kinds of bound fractions and immunosuppressive drug) has shown the amount of immunosuppressive drug in the sample like this.

Detecting the degree of agglutinating reaction can utilize the homogeneous detection form to finish, wherein unreacted reagent (being unconjugated bound fraction) does not need to separate (that is the sandwich complex and the immunosuppressive drug that, comprise two kinds of bound fractions) from the reagent that has reacted.The known homogeneous detection method of those skilled in the art system is applicable to the present invention.

According to an embodiment, two kinds of bound fractions combine with detecting particle, and after the step of measuring the aggegation amount follows hard on sample and two kinds of bound fractions contact, are measured with respect to the turbidity of reference value by working sample.The influence of aggegation degree can be determined by measuring absorbance.According to an embodiment, detecting particle is particulate, and the microparticle agglutination determination method is applied to the existence or the amount of immunosuppressive drug in the working sample.Because do not have washing step before measuring the aggegation amount that has taken place, this microparticle agglutination determination method is considered to the homogeneity determination method.The microparticle agglutination determination method utilizes automated analysis instrument such as Luo Shi/Hitachi and COBAS analyser (Luo Shi diagnosis, Indianapolis) to be convenient to very much carry out and measure.In microparticle agglutination determination method of the present invention, first bound fraction and second direct or indirect combining of bound fraction with particulate.Determined that first and second bound fractions have specificity to binding sites different on the medicine.In reaction solution, these reagent (bound fraction that combines with particulate) spread in liquid medium and form stablizes suspension.If there is immunosuppressive drug, medicine can take place combine and produce microparticle agglutination and the corresponding increase of solution turbidity generation with acceptor (bound fraction), these can come quantitative via measuring solution absorbency.The amount that the sample Chinese traditional medicine exists and the numerical value of measured absorbance are directly related.

According to an embodiment, the detection particle is a nanoshell.Nanoshell is the adjustable detection particle of being made up of electrolyte (for example, silicon dioxide, alginates or the calcium phosphate) nuclear that is coated with super thin metal (for example gold) layer of optics.The gold nanoshell has the physical property similar to collaurum, and especially the gathering electron reaction of metal pair light produces strong light absorption.The thickness of the relative size of nanoparticle core and gold shell has determined the light reaction of gold nanoshell to a great extent.By changing the relative thickness of nuclear and shell, the color of gold nanoshell can change in very wide spectral range, strides visible range and near infrared spectrum district.The gold nanoshell can be come preferential absorption or scattered light by the size that changes particulate with respect to their light wavelength of optical resonance.In one embodiment, the diameter of nanoshell core is selected from about 50nm to 200nm, and in the embodiment of about 120nm, the thickness of nanoshell bag quilt or shell is selected from about 5nm to 30nm.

The whole blood immunity determination method (Anal.Chem.75:2377-2381,2003) of utilizing the gold nanoshell has been described.It has for example indicated can replace antibody in conjunction with albumen as immunophilin and so in the nano particle determination method.The principle of nano particle aggegation is similar to microparticle agglutination technology.The high turbidity of blood and strong visible delustring make the detection of complexization of aggegation in the CA.Yet the selection of desired wavelength and the pre-service of blood sample can overcome these complicated factors.Being also advantageous in that of nanoshell, with regard to the agglutinating reaction in the whole blood, they are very stable, and make nano particle generation dimerization or the congregation can be by detecting at nearly far red spectral shift with sandwich assay form (immunosuppressive drug with combine albumen), whole blood has better conduction herein, helps detecting the gathering of nanoshell in the whole blood.

According to an embodiment, detection and quantitative immunosuppressive drug are selected from: cyclosporin, tacrolimus, rapamycin and everolimus, and bound fraction is an immunosuppressive drug bind receptor albumen.In one embodiment, acceptor is represented the natural immunophilin of target immunosuppressive drug, or the binding fragment of described natural immunophilin or derivant.In one embodiment, the target immunosuppressive drug is rapamycin or everolimus, and first acceptor comprises FK506 and comprises rapamycin target (TOR) albumen in conjunction with the albumen (FKBP) and second acceptor.In another embodiment, FKBP is FKBP12 or FKBP25, and TOR is selected from the FRBDs of mTOR, yTOR11, yTOR2 and mTOR, yTOR1 and yTOR2.

In an alternate embodiment, the immunosuppressive drug tacrolimus in the sample is detected and quantitative, and wherein first acceptor comprises FK506 second acceptor comprises calcineurin or its binding fragment or derivant in conjunction with albumen (FKBP).In another embodiment, the calcium ion that second acceptor also comprises calmodulin and combines with calcineurin.In another embodiment, detected and quantitative immunosuppressive drug is a cyclosporin, wherein the cyclophilin that comprises of first acceptor is selected from: cyclophilin A (CypA), cyclophilin B (CypB), cyclophilin C (CypC), at Mikol, Deng having described described structure among the .PNAS 91:5183-5186 (1994), and second acceptor comprises calcineurin.In another embodiment, the calcium ion that second acceptor also comprises calmodulin and combines with calcineurin.

According to an embodiment, the bind receptor albumen of immunosuppressive drug combines with detecting particle, and wherein detecting particle is nanoshell or particulate.Detecting particle can combine with receptor protein by any standard technique that those skilled in the art is known.For example, detect particle and can utilize modular connection to combine with receptor protein by direct covalent bonds, perhaps albumen can pass through ionic link, hydrogen bond, hydrophobic/hydrophilic interacts or their any combination combines with the detection particle.In one embodiment of the invention, receptor protein by direct coupling or by label attached to detecting on the particle.

In one embodiment, detect particle can attached on the anti-tag antibody and in conjunction with albumen attached on the corresponding label.Perhaps, first receptor protein is attached on the label (haptens), comprises the biological example element, can or be incorporated into the antibodies of label (haptens) with streptavidin and detect particle.Medicine second in conjunction with albumen also attached on the identical label, perhaps in another embodiment, it is attached on the different labels.Number change attached to biotin on the protein or tag molecule can be very big, but most preferred, per molecule adheres to a biotin (or other labels) in conjunction with albumen thereby drops to the spontaneous agglutination of streptavidin particulate (or part of described mark) minimum.

According to an embodiment, streptavidin, avidin or anti-biotin antibodies (or antihapten antibody) are attached to detecting on the particle.If there is immunosuppressive drug to exist, medicine and two kinds are biotinylated so will form sandwich construction simultaneously in conjunction with albumen.If have the detection particle of streptavidin mark to exist, agglutinating reaction can take place, and can measure by light scattering.The medicine that exists in the sample is many more, and the agglutinating reaction that can observe is also just obvious more.If there is not medicine to exist, then can not form any sandwich structure and detect particle can aggegation yet.According to an embodiment, detecting particle is the particulate of streptavidin mark, and biotinylated receptor protein with contain the immunosuppressive drug matter sample and contact before all in advance attached to the streptavidin particulate on.This step provides optimal result for detection.

In the same way, when two receptor proteins by a member institute mark in the paired fluorophore of fluorescence resonance energy transmission (FRET), the immunosuppressive drug of existence can be detected respectively.If there is an immunosuppressive drug, medicine and two kinds are biotinylated will to form sandwich structure simultaneously in conjunction with albumen.The formation of this compound, stimulate the donor fluorophore to produce the fluorescent energy transmission by exciting light after two fluorophores of guiding enough near each other.This activity can via detect the emission of donor fluorophore weaken or the enhancing of acceptor fluorescence group emission is measured.

According to an embodiment, receptor protein can be expressed as recombination fusion protein, has comprised the peptide sequence that shows as part or label, or for adhering to of biotin molecule providing desirable site.For example receptor protein can be the recombinant protein that includes the fusion partner of biotinylation burst.This fusion in Escherichia coli or cell free system with biotin ligase co expression, as the Avi label, catalysis the biotin moiety covalency append on the lysine residue in the burst.In one embodiment, two kinds of biotinylated proteins that different receptor proteins is the Avi mark in the determination method.Can pass through c-terminus or aminoterminal attached on the Avi sequence label.The model of sandwich notion is as having hinted that at least one label should be positioned on the aminoterminal to prevent steric hindrance.Yet two kinds of labels of discovery that the applicant is surprised are all fine in the determination method effect of c-terminus.

