CN1869701A

CN1869701A - Method for joint investigating hepatitis B virus pro S1 antigen and nuclear antigen and diagnostic kit

Info

Publication number: CN1869701A
Application number: CNA2006100905127A
Authority: CN
Inventors: 葛胜祥; 袁权; 赵昱; 罗文新; 张军; 夏宁邵
Original assignee: Xiamen University; Beijing WanTai Biological Pharmacy Enterprise Co Ltd
Current assignee: Xiamen University; Beijing WanTai Biological Pharmacy Enterprise Co Ltd
Priority date: 2006-06-27
Filing date: 2006-06-27
Publication date: 2006-11-29
Anticipated expiration: 2026-06-27
Also published as: HK1099361A1; WO2008003236A1; CN100582781C

Abstract

The invention discloses a method of diagnosing hepatitis B virus (HBV) infection by combined detection for HBV related pre-S1 antigen (HBV PreS1) and core antigen (HBcAg). And the invention also discloses a method for detecting HBV PreS1 by double-antibody sandwich method. And the invention also discloses a reagent box and virus lysate for the above detection and their uses.

Description

The method and the diagnostic kit of a kind of joint investigating hepatitis B virus pre S 1 antigen and cAg

Invention field

The present invention relates to the hepatitis type B virus detection range, relate more specifically to by pre S 1 antigen (HBV PreS1) and cAg (HBcAg) joint-detection relevant, the method for diagnosis hepatitis B virus infection to hepatitis type B virus.The present invention discloses hepatitis B virus pre S 1 antigen and cAg combined detection kit (particularly euzymelinked immunosorbent assay (ELISA) and chemoluminescence method).The invention still further relates to detection method, virolysis method and the relevant kit of pre S 1 antigen.

Background of invention

It is one of most important public health problem in the whole world that hepatitis type B virus (Hepatitis B virus) infects.

At present, HBV blood serum designated object (conventional as: HBsAg, HBsAb, HBeAg, HBeAb, HBcAb promptly are commonly called as " two double " and detect) extensively infects as existing disease HBV and the conventional sense standard that infects of HBV previously.But the false negative that the huge radix that high aberration rate is added the HBV carrier causes is conventional " two double " detects is occurred frequently.And common " two double " detect can't be from measuring reflection virus replication and infectiousness size, and normal appearance can suspect and be difficult to resolve the result who releases, and also can't determine that directly the detection individuality does not infect HBV from " two double " index.

HBV DNA is the direct indicator of hbv replication, and it is that hepatitis B patient and HBV carrier infect the goldstandard of judging with infectiousness that its dot hybridization detects (or PCR detects).PCR detects with the dot hybridization detection and all can be used as HBV infection and communicable direct indicator, but is not suitable for extensive generaI investigation or routine use.

Might with the serum mark antigen (HBV PreS1, HBcAg, HBxAg, DNAP, HBV PreS2 etc.) of HBV DNA height correlation in: HBcAg is considered to the antigen directly related with HBVDNA always, detects at HBcAg in duplicating virus peculiar diagnostic significance quantitatively and among HBV the infected of HBsAg feminine gender and the HB patient.But in view of all difficulties that detect HBcAg, before the current HBcAg immunological diagnostic reagent of developing or having reported is taked to detect usually sample is carried out pre-treatment (lytic virus, rupture of membranes and deactivation HBcAb) or adopt the mode of the circuitous HBcAg-HBcAb of detection immune complex to carry out, the former complicated operation program is loaded down with trivial details, be difficult for being accepted, also be not suitable for extensive blood donor's examination and epidemiology survey by clinical client.The latter is then because the singularity of detection mode is difficult to the specificity and the sensitivity that reach desirable.And HBV PreS1 and HBV DNA also have good correlativity (D.Buffello, 2000,96%; Kuijpers, 1989,88%; Theilman, 1986).To with the diagnostic method of HBV PreS1 antigen, the current PreS1 immunological diagnostic reagent of developing or having reported is considered low many of the relative HBsAg absolute concentration of PreS1, conventional two PreS1 antibody sandwich method difficulty reaches desirable sensitivity, the normal employing detected whole LHBs (promptly by the HBsAg mediated detection on the LHBs, use at the specific antibody of HBsAg as capture antibody or report antibody, use specific antibody to match with it again) at PreS1, this detecting pattern has amplified the detection sensitivity of PreS1 effectively, but with the detection of the PreS1 HBsAg that places one's entire reliance upon, do not form an independently detection system, may occur causing the PreS1 false negative to occur because of HBsAg sudden change, can not the accurate response mutated viruses or the PreS1 of different subtype virus infections dynamic.

The direct ELISA diagnostic reagent of developing the existing disease infection of a kind of simple, accurate and highly sensitive HBV infects epidemic disease control at HBV, and antiviral curative effect of HB patient and course of disease prognosis aspect all have urgent realistic meaning.

Therefore, this area still lacks the method for effective detection hepatitis B.

Summary of the invention

Purpose of the present invention is to wish to set up simply, effectively, the HBcAg detection method that need not sample pretreatment process and the highly sensitive pair of Pres1 antibody sandwich method detect the method for PreS1, and remedy and to cause false-negative shortcoming because of single HBcAg or PreS1 sudden change or omission by joint-detection HBcAg and PreS1 pattern, make up a kind of and associating (HBcAg and PreS1) detecting pattern HBV DNA height correlation, and this pattern is applied to euzymelinked immunosorbent assay (ELISA), chemiluminescence, time resolution, colloidal gold method, qualitative or quantitative immunology diagnosiss such as enzyme linked immunological percolation.

The present invention relates to a kind of pre S 1 antigen/cAg by the joint-detection hepatitis B that set up with the amynologic diagnostic method nucleic acid detection method height correlation.

The present invention relates to the kit and preparation method thereof of the pre S 1 antigen/cAg of joint-detection hepatitis B.

Particularly, the enzyme linked immunological kit of pre S 1 antigen/cAg of joint-detection hepatitis B and chemical luminescence reagent kit and preparation method thereof have been the present invention relates to.

Joint-detection pattern of the present invention has not only effectively embodied the complementary advantage of two kinds of antigens of joint-detection, and joint detection results form New Set---" nucleic acid related antigen " embodies the correlativity with hbv nucleic acid index height.

In addition, the present invention has announced the method for the two sandwich detection pre S 1 antigens of former S 1 immune body of a kind of high sensitivity first, and this method is different from the method for other detection pre S 1 antigens of having announced.

In particularly preferred embodiment of said method, the present invention has used a series of buffer solution that contain the different types of surfactant of various dose to be used for lytic virus first, improves the detection sensitivity of pre S 1 antigen and cAg Joint Index and the independent index of pre S 1 antigen greatly.

Description of drawings

Fig. 1 has shown the building process of the recombinant plasmid pT-2147*3 of preS1 district 21-47AA fragment triplex tandem.

Fig. 2 shows the SDS-PAGE electrophoresis result of the recombinant protein GST-2147*6 of expression and purifying.The result shows that the purity of destination protein behind the purifying surpasses 85%.Swimming lane 1: albumen marker; Swimming lane 2: the pGEx-2147*6 of expression; Swimming lane 3: the pGEX-2147*6 behind the purifying.

Fig. 3 shows chemiluminescence detection and quantitative fluorescent PCR correlativity.As described in embodiment 14, to the viral level of each sample and the luminous intensity laggard line linearity correlation analysis of taking the logarithm respectively.

Detailed Description Of The Invention

What unless otherwise defined, used all technology and scientific term all expressed here is the common implication of understanding the those of skill in the art that the present invention relates in the field. Here used name and be widely used conventional steps in the corresponding field in cell cultivation, molecular genetics, nucleic acid chemistry, Immunology Lab operating procedure.

The present invention relates to a kind of pre S 1 antigen/cAg by the joint-detection hepatitis B that set up with the amynologic diagnostic method nucleic acid detection method height correlation. In a specific embodiments of the present invention, by the comparison of S1, cAg and joint-detection pre S 1 antigen/cAg (ELISA) before the independent detection, joint-detection pre S 1 antigen/cAg has obvious advantage than S1 before detecting separately and cAg.

In the another embodiment of the present invention, pre S 1 antigen/the cAg (ELISA) of joint-detection hepatitis B is compared with conventional HBV markers detection, this detection method contains virulent sample to part can be accurately qualitative, and that two double detections are failed to this type of sample is accurately qualitative.

In the another embodiment of the present invention, the detection method of the pre S 1 antigen/cAg of this joint-detection hepatitis B is applied in the chemiluminescence diagnosis, and detection quantitative result and the viral level of the pre S 1 antigen/cAg of joint-detection hepatitis B have preferably linear dependence.

In the another embodiment of the present invention, detection method of the present invention adopts enzyme-linked immunoassay method to realize. Correspondingly, the invention provides the preparation method of relevant enzyme linked immunological kit and the kit for preparing thus.

In the another embodiment of the present invention, detection method of the present invention adopts chemiluminescence method to realize. Correspondingly, the invention provides the preparation method of relevant chemical luminescence reagent kit and the kit for preparing thus.

The below is described in more details above-mentioned aspect and other side.

The pre S 1 antigen of joint-detection hepatitis B and cAg (abbreviation joint-detection)

On the one hand, the present invention relates to a kind ofly in sample, detect hepatitis type B virus or detect hepatitis b virus infected method, comprise joint investigating hepatitis B virus pre S 1 antigen and cAg.

At this, for the purposes of the present invention, " joint-detection " comprises to the detection of pre S 1 antigen with to the detection of cAg and separately carries out or merge carrying out, and carry out simultaneously and successively carry out with any order, as long as unite to the detection of pre S 1 antigen with to the signal Processing of the detection of cAg.

Uniting and carry out signal Processing and be meant, is the hepatitis type B virus or the hepatitis b virus infected positive when still negative at judgement sample, pre S 1 antigen is detected the signal that obtains and cAg detect the signal that obtains and merge and become a single index and judge.For example, can simple addition with the signal that two Detection of antigen obtain, compare with predetermined value and to judge.Perhaps, the signal that two Detection of antigen can be obtained is merged into new single index by suitably handling or computing (for example one of them signal is weighted or two all be weighted), compares with predetermined value and judges.Can improve sensitivity and recall rate etc. like this.This may be the omission that for example causes owing to reasons such as single antigen first feminine genders owing to being avoided.But the present invention is not subjected to the restriction of such mechanism.

Therefore, the stage before signal Processing can be separate to the detection of pre S 1 antigen with to the detection of cAg, that is to say that they can occur in same or different time, the same or different test.For example, can carry out with identical method with detection, also can carry out with diverse ways to cAg to the detection of pre S 1 antigen; For example a kind of detection of antigens adopts a kind of the carrying out in the following method, and another detection of antigens adopts the method for different (perhaps identical) to carry out: enzyme linked immunosorbent assay, immunity percolation method, immunochromatographic method, chemoluminescence method, time resolution method.Preferred two kinds of antigens adopt identical method to detect.

For example, to the detection of pre S 1 antigen can occur in cAg detect before, afterwards or simultaneously.

When two kinds of antigens adopt identical methods to detect, can be in identical or different test batch, in the identical or different reaction vessel (as reacting hole), carry out on the identical or different solid phase carrier.The two can adopt identical or different signals to produce body.

In a preferred embodiment, simultaneously, merge two kinds of antigens of detection.For example, in same reaction container (as reacting hole), carry out two kinds of detection of antigens simultaneously.This can implement by for example following method: the capture antibody of two kinds of antigens of fixed trapped on identical carrier, utilize same virolysis thing, in same medium, carry out catching of two kinds of antigens simultaneously, not necessarily in same medium, carry out the combination and the detection of second antibody simultaneously.Perhaps, also can implement: the capture antibody of two kinds of antigens of fixed trapped on different carriers by for example following method, then that two kinds of carriers are mixed, utilize same virolysis thing, in same medium, carry out catching of two kinds of antigens simultaneously, not necessarily in same medium, carry out the combination and the detection of second antibody simultaneously.

In a preferred embodiment of the method for the invention, described sample is the biological sample from the separation of individuality to be checked, and preferred described sample is a serum.

In another embodiment, described method be used for judging the serum hbv nucleic acid existence whether.

In another embodiment, the joint-detection of described hepatitis B virus pre S 1 antigen and cAg realizes by one or several methods of selecting from following method: enzyme linked immunosorbent assay, immunity percolation method, immunochromatographic method, chemoluminescence method, time resolution method; Preferred enzyme linked immunosorbent assay or chemoluminescence method.

In another embodiment, in same reaction container (as reacting hole), in same reaction, detect pre S 1 antigen and cAg simultaneously.

In another embodiment, adopt double antibody sandwich method to detect pre S 1 antigen and cAg.

In another preferred embodiment, do not need to sample for example serum carry out pre-service.For example, do not need in advance with denaturation of virus.

In a specific embodiments, being captured in the same reaction container (for example reacting hole) of cracking of hepatitis type B virus and pre S 1 antigen and cAg carried out in same media simultaneously.In this embodiment, preferably, use the present invention to be used for the lysate of joint-detection as described medium.

In a specific embodiments of method of the present invention, associated detecting method of the present invention may further comprise the steps:

A) provide can with the first anti-HBV PreS1 antibody and the second anti-HBV PreS1 antibody of hepatitis B virus pre S 1 antigen (HBV PreS1) specific bond, and can with the first anti-HBc antibody and the second anti-HBc antibody of hepatitis B virus core antigen (HBc) specific bond;

B) the first anti-HBV PreS1 antibody is attached on the identical solid phase carrier as capture antibody with the first anti-HBc antibody (both are referred to as first antibody), forms first antibody-carrier conjugates; Preferably, described carrier is an elisa plate;

C) testing sample is contacted with first antibody-carrier conjugates, make if present, to be hunted down hepatitis B virus pre S 1 antigen and cAg in the sample, form antigen-first antibody-carrier conjugates; Described sample can be through pretreated or do not pass through pretreated; Preferably, described sample is not through virolysis processing in advance, and testing sample contacts with first antibody-carrier conjugates under the condition that allows virolysis and antigen capture and carries out; More preferably testing sample contacts with first antibody-carrier conjugates at the lysate that is used for joint-detection of the present invention and carries out;

D) the second anti-HBV PreS1 antibody is contacted with the product (carrier) of step c) with the second anti-HBc antibody (both are referred to as second antibody), this contact is carried out under second antibody and the condition that captive antigen combines allowing;

E) detect the amount of combined second antibody.

Described antigen-the first antibody of step c)-carrier conjugates comprises hepatitis B virus pre S 1 antigen-first anti-HBV PreS1 antibody-carrier conjugates and the anti-HBc antibody-carrier conjugates of cAg-first.

