CN101598730B - In vitro diagnosis kit of hepatitis B virus E antibody through dual-antigen sandwich method and preparation method thereof - Google Patents
In vitro diagnosis kit of hepatitis B virus E antibody through dual-antigen sandwich method and preparation method thereof Download PDFInfo
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Abstract
The invention provides an in vitro diagnosis kit (an ELISA method) of a hepatitis B virus E antibody through a dual-antigen sandwich method, which comprises the following components: 1) a recombinant HBeAg coated in a solid phase carrier; 2) an enzyme-labeled recombinant HBeAg; and 3) a substrate solution, for example, a chromogenic substrate solution applied to the ELISA, or an enzymatic chemiluminescent substrate solution. Besides, the invention provides a method for preparing the kit, which comprises the following steps: 1) coating the recombinant HBeAg into the solid phase carrier; 2) performing enzyme labeling on the recombinant HBeAg; 3) preparing the substrate solution; and 4) assembling the components into a finished kit. The kit has the advantages of high sensitivity, good specificity, accurate result and strong clinical applicability, and can accurately reflect the amount of the E antibody in the body of a hepatitis B patient.
Description
Technical field
The present invention relates to immune field of medical analysis, particularly, the present invention relates to hepatitis B virus E antibody external diagnosis reagent case and preparation method.
Background technology
Hepatitis type B virus (Hepatitis B Virus) belongs to has a liking for liver venereal disease poison family, can cause oxyhepatitis, chronic hepatitis and cirrhosis, and closely related with the generation of liver cancer.It is to infect in the world at present one of deadly virus the most widely.According to statistics, the whole world has the crowd over 3,500,000,000 to be the HBVer or to send out patient [Zucherman J.N., et al., J.Infect, 2000,41 (2): 130～136].China is district occurred frequently and the Endemic Area of hepatitis B, and 1.2 hundred million hepatitis B patients [Zhang Jianying chief editor, viral liver disease progress, first published, China Science Tech Publishing House, 1992] are approximately arranged.And, 1,000,000 people are approximately arranged in the world because infecting HBV dead [Parkin D.M., etal., Cancer Surv, 1999,33:5～33] every year.There is no at present and cure the effective ways that Chronic Hepatitis B Virus infects, so vaccine inoculation and early diagnosis be the important means of prevention, to the detection of dynamic of Viral infection also can develop for hepatitis B, treatment and prognosis provide important information.
Mainly contain three kinds of antigens such as hepatitis b virus s antigen (HBsAg), hepatitis B virus core antigen (HBcAg) and hepatitis B virus E antigen (HBeAg) [Zhang Jianying chief editor by the expressed albumen of hepatitis B, the viral liver disease progress, first published, China Science Tech Publishing House, 1992].Be the opposing hepatitis B, the human immune system understands corresponding generation for the antibody of these three kinds of antigens, and they are respectively hepatitis B surface antibody, hepatitis B core antibody and hepatitis B E antibody, and is secreted into (blood is that main antibody exists the place) in body fluid.Because the cAg of hepatitis B can't be secreted in blood, so when traditional hepatitis B virus infection examination immunology detection, only detect hepatitis B virus surface antigen, hepatitis B surface antibody, hepatitis B E antigen, totally five of hepatitis B E antibody and hepatitis B core antibodies etc. are called in the industry " Hepatitis B virus ".