In a kind of alternate embodiment, the antibodies of anti-receptor protein wherein combines with receptor protein itself or combines with the label that is incorporated into receptor protein to the antibody specificity of combination in the surface of detecting particle.As if existing immunosuppressive drug then can form sandwich structure and producing aggegation.These antibody are can not design with the principle that the receptor protein binding site of immunosuppressive drug combines and or select according to it.

According to an embodiment, measuring reagent provides for the amount of the immunosuppressive drug that detects and quantitatively exist in the sample.Sample can be the fluid sample that any suspection contains immunosuppressive drug.Typical sample gives immunosuppressive drug patient's biological fluid for collecting to control oneself.In one embodiment, sample is whole blood or its derivative products.If utilize the detection method of microparticle agglutination as sample and determination method with whole blood, be incorporated into intracellular medicine in order to discharge, blood sample usually can sample with carry out pre-service before detectable contacts.This step comprises allows whole blood sample combine with precipitation agent.This precipitation agent is selected from well known in the art, comprises for example containing aqueous sulfuric acid copper or zinc sulfate at methyl alcohol, ethanol, ethylene glycol, acetonitrile or similar water miscibility organic solvent.This then whole blood potpourri is fully mixed and is centrifugal.Shift limpid supernatant in sample cup and be placed on the analyser and analyze, in one embodiment, the potpourri that is used for the pre-service whole blood sample comprises methyl alcohol and zinc sulfate.After sample is pretreated, before adding detectable, shift out cell fragment.(about 12,000rpm) shifted out in 5 minutes by centrifuging by low speed for cell fragment in an embodiment.

According to an embodiment, measure reagent and comprise first acceptor and second acceptor, wherein the binding site specificity of separating with drug target at reagent and medicine contact back first and second acceptors in conjunction with and form sandwich complex.According to an embodiment, first and second acceptors all have can be for receptors bind to the label that detects on the particle.In another embodiment, first and second acceptors are equipped with the detection particle with first and second receptors bind.In one embodiment, first and second acceptors also comprise biotinylated label and detect particle and are incorporated into streptavidin.In another embodiment, each first and first acceptor all is equipped with single biotin molecule.In one embodiment, detecting particle is the particulate that is incorporated into streptavidin or biotin specific antibody.In one embodiment, the immunoassay reagent that provides is two kinds and comprises by biotin-streptavidin and connect the compound that key is incorporated into the acceptor of particulate.

In one embodiment, the CA reagent that is provided is the amount for rapamycin or everolimus in the quantitative sample.In this embodiment, first acceptor comprise be incorporated into detect particle FK506 in conjunction with albumen, and described second acceptor comprises and is incorporated into rapamycin target (TOR) albumen that detects particle.In one embodiment, first acceptor comprises FKBP12 or FKBP25 and second acceptor and is selected from: mTOR, yTOR1, yTOR2, and the FRBDs of mTOR, yTOR1 and yTOR2.In an alternate embodiment, the CA reagent that is provided is the amount for the tacrolimus that exists in the quantitative sample, wherein reagent comprises and detects FK506 that particle combines in conjunction with first acceptor of albumen (FKBP) with comprise the calcineurin that combines with the detection particle, or second acceptor of its binding fragment or derivant.In another embodiment, second acceptor also comprises calmodulin and is incorporated into the calcium ion of calcineurin.In another embodiment, the CA reagent that is provided is used to the amount of quantitative cyclosporin, wherein reagent comprises and contains first acceptor that is incorporated into the cyclophilin that detects particle, this cyclophilin is selected from: cyclophilin A (CypA), cyclophilin B (CypB) and cyclophilin C (CypC) and comprising is incorporated into the calcineurin that detects particle or second acceptor of calcineurin fragment.In another embodiment, second acceptor also comprises calmodulin and is incorporated into the calcium ion of calcineurin.Detect particle normally nanoshell or particulate.

In an optional embodiment, the mensuration reagent of the mensuration immunosuppressive drug that is provided is the determination method of homogeneity FRET for the basis.In this embodiment, measure reagent and comprise first acceptor and second acceptor, wherein when medicine contacts with reagent, the binding site specificity of separating on the first and second receptor-specific ground and the target immunosuppressive drug in conjunction with and form sandwich complex, wherein the donor fluorophore is marked on first acceptor and acceptor fluorescence group is marked on second acceptor.

In an embodiment, the method for the amount of immunosuppressive drug in the working sample that provides.The immunosuppressive drug that this method comprises the step of isolating sample and is incorporated into cell for release is chosen wantonly sample is carried out pre-service.Sample contacts and forms suspending liquid with the mensuration reagent composition that comprises first and second acceptors then, wherein is incorporated on the different position of target immunosuppressive drug first and second receptor-specifics.In one embodiment, reagent composition also comprises the detection particle that is incorporated into first and second acceptors.Perhaps, in one embodiment, the composition of the detection particle that is provided for separating, added first and second acceptors before, central or add the liquid that hovers afterwards.Measure reagent mix and induce the aggegation that has drug target together, wherein be combined on the binding site that described medicine separates each first and second receptor-specific, and detect particle can detected sandwich complex with each receptors bind (if not being incorporated into acceptor as yet) formation.Directly measured the amount of agglutinator then, meaning does not need further purifying or washing step, and therefore this mensuration is the homogeneity determination method.The homogeneity determination techniques of utilizing those skilled in the art to be familiar with is associated the amount of immunosuppressive drug with detected particle agglutination amount.According to an embodiment, utilize absorbance measurement to measure the degree of particle agglutination and the amount of existing medicine is determined by the normative reference curve.

In one embodiment, detecting particle is not that particulate is exactly a nanoshell, and the detection particle all is a particulate in an embodiment scheme.By selecting to be applied in the acceptor in the mensuration, described method can be used to measure the amount of immunosuppressive drug, and medicine is selected from cyclosporin, tacrolimus, rapamycin and everolimus.For detecting rapamycin or everolimus, first acceptor comprises the FK506 that combines with the first detection particle and comprises rapamycin target (TOR) albumen that combines with the second detection particle in conjunction with the albumen (FKBP) and second acceptor.Be to detect tacrolimus, first acceptor comprises with first and detects FK506 that particle combines in conjunction with albumen (FKBP), and second acceptor comprises and is incorporated into second calcineurin that detects particle.For detecting cyclosporin A, first acceptor comprises the cyclophilin that is incorporated into the first detection particle, and second acceptor comprises the calcineurin that is incorporated into the second detection particle.With regard to regard to the method for tacrolimus of measuring and quantitatively existing or cyclosporin A, second acceptor (calcineurin) can further be chosen wantonly with calmodulin and calcium ion compound.

Quantitatively the method for immunosuppressive drug can any think carry out in the aqueous specimen that contains immunosuppressive drug, as the existence of determining medicine in the sample with measure the method for its concentration.In one embodiment, sample is the body fluid that the patient exsomatizes, and these liquid comprise blood, blood plasma, serum, urine, seminal fluid, celiolymph, saliva or the like.In one embodiment, sample is a whole blood.Be combined in intracellular immunosuppressive drug in order to discharge, usually blood sample with can carry out pre-service before detectable contacts.In one embodiment, blood sample comes out by using the solution extraction that comprises methyl alcohol and zinc sulfate, and cell fragment is moved out of before described blend step.In one embodiment, the detection particle all is nanoshell and does not have the pre-service blood sample before adding detectable.

In one embodiment, in CA, can replace one or whole receptor proteins with the antibody of anti-immunosuppressive drug.

In one embodiment, the antibody that is produced (and at one among the monoclonal antibody embodiment) can resist the compound that comprises immunosuppressive drug and immunophilin, wherein according to its with immunosuppressive drug combine selection antibody.By this way, compared to the nonactive metabolic product of immunosuppressive drug, strengthened the specificity of antibody to active immne inhibition medicine.For example, the antibody that can create antagonism FKBP12 binding site (use therein immunogene comprises the compound of rapamycin and rapamycin target (TOR) albumen) is had the part of specific rapamycin molecule.Perhaps, the antibody that can create antagonism FKBP-rapamycin associated protein binding site (use therein immunogene comprises the compound of rapamycin and FKBP12) is had the part of specific rapamycin molecule.Similarly method can be applied on other immunosuppressive drugs that comprise cyclosporin, tacrolimus and everolimus etc.For example, create antagonism the FK-506 that is incorporated into FKBP12 antibody and be used for FK-506 in the working sample.FK-506 and FKBP12 combine very closely and the FK-506/FKBP12 compound can form immunogene with the KLH pairing, it is injected in the mouse body can causes immune response.(in this case, can finish coupling by the protein-protein coupling mode of standard between FKBP12 and the KLH).Separating the suitable antibody and the selection of monoclonal antibody can finish by the standard method that present technique field personnel know.This method also can be used for making the anti-antibody that is incorporated into the cyclosporin A of cyclophilin.