In step d), if have hepatitis B virus pre S 1 antigen and cAg, to form second antibody-antigen-first antibody-carrier conjugates, comprise second anti-HBV PreS1 antibody-hepatitis B virus pre S 1 antigen-first anti-HBV PreS1 antibody-carrier conjugates and the anti-HBc antibody-carrier conjugates of second anti-HBc antibody-cAg-first

The first anti-HBV PreS1 antibody can be identical or different with the second anti-HBV PreS1 antibody, can be monoclonal antibody or polyclonal antibody, preferably different monoclonal antibodies.The first anti-HBc antibody can be identical or different with the second anti-HBc antibody, can be monoclonal antibody or polyclonal antibody, preferably different monoclonal antibodies.

Step e) detects the amount of combined second antibody and can be undertaken by for example following method: provide and can produce body and can produce body with the effective secondary signal that combines of the described second anti-HBc antibody with effective first signal that combines of the described second anti-HBV PreS1 antibody, it is only relevant with the amount of the second anti-HBV PreS1 antibody and the second anti-HBc antibody respectively that first and second signals produce the signal intensity that body produced; First and second signals are produced body to be contacted with second antibody; Detection produces the signal that body produces by signal.First signal produces body can be identical or different with secondary signal generation body.In a preferred embodiment, it is identical with secondary signal generation body that first signal produces body, and perhaps first signal generation body is identical with the signal that secondary signal produces the body generation, can detect with identical method; More preferably, detect first and second signals and produce the index of the summation of the signal that body produces as judged result.

Step e) detects the amount of combined second antibody and can also be undertaken by for example following method: second antibody directly is marked with signal and produces body, detects by signal and produces the signal that body produces.The second anti-HBV PreS1 antibody can produce body by the identical signal of mark with the second anti-HBc antibody, and perhaps different signals produces body.In a preferred embodiment, it is identical that the signal of the second anti-HBVPreS1 antibody and the second anti-HBc antibody labeling produces body, and perhaps the signal of their generations is identical, can detect with identical method; More preferably, the signal that detects the second anti-HBV PreS1 antibody and the second anti-HBc antibody labeling produces the index of the summation of the signal that body produces as judged result.

In the method for the present invention, signal produce body can be any can for example can be enzyme, fluorescein, chemiluminescent substance, isotope, rare earth element by the material that detects by proper method.

In a preferred embodiment, use the monoclonal antibody of anti-hepatitis B virus pre S 1 antigen and anti-cAg to carry out immune detection.

In a preferred embodiment, the monoclonal antibody of anti-hepatitis B virus pre S 1 antigen is selected from 3H5,7H11,2A7,4D11,6F1,13G2 and 16F5; Preferably, use the 7H11 monoclonal antibody to use 4D11-HRP to detect HBV PreS1 as the first anti-HBV PreS1 antibody as the second anti-HBVPreS1 antibody pairing.

In a preferred embodiment, described method also comprises step: at testing sample with before first antibody-carrier conjugates contacts, use the virus in the lysate lysate sample, wherein said lysate preferably the present invention is used for the employing virus cracking liquid of joint-detection.

In another embodiment, (for example at step b) and c) between each step and/or c) and d) and/or d) and e) between) can also contain washing, drying and other steps.

The invention still further relates to the employing virus cracking liquid that can be used for joint investigating hepatitis B virus pre S 1 antigen and cAg, it contains surfactant and suitable buffering agent, described surfactant is selected from: Chaps, sulfobetain 8-18 (SB8, SB10, SB12, SB14, SB16, SB18), Bu Lijie 35, Tween series and/or HP-β-CD are preferably selected from SB14, SB16, SB18, HP-β-CD, TWEEN20, TWEEN40, TWEEN60, TWEEN80.

In above-mentioned employing virus cracking liquid, preferably, surfactant can be the combination of one or more (2,3,4,5 kind) in the above surfactant, for example a kind of combination of or two kinds or three kinds in HP, TWEEN series and the SB series.For example HP+TWEEN series, TWEEN series+SB series, HP+SB series or HP+TWEEN series+SB series, preferred HP+TWEEN40, HP+TWEEN40+SB14, HP+TWEEN40+SB16, HP+TWEEN40+SB18, HP+TWEEN40+SB18, HP+TWEEN20+SB14, HP+TWEEN60+SB14, HP+TWEEN80+SB14.Preferably, surfactant can be HP:0.5%-4%; Tween series: 0.1%-6%; SB14%:1%-10%; SB16:0.5%-1%; SB18:0.5%-1%; More preferably HP:1%-2%; Tween series: 0.5%-2%; SB14:2%-8%; SB16:1%; SB18:1%; As: 1%-2%HP+0.5%-1%Tween+1%-2%SB14.

Preferably, surfactant is selected from lysis1-lysis24 shown in the table 4, more preferably lysis1, lysis10, lysis11, lysis12, lysis13, lysis14, lysis15, lysis16, lysis17, lysis18, lysis19, lysis20, lysis21, lysis22, lysis23 and lysis24, more preferably lysis10, lysis13, lysis16, lysis17, lysis18, lysis19, lysis20, lysis21 and lysis24, the most preferably surfactant described in the lysis10,13,16,18.

Preferably, buffering agent is selected from citrate buffer, phosphate buffer, Tris damping fluid, buffered with amino acid liquid, acetate buffer, HEPES damping fluid, carbonate buffer solution.Buffering agent can be the combination of one or several (2,3,4,5 kind) in the above-mentioned damping fluid.Buffering agent optimization citric acid salt buffer, phosphate buffer.The pH scope and the concentration range of buffering agent can be: phosphate buffer: the preferred 20mM pH of 10-50mM pH value 6.0-7.0 value 7.4; Citrate buffer: the preferred 10mM pH of 10-50mM pH value 3.0-6.6 value 6.6; Tris damping fluid: the preferred 20mM pH of 10-50mM pH value 7.0-9.0 value 7.0; Acetate buffer: the preferred 10mM pH of 10-50mM pH value 3.8-6.0 value 6.0.

Not necessarily, lysate can also contain one or more (as 1,2,3,4,5,6-10 kind) other compositions, for example (salt for example is as NaCl for reductive agent (for example DTT, DTE, beta-mercaptoethanol), sequestrant (for example EDTA), osmotic pressure instrumentality; Sucrose).For example lysate can also contain one or several the combination in the following composition: the preferred 0.1%-0.5% of beta-mercaptoethanol: 0.01%-1%, more preferably 0.4%; The preferred 5-10mM of DTT:1-30mM, more preferably 5mM; The preferred 5-10mM of DTE:1-30mM, more preferably 5mM; The preferred 5mM of EDTA:5mM-20mM; NaCl:50mM-300mM is more preferably: 50-150mM, more preferably 100mM; The preferred 5%-10% of sucrose: 2%-20% more preferably 10%.

Preferably, lysate is selected from the lysis1-lysis24 shown in the table 4, more preferably, lysate is selected from lysis1, lysis10, lysis11, lysis12, lysis13, lysis14, lysis15, lysis16, lysis17, lysis18, lysis19, lysis20, lysis21, lysis22, lysis23, and lysis24, more preferably lysis10, lysis13, lysis16, lysis17, lysis18, lysis19, lysis20, lysis21, and lysis24, most preferably, lysate is selected from: lysis10---the citrate buffer of pH6.6,2%SB14,5mM DTE, 100mM NaCl, 5mM EDTA; The citrate buffer of lysis13---pH6.6,2%SB16,5mM DTE, 100mM NaCl, 5mM EDTA; The citrate buffer of lysis16---pH6.6,2%SB18,5mM DTE, 100mM NaCl, 5mM EDTA; The citrate buffer of and lysis18---pH6.6,2%HP, 0.5%Tw40,0.4%Me, 10% sucrose.

The employing virus cracking liquid that the present invention is used for joint investigating hepatitis B virus pre S 1 antigen and cAg can also be used for detecting separately cAg, perhaps detects pre S 1 antigen separately.Therefore, the present invention also provides the above-mentioned employing virus cracking liquid that is used to detect cAg.The present invention also provides the above-mentioned employing virus cracking liquid that is used to detect pre S 1 antigen.The present invention also provides the purposes of above-mentioned lysate in detecting cAg, and the purposes of above-mentioned lysate in detecting pre S 1 antigen.

The lysate of having reported only uses when detecting cAg, and generally uses NP40 to be used for brokenly peplos, but these known lysates are not suitable for the joint-detection of pre S 1 antigen and cAg, as CN1352392A.

A significant advantage of employing virus cracking liquid of the present invention is to be used as the medium of catching viral antigen.Employing virus cracking liquid of the present invention has abundant lytic virus but enough gentle so that characteristics of the antibody of bag quilt on the inactivation carrier substantially not.The use of employing virus cracking liquid of the present invention makes that can carry out effective antigens in lytic virus catches.But the present invention is not limited by this mechanism of action.

Therefore, in an especially preferred embodiment, adopt the employing virus cracking liquid that is used for joint-detection of the present invention, in same reaction medium, carry out the cracking of virus and catching of antigen (pre S 1 antigen and cAg) simultaneously, and need be in advance with virolysis before antigen capture.This also is a significant advantage of the present invention.

The invention still further relates to a kind of kit that is used for joint-detection, it comprises the reagent that is used for above-mentioned associated detecting method joint investigating hepatitis B virus pre S 1 antigen and cAg according to the present invention.

In a specific embodiments, contain anti-HBVPreS1 antibody in the combined detection kit of the present invention and (preferably comprise two kinds of different anti-HBV PreS1 antibody, monoclonal antibody particularly) (preferably comprises two kinds of different anti-HBc antibody with anti-HBc antibody, and/or the present invention is used for the employing virus cracking liquid of joint-detection monoclonal antibody particularly).

Preferably, at least a anti-HBV PreS1 antibody and at least a anti-HBc antibody are produced body tag by signal in the described antibody, can produce perhaps that body is effective to be combined with signal.The signal generation body of anti-HBVPreS1 antibody and anti-HBc antibody labeling or the signal of combination produce body can be identical or different, preferably can adopt identical detection method to detect.Described antibody is monoclonal antibody preferably.Preferably anti-HBV PreS1 antibody is selected from: 3H5,7H11,2A7,4D11,6F1,13G2 and 16F5, particularly 7H11 and 4D11.

Preferably also contain the reagent (for example detecting the detectable of second antibody) that is suitable for detecting described antigen-antibody reaction in the described kit.

In a preferred embodiment, comprise in the combined detection kit of the present invention:

(1) solid phase carrier is fixed with the first anti-HBV PreS1 antibody and the first anti-HBc antibody on the described solid phase carrier; And/or (2) the present invention is used for the employing virus cracking liquid of joint-detection; (3) dispensable by signal generation second anti-HBVPreS1 antibody body tag or that be not labeled and the second anti-HBc antibody; (4) detectable of dispensable detection second antibody; (5) dispensable damping fluid, cleansing solution, operation instructions (preferred operation instructions have been put down in writing the method for joint-detection of the present invention).

In a preferred embodiment, solid phase carrier is porous reaction plate (a for example elisa plate) in the kit of the present invention, is fixed with the first anti-HBV PreS1 antibody and the first anti-HBc antibody in the identical or different hole of porous reaction plate.

The invention still further relates to solid phase carrier, be fixed with anti-HBV PreS1 antibody and anti-HBc antibody on it.Preferred solid phase carrier is porous reaction plate (for example ELISA Plate, a chemiluminescence plate), is fixed with anti-HBV PreS1 antibody and anti-HBc antibody in the identical or different hole of porous reaction plate.Described antibody is monoclonal antibody preferably, antibody specific particularly disclosed by the invention.

The reagent that the invention still further relates to joint investigating hepatitis B virus pre S 1 antigen and cAg is used for checking the purposes of hepatitis b virus infected kit in preparation.Preferably, described reagent is joint-detection reagent described in the invention.Joint-detection described in the invention has with the example of reagent, anti-HBV PreS1 antibody (preferably comprises two kinds of different anti-HBV PreS1 antibody, monoclonal antibody particularly) (preferably comprises two kinds of different anti-HBc antibody with anti-HBc antibody, and/or the present invention is used for the employing virus cracking liquid of joint-detection monoclonal antibody particularly).Particularly, (1) solid phase carrier is fixed with the first anti-HBV PreS1 antibody and the first anti-HBc antibody on the described solid phase carrier; And/or (2) the present invention is used for the employing virus cracking liquid of joint-detection; (3) dispensable by signal generation second anti-HBV PreS1 antibody body tag or that be not labeled and the second anti-HBc antibody; (4) detectable of dispensable detection second antibody; (5) dispensable damping fluid, cleansing solution, operation instructions (preferred operation instructions have been put down in writing the method for joint-detection of the present invention).

Double antibody sandwich method detects hepatitis B virus pre S 1 antigen

On the other hand, the present invention relates to adopt double antibody sandwich method to detect the method for hepatitis B virus pre S 1 antigen, it is characterized in that two kinds of used antibody (capture antibody and report antibody) all are the anti-HBV PreS1 antibody with the hepatitis B virus pre S 1 antigen specific bond.

In the preferred embodiment in the methods of the invention, do not need sample is carried out pre-service.

In a further preferred embodiment, being captured in the same reaction container (for example reacting hole) of cracking of hepatitis type B virus and pre S 1 antigen carried out in same media simultaneously.

In a further preferred embodiment, be used to detect the lysate of pre S 1 antigen as described medium described in use the present invention.

In a specific embodiments, method of the present invention comprises:

A) provide can with the first anti-HBV PreS1 antibody and the second anti-HBV PreS1 antibody (two kinds of antibody can be identical or different, and are preferably different, particularly monoclonal antibody) of hepatitis B virus pre S 1 antigen specific bond;

B) the first anti-HBV PreS1 antibody is attached on the solid phase carrier as capture antibody, forms first anti-HBV PreS1 antibody-carrier conjugates; Preferably, described carrier is an elisa plate;

C) testing sample is contacted with first anti-HBV PreS1 antibody-carrier conjugates, make if present, to be hunted down hepatitis B virus pre S 1 antigen in the sample, form the anti-HBV PreS1 of antigen-first antibody-carrier conjugates;

D) the second anti-HBV PreS1 antibody is contacted with the product of step c), this contact combines with captive antigen under the condition that forms the anti-HBV PreS1 of second anti-HBV PreS1 antibody-antigen-first antibody-carrier conjugates at the permission second anti-HBV PreS1 antibody to be carried out;

E) detect the amount of the second combined anti-HBV PreS1 antibody.