The genome of hepatitis B is that one small and exquisite and only there is 3.2kb in efficient system, only has 4 main open reading frames, but but bearing nearly ten kinds of protein expression tasks [GalibertF., Mandrat E., et al., Nature, 1979,281:646～650; Mandrat E., Kay A., J.Virol., 1984,49:782～792].Its genome generally is divided into: the parts such as front S district, S district, CORE district (pre-c/c) and corresponding promoter, wherein CORE is responsible in district two kinds of albumen of expression: hepatitis B virus core albumen and hepatitis B E albumen.That is to say, HBeAg and HBcAg share genomic same section.More and more studies have shown that, HBeAg is the product of HBcAg after proteases for decomposing.The molecular weight of HBcAg is 21000 dalton, and the molecular weight of HBeAg is 14000 dalton.[Ou J.H.，O.Laub，et al.，Proc.Natl.Acad.Sci.USA，83：1578～1586；Takahashi，K.，et al.，J.Immunol.，1983，130：2903～2907；Nassal，M.，and Schaller，H.，Trends Microbiol.，1993，1：221～228；Psaek M.，et al.，NAture，1979，282：575～579；Murray K.，Proc.R.Soc.London B，1987，230：107～146；Strandring D.H.，et al.，Proc Natl.Acad.Sci.USA，1988，85：8405～8409；]
Hepatitis B E antibody (anti-HBe) is the antibody of specificity anti-hepatitis virus E antigen (HBeAg).It is generally acknowledged, the appearance of anti-HBe is the sign that hbv replication weakens or stops in patient body, indicating patient's self-healing preferably or result for the treatment of [will Fang Junfu, clinical patient is learned, 1978,8 (1); Wang Ying, medical test education magazine, 1997,4 (11): 167; Tang Qingbin, Hengyang Medical College's journal, 1996,24 (4): 314～315; Ren Jianlin etc., Xian Medical Univ's journal, 1999,20 (2): 226～228; Wu Shuhuan, Wang Lixing, Hepatitis B virus explored secrets (three)].Therefore the detection of anti-HBe is during " Hepatitis B virus " detects important one, is the important evidence that the clinician judges the patient treatment effect.
the detection method of " hepatitis B E antibody " has a variety of, from initial Ago-Gel immune double diffusion method (AGD), counter immunoelectrophoresis (CIEP), complement fixation test (CFT) (CF), passive hemagglutination assay (PHA), reversed passive hemagglutination test (RPHA), lower method [the Liu Xiguang of hemagglutination test (IAHA) isosensitivity is sticked in immunity, the chief editors such as Hu Zonghan, the virus hepatitis laboratory diagnosis, first published, the People's Health Publisher, 1986], the radiommunoassay based on immune absorption principle (IA) finally (RIA), enzyme linked immunosorbent assay (ELISA) (ELISA), gold test strip is measured (GIA), the method of the high sensitives such as chemiluminescent immunoassay(CLIA) (CLIA) and time-resolved fluoroimmunoassay determining adsorption (TRFIA).Present routine clinical assay method all is based on the method for IA, and is more complicated and the radioactivity risk is arranged due to the use of RIA method, basically is eliminated.The ELISA method because of easy to use and low price, is to use maximum methods; The gold test strip method is easy-to-use and quick due to it, and its market use amount is increasing; Chemiluminescent immunoassay and time-resolved fluorescent immunoassay are higher and be easy to robotization and use because of its susceptibility, also have been subject to clinical welcome.