According to an embodiment, the method for immunosuppressive drug amount in the working sample is provided, wherein measure reagent and be selected from the antibody that is incorporated into the receptor protein that detects particle and is incorporated into the detection particle.Antibody is monoclonal antibody in one embodiment.In one embodiment, measure and utilized two kinds of antibody to finish, wherein the antibody that is produced all at the compound of immunosuppressive drug/immunophilin, still has specificity to two different sites on the immunosuppressive drug.In this embodiment, two kinds of antibody with detect the particle coupling.In another embodiment, measure and finish with two kinds of antibody, wherein the antibody that is produced is all resisted the compound of immunosuppressive drug/immunophilin, but two different sites on the immunophilin are had specificity.In another embodiment, mensuration reagent may comprise the acceptor and the another kind of receptor-specific that combine with the target immunosuppressive drug specifically and be incorporated on the diverse location of target immunosuppressive drug.Receptor protein and antibody all combine with the detection particle in this embodiment.

In another embodiment, measure reagent may comprise first acceptor, second acceptor and specifically with the antibody of second receptors bind.In this embodiment, first acceptor with detect that particle combines and the first receptor-specific ground combines with the target immunosuppressive drug.Second receptor protein is combined in the position that is different from first receptor protein on the target immunosuppressive drug.In this embodiment, second receptor protein with detect that particle does not combine and antibody is not labeled usually.Yet antibody may be chosen wantonly and detect particle and combine.In one embodiment, the antibody that combines with second receptor protein specifically that is produced, opposing comprises the immunogene of second acceptor/immunosuppressive drug compound.Therein in embodiment, wherein a kind of acceptor, for example FKBP12 (for rapamycin) is labeled label, the biological example element.When sample mixes with a spot of biotinylated first receptor protein and second receptor protein, existing drug target forms sandwich complex in the sample.Gained biotinylation sandwich complex and unreacted FKBP12 are added in second reagent of the antibody that contains the mould emulsion particle of antibiosis protein chain bacterium and combine with second receptor protein specifically.The agglutinating reaction that takes place be incorporated into the mould latex of antibiosis protein chain bacterium and proportional by the quantity of the biotinylation sandwich complex of antibody aggegation, it is directly related with the medication amount that exists again.Antibody can not produce any signal with free non-second compound acceptor gametophyte reaction.It is to be noted that in this alternatives, antibodies is not the receptor binding site of medicine in the site of second acceptor.US 6,464, described specificity in 974 to be incorporated into the FKBP/ rapamycin in conjunction with the anti-immunophilin antibody on the territory.

According to an embodiment, quantitatively the method for immunosuppressive drug comprises sampling and biased sample and reagent composition and forms suspending liquid.Reagent composition comprises acceptor and antibody, each all with detect that particle combine and wherein each described acceptor and antibody while combine with immunosuppressive drug specifically.Utilize the homogeneity determination techniques to measure the amount of existing agglutinating particle in the suspending liquid then, for example measure absorbance, again the amount of the immunosuppressive drug that exists in the particle agglutination amount of measuring and the sample is associated.

According to another embodiment, quantitatively the method for immunosuppressive drug comprises sampling, biased sample and reagent composition also form suspending liquid, wherein reagent composition comprises first and second antibody, and they all combine with the detection particle and wherein each described first and second antibody while combines with immunosuppressive drug specifically.Utilize the homogeneity determination techniques to measure the amount of existing agglutinating particle in the suspending liquid then, for example measure absorbance, again the particle agglutination amount of measuring is associated with the amount of existing immunosuppressive drug in the sample.

According to another embodiment, quantitatively the method for immunosuppressive drug comprises sampling, test agent mixed with reagent composition and form suspending liquid, wherein reagent composition comprise first and second acceptors and specifically with the antibody of second receptors bind, wherein each described first and second acceptor combines with immunosuppressive drug simultaneously specifically, and first acceptor combines with the detection particle.In one embodiment, antibody also combines with the detection particle.Utilize the homogeneity determination techniques to measure the amount of the agglutinating particle that exists in the suspending liquid then, for example measure absorbance, again the particle agglutination amount of measuring is associated with the amount of existing immunosuppressive drug in the sample.

The present invention has expected that also the generation of acceptor mutant can strengthen the selectivity of parent drug.According to an embodiment, utilize directed mutagenesis to produce the mutant of yTOR2 FRBD and FKBP12 peptide, thereby strengthen the specificity that the rapamycin parent drug is measured.Thereby make the receptor protein sudden change strengthen the specific notion of the receptor complex of rapamycin parent drug, can be applied on any receptor protein disclosed herein.The concentration of patient's sample rapamycin can be monitored and be used for measuring to the formation of yTOR2 FRBD/ rapamycin/FKBP12 compound by the several method of mentioning herein.

The research that is disclosed as mutagenesis of crystal structure provides help.The protein-bonded generation of variation can be finished by the recombination method that present technique field personnel know, and the recombination method affix affinity tag of also knowing by those skilled in the art in their purge process is offered help.Correspondingly, with reference to following form, can expect and modify the specificity that following amino acid residue can strengthen molecule: the glutamic acid on the FKBP12 position 54, the asparagine on the yTOR2 position 2038, arginine on aspartic acid on the yTOR2 position 1972 and the yTOR2 position 1976.

Following table has shown carbon-to-carbon distance between measured rapamycin metabolic product FKBP12 and the FRBD.Measure rapamycin metabolic product carbon-to-carbon apart from the 1fap crystal structure (12) that has utilized RCSB PDB (Protein Data Bank) to announce.

In one embodiment, utilize the fluorescence resonance of fluorophore can transmit (FRET) to measuring the formation of compound.FRET is based on excited energy is delivered to acceptor fluorescence group from the donor fluorophore the transmission apart from dependent form.If the donor fluorophore is excited by incident light and the size of acceptor exists

Extremely

In, excited energy could be uploaded from donor and hand over.The optimum range that exceeds intermolecular distance, successively decreasing of energy transmission efficiency is proportional with 1/6 power (inverse sixth power) of distance.This transmission causes the fluorescence intensity of donor and excited state lifetime to reduce, and strengthened acceptor emissive porwer (Selvin, P.R., Nature Structural Biology, 2000,7,9,730-734).This mensuration form needs a pair of FRET fluorophore and a pair of bind receptor coupling.Utilization detects in the optical excitation and the emission at the 665nm place at 340nm place, and use donor fluorophore (as europium) and acceptor fluorescence group (as Cy5) (Pulli, T., et.al, Analytical Chemistry, 2005,77,2637-2642).Correspondingly, first and second receptor proteins that combine with immunosuppressive drug specifically can come mark with donor and acceptor fluorescence group respectively.For example in the mensuration of rapamycin, can with europium donor fluorophore come mark first acceptor (for example, FKBP12) and second acceptor (for example, yTOR2FRBD) can come mark with Cy5 acceptor fluorescence group.Following form has been listed the measuring distance between selected amino acid in mTOR FRBD-rapamycin-FKBP12 compound (1fap, Protein Data Bank).A kind of possible combination is, is marked in the crystal structure apart

The aminoterminal of yTOR2 (R1959) and the aminoterminal of FKBP12 (G1), suppose yTOR2 adopted to mTOR FRBD-rapamycin-FKBP12 compound in the similar conformation of mTORFRBD.