In a preferred embodiment, step e) detects the amount of combined second antibody and carries out by the following method: provide and can produce body with the effective signal that combines of the described second anti-HBV PreS1 antibody, it is only relevant with the amount of the second anti-HBV PreS1 antibody that this signal produces the signal intensity that body produced; Signal is produced body to be contacted with second antibody; Detection produces the signal that body produces by signal.

In a further preferred embodiment, step e) detects the amount of combined second antibody and carries out by the following method: second antibody directly is marked with signal and produces body, detects by signal and produces the signal that body produces.

Preferably, signal generation body is enzyme, fluorescein, chemiluminescent substance, isotope, rare earth element.

In one embodiment, the detection of described hepatitis B virus pre S 1 antigen realizes by one or several methods of selecting from following method: enzyme linked immunosorbent assay, immunity percolation method, immunochromatographic method, chemoluminescence method, time resolution method; Preferred enzyme linked immunosorbent assay or chemoluminescence method.

In another embodiment, (for example at step b) and c) between each step of the present invention and/or c) and d) and/or d) and e) between) can also contain the washing and/or drying and other steps.

The invention still further relates to the employing virus cracking liquid that can be used for detecting pre S 1 antigen, it contains surfactant and suitable buffering agent, described surfactant is selected from: Chaps, sulfobetain8-18, Bu Lijie 35, TritonX100, Tween series and/or HP-β-CD, be preferably selected from SB8, SB12, SB14, SB16, SB18, HP-β-CD, TWEEN20, TWEEN40, TWEEN60, TWEEN80, BRJ35, more preferably SB12, SB14, SB16, SB18, TWEEN80.

Preferably, surfactant can be the combination of one or more (2,3,4,5 kind) in the above surfactant, preferred SB16+SB14, SB18+SB14.Preferably, the surfactant concentrations scope can be SB14%:1%-10%; SB16:0.5%-1%; SB18:0.5%-1%; More preferably: SB14:2%-8%; SB16:1%; SB18:1%; As: 1%SB14+0.5%SB16 or 1%SB14+0.5%SB18.

Preferably, surfactant is selected from the A-P lysate shown in the table 2, more preferably C-M lysate and P lysate, more preferably E, F, G, H, J lysate, most preferably G lysate, H lysate, the perhaps surfactant described in the ε lysate shown in the table 6, ζ lysate, η lysate, the θ lysate.

Preferably, buffering agent is selected from citrate buffer, phosphate buffer, Tris damping fluid, buffered with amino acid liquid, acetate buffer, HEPES damping fluid, carbonate buffer solution.Buffering agent can be the combination of one or several (2,3,4,5 kind) in the above-mentioned damping fluid.Buffering agent optimization citric acid salt buffer, phosphate buffer.The pH scope and the concentration range of buffering agent can be phosphate buffer: the preferred 20mM pH of 10-50mM pH value 6.0-7.0 value 7.4; Citrate buffer: the preferred 10mM pH of 10-50mM pH value 3.0-6.6 value 6.6; Tris damping fluid: the preferred 20mM pH of 10-50mM pH value 7.0-9.0 value 7.0; Acetate buffer: the preferred 10mM pH of 10-50mM pH value 3.8-6.0 value 6.0.

Not necessarily, lysate can also contain one or more (as 1,2,3,4,5,6-10 kind) other compositions, for example (salt for example is as NaCl for reductive agent (for example DTT, DTE, beta-mercaptoethanol), sequestrant (for example EDTA), osmotic pressure instrumentality; Sucrose).For example lysate can also contain one or several compositions that are selected from from following group: the preferred 0.1%-0.5% of beta-mercaptoethanol: 0.01%-1%, more preferably 0.2%; The preferred 5-10mM of DTT:1-30mM, more preferably 5mM; The preferred 5-10mM of DTE:1-30mM, more preferably 5mM; The preferred 5mM of EDTA:5mM-20mM.

Preferably, lysate is selected from the A-P lysate shown in the table 2, more preferably C-M lysate and P lysate, more preferably E, F, G, H, J lysate, most preferably G lysate, H lysate; Perhaps be selected from the ε lysate shown in the table 6, ζ lysate, η lysate, θ lysate.

The invention still further relates to the kit that is used to detect hepatitis B virus pre S 1 antigen, it comprises the reagent that is used for detecting according to method of the present invention hepatitis B virus pre S 1 antigen.

In a preferred embodiment, kit of the present invention comprises: the lysate that can be used to detect hepatitis B virus pre S 1 antigen with the first anti-HBV PreS1 antibody of hepatitis B virus pre S 1 antigen specific bond and the second anti-HBV PreS1 antibody (two kinds of antibody can be identical or different, can be monoclonal antibody or polyclonal antibody) and/or the present invention.Preferably also contain the reagent (for example detecting the detectable of second antibody) that is suitable for detecting described antigen-antibody reaction in the described kit.At least a anti-HBV PreS1 antibody is produced body tag by signal in the preferably described antibody, can produce perhaps that body is effective to be combined with signal.Described antibody is monoclonal antibody preferably.Preferably anti-HBV PreS1 antibody is selected from: 3H5,7H11,2A7,4D11,6F1,13G2 and 16F5, particularly 7H11 and 4D11.

In a specific embodiments, described kit comprises: (1) solid phase carrier is fixed with the first anti-HBV PreS1 antibody on the described solid phase carrier; And/or (2) the present invention is used to detect the lysate of hepatitis B virus pre S 1 antigen; (3) dispensable by signal generation second anti-HBV PreS1 antibody body tag or that be not labeled; (4) detectable of dispensable detection second antibody; (5) dispensable damping fluid, cleansing solution, operation instructions (preferred operation instructions have been put down in writing the method that the present invention detects hepatitis B virus pre S 1 antigen).

Preferably, solid phase carrier is porous reaction plate (for example ELISA Plate, a chemiluminescence plate), is fixed with anti-HBV PreS1 antibody in the hole of porous reaction plate.

The reagent that detects hepatitis B virus pre S 1 antigen is used for checking the purposes of hepatitis b virus infected kit in preparation; Preferably, described reagent is the reagent of detection hepatitis B virus pre S 1 antigen described in the invention.For example, described reagent comprises: can (two kinds of antibody can be identical or different with the first anti-HBV PreS1 antibody of hepatitis B virus pre S 1 antigen specific bond and the second anti-HBV PreS1 antibody, can be monoclonal antibody or polyclonal antibody) and/or the present invention be used to detect the lysate of hepatitis B virus pre S 1 antigen, the nonessential reagent (for example detecting the detectable of second antibody) that is suitable for detecting described antigen-antibody reaction; Particularly: (1) solid phase carrier is fixed with the first anti-HBV PreS1 antibody on the described solid phase carrier; And/or (2) the present invention is used to detect the lysate of hepatitis B virus pre S 1 antigen; (3) dispensable by signal generation second anti-HBVPreS1 antibody body tag or that be not labeled; (4) detectable of dispensable detection second antibody; (5) dispensable damping fluid, cleansing solution, operation instructions (preferred operation instructions have been put down in writing the method that the present invention detects hepatitis B virus pre S 1 antigen).

The invention still further relates to the method for lytic virus, be included in not in fact denatured protein (deactivation antibody in fact not for example, for example pre S 1 antigen or cAg antibody) temperate condition under lytic virus, preferably adopt the present invention to detect the lysate of hepatitis B virus pre S 1 antigen or the lysate lytic virus of joint-detection of the present invention; Described virus is hepatitis type B virus preferably." in fact " be meant that at least 70%, for example at least 80%, at least 90%, at least 95% things takes place.For example, " not denatured protein " in fact or " not deactivation antibody " in fact is meant that at least 70%, for example at least 80%, preferred at least 90% or at least 95% protein or antibody is not by sex change or deactivation.

The invention still further relates to a kind of employing virus cracking liquid of gentleness, its can be not denatured protein (for example not deactivation antibody, for example pre S 1 antigen or cAg antibody) in fact in fact, and lytic virus.

The invention still further relates to the method that detects hepatitis B virus pre S 1 antigen and/or cAg, comprise: at denatured protein (deactivation antibody in fact not for example in fact not, for example pre S 1 antigen or cAg antibody) temperate condition under lytic virus, preferably adopt the present invention to detect the lysate of hepatitis B virus pre S 1 antigen or the lysate lytic virus of joint-detection of the present invention; Detect hepatitis B virus pre S 1 antigen and/or cAg.Can adopt known method or method disclosed by the invention to detect hepatitis B virus pre S 1 antigen and cAg.

Particularly, the present invention relates to a kind of method that detects hepatitis B virus pre S 1 antigen, comprise:, preferably adopt the present invention to detect the lysate of hepatitis B virus pre S 1 antigen or the lysate lytic virus of joint-detection of the present invention lytic virus under the temperate condition of the anti-pre S 1 antigen antibody of deactivation in fact not; Detect hepatitis B virus pre S 1 antigen.The invention still further relates to the method that detects hepatitis B virus core antigen, comprise:, preferably adopt among the lysate of joint-detection of the present invention or the embodiment and detect the used lysate lytic virus of cAg lytic virus under the temperate condition of the anti-cAg antibody of deactivation in fact not; Detect hepatitis B virus core antigen.

In a specific embodiments, the present invention relates to the method for the virus in a kind of lysate sample, comprise and adopt employing virus cracking liquid lytic virus of the present invention.Correspondingly, the present invention also provides a kind of method that detects cAg, and this method comprises the virus that adopts in the above-mentioned lysate lysate sample.Can adopt known method or method disclosed by the invention to detect cAg then.The present invention also provides a kind of method that detects pre S 1 antigen, and this method comprises the virus that adopts in the above-mentioned lysate lysate sample.Can adopt known method or method disclosed by the invention to detect pre S 1 antigen then.Described virus is hepatitis B preferably.

In another embodiment, the present invention relates to the method for lytic virus, comprise the lysate lytic virus of adopting the present invention to detect hepatitis B virus pre S 1 antigen; Described virus is hepatitis type B virus preferably.The present invention also provides a kind of method that detects pre S 1 antigen, and this method comprises the virus that adopts in the above-mentioned lysate lysate sample.Can adopt known method or method disclosed by the invention to detect pre S 1 antigen then.

Below in conjunction with specific embodiment and attached chart, the present invention is further described.Described embodiment is intended to specifically illustrate the present invention with way of example.The value of the concentration of reagent, reagent, temperature and its dependent variable and carrier and host's selection just illustrates application of the present invention, and is not construed as limiting the invention.

Embodiment

One. the HBV PreS1 MONOCLONAL ANTIBODIES SPECIFIC FOR that the present invention relates to

Embodiment 1 is as the GST-2147*6 antigen preparation of antigen

The template preparation

Utilize Proteinase K-phenol: the chloroform method, extract HBV DNA in the chronic hepatitis B patients serum sample of Bao Cuning (numbering 263) internally.Design many primers, the PCR segmentation separates the HBV gene, and is cloned into pMD18-T carrier (Takara NO.CK2401) and checks order.The GenBank accession number of the total length HBV sequence that assembly unit goes out is AF233236, and this HBV pnca gene group total length 3068bp is the adw2 hypotype.Wherein cloned plasmids pT-PreS comprises the PreS1 and the PreS2 gene of total length.Sequence is as follows:

atgggaggttggtcttccaaacctcgacaaggcatggggacaaatctttctgttcc

caatcctctgggattctttcccgatcaccagttggaccctgcgttcggagccaactcaa

acaatccagattgggacttcaaccccaacaaggatcactggccagaggcaaatcaggta

ggagcgggagcattcgggccagggttcaccccaccacacggcggtcttttggggtggag

ccctcaggctcagggcatattgacaacagtgccagcagcacctcctcctgcctccacca

atcggcagtcaggaagacagcctactcccatctctccacctctaagagacagtcatcct

caggcc(PreS1)；

atgcagtggaactccacaacattccaccaagctctgctagatcccagagtgagggg

cctatattttcctgctggtggctccagttccggaacagtaaaccctgttccgactactg

cctcacccatatcgtcaatcttctcgaggactggggaccctgcaccgaac(PreS2)。

Plasmid construction

Design upstream primer 2147F1 (ATG AGA TCT CCT CTG GGA TTC TTT CC), downstream primer 2147R1 (TTA GGA TCC ACC TCC ACC CGG ATT AAA GTC CCAATC TGG ATT GTT) is a masterplate with cloned plasmids pT-PreS, access the gene order of the 21-47AA section in the preS1 district wherein with PCR method, the plasmid pT-2147*1 that oppositely inserts pMD-18T at first obtains the plasmid pT-2147*2 of 21-47 fragment two-fold series connection as setting out plasmid.Use similar method, on the basis of pT-2147*1 and pT-2147*2, be building up to the recombinant plasmid pT-2147*3 (the plasmid construction process is referring to Fig. 1 in detail) of 21-47 fragment triplex tandem.Then pT-2147*3 is the plasmid that sets out, and obtains the recombinant plasmid pT-2147*6 of PreS1 (21-47) sixfold series connection.

The recombinant plasmid pT-2147*6 of above sixfold series connection is handled with Bgl II, EcoR I double digestion, and glue reclaims kit (the Shanghai China wink NO.W5212 of bioengineering company limited) and reclaims purpose fragment 2147*6.BamH I, EcoR I double digestion are handled expression plasmid pGEX-20T, and ethanol precipitation reclaims carrier.With dna ligase the purpose fragment is connected with pGEX-20T, after enzyme is cut evaluation, obtains the GST-2147*6 recombinant expression plasmid.

The expression of polypeptide GST-2147*6 and purifying

The GST-2147*6 recombinant plasmid transformed that makes up is expressed in expression strain E.coli ER2566.SDS-PAGE electrophoretic analysis expression of results.GST-2147*6 exists with soluble form.