According to the principle of immunity absorption (IA) method, be divided into again the many measure methods such as sandwich method, indirect method, competition law (in comprising and competition law) and prize law because of the relation of test event and raw materials quality.In these all assay methods, what pattern was best is sandwich method.And competition law is the poorest pattern.[Shen Caixia, Medical Immunology, first published, Shanghai Tongji University publishing house, 1987:177; Yang Liguo etc. write, enzyme immunoassay technique, first published, publishing house of Nanjing University, 1998] advantage with competition law in is: not high with the purity requirement of HBeAg to neutralization, interfering material in antigen-like material is difficult for removing, or when being difficult to obtain enough purifying antigens, available this method detection specificity antibody.But its major defect is also clearly: sensitivity is low, poor specificity, and the large and result inconvenience interpretation of differences between batches, and result is inaccurate etc.These shortcomings cause the problems in clinical use.Such as: because sensitivity is low, produced hepatitis B E antibody in patient body, in and the competition law kit but do not measure, this provide correctly information timely just can not for the doctor when judgement result for the treatment of and formulation successive treatment scheme; Due to poor specificity, often have false-positive result, thereby cause Positive rate higher than actual positive rate [ChengBing Li, the Research on Problems of anti--HBe detection method, 302 hospital's epi chambers]; Because the CUTOFF value that the interpretation as a result of the method is used can arbitrarily be adjusted by the technician, cause the official written reply difference of product large, and different manufacturers product measured result is also inconsistent.Equally, in and competition law also measured the impact of operating habit, when sample adds the mistiming that adds with labelled antibody inconsistent, due to the hepatitis B E antibody in sample and the hepatitis B E antibody in mark in and the inequality competition of antigen, cause result inconsistent [Xu Weiping, the Chen Xiaoai of same sample in homogeneous is not measured, Li Xin, laboratory medicine and clinical, 2007,4 (10): 960; ]; Due to the inconvenient interpretation of result, result is inaccurate, and it is negative or positive causing reviewer's a sample that can't intuitively judge rightly, and detects index and comes auxiliary judgment and must seek other.All above shortcomings allow this detection kit lose the basic function that detects.
Dual-antigen sandwich method is surveyed hepatitis B E antibody but can overcome these problems.The one, sandwich method highly sensitive can detect the antibody that occurs in blood in advance than competition law; The 2nd, the antibody in sample and labelled antigen are not competed envelope antigen, thereby it is inconsistent the results that cause because the application of sample time difference is different can not occur; The 3rd, the CUTOFF value of interpretation as a result can't arbitrarily change because of the artificial origin, has guaranteed that different batches product or different manufacturers product are to the comparability of same pattern detection result.The 4th, the color difference of yin and yang attribute is obvious, almost can rely on range estimation to come interpretation yin and yang attribute result.
At present, clinically the mensuration of hepatitis B E antibody adopt be in and competition law.From analysis principle, the detection of hepatitis B E antibody equally also can be used dual-antigen sandwich method.But, since the hepatitis B E antibody detection kit is born by now, in but adopting and the very poor method of this pattern of competition law always, the quality of this and a kind of most important raw material hepatitis B E antigen used is relevant, and [Huang is helped, gold-tinted China and Chen Jiangping, shanghai Medicine, 1997,20 (2): 100～102; Zhao Xiangsheng and Liu Yu etc., Chinese traditional Chinese medicine magazine, 2000,24 (2): 110～111; Liang Baoduo, Ceng Zhen, Zhao Xiangsheng, Chinese journal of medical examination, 1985,18 (4): 232; ].
In the 1970s and 1980s in 20th century, " Hepatitis B virus " kit is developed successfully, and is applied to the clinical examination use.At that time, the Antigens raw material that is used for these kit productions was all that blood or the liver from hepatitis B patient proposes, and the source is rare, and quality is unstable.Along with the demand to " Hepatitis B virus " kit clinically significantly increases, the source problem that comes of these Antigens raw materials becomes the key of restriction kit output and cost.Along with the development of biotechnology, the production that the genetic engineering recombinant technique is applied to these antigens comes up, and has successfully built HBeAg and the HBcAg[Zhu Qiu duckweed of restructuring, Gu Shuyan, Chinese J.Exp.Clin.Virol., 1998,12 (3): 276～278; Scape is new, Zhu Jiming, viral journal, 1989,5 (3): 201～210; Edman J.C., et al.Nature, 1981,291:503～506; Ma Xiankai etc., Shanghai Journal of Immunology, 1984,4 (2): 129～130].But, researchist's discovery, the quality of the HBeAg of restructuring can not show a candle to the quality of HBcAg, and has found reason.Because the homology of HBeAg and HBcAg, and HBeAg is the part of HBcAg, use the HBeAg of Bacillus coli expression, because lack the relevant proteinase that the human body inner virus can be expressed, can't decompose accurately folding with three-dimensional structure to HBcAg, this has just caused and has always existed the active component of HBcAg in HBeAg, and HBeAg is also unstable.[David R.Milich, etal, J.Immunol., 1988,141 (10): 3617～3624; FlorianSchodel, et al, J.Biolog.Chem.1993,268 (2): 1332～1337; Paul T.Wingfield, Biochem., 1995,34:4919～4932; ] for these reasons, the researchist finds, HBeAg is difficult to realize the mark of conventional method, also is difficult to get rid of the activity of the HBcAg in preparation antigen coated.So, just can't prepare the hepatitis B E antibody diagnostic kit with the double antigens sandwich law technology, thus have to have to take the second best adopted in and competition law.