Several commercial reagents boxes can come the amino acid of mark yTOR2 and FKBP12 with donor and acceptor fluorescence group.For example, LANCE Eu-W1024-ITC chelate (Perkin Elmer, ADOO 13) is optimized to the albumen that the enough europium covalent labelings of energy have at least one one-level fatty amine (aminoterminal or lysine base).In addition, CyDye Cy5 single reaction dyeing bag (Amersham, Product CodePA23500) can be used the amido on the Cy5 covalent labeling protein.Other adoptable chemical reagent can be by amido (utilizing the dyeing of NHS ester), sulfydryl (utilizing maleimide dyeing), or aldehyde radical (utilizing hydrazides dyeing) comes mark.Therefore can mark yTOR2 and FKBP12 acceptor, and can from the acceptor of mark, separate the fluorophore on unmarked by column chromatography.

Mensuration can be by rapamycin being added to suitable mark yTOR2 and carried out among the FKBP12 of mark.The existence of rapamycin can promote the formation with the compound of the yTOR2/ rapamycin/FKBP12 under the rapamycin dependent form mode.Utilize photofluorometer can measure the fluorescence that optical excitation produced by the yTOR2/ rapamycin/the FKBP12 compound forms.In this example, the measurement of fluorescence concentration direct and rapamycin is proportional.Can utilize known rapamycin concentrations to make up typical curve.Like this, unknown rapamycin concentrations can be calculated according to typical curve in patient's sample.Similar, other immunosuppressive drugs can be used to measure the receptor protein FKBP12 and the calcineurin of tacrolimus respectively with donor and acceptor fluorescence group mark, and the cyclophilin and the calcineurin that perhaps are used to measure cyclosporin are measured.

The present invention has also comprised the dual receptor determination method of tacrolimus (FK506), has wherein utilized the calcineurin A/B fusion that combines with FKBP12.Calcineurin A/B fusion may be the truncated sequence that has the native protein of amino or carboxyl terminal label.Therefore, in one embodiment, based in Escherichia coli and cell free system, expressing and the screening (screen) of solubility, carry out having selected the calcineurin A/B fusion that molecular weight is respectively the carboxyl terminal his mark of 64kDa (long CNtAB) and 26kDa (lacking CNtAB) in conjunction with determination of activity.If at expression in escherichia coli, long CNtAB and short CNtAB fusions have shown the stable and solubilized of fusion of strongly expressed level and expression.Utilize the fusion purity that purification step obtained of single affinity capture and SEC all to reach more than 90%.Importantly, in the mode of FK506 dependent form, the long CNtAB of purifying and combining of FKBP12 show that the EC50 value is 0.77nM under reference time resolution fluorescence (TRF) determination method.Yet, being combined in of the short CNtAB of purifying and FKBP12-FK506 almost detect in the mensuration less than.Plasmon resonance (SPR) determination method in Biacore surface can confirm the observed result of above TRF determination method.

The kit of the mensuration reagent that comprises immunosuppressive drug in the measuring samples is also contained in the present invention.This kit has also comprised many kinds of containers, for example phial, test tube, bottle or the like.Preferably, this kit also will comprise operation instructions.In one embodiment, kit comprises first and second bound fractions, wherein at reagent with after medicine contacts, the first and second bound fraction specificitys are incorporated on the binding site of separation of drug target and form sandwich complex.First and second bound fractions are represented the compound that independently is selected from receptor protein and antibody.In one embodiment, kit comprises first and second receptor proteins and antibody, wherein first receptor protein with detect that particle combine and antibody specificity combine with second receptor protein.In this embodiment, can choose wantonly with second the specific antibody of second acceptor and detect particle and combine.Single bound fraction can be provided in the independent container or is blended in the single container.

In an alternate embodiment, kit comprises first acceptor and second acceptor, wherein each first and second acceptor combines with at least one detection particle, and with after medicine contacts, first and second receptor-specifics are incorporated on the binding site that drug target separates and form sandwich complex at acceptor.In one embodiment, the receptor protein in the kit each all be equipped with can be with receptors bind in the label that detects particle, and detect particle and be contained in the independent container.In another embodiment, first and second acceptors will be equipped with the detection particle with first and second receptors bind.First and second receptor proteins can be contained in separately independently in the container or mix and be contained in the single container.In one embodiment, first and second acceptors comprise biotinylated label, detect particle and are incorporated into streptavidin.In one embodiment, each first and second acceptor all combines with single biotin molecule.In one embodiment, detecting particle is to be incorporated into streptavidin or to be incorporated into the particulate that biology is have specific antibody.In one embodiment, kit comprises two kinds of compounds, and each comprises by biotin-streptavidin and connects the acceptor that key combines with particulate.

The kit of rapamycin in the quantitative sample or everolimus amount is provided in one embodiment.In this embodiment, kit comprises: comprise second acceptor that is incorporated into protein-bonded first acceptor of FK506 that detects particle and comprises rapamycin target (TOR) albumen that is incorporated into second detection particle.In one embodiment, first acceptor comprises FKBP12 or FKBP25 and second acceptor and is selected from: mTOR, yTOR1, yTOR2, and the FRBDs of mTOR, yTOR1 and yTOR2.In an alternative embodiment, the kit of existing tacrolimus amount in the quantitative sample is provided, and wherein the reagent of kit comprises: comprise and be incorporated into the FK506 that detects particle in conjunction with first acceptor of albumen (FKBP) with comprise and be incorporated into the calcineurin that detects particle or second acceptor of binding fragment or its binding fragment derivant.In another embodiment, kit also comprises calmodulin and the calcium ion that is contained in the independent container or combines with calcineurin.

In the another one embodiment, the kit of quantitative cyclosporin amount is provided, wherein reagent comprises: comprise first acceptor that is incorporated into the cyclophilin that detects particle and comprise second acceptor that is incorporated into the calcineurin that detects particle.In another embodiment, kit also comprises calmodulin and the calcium ion that is contained in the autonomous container or combines with calcineurin.

The detection particle of kit is not that nanoshell is exactly a particulate usually, and to detect particle in one embodiment be emulsion particle.

The following example is used for illustrating specific embodiment and should be understood that to have limited the defined scope of the present invention of claims.

Specific embodiments

Embodiment 1: the cDNA's of coding yTOR1 FRBD and yTOR2 FRBD is synthetic

90 amino acid FKBP12 rapamycins of coding yeast TOR1 and TOR2 are in conjunction with the cDNA of territory (FRBD), utilize the following nucleotide sequence of optimizing by blue aigret biotechnology (Seattle, WA) chemosynthesis:

SEQ?ID?NO：1(yTOR1?FRBD)：

5′GAACTTTGGTATGAAGGACTCGAAGATGCATCTCGCCAATTTTTTGTTGAACACAATATC

GAAAAAATGTTTTCTACACTTGAACCCTTACACAAACACCTTGGAAATGAACCACAAACCCTC

TCTGAAGTCTCCTTTCAAAAATCCTTTGGCCGTGACCTTAACGACGCATATGAATGGCTTAAC

AACTACAAAAAATCTAAAGATATTAATAACCTGAATCAAGCATGGGACATCTATTACAACGTA

TTTCGCAAAATTACTCGTCAA?3′

SEQ?ID?NO：2(yTOR2?FRBD)：

5′GAACAATGGTATGAAGGCCTGGATGATGCCTCTCGTCAATTCTTTGGTGAACACAATACA

GAAAAAATGTTTGCAGCGCTCGAACCCTTATATGAAATGTTAAAACGTGGTCCAGAAACTTTA

CGGGAAATTTCTTTTCAAAATTCTTTTGGTCGTGATCTCAATGATGCCTATGAATGGTTAATG

AATTATAAAAAAAGCAAAGATGTTAGCAACCTTAATCAAGCCTGGGATATTTACTATAACGTA

TTTCGCAAAATCGGTAAACAG?3′

Embodiment 2: the structure of expression vector

Synthetic cDNAs is together with two groups of Oligonucleolide primers, the dna fragmentation that is used for the about 300-bp of pcr amplification, it is corresponding to yTOR1 FRBD and yTOR2 FRBD, and these dna fragmentations are cloned among expression vector pIVEX2.7d or the pFVEX2.8d (Luo Shi diagnoses GmbH, Germany).The plasmid that obtains confirms via determined dna sequence, can express the target protein of Avi mark, its FKBP12 that can not only combine with rapamycin in conjunction with can also by biotin ligase BirA specifically biotin labeling to single lysine residue.