With Glutathione-Sepharose 4B affinity chromatography solubility recombinant protein GST-2147*6 is carried out purifying.The SDS-PAGE electrophoresis result shows that the purity of destination protein surpasses 85%.(see figure 2)

The generation of embodiment 2 hybridoma cell line 3H5,7H11,2A7,4D11,6F1,13G2,16F5

The fusion of mouse immune and splenocyte and hybridoma

Get female Balb/c mouse in 6～8 ages in week, the 5 μ g GST-2147*6 recombinant antigens (cumulative volume 50 μ l) of every mouse muscle injection of initial immunity Fu Shi Freund's complete adjuvant emulsification.Carry out the immunity of secondary base plinth after 15 days, method is the antigen freund 's incomplete adjuvant emulsification of getting same amount, intramuscular injection.After 30 days, tail vein booster shots 5 μ g do not add the antigen of adjuvant, and behind booster immunization 72 hours, kill mouse, collect blood, get spleen and prepare splenocyte suspension (being suspended from RPMI 1640 nutrient culture media), cell counting count board cell count.Get the SP2/0 murine myeloma cell of cultivation by 1/6 in the quantity of splenocyte, centrifugal after mixing, utilize polyglycol (PEG1500) that splenocyte and murine myeloma cell SP2/0 are merged.With after isopyknic feeder cells (Balb/c mouse macrophage and thymocyte) mix, (200 μ l/ hole) is in 37 ℃ of cultivations of 5% CO2gas incubator (ESPEC BNA-311) in the 96 porocyte culture plates that are placed in cell suspension.After 3 days, ((hypoxanthn, H) (thymidine, T) 0.388mg add RPMI1640 (GIBCO company) nutrient culture media to 100mL to the 1.361mg+ thymidine to xanthine with the HT nutrient culture media.After dissolving under 45～50 ℃ of conditions, filtration sterilization.) partly keep and change liquid.After 7 days, by plate, utilize as following enzyme linked immunosorbent assay (ELISA) and detect gained hybridoma supernatant nutrient solution in the 96 porocyte culture plates with the synthetic peptide bag of HBV PreS1 21-47.For the cell clone of ELISA test positive, utilize limiting dilution assay to carry out cloning.

The ELISA detection method

1. envelope antigen: HBV PreS1 21-47 synthesizes peptide.

Sequence is: PLGFFPDHQLDPAFGANSNNPDWDFNP

2. bag is cushioned the 0.05mol/L carbonate buffer solution (20.02gNa of liquid: pH 9.6 ₂CO ₃, 2.52g NaHCO ₃Add deionized water to 1 liter).

3. coating buffer preparation: HBV PreS1 21-47 polypeptide is dissolved in bag is cushioned in the liquid, concentration is 1 μ g/ml,

4. bag quilt:

The coating buffer that adds 100 μ l in the every hole of 96 hole ELISA Plate, 37 ℃ of bags be by 2 hours, change over to then 2～8 ℃ 16 hours.

5. sealing:

(0.1%Tween20) washing is 1 time for 20mM PB7.4,150mM NaCl with the PBST cleansing solution.The confining liquid that every then hole adds 200 μ l (contains 20% calf serum and 1% caseic pH value and is 7.4 20mM Na ₂HPO ₄/ NaH ₂PO ₄Buffer soln), put into 37 ℃ of sealings 2 hours; Get rid of deblocking liquid.2～8 ℃ of preservations of aluminium foil bag of packing into after the drying.

6. detect:

In sample well, add 100 μ l cell culture fluid supernatants, every ELISA Plate is established 1 hole positive control (mouse anti GST-2147*6 polyvalent antibody (obtaining as embodiment 2), dilution in 1: 100 is used), other establishes 1 hole negative control (fresh cell culture fluid), and 37 ℃ of temperature were bathed 30 minutes; (20mM PB7.4,150mM NaCl 0.1%Tween20) wash 5 times, pat dry the back and add 100 μ l and (contain 20% calf serum and 1% caseic pH value and be 7.4 20mM Na with the enzyme dilution with the PBST cleansing solution ₂HPO ₄/ NaH ₂PO ₄The horseradish peroxidase mark sheep anti mouse immunoglobulin (Ig) (HRP-GAM Ig, DAKO company) of dilution in buffer soln) 1: 1000,37 ℃ of temperature were bathed 30 minutes; (0.1%Tween20) cleansing solution is washed 5 times for 20mM PB7.4,150mM NaCl, pats dry the back and adds each 50 μ l of A, B colour developing liquid (Beijing Tso Biological Pharmaceutical Co provides), and 37 ℃ were developed the color 10 minutes with PBST; Add 50 μ l stop buffer (2M H ₂SO ₄) cessation reaction, and in microplate reader (TECAN company, Sunrise) the last A that detects each hole ₄₅₀Value is usually with A ₄₅₀Value be higher than negative control more than 2 times the person be considered as the positive.

The acquisition of monoclonal antibody and purifying

1. monoclonal antibody ascites obtains

Get the healthy Balb/c mouse in 10 ages in week, lumbar injection paraffin oil, every 0.5ml.After 2～7 days, collect the hybridoma of cloning, the centrifugal supernatant that goes adds the nutrient culture media (1640HT) that does not contain serum, regulates cell density to 2 * 10 ⁵～2 * 10 ⁶Individual/ml, every injected in mice 0.5ml.Mouse web portion increases after 7～10 days, begins to collect ascites.Centrifugal 15 minutes of 3000rpm, the liquid of clarification part in the middle of drawing, the filtering with microporous membrane degerming of 0.45 μ m ,-20 ℃ of preservations after the packing.

2. monoclonal antibody obtains

The ascites of handling well is used PBS (the 81ml 0.2mol/LNa of 0.02mol/L, pH 7.4 ₂HPO ₄, 19ml 0.2mol/L NaH ₂PO ₄, 20ml 3mol/L NaCl adds water to 100ml) and the two-fold dilution, dropwise slowly add saturated ammonium sulfate (concentration reaches 50% saturation degree) under stirring, 2～8 ℃ are spent the night.4 ℃, centrifugal 15 minutes of 12000rpm abandons supernatant, precipitation is dissolved among the PBS of 2 times of former ascites volumes.Dropwise slowly add saturated ammonium sulfate under stirring, make ammonium sulfate concentrations reach 33% saturation degree, 2～8 ℃ of standing over night.4 ℃, centrifugal 15 minutes of 12000rpm collects supernatant.Dropwise slowly add saturated ammonium sulfate under stirring, (concentration reaches 50% saturation degree), 2～8 ℃ are spent the night.4 ℃, centrifugal 15 minutes of 12000rpm abandons supernatant.Precipitation is dissolved among an amount of PBS, in packing into the dialysis band, puts under 50～100 times of 2～8 ℃ of stirrings of 0.02M PB damping fluid that contain pH7.4 desalination about 12 hours, during change dislysate more than 3 times.This moment, monoclonal antibody purity can reach about 80%.

3. monoclonal antibody purifying

Monoclonal antibody after dialysis finished is collected and is penetrated the peak with DE52 post (Whatman NO.4057050) purifying under FPLC.This moment, monoclonal antibody purity can reach more than 90%.The monoclonal antibody that purifying is good is in-20 ℃ of preservations.

Filter out 7 strains with said method and secreted the hybridoma cell strain (3H5,7H11,2A7,4D11,6F1,13G2 and 16F5) (not preservation) of anti-HBV PreS1 monoclonal antibody

Two. the HBV PreS1 monoclonal antibody Preliminary Identification that the present invention relates to

Anti-and the enzyme mark monoclonal antibody of embodiment 3 screening high-affinity HBV PreS1 Sheets

The preparation of HBV PreS1 enzyme mark monoclonal antibody:

Adopt improvement sodium periodate method.With mark 20mg 3H5 monoclonal antibody is example: accurately take by weighing HRP40mg, add 0.2M pH5.6 acetate buffer 2ml, the dissolving back adds 0.06M NaIO ₄Solution 2ml, room temperature reaction 20 minutes; Add 0.16M ethylene glycol-10%NaCl solution 2ml, room temperature reaction 20 minutes is packed enzyme liquid in the bag filter into, to 0.001M pH4.0 acetate buffer dialysed overnight; Add 2M pH9.6 carbonate buffer solution 0.8ml, add 3H5 monoclonal antibody 20mg, 4 ℃ of stirring reactions 2 hours; The NaBH that adds new preparation ₄Solution (5mg/ml) 0.4ml, 4 ℃ of stirring reactions 2 hours; Drip saturated (NH ₄) ₂SO ₄Solution 9.2ml; 4 ℃ of stirring reactions 30 minutes, 4 ℃ 3500 left the heart 20 minutes; Abandon supernatant, precipitation is dissolved in the phosphate buffer of 2ml 0.02M pH7.4, dialysed 24 hours, add 2ml glycerine ,-20 ℃ of preservations behind the mixing with the phosphate buffer of 0.02M pH7.4.

Respectively 3H5,7H11,2A7,4D11,6F1,13G2 and 16F5 monoclonal antibody are carried out the HRP mark with said method.

The ELISA Plate preparation:

1. coating buffer preparation:

20mM PB damping fluid (Na with HBV PreS1 monoclonal anti body and function pH7.4 ₂HPO ₄/ NaH ₂PO ₄Damping fluid, final concentration are 20mM, and the pH value is 7.4) dilution, final concentration is 5 μ g/ml.

2. bag quilt:

Add the coating buffer of 100 μ l in the every hole of 96 hole ELISA Plate, 2～8 ℃ of bags were by 16～24 hours.

3. sealing:

Respectively 3H5,7H11,2A7,6F1,13G2 and 16F5 monoclonal antibody are wrapped quilt with said method.

Experimental technique:

1. sample:

The clinical serum specimen of HBV virus-positive (PCR detection), the negative clinical serum specimen of HBV virus.

2. application of sample:

Get one of the ELISA Plate of wrapping quilt, every hole adds the 50 μ l lysate (Na that contain 1%TritonX100 ₂HPO ₄/ NaH ₂PO ₄Damping fluid, final concentration are 20mM, and the pH value is 7.4), every hole adds the corresponding sample of 50 μ l, behind the concussion mixing, places 37 ℃ of incubator reactions 1 hour.

3. enzyme-added:

(0.1%Tween20) washing is 5 times for 20mM PB7.4,150mM NaCl, and every hole adds 100 μ l and (contains 20% calf serum and 1% caseic pH value and be 7.4 20mM Na with the enzyme dilution with the PBST washing lotion with reacted ELISA Plate ₂HPO ₄/ NaH ₂PO ₄The enzyme labeling monoclonal antibody of dilution in buffer soln) 1: 1000 (adopts NaIO ₄The oxidation labelling method carries out the HRP mark to corresponding monoclonal antibody), place 37 ℃ of incubator reactions 30 minutes.

4. colour developing:

(0.1%Tween20) washing is 5 times for 20mM PB7.4,150mM NaCl, and every hole adds A, each 50 μ l of B developer, places 37 ℃ of incubator reactions 15 minutes with the PBST washing lotion with reacted ELISA Plate.

5. stop, the value of reading:

Every hole adds stop buffer (2M H in the ELISA Plate of having reacted ₂SO ₄) 50 μ l, and on microplate reader, detect the A450/630 value in each hole.

Match with enzyme mark monoclonal antibody so that each Sheet of said method quadrature detection is anti-, ask P/N (positive sample detects average and negative sample detects average ratio) value.See Table 1

Each monoclonal antibody of table 1 and enzyme mark monoclonal antibody P/N ratio statistics

Sheet is anti-
Sheet is anti-							3H5	7H11	2A7	4D11	6F1	13G2	16F5
Enzyme mark monoclonal antibody	3H5-HRP 7H11-HRP 2A7-HRP 4D11-HRP 6F1-HRP 13G2-HRP 16F5-HRP	12.6 13.9 12.0 27.6 12.0 12.4 13.2	12.3 17.4 13.4 35.1 13.1 12.9 14.7	11.0 6.4 10.7 12.7 1.4 1.6 10.9	18.4 10.9 1.5 13.5 12.1 2.3 6.4	11.5 3.4 2.9 14.1 1.1 2.8 5.3	3H5	7H11	2A7	4D11	6F1	13G2	16F5	15.3 6.3 3.1 13.2 3.9 11.7 3.8	16.2 5.1 1.2 11.8 1.4 11.5 4.1

6. interpretation of result:

By last table as seen, 7H11 monoclonal antibody bag is a best pairing by detecting HBV PreS1 with the 4D11-HRP pairing.

Three. the reactive system that the present invention relates to is set up

The foundation of the two sandwich detection pre S 1 antigen of the former S 1 immune body systems of embodiment 4 high sensitivity

The ELISA Plate preparation:

1. coating buffer preparation:

With the 20mMPB damping fluid dilution of HBV PreS1 monoclonal antibody 7H11 with pH7.4, final concentration is 5 μ g/ml.

2. bag quilt:

3. sealing:

4. contain the preparation of the lysate of different surfaces activating agent:

The preparation of the lysate of table 2:A-P different component

A lysate: 2%Chaps+5mM EDTA+20mM PB7.4

B lysate: 2%Chaps+5mM EDTA+20mM PB7.4+0.2%Me

C lysate: 2%HP+0.2%Me+20mM PB7.4

D lysate: 2%SB8+0.2%Me+5mM EDTA+20mM PB7.4

E lysate: 2%SB12+0.2%Me+5mM EDTA+20mM PB7.4

F lysate: 2%SB14+0.2%Me+5mM EDTA+20mM PB7.4

G lysate: 1%SB16+0.2%Me+5mM EDTA+20mM PB7.4+0.5%SB14

H lysate: 1%SB18+0.2%Me+5mM EDTA+20mM PB7.4+0.5%SB14

I lysate: 2%SB14+5mMEDTA+20mM PB7.4

J lysate: 2%Tw80+0.2%Me+5mM EDTA+20mM PB7.4

K lysate: 2%Tw60+0.2%Me+5mM EDTA+20mM PB7.4

L lysate: 2%Tw40+0.2%Me+5mM EDTA+20mM PB7.4

M lysate: 2%Tw20+0.2%Me+5mM EDTA+20mM PB7.4

N lysate: 2%TX100+0.2%Me+5mM EDTA+20mM PB7.4

O lysate: 2%BRJ35+0.2%Me+5mM EDTA+20mM PB7.4

P lysate: 1%BRJ35+0.2%Me+5mM EDTA+20mM PB7.4

Annotate:

HP:HP-β-CD, and HYDROXYPROPYL BETA-CYCLODEXTRIN (Hydroxypropyl-β-Cyclodextrin);

SB8：sulfobetain8，

3-(Octodecyldimethylammonic)propanesulfonate；

SB10：sulfobetain10，

3-(Decyldimethylammonic)propanesulfonate；

SB12：sulfobetain12，

3-(N，N-Dimethyldodecylammonio)-propanesulfonate；SB14：sulfobetain14，

3-(N，N-Dimethylmyristylammonio)-propanesulfonate；SB16：sulfobetain16，

3-(N，N-Dimethylpalmitylammonio)-propanesulfonate；SB18：sulfobetain18，

3-(N，N-Dimethylstearylammonio)-propanesulfonateChaps：

3-(3-(cholamidopropyl) dimethylammonio)-1-propanesulfonateMe: beta-mercaptoethanol; Tw80:Tween-80; Tw40:Tween-40; Tw20:Tween-20; TX100:TritonX100; BRJ35: Bu Lijie 35 (poly-oxirene lauric acid acyl ether 35); 20mM PB7.4:Na ₂HPO ₄/ NaH ₂PO ₄Damping fluid, final concentration are 20mM, and the pH value is 7.4

Experimental technique:

1. sample:

The clinical serum specimen of HBV virus-positive (PCR detection), the normal human serum sample.