The coupling of high molecular weight protein and enzyme, along with the progress of technology with according to the characteristics of albumen used and enzyme, a lot of methods have been developed, as: the chemical coupling method of glutaraldehyde method, sodium periodate method, 1,4-benzoquinone method, adjacent phenylenedimaleimide method etc. and immunology coupling method [Molin S.O.et al., J.Histochem.Cytochem., 1978,26 (12): 1053～1059; Nagren H., J.Histochem.Cytochem.1974,22 (1): 1084～1093; Termumck T.and Avrameas S., Immunochem., 1977,14:767～773; Mason D.Y., et al.Immunocytochem.Bristol., Wright PSG, 1983,113～118].The physicochemical property of protein show: after two kinds of protein molecular generation couplings, the space conformation of protein molecular, intermolecular Van der Waals force and electrostatic force all can exert an influence to two molecules after coupling.Practice for many years thinks, during to the enzyme that contains glycosyl and large molecule coupling, the periodic acid method be effect better and the better simply method of technique, and be widely adopted [P.Tijssen and E.Kurstak, Anal.Biochem., 1984,136:451～457; Antoine Cuvelier et al., J.Immunol.; 1996,17 (4): 371～382].But the shortcoming of sodium periodate method is also significantly, and it needs the amino combination of enzyme and coupling protein, and be randomly with albumen on any amino coupled.Even the enzyme molecule also can coupling from amino with it.For HBeAg, molecular weight, and also antigenic determinant also has two, so the space structure of its molecule is huge on the activity performance impact of HBeAg.When adopting the sodium periodate method with HBeAg and enzyme coupling, its probability that causes the amino in the determinant scope of enzyme and HBeAg to be combined with enzyme is larger.After HBeAg is combined, on the one hand, when enzyme is combined with the amino on the HBeAg antigenic determinant, can cause the inactivation of this antigenic determinant when the enzyme molecule; On the other hand, if HBeAg and the coupling of more than one enzyme molecule are more much bigger than HBeAg because the molecular weight of enzyme is 43000 dalton, thereby produce sterically hinderedly, cause most of inactivation of HBeAg.Therefore, if adopt inappropriate coupling mode to make HBeAg and enzyme coupling, will cause the inactivation of HBeAg, apparent upper reflection seemingly HBeAg can't with the enzyme coupling.
In addition, owing to being mixed with the activity of HBcAg in prepared now restructuring HBeAg, after enzyme molecule and HBeAg coupling, the activity of HBcAg still exists, it still can produce immune response with the hepatitis B core antibody that is adsorbed onto on solid phase, thereby causes the Sample producing cross reaction to the hepatitis B core antibody positive.
Summary of the invention
The invention solves the mark of HBeAg and HBcAg activity to the difficult problem that affects of kit, thereby prepare the hepatitis B E antibody external diagnosis reagent case based on the dual-antigen sandwich method principle.