SEQ ID NO:3 (forward primer of yTOR1 FRBD):

5’CTTTAAGAAGGAGATATACCATGGAACTTTGGTATGAAGGACTC?3’

SEQ ID NO:4 (reverse primer of yTOR1 FRBD):

5’AAGATGTCGTTCAGGCCCCCTTGACGAGTAATTTTGCGAAA?3’

SEQ ID NO:5 (forward primer of yTOR2 FRBD):

5’CGCTTAATTAAACATATGACCGAACAATGGTATGAAGGCCTG?3’

SEQ ID NO:6 (reverse primer of yTOR2 FRBD):

5’TTAGTTAGTTACCGGATCCCTTACTGTTTACCGATTTTGCGAAA?3’

Embodiment 3: the generation of biotinylated yTOR1 FRBD and biotinylated yTOR2FRBD in the Escherichia coli

After utilizing acellular quick translation system (RTS) selection of Luo Shi diagnostic companies, best expression construct and BirA expression vector are transformed in the BL21-A1 cell together, and inductive condition is that 50 μ M biotins, 0.2% arabinose and 0.2mM isopropyl B-D-thiogalactoside (IPTG) act on 16 hours down at 30 ℃.Recombinant protein by the avidin globule and the SEC purifying of improvement are expressed makes its purity reach about 90%.

Following is the amino acid sequence (106 amino acid) of reorganization yTOR1FRBD.Lysine 101 is the biotin labeled specific site of BirA dependent form.This albumen and rapamycin are combined on the FKBP12 binding site.The Avi sequence label represented by runic, and lysine 101 ( K) be the biotin labeled specific site of BirA dependent form.

SEQ ID NO:7 (yTOR1FRBD of Avi mark):

1 10 20 30 40 50 60

ELWYEGLEDASRQFFVEHNIEKMFSTLEPLHKHLGNEPQTLSEVSFQKSFGRDLNDAYEWL

70 80 90 100

NNYKKSKDINNLNQAWDIYYNVFRKITRQGGLNDIFEAQ KIEWHE

Be the amino acid sequence (119 amino acid) of reorganization yTOR2FRBD below.Lysine 12 is biotin labeled specific sites of BirA dependent form.This albumen and rapamycin are combined on the FKBP12 binding site.The Avi sequence label represented by runic, and lysine 101 ( K) be the biotin labeled specific site of BirA dependent form.

The yTOR2FRBD of SEQ ID NO:8:(Avi mark):

1 10 20 30 40 50 60

MSGLNDIFEAQ KIEWHEIEGRGRLIKHMTEQWYEGLDDASRQFFGEHNTEKMFAALEPLYE

70 80 90 100 110 120

MLKRGPETLREISFQNSFGRDLNDAYEWLMNYKKSKDVSNLNQAWDIYYNVFRKIGKQ

Embodiment 4: the structure of expression vector

Utilize two kinds of Oligonucleolide primers to produce dna fragmentation by PCR, it is coded in c-terminus and has a plurality of glycocoll and the amino acid whose FKBP12 of histidine, and is cloned on the Nco I/Sma I site of expression vector pIVEX2.7d.The plasmid of gained confirms via determined dna sequence, can express can combine with rapamycin and nickel NTA matrix and expect that molecular weight is the fusion of 15.0kDa, and carry out biotin labeling with biotin ligase BirA on the special lysine of Avi label.

Embodiment 5: the generation of biotinylation FKBP12 in the Escherichia coli (molecular weight 15kDa)

The BL21-A1 cell that transforms comprises above expression vector and expresses the carrier of BirA, under 30 ℃, induces described cell in 16 hours by 0.2% arabinose and 0.4mM isopropyl IPTG effect.The recombinant protein of expressing is respectively by reaching about 95% purity with Ni-NTA agarose and monoclonal antibody biotin protein globule (Pierce) through continuous affine purification.

Below be the protein-bonded amino acid sequence of this reorganization rapamycin (134 amino acid).Lysine 129 is the biotin labeled specific site of BirA dependent form.This albumen combines with rapamycin.

The FKBP12 of SEQ DD NO:9:(Avi mark):

1 10 20 30 40 50

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRG

60 70 80 90 100 110

WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSHHHHHHG

120 130

GLNDIFEAQ KIEWHE

The preparation of the particulate of embodiment 6:FKBP12 bag quilt

Streptavidin particulate (127nm) (SA-latex Universalreagenz ZLF, Mat. coding 03140431001) is from German Luo Shi diagnosis GmbH.At 50mM MOPS (3-(N-morpholino) propane sulfonic acid), among the pH7.9, ultimate density is 0.3% (w/v).

(32 μ L 3.27mg/mL) join in the 50nM MOPS damping fluid (pH7.9) of 5.2ml with the biotinylated FKBP12 among the embodiment 5.In the suspending liquid of this biotinylated protein solution stirring of the 50nM MOPS that contains 0.3% streptavidin particulate (127nm) of the disposable 5.2ml of joining at room temperature.At room temperature stir and spent the night in 2 hours and 4 ℃, then 16,000rpm centrifugal 1 hour 45 minutes.Particulate is resuspended in the 50mM MOPS damping fluid (pH7.9), uses 16 then, 000rpm carries out centrifugal.Repeat once resuspended and centrifugal program again, the ultimate density of the particulate of FKBP12 bag quilt is adjusted to 0.15% (w/v).

The preparation of the particulate of embodiment 7:yTOR1 FRBD and yTOR2 FRBD bag quilt

Biotinylated yTOR1 FRBD among the embodiment 3 (concentration is 0.35mg/ml, 300 μ l) is joined in the 50mM MOPS damping fluid (pH7.9).This biotinylated protein solution at room temperature joins 5.2ml once and contains in the suspending liquid of stirring of 50nMMOPS of 0.3% streptavidin particulate (127nm).At room temperature stir and spent the night in 2 hours and 4 ℃, then 16,000rpm centrifugal 1 hour 45 minutes.Particulate is resuspended in the 50mM MOPS damping fluid (pH7.9), uses 16 then, 000rpm carries out centrifugal.Repeat once resuspended and centrifugal program again, and the ultimate density of the particulate of yTOR1FRBD bag quilt is adjusted to 0.15% (w/v).

According to top description, can also prepare the particulate of yTOR2 FRBD bag quilt with the yTOR2 FRBD that makes among the embodiment 3.

Embodiment 8: the rapamycin calibration curve that utilizes the whole blood sample drafting of admixture (contrast)

Preparation contains the 175mM PIPES damping fluid of 0.5% polyacrylic acid (PAA), and pH7.4 comes the preparation feedback buffer reagent.This is the first operation reagent (working reagent).

The particulate that FKBP12 is wrapped the particulate of quilt and yTOR1 FRBD bag quilt mixes as the second operation reagent with the ratio (v/v) of 1:1.The second alternative operation reagent is to mix with the ratio (v/v) of 1:1 by the particulate that particulate that FKBP12 is wrapped quilt and yTOR2FRBD wrap quilt to get.

Calibration solution (calibrator) is by EDTA whole blood pond, with the methyl alcohol stock solution of rapamycin that contains 1 μ g/ml, by the admixture level be 0,5,10,20 and the rapamycin of 30ng/ml get.The pre-service of blood sample is finished with the albumen precipitation reagent of 4:1 ratio mixing methyl alcohol and aqueous sulfuric acid zinc (300mM) by use.

The whole blood of 200 μ l is joined in the precipitation reagent of 200 μ l and vortex 15-30 second, at room temperature left standstill 5 minutes, then 12,000rpm rotation 5 minutes down.Move to supernatant in the sample cup and put into analyser.

The first operation reagent (reaction buffer) is placed on the R1 reagent spinner, and the second operation reagent (particulate reagent) is placed on the R2 reagent spinner.With the sample of 35 μ l, the second operation reagent of the first operation reagent of 150 μ l and 80 μ l was measured (place is measured at the 480nm wavelength) 10 minutes by Luo Shi/Hitachi's 917 automatic analyzers (Luo Shi diagnostic companies, Indianapolis).Fig. 1 has shown that the rapamycin of drawing with yTOR1 FRBD detects calibration curve.Fig. 2 has shown that the rapamycin of drawing with yTOR2FRBD detects calibration curve.