2. application of sample:

Get the ELISA Plate of wrapping quilt, every hole adds 50 μ l different formulations lysates, and every hole adds the corresponding sample of 50 μ l, and (not cracking contrast does not add any lysate for only adding the corresponding sample of 50 μ l.) behind the concussion mixing, place 37 ℃ of incubators reaction 1 hour.

3. enzyme-added:

(0.1%Tween20) washing is 5 times for 20mM PB7.4,150mM NaCl, and every hole adds 100 μ l and (contains 20% calf serum and 1% caseic pH value and be 7.4 20mM Na with the enzyme dilution with the PBST washing lotion with reacted ELISA Plate ₂HPO ₄/ NaH ₂PO ₄The 4D11 enzyme labeling monoclonal antibody of dilution in buffer soln) 1: 1000 places 37 ℃ of incubator reactions 30 minutes.

4. colour developing:

5. stop, the value of reading:

Every hole adds stop buffer (2M H in the ELISA Plate of having reacted ₂SO ₄) 50 μ l, and on microplate reader, detect the A in each hole _450/630Value.

6. positive sample detects the calculating of titre:

Positive sample detects titre and is meant that positive until not being detected (positive judgement is with the negative sample A with a kind of cracking condition correspondence by dilution doubly with positive sample _450/630* 2.1 is Cutoff, is judged to be more than or equal to Cutoff and detects the positive, detects feminine gender otherwise be judged to be), sample can be detected the detection titre that positive maximum dilution multiple is positive sample.

Experimental result:

Table 3 A-P is different, and lysate detects data statistics

	The detection titre of 4 parts of different positive samples
						Positive sample 1	Positive sample 2	Positive sample 3	Positive sample 4	Effect assessment
	Not cracking contrast	256	128	512	32	Positive sample 1	Positive sample 2	Positive sample 3	Positive sample 4	Effect assessment	+
The A lysate	Not cracking contrast	256	128	512	32	256	256	1024	128	+	+
The A lysate	The B lysate	512	1024	1024	512	256	256	1024	128	+	+
The C lysate	The B lysate	512	1024	1024	512	1024	1024	2048	512	++	+
The C lysate	The D lysate	2048	2048	4096	1024	1024	1024	2048	512	++	++
The E lysate	The D lysate	2048	2048	4096	1024	4096	2048	8192	2048	+++	++
The E lysate	The F lysate	2048	4096	4096	4096	4096	2048	8192	2048	+++	+++
The G lysate	The F lysate	2048	4096	4096	4096	16384	8192	32768	8192	++++	+++
The G lysate	The H lysate	32768	16384	32768	4096	16384	8192	32768	8192	++++	++++
The I lysate	The H lysate	32768	16384	32768	4096	1024	1024	2048	512	++	++++
The I lysate	The J lysate	4096	4096	8192	1024	1024	1024	2048	512	++	+++
The K lysate	The J lysate	4096	4096	8192	1024	1024	1024	1024	512	++	+++
The K lysate	The L lysate	2048	1024	2048	512	1024	1024	1024	512	++	++
The M lysate	The L lysate	2048	1024	2048	512	2048	2048	4096	512	++	++
The M lysate	The N lysate	512	256	1024	256	2048	2048	4096	512	++	+
The O lysate	The N lysate	512	256	1024	256	256	128	256	64	+	+
The O lysate	The P lysate	1024	512	1024	256	256	128	256	64	+	++

In experimental result as can be seen, use a series of buffer solution that contain the different types of surfactant of various dose lytic virus effectively, improve the detection sensitivity that two former S 1 immune bodies detect pre S 1 antigen, effectively composition comprises: Chaps, sulfobetain8-18, Bu Lijie 35, Tween series, HP-β-CD, TritonX100 etc.

The foundation of embodiment 5 pre S 1 antigens and cAg joint-detection cracking system

The preparation of the lysate of different joint-detection prescriptions

Press the lysate of 24 kinds of different components of table 4 preparation

The 10mM citrate buffer of pH6.6: citric acid/sodium citrate damping fluid, final concentration are 10mM, and the pH value is 6.6, and other drug chemistry titles are seen embodiment 4.</entry></row></tbody></tgroup></table></tables>

The ELISA Plate preparation:

1. coating buffer preparation:

7H11 monoclonal antibody, anti-HBc monoclonal antibody G8 (the Xiamen Bo Sheng NO.050824 of biotech company) are diluted with the 20mMPB damping fluid of pH7.4, and final concentration is respectively 5 μ g/ml and 4 μ g/ml.

2. bag quilt:

3. sealing:

Experimental technique:

7. sample:

8. application of sample:

Get one of the ELISA Plate of wrapping quilt, every hole adds 50 μ l different formulations lysates, and every hole adds the corresponding sample (every kind of cracking condition all detects identical 4 parts of positive serums and 4 parts of negative serums) of 50 μ l, behind the concussion mixing, places 37 ℃ of incubators reactions 1 hour.

9. enzyme-added:

(0.1%Tween20) washing is 5 times for 20mM PB7.4,150mM NaCl, and every hole adds 100 μ l and (contains 20% calf serum and 1% caseic pH value and be 7.4 20mM Na with the enzyme dilution with the PBST washing lotion with reacted ELISA Plate ₂HPO ₄/ NaH ₂PO ₄4D11 enzyme (HRP) the mark monoclonal antibody of dilution in buffer soln) 1: 1000 and enzyme (HRP) mark anti-HBc monoclonal antibody CZ (Xiamen Bo Sheng biotech company, NO.04042901) place 37 ℃ of incubator reactions 30 minutes.

10. colour developing:

11. termination, the value of reading:

With the lysate of 24 kinds of different formulations of said method detection, ask P/N (positive sample detects average and negative sample detects average ratio) value.See Table 5

12 kinds of lysates of table 5 detect data statistics

	Positive average P	Negative average N	P/N
	Positive average P	Negative average N	P/N	Not cracking contrast	0.221	0.044	5
lysis1	2.426	0.031	78	Not cracking contrast	0.221	0.044	5
lysis1	2.426	0.031	78	lysis2	1.446	0.044	33
lysis3	1.574	0.046	34	lysis2	1.446	0.044	33
lysis3	1.574	0.046	34	lysis4	1.521	0.059	26
lysis5	1.534	0.071	22	lysis4	1.521	0.059	26
lysis5	1.534	0.071	22	lysis6	1.523	0.037	41
lysis7	2.765	0.286	10	lysis6	1.523	0.037	41
lysis7	2.765	0.286	10	lysis8	1.534	0.053	29
lysis9	1.409	0.031	45	lysis8	1.534	0.053	29
lysis9	1.409	0.031	45	lysis10	2.428	0.011	221
lysis11	1.324	0.019	70	lysis10	2.428	0.011	221
lysis11	1.324	0.019	70	lysis12	1.468	0.016	92
lysis13	3.468	0.013	267	lysis12	1.468	0.016	92
lysis13	3.468	0.013	267	lysis14	1.566	0.018	87
lysis15	1.344	0.02	67	lysis14	1.566	0.018	87
lysis15	1.344	0.02	67	lysis16	2.441	0.012	203
lysis17	2.444	0.015	163	lysis16	2.441	0.012	203
lysis17	2.444	0.015	163	lysis18	2.422	0.011	220
lysis19	2.553	0.015	170	lysis18	2.422	0.011	220
lysis19	2.553	0.015	170	lysis20	1.787	0.014	128
lysis21	1.566	0.012	131	lysis20	1.787	0.014	128
lysis21	1.566	0.012	131	lysis22	1.776	0.025	71
lysis23	1.781	0.02	89	lysis22	1.776	0.025	71
lysis23	1.781	0.02	89	lysis24	1.819	0.015	121

12. interpretation of result:

By last table as seen, lysis10, lysis13, lysis16, lysis18 are best lysate prescription.

Embodiment 6 adds the function analysis of cracking system in traditional hepatitis B virus pre S 1 antigen detects

Traditional hepatitis B virus pre S 1 antigen detecting pattern generally also is to adopt double antibody sandwich method ELISA to detect, but be to use anti-HBsAg (anti-HBsAg of signal mark) as capture antibody (report antibody) and pairing with the anti-PreS1 (anti-PreS1) of signal mark report antibody (capture antibody) as correspondence, improve the detection sensitivity of pre S 1 antigen by the high abundance of HBsAg on hepatitis B virus particles.

The ELISA Plate preparation:

1. coating buffer preparation:

With the 20mMPB damping fluid dilution of HBV PreS1 monoclonal antibody 4D11 with pH7.4, final concentration is 5 μ g/ml; Use the 20mMPB damping fluid of pH 7.4 to dilute the anti-HBsAg (Xiamen Bo Sheng biotech company) that buys, final concentration is 3 μ g/ml.

2. bag quilt:

Add the coating buffer of 100 μ l in the every hole of 96 hole ELISA Plate, 2～8 ℃ of bags were by 16～24 hours; The ELISA Plate of 4D11 monoclonal antibody bag quilt is labeled as the A plate, and the ELISA Plate of anti-HBsAg monoclonal antibody bag quilt is labeled as the B plate.

3. sealing:

Bag is washed 1 time with PBST cleansing solution (the PBS solution that contains 0.1%Tween20) by intact A plate and B plate.The confining liquid that every then hole adds 200 μ l (contains 20% calf serum and 1% caseic pH value and is 7.4 20mM Na ₂HPO ₄/ NaH ₂PO ₄Buffer soln), put into 37 ℃ of sealings 2 hours; Get rid of deblocking liquid.2～8 ℃ of preservations of aluminium foil bag of packing into after the drying.

The preparation of the lysate of table 6: α-o different component

The α lysate is: 2%Chaps+5mMEDTA+20mMPB7.4

βLie Xie liquid is: 2%HP+20mMPB7.4

The γ lysate is: 2%SB8+5mMEDTA+20mMPB7.4

The δ lysate is: 2%SB12+5mMEDTA+20mMPB7.4

The ε lysate is: 2%SB14+5mMEDTA+20mMPB7.4

The ζ lysate is: 1%SB16+5mMEDTA+20mMPB7.4+0.5%SB14

The η lysate is: 1%SB18+5mMEDTA+20mMPB7.4+0.5%SB14

The θ lysate is: 2%SB14+5mMEDTA+20mMPB7.4

The ι lysate is: 2%Tw80+5mMEDTA+20mMPB7.4

The κ lysate is: 2%Tw60+5mMEDTA+20mMPB7.4

The λ lysate is: 2%Tw40+5mMEDTA+20mMPB7.4

The μ lysate is: 2%Tw20+5mMEDTA+20mMPB7.4

The ν lysate is: 2%TX100+5mMEDTA+20mMPB7.4

The ξ lysate is: 2%BRJ35+5mMEDTA+20mMPB7.4

The o lysate is: 1%BRJ35+5mMEDTA+20mMPB7.4

Annotate: used drug chemistry name is seen embodiment 4 and embodiment 5

Experimental technique:

5. sample:

6. application of sample:

Get the A plate and the B plate that wrap quilt, every hole adds 50 μ l different formulations lysates, and every hole adds the corresponding sample of 50 μ l, and (not cracking contrast does not add any lysate for only adding the corresponding sample of 50 μ l.) behind the concussion mixing, place 37 ℃ of incubators reaction 1 hour.

7. enzyme-added:

With PBST washing lotion washing 5 times, the every hole of A plate adds 100 μ l and (contains 20% calf serum and 1% caseic pH value and be 7.4 20mMNa with the enzyme dilution with reacted A plate and B plate ₂HPO ₄/ NaH ₂PO ₄Buffer soln) anti-HBsAg of HRP mark (1: 10000); The every hole of B plate adds 100 μ l and (contains 20% calf serum and 1% caseic pH value and be 7.4 20mM Na with the enzyme dilution ₂HPO ₄/ NaH ₂PO ₄Buffer soln) 4D11 of HRP mark (1: 1000) places 37 ℃ of incubator reactions 30 minutes.

8. colour developing:

Reacted A plate and B plate are washed 5 times with the PBST washing lotion, and every hole adds A, each 50 μ l of B developer, places 37 ℃ of incubators reactions 15 minutes.

9. stop, the value of reading:

Every hole adds stop buffer 50 μ l in A plate that has reacted and B plate, and detects the A in each hole on microplate reader _450/630Value.

10. positive sample detects the calculating of titre:

Experimental result:

The lysate of table 7 α-o different component detects data statistics

	The detection titre of 4 parts of different positive samples
						Positive sample 5	Positive sample 6	Positive sample 7	Positive sample 8	Effect assessment
	Not cracking contrast	128	2048	256	256	Positive sample 5	Positive sample 6	Positive sample 7	Positive sample 8	Effect assessment	+
The α lysate	Not cracking contrast	128	2048	256	256	128	1024	128	128	+′/-	+
The α lysate	βLie Xie liquid	512	4096	512	1024	128	1024	128	128	+′/-	++
The γ lysate	βLie Xie liquid	512	4096	512	1024	128	1024	128	128	+′/-	++
The γ lysate	The δ lysate	1024	4094	512	512	128	1024	128	128	+′/-	++
The ε lysate	The δ lysate	1024	4094	512	512	8192	8192	16384	4096	++++	++
The ε lysate	The ζ lysate	8192	8192	16384	8192	8192	8192	16384	4096	++++	++++
The η lysate	The ζ lysate	8192	8192	16384	8192	4096	4096	8192	8192	+++	++++
The η lysate	The θ lysate	4096	4096	16384	8192	4096	4096	8192	8192	+++	++++
The ι lysate	The θ lysate	4096	4096	16384	8192	512	4096	512	512	++	++++
The ι lysate	The κ lysate	128	1024	128	256	512	4096	512	512	++	+′/-
The λ lysate	The κ lysate	128	1024	128	256	256	1024	128	256	+	+′/-
The λ lysate	The μ lysate	512	4096	512	512	256	1024	128	256	+	+
The ν lysate	The μ lysate	512	4096	512	512	128	1024	128	128	+′/-	+
The ν lysate	The ξ lysate	128	1024	128	256	128	1024	128	128	+′/-	+′/-
The o lysate	The ξ lysate	128	1024	128	256	128	2048	128	256	+′/-	+′/-

Interpretation of result:

Know by above experimental result: ε lysate, ζ lysate, η lysate, θ lysate have very great help to traditional hepatitis B virus pre S 1 antigen detection system sensitivity raising

Four. the diagnostic kit that the present invention is correlated with

Embodiment 7 is used to detect the diagnostic kit preparation and the detection method (euzymelinked immunosorbent assay (ELISA)) thereof of hepatitis B virus pre S 1 antigen (HBV PreS1).