In another kind of the mensuration, obtained 13 individual whole blood samples there, in having contained the 0-48ng/ml scope, gone into the rapamycin of various levels (having used 26 samples altogether) to admixture wherein from 13 volunteers.Utilize Luo Shi/Hitachi's 917 automatic analyzers to measure whole 26 samples according to above-mentioned step.The data presentation that is obtained is in Fig. 3, and is used for the mensuration of carrying out with yTOR2 FRBD.

Embodiment 9: the cDNA's of coding mTOR FRBD is synthetic

The cDNA in conjunction with territory (FRBD) of 90 amino acid whose FKBP12 rapamycins of coding mTOR, be utilize the following nucleotide sequence of optimizing by blue aigret biotechnology (Seattle, WA) chemosynthesis:

SEQ?ID?NO：10(mTOR?FRBD)：

5′gaaatgtggcatgaaggacttgaagaagctagccgtctctattttggtgaacgcaacgta

aaaggaatgtttgaagtacttgaacctttacacgcaatgatggaacgtggaccccaaacctta

aaagaaacctcctttaatcaagcatatggtcgtgacttaatggaagctcaagaatggtgtcgt

aaatatatgaaatctggtaatgtaaaagatttaactcaagcctgggatttatactatcacgtt

ttccgccgtatctcaaaacaa?3′

Embodiment 10: the structure of expression vector

SEQ ID NO:11 (primer 1 of mTOR FRBD):

5′CTTTAAGAAGGAGATATACCATGGAAATGTGGCATGAAGGACTTGAA?3′

SEQ ID NO:12 (primer 2 of mTOR FRBD):

5′AAGATGTCGTTCAGGCCCCCTTGTTTTGAGATACGGCGGAA?3′

Utilize the dna fragmentation of the cDNA of above two kinds of Oligonucleolide primers and above-mentioned coding mTOR FRBD, and it is cloned on the NcoI/Sma I site of expression vector pIVEX2.7d by pcr amplification 310bp.The plasmid of gained confirms by determined dna sequence, can express can combine with the FKBP12 that is incorporated into rapamycin and can carry out biotin labeled fusion with biotin ligase BirA.

Embodiment 11: the generation of biotinylation mTOR FRBD in the Escherichia coli

Utilize the acellular quick translation system (RTS) of Luo Shi to make the mTOR FRBD of biotin labeled Avi mark.The plasmid DNA of purifying in RTS 500 ProteoMaster reaction units with RTS reaction and biotinylation reagent incubation 18 hours.The recombinant protein that produces makes purity reach about 95% by the avidin globule purifying of improvement.

Following is the amino acid sequence (107 amino acid) (lysine 102 is biotin labeled specific sites of BirA dependent form) of this reorganization FRBD:

SEQ ID NO:13 (the mTOR FRBD of Avi mark):

1 10 20 30 40 50

MEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQ

60 70 80 90 100

EWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQGGLNDIFEAQ KIEWHE

The preparation of the particulate of embodiment 12:FKBP12 bag quilt

The particulate of FKBP12 bag quilt prepares by describe similar step in embodiment 6.Streptavidin particulate (SA-latex Universalreagenz ZLF, Mat. number 03140431001) is from German Luo Shi diagnosis GmbH (127nm).At 50mM MOPS (3-(N-morpholino) propane sulfonic acid), among the pH7.9, ultimate density is 0.3% (w/v).

Biotinylated FKBP12 (concentration is 2.5mg/ml, 40 μ l) is joined in the 50mMMOPS damping fluid of 5ml (pH7.9).This biotinylated protein solution is at room temperature joined 5ml once to be contained in the suspending liquid of stirring of 50mM MOPS (pH7.9) of 0.3% streptavidin particulate.At room temperature stirred 2 hours and under 4 ℃, spend the night, then 16,000rpm centrifugal 1 hour 45 minutes.Particulate is resuspended in the 50mM MOPS damping fluid (pH7.9), and with 16,000rpm is centrifugal.Repeat resuspended and centrifugal step more once, and the ultimate density that FKBP12 wraps the particulate of quilt is transferred to 0.15% (w/v).

The preparation of the particulate of embodiment 13:mTOR FRBD bag quilt

Biotinylated mTOR FRBD among the embodiment 11 (concentration is 2mg/ml, 50 μ l) is joined in the 5ml 50mM MOPS damping fluid (pH7.9).This biotinylated protein solution is joined 5ml once to be contained in the suspending liquid of stirring of 50mMMOPS (pH7.9) of 0.3% streptavidin particulate (127nm).At room temperature stir and made its reaction in 2 hours, then 16,000rpm centrifugal 1 hour 45 minutes, and the ultimate density of albumen-streptavidin particulate transferred to 0.15% (w/v).

Embodiment 14: the rapamycin calibration curve of using the whole blood sample drafting of admixture (contrast)

The particulate that FKBP12 is wrapped the particulate of quilt and mTOR FRBD bag quilt is mixed into the first operation reagent with the ratio (v/v) of 1:1.This reagent ultimate density in the 50mM of pH7.9 MOPS damping fluid is use in 0.15% o'clock.

The 175mM PIPES damping fluid (pH7.4) that contains 0.8% polyacrylic acid (PAA) by preparation prepares the second operation reagent.

Utilize admixture have 12.5,25 and the EDTA whole blood sample of the rapamycin (is the methyl alcohol stock solution of 10 μ g/mL from rapamycin concentrations) of 50ng/ml draw calibration curve.By using methyl alcohol and moisture CuSO ₄The albumen precipitation of (copper sulphate) solution is finished the pre-service of blood sample.

The whole blood of 200 μ l is joined in the methyl alcohol of 200 μ l, then add the 300mMCuSO of 50 μ l immediately ₄This solution of vortex 15-30 second, and at room temperature leaving standstill 5 minutes, then 12,000rpm rotation 5 minutes.Shift supernatant to sample cup and be loaded on Luo Shi/Hitachi's 917 automatic analyzers (Luo Shi diagnostic companies, Indianapolis).

Detect sample with 20 μ l, the first operation reagent of 80 μ l, the second operation reagent of 180 μ l carries out, and measures at wavelength 480nm place.Fig. 4 has shown this result.

Embodiment 15: utilize the rapamycin of whole blood sample to detect

Human blood sample admixture has the rapamycin of 25ng/ml, and extracts (extracted) by above-mentioned step, utilize the calibration curve of Fig. 4 to determine concentration, and concentration is defined as 24.7ng/ml and 24.6ng/ml in twice parallel experiment.

Embodiment 16: the generation of the reorganization calcineurin A of brachymemma in the Escherichia coli and B fusion (CNtABs)

The calcineurin A of brachymemma and B cDNAs

The nucleotide sequence (SEQ ID NO:14) that the cDNA of coding human calcineurin A (amino acid/11 2-392) of brachymemma and wild type chain B (amino acid/11-170) has utilized following optimization by blue aigret biotechnology (Seattle, WA) chemosynthesis:

5’TCAACCACTGATCGTGTCGTTAAAGCTGTCCCGTTTCCACCGAGCCACCGCTTAACT

GCAAAAGAAGTTTTTGATAACGACGGAAAACCGCGTGTTGATATCCTTAAAGCACATC

TGATGAAGGAAGGCCGTTTAGAAGAATCAGTAGCGCTGCGTATCATTACCGAAGGAGC

GTCAATCTTACGTCAAGAAAAAAATCTGCTCGACATCGATGCACCGGTTACCGTATGTG

GTGATATTCATGGTCAGTTTTTCGACTTAATGAAATTATTTGAAGTGGGTGGTTCTCCGG

CTAACACTCGTTATCTCTTTCTGGGTGATTACGTCGATCGTGGCTACTTTTCTATTGAGT

GTGTTCTGTACCTGTGGGCATTAAAAATTCTTTATCCAAAAACTCTTTTCTTACTGCGTG

GCAATCATGAATGTCGTCATCTGACCGAATACTTCACTTTTAAACAAGAATGTAAAATC

AAATATAGCGAACGTGTGTATGATGCTTGTATGGATGCCTTTGATTGCTTACCCTTAGCC

GCCTTAATGAACCAACAATTCCTGTGTGTACATGGTGGCCTTAGCCCCGAAATTAACAC

CTTAGATGATATCCGTAAATTAGATCGTTTTAAAGAACCACCCGCTTATGGCCCGATGTG

TGATATCTTGTGGTCGGATCCTCTGGAAGATTTTGGTAACGAAAAAACTCAGGAACATT

TTACTCATAACACCGTTCGTGGCTGTAGTTATTTCTATTCTTACCCAGCCGTGTGCGAAT

TCCTTCAACACAACAACCTGCTGAGTATTCTCCGCGCCCATGAAGCCCAAGATGCCGG

TTATCGTATGTATCGTAAATCACAGACCACCGGGTTCCCATCTCTCATTACGATTTTTTCT

GCCCCGAACTACCTTGACGTTTACAATAATAAAGCCGCTGTTCTGAAATATGAAAACAA

CGTCATGAATATTCGCCAATTCAATTGCAGCCCTCACCCTTATTGGCTTCCCAATTTTAT

GGACGTGTTTACCTGGTCACTGCCATTTGTCGGTGAGAAAGTTACTGAAATGCTTGTTA

ATGTTCTGAACATCTGTAGCGATGATGAATTAGGGTCCGAAGAAGACGGGTTTGACGG

TGCCACAGCTGCCGCGCGCAAAGAAATGGGCAACGAAGCATCCTATCCACTTGAAATG

TGTTCTCATTTTGATGCCGATGAAATTAAACGCTTAGGTAAACGGTTTAAAAAACTTGA

TCTTGATAACTCTGGCTCCCTTTCTGTGGAAGAATTTATGTCCTTACCGGAGCTGCAGC

AGAACCCTCTGGTCCAACGCGTAATTGATATCTTTGACACCGATGGAAATGGCGAAGT

CGATTTTAAAGAATTTATTGAAGGTGTTTCTCAATTTAGCGTAAAGGGTGACAAAGAAC

AGAAACTGCGTTTCGCATTCCGCATCTATGACATGGATAAAGATGGTTATATCTCAAAC

GGGGAATTGTTCCAGGTTTTAAAAATGATGGTAGGCAACAATTTAAAAGATACCCAATT

ACAACAAATTGTAGATAAAACGATTATTAACGCAGATAAGGATGGTGATGGTCGCATTT

CTTTCGAAGAGTTCTGCGCCGTTGTGGGAGGTCTTGATATTCATAAAAAAATGGTCGTC

GATGTC?3’

The structure of expression vector

He Cheng cDNA is together with two groups of Oligonucleolide primers above, by PCR respectively according to long CNtAB and 2 dna fragmentations of short CNtAB amplification.These dna fragmentations are cloned on the acellular expression vector pFVEX2.3d of Luo Shi, thereby give expression to the chimera calcineurin A/B albumen of the His mark of two different editions.

The forward primer of long CNtAB (SEQ ID NO:15):

5’CTTTAAGAAGGAGATATACCATGTCAACCACTGATCGTGTCGT?3’

The reverse primer of long CNtAB (SEQ ID NO:16):

5’TGATGATGAGAACCCCCCCCGACATCGACGACCATTTTTTT?3’

The forward primer (SEQ ID NO:17) of short CNtAB:

5’CTTTAAGAAGGAGATATACCATGCCTTATTGGCTTCCCAATTTT?3’

The reverse primer (SEQ ID NO:18) of short CNtAB:

5’TGATGATGAGAACCCCCCCCGACATCGACGACCATTTTTTT?3’

The human calcineurin A of His mark and the generation of B fusion

Behind the acellular quick translation system of Luo Shi (RTS) prescreen, the optimum expression construction of choosing is transformed in the BL21-A1 cell, and by under 37 ℃, arabinose with 0.2% and 0.4mMIPTG incubation were induced the expression of target protein in 15 hours.The recombinant protein of expressing, long CNtAB and short CNtAB by using the Ni-NTA agarose affine purification and SEC (Snperdex 200, GE) are purified to 85% and 95% respectively.

Following is the amino acid sequence (564 amino acid) (SEQ ID NO:19) of the long CNtAN albumen of reorganization:

1 10 20 30 40 50 60

MSTTDRVVKAVPFPPSHRLTAKEVFDNDGKPRVDILKAHLMKEGRLEESVALRIITEGAS

70 80 90 100 110 120

ILRQEKNLLDIDAPVTVCGDIHGQFFDLMKLFEVGGSPANTRTLFLGDYVDRGYFSIECV

130 140 150 160 170 180

LYLWALKILYPKTLFLLRGNHECRHLTEYFTFKQECKIKYSERVYDACMDAFDCLPLAAL

190 200 210 220 230 240

MNQQFLCVHGGLSPEINTLDDIRKLDRFKEPPAYGPMCDILWSDPLEDFGNEKTQEHFTH

250 260 270 280 290 300

NTVRGCSYFYSYPAVCEFLQHNNLLSILRAHEAQDAGYRMYRKSQTTGFPSLITIFSAPN

310 320 330 340 350 360

TLDVYNNKAAVLKYENNVMNIRQFNCSPHPYWLPNFMDVFTWSLPFVGEKVTEMLVNVLN

370 380 390 400 410 420

ICSDDELGSEEDGFDGATAAARKE MGNEASYPLEMCSHFDADEIKRLGKRFKKLDLDNSG

430 440 450 460 470 480

SLSVEEFMSLPELQQNPLVQRVIDIFDTDGNGEVDFKEFIEGVSQFSVKGDKEQKLRFAF

490 500 510 520 530 540

RIYDMDKDGYISNGELFQVLKMMVGNNLKDTQLQQIVDKTIINADKDGDGRISFEEFCA

V

550 560

VGGLDIHKKMVVDVGGGSHHHHHH

CNtA1 corresponding to the amino acid/11 2-394 of human calcineurin A, is (in the 2-384 position) of italic.CNB corresponding to the amino acid/11-170 of human calcineurin B, is underlined (in the 385-554 position).The His label is in the 555-564 position.

Be the amino acid sequence (236 amino acid) (SEQ ID NO:20) of the short CNtAB of reorganization below:

1 10 20 30 40 50 60

MPYWLPNFMDVFTWSLPFVGEKVTEMLVNVLMCSDD?ELGSEEDGFDGATAAARKE MGNE

70 80 90 100 110 120

ASYPLEMCSHFDADFIKRLGKRFKKLDLDNSGSLSVEEFMSLPELQQNPLVQRVIDIFDT

130 140 150 160 170 180

DGNGEVDFKEFIEGVSQFSVKGDKEQKLRFAFRIYDMDKDGYISNGELFQVLKMMVGN

NL

190 200 210 220 230

KDTQLQQIVDKTIINADKDGDGRISFEEFCAVVGGLDIHKKMVVDVGGGSHHHHHH

CNtA2 corresponding to the amino acid 340-394 of human calcineurin A, is (in the 2-56 position) of italic.CNB, corresponding to the amino acid/11-170 of human calcineurin B, (in the 57-226 position) of underscore that be picture.The His label is in the 227-236 position.

The interaction of FK506 dependent form between embodiment 17:FKBP12 and the long CNtAB

The calcineurin A/B fusions CNtABs of brachymemma and the FKBP12 of FK506 combination evaluate by reference time resolution fluorometry (TRF) and other mensuration in conjunction with active.In TRF measures, will contain purifying the His mark long CNtAB or lack the 0.1M NaHCO of CNtAB ₃Being coated on the last 40C of black 96 orifice plates (Perkin Elmer) spends the night.The plate of albumen bag quilt is with containing 7.5%BSA, 0.1M Tris hydrochloric acid, and 0.15M NaCl and 20 μ M diethylene-triamine pentaacetic acids (diethylenetriamine-pentaacetic acid) buffer A (DTPA) are sealed.Simultaneously, the preparation of FKBP12-europium (Eu) conjugate be by with the biotin labeled FKBP12 among the embodiment 5 on ice with streptavidin (Perkin-Elmer) incubation of Eu mark 30 minutes.After sealing is finished, with containing 0.1M Tris hydrochloric acid, 0.15M NaCl, 0.1% tween Tween (ICI Americas, Inc.) and the buffer B of 20 μ M DTPA wash 96 orifice plates that wrapped quilt.Then the FKBP12-Eu conjugate for preparing is joined on the plate of long CNtAB and short CNtAB bag quilt.The FK506 of a series of variable concentrations is added to the formation that starts the FKBP12-FK506-CNtABs compound on the plate.At room temperature incubation is washed plate for several times with buffer B after 1 hour 30 minutes.At last, will strengthen solution (Perkin-Elmer) adds in the entering plate and at room temperature shakes gently and incubation 5 minutes.Use the VICTOR of Wallac company then ²Many plates of V microtitration reader is read plate.Mean value by three umber strong points draws the relative fluorescence value.