The kit preparation

The composition of the hepatitis B virus pre S 1 antigen diagnostic kit of HBV PreS1 monoclonal antibody 7H11, the 4D11 that the present invention comprises (also can use other anti-pre S 1 antigen antibody) and being prepared as follows:

1. ELISA Plate preparation:

3 preparations are fit to the coating buffer of concentration as embodiment, and the bag quilt is in ELISA Plate.

2. sample dissociation liquid:

As the F among the embodiment 4 or G or H lysate

3. enzyme marking reagent preparation:

HRP mark 4D11 is mixed with the enzyme marking reagent of suitable concentration (1: 1000), and dilution is that to contain 20% calf serum and 1% caseic pH value be 7.4 20mM Na ₂HPO ₄/ NaH ₂PO ₄Buffer soln.

4. positive and negative contrast, developer A, B liquid, 20 * concentrated cleaning solution, stop buffer (2MH ₂SO ₄):

The Beijing Tso Biological Pharmaceutical Co provides.

Kit test method

The detection method of diagnostic kit that is used to detect hepatitis B virus pre S 1 antigen (HBV PreS1) is as follows:

1. prepare: reagent was positioned under the room temperature (18～30 ℃) balance 15～30 minutes.

2. dosing: it is standby that 50ml concentrated cleaning solution (20 *) is diluted to 1000ml with distilled water or deionized water.

3. number: the corresponding micropore of sample is numbered in order.

4. cracking: add 50 μ l sample dissociation liquid (F or G or H cracking liquid in as embodiment 4) at corresponding aperture with pipettor.

5. application of sample: in respective aperture, add testing sample 50 μ l respectively, the concussion mixing.

6. incubation: with 37 ± 1 ℃ of incubations of shrouding film shrouding postposition 60 minutes.

7. washing: behind the incubation shrouding film is taken off, blotted liquid in the hole,, soaked 30～60 seconds at every turn with cleansing solution (1x) washing 5 times.

8. enzyme-added: as in respective aperture, to add enzyme marking reagent 100 μ l respectively, except the blank well.

9. incubation: with 37 ± 1 ℃ of incubations of shrouding film shrouding postposition 30 minutes.

10. washing: behind the incubation shrouding film is taken off, blotted liquid in the hole,, soaked 30～60 seconds at every turn with cleansing solution (1x) washing 5 times.

11. colour developing: every hole adds developer A, each 50 μ l of B liquid, and the light shaking mixing was with 37 ± 1 ℃ of lucifuge colour developings of shrouding film shrouding postposition 15 minutes.

12. stop: every hole adds stop buffer (2M H ₂SO ₄) 50 μ l, the light shaking mixing.

13. measure: set the microplate reader ripple and be longer than the 450/630nm detection, measure each hole OD value.

14. the result judges:

Critical value: Cut Off (C.O.)=0.105

The result judges: (absorbance in the every hole of S=)

Negative findings (S/C.O.＜1): the absorbance of sample is negative less than Cut Off value, represents and does not detect HBV PreS1 antigen in this sample.

Positive findings (S/C.O. 〉=1): the absorbance of sample is positive more than or equal to Cut Off value, represents and detects HBV PreS1 antigen in this sample.

Embodiment 8 is used to detect the diagnostic kit preparation and the detection method (euzymelinked immunosorbent assay (ELISA)) thereof of hepatitis B virus core antigen (HBcAg).

The kit preparation

The composition of the hepatitis B virus core antigen diagnostic kit that the present invention comprises and being prepared as follows:

1. ELISA Plate preparation:

Anti-HBc monoclonal antibody G8 (the Xiamen Bo Sheng NO.050824 of biotech company) dilutes with the 20mM PB damping fluid of pH7.4, and final concentration is 4 μ g/ml.

2. sample dissociation liquid:

As the lysis10 among the embodiment 5 or lysis13 or lysis16 or lysis18

3. enzyme marking reagent preparation:

With the HRP mark of buying anti--HBc monoclonal antibody CZ (Xiamen Bo Sheng biotech company, NO.04042901) is mixed with the enzyme marking reagent of suitable concentration (dilution in 1: 1000), dilution is that to contain 20% calf serum and 1% caseic pH value be 7.4 20mM Na ₂HPO ₄/ NaH ₂PO ₄Buffer soln.

Positive and negative contrast, developer A, B liquid, 20 * concentrated cleaning solution, stop buffer (2M H ₂SO ₄).

Kit test method

The detection method of diagnostic kit that is used to detect hepatitis B virus core antigen (HBcAg) is as follows:

3. number: the corresponding micropore of sample is numbered in order.

4. cracking: with pipettor corresponding aperture add 50 μ l sample dissociations (lysis10 in as embodiment 5, lysis13, lysis16, lysis18).

7. washing: behind the incubation shrouding film is taken off, blotted liquid in the hole,, soaked 30～60 seconds at every turn with cleansing solution washing 5 times.

10. washing: behind the incubation shrouding film is taken off, blotted liquid in the hole,, soaked 30～60 seconds at every turn with cleansing solution washing 5 times.

14. the result judges:

Critical value: Cut Off (C.O.)=0.105

The result judges: (absorbance in the every hole of S=)

Negative findings (S/C.O.＜1): the absorbance of sample is negative less than Cut Off value, represents in this sample and does not detect HBcAg.

Positive findings (S/C.O. 〉=1): the absorbance of sample is positive more than or equal to Cut Off value, represents in this sample and detects HBcAg.

The preparation and the detection method thereof of embodiment 9 joint investigating hepatitis B virus pre S 1 antigens and cAg diagnostic kit (euzymelinked immunosorbent assay (ELISA))

The kit preparation

The composition of S1 and cAg diagnostic kit (euzymelinked immunosorbent assay (ELISA)) and being prepared as follows before the joint investigating hepatitis B virus that the present invention comprises:

1. ELISA Plate preparation:

As embodiment 4 with 7H11 (also can use other anti-pre S 1 antigen antibody) and the anti--HBc monoclonal antibody G8 (the Xiamen Bo Sheng NO.050824 of biotech company) that buys) (also can use other anti--HBc antibody) be diluted to suitable concentration (final concentration is respectively 5 μ g/ml and 4 μ g/ml) combined packet quilt in ELISA Plate with the 20mMPB damping fluid of pH7.4.

2. sample dissociation liquid preparation:

As the lysis10 among the embodiment 5 or lysis13 or lysis16 or lysis18

3. enzyme marking reagent preparation:

As embodiment 4 anti--HBc monoclonal antibody CZ (Xiamen Bo Sheng biotech company, NO.04042901) with 4D11-HRP (also can use other anti-pre S 1 antigen antibody) and HRP mark) (also can use other anti--HBc antibody) be mixed with the enzyme marking reagent of suitable concentration (dilution in each 1: 1000), and dilution is that to contain 20% calf serum and 1% caseic pH value be 7.4 20mM Na ₂HPO ₄/ NaH ₂PO ₄Buffer soln.

4. positive and negative contrast, developer A, B liquid, 20 * concentrated cleaning solution, stop buffer (2MH ₂SO ₄).

Kit test method

The detection method of the diagnostic kit of joint investigating hepatitis B virus pre S 1 antigen and cAg (HBcAg) is as follows:

3. number: the corresponding micropore of sample is numbered in order.

4. dilution: add 50 μ l sample diluting liquids at corresponding aperture with pipettor.

14. the result judges:

Critical value: Cut Off (C.O.)=0.105

The result judges: (absorbance in the every hole of S=)

Negative findings (S/C.O.＜1): the absorbance of sample is negative less than Cut Off value, represents and does not detect HBV PreS1 and HBcAg in this sample.

Positive findings (S/C.O. 〉=1): the absorbance of sample is positive more than or equal to Cut Off value, represent detect in this sample HBV PreS1 and (or) HBcAg.

The diagnostic kit that five. Ben Duming are relevant is used and interpretation of result

Embodiment 10 joint investigating hepatitis B virus pre S 1 antigens detect, reach the parallel comparison that similar independent pre S 1 antigen detects with cAg (nucleic acid related antigen) with hbv nucleic acid

Kit is prepared

The preparation of hepatitis B virus pre S 1 antigen diagnostic kit (euzymelinked immunosorbent assay (ELISA)) such as embodiment 7; The preparation of hepatitis B virus core antigen diagnostic kit (euzymelinked immunosorbent assay (ELISA)) is as embodiment 8; The preparation of joint investigating hepatitis B virus pre S 1 antigen and cAg diagnostic kit (euzymelinked immunosorbent assay (ELISA)) such as embodiment 9.The independent hepatitis B virus pre S 1 antigen diagnostic kit of commercialization (euzymelinked immunosorbent assay (ELISA)) is respectively available from multiple star Long March Pharmaceutical Technology Co., Ltd (NO.3150480) in Shanghai and Shanghai Alpha biotech company.

The hbv nucleic acid quantitative PCR detecting reagent, available from ShenZhen PiJi Biology Engineering Co., Ltd (NO.06010121), Yingke Xinchuang (Ximen) Sci. ﹠ Tech. Co., Ltd. and reach the peace gene incorporated company (NO.#DA-B051).

Hepatitis type B virus " two double " enzyme linked immunosorbent detection reagent available from Beijing Tso Biological Pharmaceutical Co (WB2196), Yingke Xinchuang (Ximen) Sci. ﹠ Tech. Co., Ltd. (IPT21001, IPT21201, IPT21301, IPT21401, IPT21501) and Shanghai industry section China biotechnology (NO.267 265 266 263) company limited.

Detect sample

Collect 116 parts of clinical hepatitis b virus infected related diseases human serum samples from Xiamen City Traditional Chinese Medicine Hospital and First People's Hospital, Quanzhou City ,-20 ℃ of freezing preservations year March in October, 2005-2006.

Test item

Every part of serum specimen is carried out the hbv nucleic acid quantitative PCR detection, adopt three kinds of different manufacturers reagent (ShenZhen PiJi Biology Engineering Co., Ltd, Yingke Xinchuang (Ximen) Sci. ﹠ Tech. Co., Ltd. and reach peace gene incorporated company) to carry out parallel detection, trace routine and result judge and carry out in strict accordance with each reagent instructions.For the qualitative judgement of hbv nucleic acid with identical being as the criterion of two tame reagent testing results.

Every part of serum specimen is carried out hepatitis type B virus " two double " enzyme linked immunosorbent detection, adopt three kinds of different manufacturers reagent (Beijing Tso Biological Pharmaceutical Co, Yingke Xinchuang (Ximen) Sci. ﹠ Tech. Co., Ltd. and Shanghai Shiye Kehua Biotechnology Co., Ltd) to carry out parallel detection, trace routine and result's judgement are carried out in strict accordance with each reagent instructions.For the qualitative judgement of hepatitis type B virus HBsAg, HBsAb, HBeAg, HBeAb, HBcAb with identical being as the criterion of two tame reagent testing results.

Every part of serum specimen is carried out similar commercialization hepatitis B virus pre S 1 antigen enzyme linked immunosorbent detection, adopt two kinds of different manufacturers reagent (multiple star Long March Pharmaceutical Technology Co., Ltd in Shanghai and Shanghai Alpha biotech company) to carry out parallel detection, trace routine and result's judgement are carried out in strict accordance with each reagent instructions.

Every part of serum specimen is carried out the hepatitis B virus pre S 1 antigens detection of being announced as enforcement 6, enforcement 7, enforcement 8 respectively, hepatitis B virus core antigens detection, joint investigating hepatitis B virus pre S 1 antigen and the cAg (being collectively referred to as the nucleic acid related antigen) that the present invention announced.Trace routine and result's judgement are carried out in strict accordance with corresponding embodiment explanation.

Testing result

Finish statistics after all projects to be detected, as table 8.

The clinical hepatitis B serum of table 8:116 part statistics

The sample source	Quantity	HBV nucleic acid (PCR) is confirmed the result	HBV combo ELISA of the present invention						S1 detectable separately
			HBV combo ELISA of the present invention						S1 detectable separately				The joint inspection result		PreS1		Core		Homemade 1		Homemade 2
			Quantity	+	-	+	-	+	-	+	-	+	The joint inspection result		PreS1		Core		Homemade 1		Homemade 2		-
		Clinical samples	Quantity	+	-	+	-	+	-	+	-	+	116	Negative 6 sensitivity 100% and nucleic acid coincidence rate 100% of positive 110 nucleic acid of nucleic acid	107 0	3 6	103 0	7 6	70 0	40 6	66 0	44 6	-	94 0	16 6
97.27％ 97.41％			93.64％ 93.97％		63.64％ 65.52％		60.00％ 62.07％		85.45％ 86.21％						107 0	3 6	103 0	7 6	70 0	40 6	66 0	44 6		94 0	16 6

Annotate: " homemade 1 " reagent: the hepatitis B virus pre S 1 antigen diagnostic kit (euzymelinked immunosorbent assay (ELISA)) that Shanghai Alpha biotech company produces

" homemade 2 " reagent: the hepatitis B virus pre S 1 antigen diagnostic kit (euzymelinked immunosorbent assay (ELISA)) that the multiple star Long March Pharmaceutical Technology Co., Ltd in Shanghai produces

" joint inspection result " reagent: associating hepatitis B virus pre S 1 antigen and cAg detectable (being collectively referred to as HBV combo ELISA) that the present invention announces, as embodiment 8

" PreS1 " reagent: the hepatitis B virus pre S 1 antigen detectable that the present invention announces, as embodiment 6

" Core " reagent: the hepatitis B virus core antigen detectable that the present invention announces, as embodiment 7

Sensitivity and with the computing method of nucleic acid coincidence rate:

Sensitivity=detect positive sum/true positives (the nucleic acid positive) sum

With nucleic acid coincidence rate=(detect total sample number-two kind of a testing result and be not inconsistent sum)/detection total sample number

Interpretation of result:

From the statistics of table 2 as can be seen, hepatitis B virus pre S 1 antigen detectable as embodiment 7 has bigger advantage than commercialization pre S 1 antigen detectable in sensitivity, this is to be different from traditional commercialization reagent because of the hepatitis B virus pre S 1 antigen detectable as embodiment 7, it adopts two former S 1 immune body sandwich method detecting patterns, detect whole LHBs (promptly by the HBsAg mediated detection on the LHBs and no longer continue to use traditional dependence, use at the specific antibody of HBsAg as capture antibody or report antibody, use specific antibody to match with it again) at PreS1, form an independently pre S 1 antigen detectable, can not cause the viral Dane particle S1 antigen detection miss of going forward because LHBs goes up the sudden change of HBsAg, and have the correlativity of height with hbv nucleic acid.