Among Fig. 5, the curve representation of FK506 dose dependent the value of the EC50 that combines with FKBP12 of long CNtAB be 0.77nM.On the contrary, the combination of lacking the FKBP12 that CNtAB combines with FK506 almost detect less than.In addition, consistent by the result of surperficial plasmon resonance BLACORE determination method (BiacoreAG) gained and time-resolved fluorometry (data not shown).As a whole, these tables of data understand that the long CNtAB of reorganization has the still short CNtAB of function may not have functional.

Claims (34)

1. the assay method of the existence of immunosuppressive drug or amount in the working sample may further comprise the steps:

Sampling;

This sample is mixed formation suspending liquid with first acceptor and second acceptor, wherein said first and second acceptors combine with the detection particle, and each described first and second receptor-specific is incorporated into the binding site that separates on the described medicine, and the existence of its Chinese traditional medicine causes detecting particle agglutination;

Directly detect or measure the amount of particle agglutination in the described suspending liquid; And

The amount of particle agglutination is associated with the existence or the amount of immunosuppressive drug in the sample.

2. the process of claim 1 wherein that immunosuppressive drug is selected from cyclosporin, tacrolimus, rapamycin and everolimus, and described first acceptor is immunophilin or its binding fragment.

3. the process of claim 1 wherein that detecting particle is selected from particulate and nanoshell.

4. the process of claim 1 wherein

Immunosuppressive drug is rapamycin or everolimus;

First acceptor comprises FK506 in conjunction with albumen (FKBP); And

Second acceptor comprises rapamycin target (TOR) albumen.

5. the method for claim 4, wherein FKBP comprises one of them of FKBP12 or FKBP25, and second acceptor FKBP that is selected from mTOR, mTOR in conjunction with the FKBP of territory, yTOR1, yTOR1 in conjunction with the FKBP of territory, yTOR2 and yTOR2 in conjunction with the territory.

6. the process of claim 1 wherein

Immunosuppressive drug is a tacrolimus;

First acceptor comprises FK506 in conjunction with albumen (FKBP); And

Second acceptor comprises calcineurin or its binding fragment.

7. the method for claim 6 wherein is combined with calmodulin and calcium ion on the calcineurin.

8. the process of claim 1 wherein

Immunosuppressive drug is a cyclosporin;

First acceptor comprises cyclophilin; And

Second acceptor comprises calcineurin.

9. the method for claim 8 wherein is combined with calmodulin and calcium ion on the calcineurin.

10. the method for claim 3, wherein sample is a blood sample of having used the patient of at least a immunosuppressive drug.

11. the method for claim 10, wherein the processing of blood sample is prior to described blend step.

12. the method for claim 3, wherein detecting particle is nanoshell.

13. the method for claim 3 wherein detects particle and connects key and described first and second receptors bind by biotin-streptavidin or biotin-avidin.

14. the method for claim 3 wherein detects particle directly or indirectly by joint and the described first and second acceptor covalent bond.

15. the method for claim 14, its center tap comprise the antibody of first acceptor and the antibody of second acceptor.

16. the method for claim 13, wherein first and second acceptors are the recombination fusion protein that comprises the biotin molecule attachment site.

17. the method for claim 16, wherein attachment site comprises biotinylated burst.

18. the method for claim 17, wherein fusion and biotin ligase coexpression or express in the presence of the biotin ligase are on the residue of the plain molecule covalency adduction of described biotin ligase catalysis biological in burst.

19. the method for claim 18, wherein the biotin ligase comprises the Avi label and residue is a lysine.

20. the method for claim 17, wherein fusion fusion partner is to have c-terminus and N-terminal recombinant receptor albumen, and a fusion partner comprises the biotinylation burst, in addition, wherein when the attachment site of first acceptor during at c-terminus, then the attachment site of second acceptor is at aminoterminal or at c-terminus.

21. the reagent of immunosuppressive drug in the working sample, described reagent comprises

First medicine comprises with first and detects first acceptor that particle combines in conjunction with compound;

Second medicine is in conjunction with compound, comprise and be selected from following bound fraction: be incorporated into second acceptor that detects particle, be incorporated into the immunosuppressive drug specific antibody of the second detection particle, and with second second receptor specific antibody that is subjected to bluk recombination, wherein every kind of medicine is incorporated into the binding site that separates on the described immunosuppressive drug in conjunction with the compound specificity.

22. the reagent of claim 21, wherein bound fraction is to be incorporated into second acceptor that detects particle.

23. the reagent of claim 22, immunosuppressive drug wherein to be determined is a rapamycin, and described first medicine comprises FK506 in conjunction with albumen in conjunction with compound, and described second medicine comprises rapamycin target (TOR) albumen in conjunction with compound.

24. the reagent of claim 21, wherein detecting particle is particulate.

25. the reagent of claim 21, wherein second medicine comprises second receptor specific antibody that is subjected to bluk recombination with second in conjunction with compound.

26. the method that immunosuppressive drug exists or measures in the working sample comprises following steps:

Sampling;

With this sample be incorporated into first and detect acceptor that particle combines and bound fraction and mix and form suspending liquid, wherein bound fraction is selected from the immunosuppressive drug specific antibody that is incorporated into the second detection particle, and with second second receptor specific antibody that is subjected to bluk recombination, wherein said receptor-specific is incorporated on the position that is different from the combination of immunosuppressive drug specific antibody on the described immunosuppressive drug, and the existence of its Chinese traditional medicine causes detecting particle agglutination;

Utilize absorbance measurement to measure the amount of particle agglutination in the described suspending liquid, and

The amount of particle agglutination is associated with the existence or the amount of immunosuppressive drug in the sample.

27. the method for claim 26, wherein immunosuppressive drug is selected from cyclosporin, tacrolimus, rapamycin and everolimus, and bound fraction is the specific antibody of immunosuppressive drug, and wherein antibodies is in detecting particle.

28. the method for claim 26, wherein

Immunosuppressive drug is rapamycin or everolimus;

First acceptor comprises FK506 in conjunction with albumen (FKBP); And

Bound fraction comprise rapamycin target (TOR) albumen or specifically with the protein bound antibody of rapamycin target (TOR).

29. the method for claim 26, wherein

Immunosuppressive drug is a tacrolimus;

First acceptor comprises FK506 in conjunction with albumen (FKBP); And

The antibody that second acceptor comprises calcineurin or combines with calcineurin specifically.

30. the method for claim 24, wherein

Immunosuppressive drug is a cyclosporin;

First acceptor comprises cyclophilin; And

The antibody that second acceptor comprises calcineurin or combines with calcineurin specifically.

31. measure the kit of immunosuppressive drug, described kit comprises

Be combined in first acceptor and second acceptor of two separation point positions on the immunosuppressive drug specifically with the target immunosuppressive drug, each also comprises label described first and second acceptors; With

A large amount of detection particles, each described particle remain to be combined with the material that is incorporated into described label.

32. the kit of immunosuppressive drug in the working sample, described kit comprises

First reagent comprises and is incorporated into first suspending liquid that detects the immunophilin of particle;

Second reagent, comprise the suspending liquid that is selected from following receptor protein: the antibody of FKBP-rapamycin associated protein, calcineurin and calcineurin, be incorporated into described second receptor protein that detects particle, wherein said detection particle is selected from particulate and nanoshell.

33. measure the kit of active rapamycin, described kit comprises

First reagent comprises and is incorporated into first and detects the FK506 of particle in conjunction with albumen (FKBP);

Second reagent comprises rapamycin target (TOR) albumen that is incorporated into the second detection particle, and wherein the first and second detection particles are independently selected from particulate and nanoshell.

34. the kit of claim 33, wherein FKBP albumen is FKBP12 or FKBP25, and wherein the FKBP-rapamycin associated protein FKBP that is selected from mTOR, mTOR in conjunction with the FKBP of territory, yTOR1, yTOR1 in conjunction with the FKBP of territory, yTOR2 and yTOR2 in conjunction with the territory.

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