From table 2 also as can be seen under the similar detection condition, no matter sensitivity or with the nucleic acid coincidence rate, joint-detection PreS1, HBcAg all have bigger advantage than independent detection PreS1 and HBcAg, and joint-detection HBcAg and PreS1 pattern have remedied and may cause false-negative shortcoming because of single HBcAg or PreS1 sudden change or omission.The concrete testing result of part serum specimen that exemplifies in table 9 can illustrate this point very intuitively.

</entry></row></tbody></tgroup></table></tables>

Find out from above analysis, embody the high correlation that detects with hbv nucleic acid, it is not enough detecting pre S 1 antigen separately, and the New Set that joint-detection pre S 1 antigen and cAg form has then embodied high correlation (this that detects with hbv nucleic acid

Be 97.41% among the embodiment).

The comparative analysis of embodiment 11 joint investigating hepatitis B virus pre S 1 antigens and cAg (nucleic acid related antigen) and hepatitis type B virus " two double " (HBsAg, HBsAb, HBeAg, HBeAb, HBcAb)

Kit is prepared

The preparation of joint investigating hepatitis B virus pre S 1 antigen and cAg diagnostic kit (euzymelinked immunosorbent assay (ELISA)) also can be used the kit with the similar approach preparation as described in the embodiment 9.

The hbv nucleic acid quantitative PCR detecting reagent is available from ShenZhen PiJi Biology Engineering Co., Ltd, Yingke Xinchuang (Ximen) Sci. ﹠ Tech. Co., Ltd. and reach peace gene incorporated company.

Hepatitis type B virus " two double " enzyme linked immunosorbent detection reagent is available from Beijing Tso Biological Pharmaceutical Co, Yingke Xinchuang (Ximen) Sci. ﹠ Tech. Co., Ltd. and Shanghai Shiye Kehua Biotechnology Co., Ltd.

Detect sample

Collect 116 parts of clinical hepatitis b virus infected related diseases human serum samples year March in October, 2005-2006 from Xiamen City Traditional Chinese Medicine Hospital and First People's Hospital, Quanzhou City, reach and collect from the blood station, Xiamen hepatitis b virus infected relevant serum from blood donors sample ,-20 ℃ of freezing preservations.

Test item

Screening PCR confirms the hbv nucleic acid positive, " two double " negative and half off-order mode sample of HBsAg, carries out associating hepatitis B virus pre S 1 antigen and cAg (being collectively referred to as the nucleic acid related antigen) detection as embodiment 9 that the present invention announced.Trace routine and result's judgement are carried out in strict accordance with embodiment 9 explanations.

Testing result and analysis

Finish statistics after all projects to be detected, as table 10.

</entry></row></tbody></tgroup></table></tables>

As can be seen from the above table, hepatitis B in routine " two double " detect in the sample of judging non-virus replication carrier (HBeAg feminine gender) or non-B-mode infective virus the infected (HBsAg feminine gender), still have many samples to contain a large amount of viruses, joint-detection by preceding S1 and cAg can well detect these samples, whether carrying virus for the clinical hepatitis patient can have a right judgement and understanding, thereby can better determine therapeutic scheme.Especially for this blood station conventional sense project of surface antigen (HBsAg),, can reduce the hepatitis B blood transfusion that causes owing to surface antigen variation or omission and propagate by the joint-detection of preceding S1 and cAg.

The comparative analysis of embodiment 12 joint investigating hepatitis B virus pre S 1 antigens and cAg (nucleic acid related antigen) and conventional single primer hbv nucleic acid quantitative PCR

Kit is prepared

The preparation of joint investigating hepatitis B virus pre S 1 antigen and cAg diagnostic kit (euzymelinked immunosorbent assay (ELISA)) such as embodiment 9.

Detect sample

Collect in Dec, 2005 from the calibrating of Chinese biological goods the hepatitis b virus infected serum specimen of 314 parts of HBsAg positives ,-20 ℃ of freezing preservations.

Test item

Every part of serum specimen is carried out the hbv nucleic acid quantitative PCR detection, adopt three kinds of different manufacturers reagent (ShenZhen PiJi Biology Engineering Co., Ltd, Yingke Xinchuang (Ximen) Sci. ﹠ Tech. Co., Ltd. and reach peace gene incorporated company) to carry out parallel detection, trace routine and result judge and carry out in strict accordance with each reagent instructions.Qualitative property judgement for the feminine gender of hbv nucleic acid is the quantitative PCR feminine gender with the equal feminine gender of three tame reagent testing results.

The screening quantitative PCR is confirmed hbv nucleic acid feminine gender, " two double " HBsAg positive, carries out associating hepatitis B virus pre S 1 antigen and cAg (being collectively referred to as the nucleic acid related antigen) detection as embodiment 5 that the present invention announced.Trace routine and result's judgement are carried out in strict accordance with embodiment 5 explanations.And these samples are carried out the subregion sleeve type PCR detect (method reference: Marie-Anne Petit et al., JMV 2001).

Testing result and analysis

Finish statistics after all projects to be detected, amount to 39 parts of HBsAg positives and quantitative PCR and confirm hbv nucleic acid sera negative sample.31 parts of joint-detection positives in these 39 parts of samples,

These 31 parts of samples are carried out after the subregion sleeve type PCR detects, confirm that wherein 9 parts is the hbv nucleic acid positive.

Only can detect the virus of duplicating from above result owing to conventional blood-serum P CR, and can not detect provirus, the existence of the non-replication status of virus beyond expression of words, and conventional quantitative PCR detecting reagent is because all restrictions may be because adopt single primer, do not adopt highly sensitive sleeve type PCR, may have omission to a certain degree.Often have the hepatitis B patient hepatitis symptom obvious clinically, but conventional PCR detection is the nucleic acid feminine gender.

Joint investigating hepatitis B virus pre S 1 antigen of this announcement and cAg pattern can remedy the part defective of conventional blood-serum P CR to a certain extent.

The specificity analyses of embodiment 13 joint investigating hepatitis B virus pre S 1 antigens and cAg (nucleic acid related antigen) reagent

Kit is prepared

The preparation of the diagnostic kit of joint investigating hepatitis B virus pre S 1 antigen and cAg (euzymelinked immunosorbent assay (ELISA)) such as embodiment 9.

Detect sample

In year March in October, 2005-2006, collect 5976 parts of healthy blood donor serum specimens ,-20 ℃ of freezing preservations from Xiamen CDC, blood station, Beijing, blood station, Shaoxing.

Test item

Every part of serum is carried out associating hepatitis B virus pre S 1 antigen and cAg (being collectively referred to as HBV combo ELISA) as embodiment 9 that the present invention announced to be detected.Trace routine and result's judgement are carried out in strict accordance with embodiment 9 explanations.

Result and analysis

Finish statistics after all projects to be detected, statistics such as table 11.

The sample source	Negative number of samples	The negative number of samples of joint-detection	Specificity
The sample source	Negative number of samples	The negative number of samples of joint-detection	Specificity	Xiamen CDC	4554	4547	99.90％
The blood station, Shaoxing	428	427	99.80％	Xiamen CDC	4554	4547	99.90％
The blood station, Shaoxing	428	427	99.80％	Blood station, Beijing	994	991	99.70％
Add up to	5976	5965	99.82％	Blood station, Beijing	994	991	99.70％

Table 11: the specificity analyses of joint-detection

From above result, the present invention announces that associating hepatitis B virus pre S 1 antigen and cAg (being collectively referred to as the nucleic acid related antigen) detectable specificity are good.

Embodiment 14 joint investigating hepatitis B pre S 1 antigens and the application of cAg in chemiluminescence

The chemical luminescence reagent kit preparation

The composition of S1 and cAg diagnostic kit (chemoluminescence method) and being prepared as follows before the joint investigating hepatitis B virus that the present invention comprises:

1. chemiluminescence plate preparation:

As embodiment 3 7H11 (also can use other) is diluted G8 (the Xiamen Bo Sheng NO.050824 of biotech company) (also can use the antibody of other anti-pre S 1 antigen and anti-cAg) to being fit to concentration (final concentration is respectively 5 μ g/ml and 4 μ g/ml) combined packet quilt in chemiluminescence plate (the happy Xin Mei in Xiamen company limited, NO.C0004) with anti--HBc monoclonal antibody of purchase.

2. sample dissociation liquid:

As the lysis10 among the embodiment 5 or lysis13 or lysis16 or lysis18.

3. enzyme marking reagent preparation:

(1: 10000 enzyme marking reagent, dilution are that to contain 20% calf serum and 1% caseic pH value be 7.4 20mM Na as embodiment 2 anti--HBc monoclonal antibody CZ (Xiamen Bo Sheng biotech company, NO.04042901) (also can use the antibody of other anti-pre S 1 antigen and anti-cAg) of anti--HBs monoclonal antibody of the HRP mark of 4D11-HRP and purchase, HRP mark to be mixed with suitable concentration ₂HPO ₄/ NaH ₂PO ₄Buffer soln.

4. luminous agent (PIERCE NO.1856155), cleansing solution (the PBS solution that contains 1 ‰ Tween20)

Kit test method

The detection method of the diagnostic kit (chemoluminescence method) of joint investigating hepatitis B virus pre S 1 antigen and cAg (HBcAg) is as follows:

3. number: the corresponding micropore of sample is numbered in order.

4. cracking: add 50 μ l sample dissociation liquid at corresponding aperture with pipettor.

11. luminous: every hole adds luminous agent 100 μ l, places Chemiluminescence Apparatus to measure each hole luminous intensity.

Interpretation of result

Get 20 parts and measure the hepatitis B positive sample contain the different virus amount, measure result such as table 12 with chemoluminescence method through quantitative fluorescent PCR.All distribute near logarithm because quantitative fluorescent PCR quantitatively reaches luminous intensity, we take the logarithm laggard line linearity correlation analysis respectively as shown in Figure 3 to the viral level and the luminous intensity of each sample.

Table 12 chemiluminescence detection and quantitative fluorescent PCR correlativity

Numbering	Virus load	Chemiluminescence	1g (viral level)	1g (luminous intensity)
Numbering	Virus load	Chemiluminescence	1g (viral level)	1g (luminous intensity)	C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20	3.3E+08 2.3E+08 2.0E+08 3.5E+07 3.4E+07 3.0E+07 2.8E+07 1.4E+07 1.7E+06 7.7E+05 6.4E+05 5.1E+05 4.6E+04 9.7E+03 3.5E+03 1.3E+03 1.0E+03 1.0E+03 6.0E+02 1.0E+02	633 433.1 541.4 258.3 168.6 214.4 165.5 103.6 114.2 52.18 46.93 26.33 35.07 8.608 5.762 2.114 1.771 1.998 1.467 0.227	8.52 8.35 8.29 7.55 7.53 7.48 7.44 7.16 6.22 5.88 5.81 5.71 4.66 3.99 3.55 3.11 3.00 3.00 2.78 2.00	2.80 2.64 2.73 2.41 2.23 2.33 2.22 2.02 2.06 1.72 1.67 1.42 1.54 0.93 0.76 0.33 0.25 0.30 0.17 -0.64

By table 12 and Fig. 3 as seen, S1 and cAg method and viral level have higher correlativity before the joint investigating hepatitis B virus.Can realize utilizing associating hepatitis B virus front S 1 and cAg detection by quantitative to come the nucleic acid carrying capacity of sample estimates.

Claims

1, a kind ofly detects hepatitis type B virus or detect hepatitis b virus infected method, being included in joint-detection in the sample (comprises separate detection and merges and detect, and detect simultaneously and successively detect with any order, preferred while, merging detect) hepatitis B virus pre S 1 antigen and cAg.

2, the described method of claim 1, wherein said sample is the biological sample from the separation of individuality to be checked, preferred described sample is a serum.

3, the existence that claim 1 or 2 described methods, wherein said method are used for judging the serum hbv nucleic acid whether.

4, each described method of claim 1-3, the joint-detection of wherein said hepatitis B virus pre S 1 antigen and cAg realizes by one or several methods of selecting from following method: enzyme linked immunosorbent assay, immunity percolation method, immunochromatographic method, chemoluminescence method, time resolution method; Preferred enzyme linked immunosorbent assay or chemoluminescence method.

5, the described method of each of claim 1-4 wherein detects pre S 1 antigen and cAg simultaneously in same reaction in same reaction container (as reacting hole).

6, the described method of each of claim 1-5 wherein adopts double antibody sandwich method to detect pre S 1 antigen and cAg.

7, the described method of each of claim 1-6 does not wherein need sample is carried out pre-service.

8, the described method of each of claim 1-7, wherein being captured in the same reaction container (for example reacting hole) of cracking of hepatitis type B virus and pre S 1 antigen and cAg carried out in same media simultaneously.

9, the method for claim 8 wherein uses the lysate described in the claim 17 as described medium.

10, the described method of each of claim 1-9, it may further comprise the steps:

A) providing can (can be identical or different with the first anti-HBV PreS1 antibody and the second anti-HBV PreS1 antibody of hepatitis B virus pre S 1 antigen specific bond, can be monoclonal antibody or polyclonal antibody), and can with the first anti-HBc antibody and the second anti-HBc antibody (can be identical or different, can be monoclonal antibody or polyclonal antibody) of hepatitis B virus core antigen specific bond;

B) the first anti-HBV PreS1 antibody is attached on the identical solid phase carrier as capture antibody with the first anti-HBc antibody, forms first antibody-carrier conjugates; Preferably, described carrier is an elisa plate;

C) testing sample is contacted with first antibody-carrier conjugates, make if present, to be hunted down hepatitis B virus pre S 1 antigen and cAg in the sample, form antigen-first antibody-carrier conjugates; Described sample can be through pretreated or do not pass through pretreated; Preferably, described sample is not through virolysis processing in advance, and testing sample contacts with first antibody-carrier conjugates under the condition that allows virolysis and antigen capture and carries out; More preferably testing sample contacts with first antibody-carrier conjugates in the described lysate of claim 17 and carries out;

D) the second anti-HBV PreS1 antibody is contacted with the product (carrier) of step c) with the second anti-HBc antibody, this contact combines with captive antigen under the condition that forms second antibody (the second anti-HBV PreS1 antibody/second anti-HBc antibody)-antigen (hepatitis B virus pre S 1 antigen/cAg)-first antibody (the first anti-HBV PreS1 antibody/first anti-HBc antibody)-carrier conjugates in the permission second antibody to be carried out;

E) detect the amount of combined second antibody.

11, the method for claim 10, wherein the step e) amount that detects combined second antibody is carried out by the following method: provide and can produce body and can produce body with the effective secondary signal that combines of the described second anti-HBc antibody with effective first signal that combines of the described second anti-HBV PreS1 antibody, it is only relevant with the amount of the second anti-HBVPreS1 antibody and the second anti-HBc antibody respectively that first and second signals produce the signal intensity that body produced; First and second signals are produced body to be contacted with second antibody; Detection produces the signal that body produces by signal.

12, the method for claim 10, wherein the step e) amount that detects combined second antibody is carried out by the following method: second antibody directly is marked with signal and produces body, detects by signal and produces the signal that body produces.

13, claim 11 or 12 method, wherein to produce body be enzyme, fluorescein, chemiluminescent substance, isotope, rare earth element to signal.

14, the described method of each of claim 1-13 wherein uses the monoclonal antibody of anti-hepatitis B virus pre S 1 antigen and anti-cAg to carry out immune detection.

15, the described method of claim 14, wherein the monoclonal antibody of anti-hepatitis B virus pre S 1 antigen is selected from 3H5,7H11,2A7,4D11,6F1,13G2 and 16F5; Preferably, use the 7H11 monoclonal antibody to use 4D11-HRP to detect HBV PreS1 as the first anti-HBV PreS1 antibody as the second anti-HBV PreS1 antibody pairing.

16, the method for claim 10 also comprises step: with before first antibody-carrier conjugates contacts, use the virus in the lysate lysate sample at testing sample, wherein said lysate is the employing virus cracking liquid of claim 17 preferably.

17, employing virus cracking liquid (this employing virus cracking liquid can be used for joint-detection), it contains surfactant and suitable buffering agent, described surfactant is selected from: Chaps, sulfobetain8-18 (SB8, SB10, SB12, SB14, SB16, SB18), Bu Lijie 35, Tween series and/or HP-β-CD are preferably selected from SB14, SB16, SB18, HP-β-CD, TWEEN20, TWEEN40, TWEEN60, TWEEN80;

Preferably, surfactant can be the combination of one or more (2,3,4,5 kind) in the above surfactant, for example a kind of combination of or two kinds or three kinds in HP, TWEEN series and the SB series, for example HP+TWEEN series, TWEEN series+SB series, HP+SB series or HP+TWEEN series+SB series, preferred HP+TWEEN40, HP+TWEEN40+SB14, HP+TWEEN40+SB16, HP+TWEEN40+SB18, HP+TWEEN40+SB18, HP+TWEEN20+SB14, HP+TWEEN60+SB14, HP+TWEEN80+SB14; Preferably, surfactant can be HP:0.5%-4%; Tween series: 0.1%-6%; SB14%:1%-10%; SB16:0.5%-1%; SB18:0.5%-1%; More preferably HP:1%-2%; Tween series: 0.5%-2%; SB14:2%-8%; SB16:1%; SB18:1%; As: 1%-2%HP+0.5%-1%Tween+1%-2%SB14;

Preferably, surfactant is selected from lysis1-lysis24 shown in the table 4, more preferably lysis1, lysis10, lysis11, lysis12, lysis13, lysis14, lysis15, lysis16, lysis17, lysis18, lysis19, lysis20, lysis21, lysis22, lysis23 and lysis24, more preferably lysis10, lysis13, lysis16, lysis17, lysis18, lysis19, lysis20, lysis21 and lysis24, the most preferably surfactant described in the lysis10,13,16,18;

Preferably, buffering agent is selected from citrate buffer, phosphate buffer, Tris damping fluid, buffered with amino acid liquid, acetate buffer, HEPES damping fluid, carbonate buffer solution; Buffering agent can be the combination of one or several (2,3,4,5 kind) in the above-mentioned damping fluid; Buffering agent optimization citric acid salt buffer, phosphate buffer; The pH scope and the concentration range of buffering agent can be: phosphate buffer: the preferred 20mM pH of 10-50mM pH value 6.0-7.0 value 7.4; Citrate buffer: the preferred 10mM pH of 10-50mM pH value 3.0-6.6 value 6.6; Tris damping fluid: the preferred 20mM pH of 10-50mM pH value 7.0-9.0 value 7.0; Acetate buffer: the preferred 10mM pH of 10-50mM pH value 3.8-6.0 value 6.0;

Not necessarily, lysate can also contain one or more (as 1,2,3,4,5,6-10 kind) other compositions, for example (salt for example is as NaCl for reductive agent (for example DTT, DTE, beta-mercaptoethanol), sequestrant (for example EDTA), osmotic pressure instrumentality; Sucrose), the preferred 0.1%-0.5% of beta-mercaptoethanol: 0.01%-1% for example, more preferably 0.4%; The preferred 5-10mM of DTT:1-30mM, more preferably 5mM; The preferred 5-10mM of DTE:1-30mM, more preferably 5mM; The preferred 5mM of EDTA:5mM-20mM; NaCl:50mM-300mM is more preferably: 50-150mM, more preferably 100mM; The preferred 5%-10% of sucrose: 2%-20% more preferably 10%;

18, a kind of kit, it comprises and being used for according to each the described method joint investigating hepatitis B virus pre S 1 antigen of claim 1-13 and the reagent of cAg.

19, kit described in the claim 18 wherein contains anti-HBV PreS1 antibody (preferably comprising two kinds of different anti-HBV PreS1 antibody) and anti-HBc antibody (preferably comprising two kinds of different anti-HBc antibody), and/or the employing virus cracking liquid of claim 17; Preferably also contain the reagent (for example detecting the detectable of second antibody) that is suitable for detecting described antigen-antibody reaction in the described kit; At least a anti-HBV PreS1 antibody and at least a anti-HBc antibody are produced body tag by signal in the preferably described antibody, can produce perhaps that body is effective to be combined with signal, the signal generation body of anti-HBVPreS1 antibody and anti-HBc antibody labeling or the signal of combination produce body can be identical or different, preferably can adopt identical detection method to detect; Described antibody is monoclonal antibody preferably; Preferably anti-HBV PreS1 antibody is selected from: 3H5,7H11,2A7,4D11,6F1,13G2 and 16F5, particularly 7H11 and 4D11.

20, kit described in the claim 19 wherein comprises: (1) solid phase carrier is fixed with the first anti-HBV PreS1 antibody and the first anti-HBc antibody on the described solid phase carrier; And/or the employing virus cracking liquid of (2) claim 17; (3) dispensable by signal generation second anti-HBV PreS1 antibody body tag or that be not labeled and the second anti-HBc antibody; (4) detectable of dispensable detection second antibody; (5) dispensable damping fluid, cleansing solution, operation instructions (preferred operation instructions have been put down in writing each method of claim 1-16).

21, kit described in the claim 20, wherein solid phase carrier is the porous reaction plate, is fixed with the first anti-HBV PreS1 antibody and the first anti-HBc antibody in the hole of porous reaction plate.

22, solid phase carrier is fixed with anti-HBV PreS1 antibody and anti-HBc antibody on it; Preferred solid phase carrier is porous reaction plate (for example ELISA Plate, a chemiluminescence plate), is fixed with anti-HBV PreS1 antibody and anti-HBc antibody in the hole of porous reaction plate.

23, the reagent of joint investigating hepatitis B virus pre S 1 antigen and cAg is used for checking the purposes of hepatitis b virus infected kit in preparation; Preferably, described reagent is the reagent of claim 18-22 described in each.

24, adopt double antibody sandwich method to detect the method for hepatitis B virus pre S 1 antigen, it is characterized in that two kinds of used antibody (capture antibody and report antibody) all are the HBV PreS1 antibody with the hepatitis B virus pre S 1 antigen specific bond.

25, the method for claim 24 does not wherein need sample is carried out pre-service.

26, each method of claim 24-25, wherein being captured in the same reaction container (reacting hole) of cracking of hepatitis type B virus and pre S 1 antigen carried out in same media simultaneously.

27, each method of claim 24-26 wherein uses the lysate described in the claim 33 as described medium.

28, each method of claim 24-27, it comprises

A) provide can with the first anti-HBV PreS1 antibody and the second anti-HBV PreS1 antibody (two kinds of antibody can be identical or different) of hepatitis B virus pre S 1 antigen specific bond;

C) testing sample and the-anti-HBV PreS1 antibody-carrier conjugates is contacted, make if present, to be hunted down hepatitis B virus pre S 1 antigen in the sample, form the anti-HBV PreS1 of antigen-first antibody-carrier conjugates;

E) detect the amount of the second combined anti-HBV PreS1 antibody.

29, the method for claim 28, wherein the step e) amount that detects combined second antibody is carried out by the following method: provide and can produce body with the effective signal that combines of the described second anti-HBV PreS1 antibody, it is only relevant with the amount of the second anti-HBVPreS1 antibody that this signal produces signal intensity that body produced; Signal is produced body to be contacted with second antibody; Detection produces the signal that body produces by signal.

30, the method for claim 28, wherein the step e) amount that detects combined second antibody is carried out by the following method: second antibody directly is marked with signal and produces body, detects by signal and produces the signal that body produces.

31, each method of claim 24-30, wherein to produce body be enzyme, fluorescein, chemiluminescent substance, isotope, rare earth element to signal.

32, each described method of claim 24-31, the detection of wherein said hepatitis B virus pre S 1 antigen realizes by one or several methods of selecting from following method: enzyme linked immunosorbent assay, immunity percolation method, immunochromatographic method, chemoluminescence method, time resolution method; Preferred enzyme linked immunosorbent assay or chemoluminescence method.

33, employing virus cracking liquid (this employing virus cracking liquid can be used for detecting pre S 1 antigen), it contains surfactant and suitable buffering agent, described surfactant is selected from: Chaps, sulfobetain 8-18, Bu Lijie 35, TritonX100, Tween series and/or HP-β-CD, be preferably selected from SB8, SB12, SB14, SB16, SB18, HP-β-CD, TWEEN20, TWEEN40, TWEEN60, TWEEN80, BRJ35, more preferably SB12, SB14, SB16, SB18, TWEEN80;

Preferably, surfactant can be the combination of one or more (2,3,4,5 kind) in the above surfactant, preferred SB16+SB14, SB18+SB14; Preferably, the surfactant concentrations scope can be SB14%:1%-10%; SB16:0.5%-1%; SB18:0.5%-1%; More preferably: SB14:2%-8%; SB16:1%; SB18:1%; As: 1%SB14+0.5%SB16 or 1%SB 14+0.5%SB18,2%SB14,1%SB16+0.5%SB14,1%SB18+0.5%SB14,2%SB14;

Preferably, surfactant is selected from the A-P lysate shown in the table 2, more preferably C-M lysate and P lysate, more preferably E, F, G, H, J lysate, most preferably G lysate, H lysate, the perhaps surfactant described in the ε lysate shown in the table 6, ζ lysate, η lysate, the θ lysate;

Preferably, buffering agent is selected from citrate buffer, phosphate buffer, Tris damping fluid, buffered with amino acid liquid, acetate buffer, HEPES damping fluid, carbonate buffer solution; Buffering agent can be the combination of one or several (2,3,4,5 kind) in the above-mentioned damping fluid; Buffering agent optimization citric acid salt buffer, phosphate buffer; The pH scope and the concentration range of buffering agent can be phosphate buffer: the preferred 20mM pH of 10-50mM pH value 6.0-7.0 value 7.4; Citrate buffer: the preferred 10mM pH of 10-50mM pH value 3.0-6.6 value 6.6; Tris damping fluid: the preferred 20mM pH of 10-50mM pH value 7.0-9.0 value 7.0; Acetate buffer: the preferred 10mM pH of 10-50mM pH value 3.8-6.0 value 6.0;

Not necessarily, lysate can also contain one or more (as 1,2,3,4,5,6-10 kind) other compositions, for example (salt for example is as NaCl for reductive agent (for example DTT, DTE, beta-mercaptoethanol), sequestrant (for example EDTA), osmotic pressure instrumentality; Sucrose), the preferred 0.1%-0.5% of beta-mercaptoethanol: 0.01%-1% for example, more preferably 0.2%; The preferred 5-10mM of DTT:1-30mM, more preferably 5mM; The preferred 5-10mM of DTE:1-30mM, more preferably 5mM; The preferred 5mM of EDTA:5mM-20mM; )

34, be used to detect the kit of hepatitis B virus pre S 1 antigen, it comprises the reagent that is used for detecting according to each described method of claim 24-32 hepatitis B virus pre S 1 antigen.

35, the kit of claim 34, its comprise can with the first anti-HBV PreS1 antibody of hepatitis B virus pre S 1 antigen specific bond and the lysate of second anti-HBV PreS1 antibody (two kinds of antibody can be identical or different, can be monoclonal antibody or polyclonal antibody) and/or claim 33; Preferably also contain the reagent (for example detecting the detectable of second antibody) that is suitable for detecting described antigen-antibody reaction in the described kit; At least a anti-HBV PreS1 antibody is produced body tag by signal in the preferably described antibody, can produce perhaps that body is effective to be combined with signal; Described antibody is monoclonal antibody preferably; Preferably anti-HBV PreS1 antibody is selected from: 3H5,7H11,2A7,4D11,6F1,13G2 and 16F5, particularly 7H11 and 4D11.

36, claim 34-35 kit described in each, wherein comprise: (1) solid phase carrier is fixed with the first anti-HBV PreS1 antibody on the described solid phase carrier; And/or the lysate of (2) claim 33; (3) dispensable by signal generation second anti-HBV PreS1 antibody body tag or that be not labeled; (4) detectable of dispensable detection second antibody; (5) dispensable damping fluid, cleansing solution, operation instructions (preferred operation instructions have been put down in writing each method of claim 24-32).

37, claim 34-36 kit described in each, wherein solid phase carrier is porous reaction plate (for example ELISA Plate, a chemiluminescence plate), is fixed with anti-HBV PreS1 antibody in the hole of porous reaction plate.

38, the reagent of detection hepatitis B virus pre S 1 antigen is used for checking the purposes of hepatitis b virus infected kit in preparation; Preferably, described reagent is the reagent of claim 34-37 described in each.

39, the method for lytic virus is included in not lytic virus under the temperate condition of denatured protein in fact (for example not deactivation antibody, for example pre S 1 antigen or cAg antibody) in fact, preferably adopts the lysate lytic virus of claim 17 or 33; Described virus is hepatitis type B virus preferably.

40, detect the method for hepatitis B virus pre S 1 antigen or cAg, comprise: at denatured protein (deactivation antibody in fact not for example in fact not, for example pre S 1 antigen or cAg antibody) temperate condition under lytic virus, preferably adopt the lysate lytic virus of claim 17 or 33; Detect hepatitis B virus pre S 1 antigen or cAg.