We find through consulting a lot of documents: find to have 2 free cysteine residues (Cys) on the HBeAg molecule, one of them is hidden in three-dimensional structure, only has an exposed outside [Michael Nassal and Andrea Rieger of Cys, J.Virol., 1993,67 (7) 4307～4315; Birnbaum, F., et al., J.Virol., 1990,64:3319～3330; Gallina, A., et al., J.Virol., 1989,63:4645～4652; Mark A.Baumeister; Et al., J.Med.Virol., 2000,60:256～263].According to maleimide ratio juris [Kato K., et al., J.Biochem., 1975,78 (1): 423～429; Yoshitake S., et al., J.Biochem., 1981,29 (12): 1387～1392; Kitagawa T.and Aikawa T., J.Biochem., 1976,79 (1): 233～239; Shechter T., et al., Proc.Natl.Acad.Sci., 1978,75:2135～2140; ], we develop MBS (abbreviation of m-maleimidobenzoyl-N-hydroxysuccinimide eater) method, realize the coupling of a HBeAg and an enzyme, also weaken as much as possible space steric effect simultaneously.
And we adopt affinity purification method to remove the coupling molecule that the HBcAg activity is wherein arranged after the coupling that has realized HBeAg and enzyme molecule, eliminating as much as possible in the future the cross reaction to hepatitis B core antibody, thereby improve the specificity of kit.
So the present invention is protein coupling technology and the effective combination of IA method, provide a kind of highly sensitive, specificity good, interpretation as a result accurately, production and hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent case easy to use.This kit is suitable substitute existing in and the kit of competition law, have clinically extensive use.
Hepatitis B E antibody external diagnosis reagent case based on dual-antigen sandwich method provided by the invention comprises: the restructuring HBeAg that 1) has been coated in solid phase carrier; 2) carry out the restructuring HBeAg of enzyme labeling; 3) substrate solution for example is applied to the chromogenic substrate liquid of ELISA, or is applied to the enzyme-catalyzed chemical luminescence substrate liquid of CLIA.Described kit can be applied to enzyme linked immunosorbent assay (ELISA) and enzyme-catalyzed chemical luminescence method (CLIA) etc. based on the method for Enzyme immunoassay.
In kit according to the present invention, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.The microwell plate that wherein is used for ELISA is transparent, and the microwell plate that is used for CLIA is opaque.
In kit according to the present invention, described enzyme is horseradish peroxidase or alkaline phosphatase.
In kit according to the present invention, the chromogenic substrate liquid that is applied to ELISA comprises:
Nitrite ion A: citric acid 0.86g, trisodium citrate 1.735g is dissolved in deionized water, is settled to 100ml, adds H
2O
250 μ L mixings;
Nitrite ion B: 1. citric acid 0.8256g, trisodium citrate 1.6656g and sucrose 6g are dissolved in deionized water, are settled to 96ml, and 2. tetramethyl benzidine (TMB) 0.026g is dissolved in 4ml DMSO, will be 1. and 2. mixing.
In the mentioned reagent box, preferably, described TMB is 3,3,5,5-tetramethyl benzidine, 3,3,5,5-tetramethyl benzidine sulfate or 3,3,5,5-tetramethyl biphenyl amine hydrochlorate.
In kit according to the present invention, the enzyme-catalyzed chemical luminescence substrate liquid that is applied to CLIA comprises:
Reactant liquor A:12.1g Tris, the 2.95ml concentrated hydrochloric acid, the 5g luminol, 1g phenylpropyl alcohol fluoranthene, 0.5ml Tween20, constant volume is to 1000ml.
Reactant liquor B:7.3g trisodium citrate, the 4.4g citric acid, the hydrogen peroxide of 1.2ml 30%, constant volume is to 1000ml.
In addition, the invention provides the method for preparing kit of the present invention, it comprises:
1) HBeAg that will recombinate is coated in solid phase carrier;
2) HBeAg that will recombinate carries out enzyme labeling;
3) preparation of substrate solution;
4) be assembled into the finished product kit.
According to the present invention, preferably, described step 1) HBeAg that will recombinate is coated in solid phase carrier and comprises the following steps:
A) coated
Adopt carbonate buffer solution (1.6g sodium carbonate and 2.9g sodium bicarbonate are dissolved in 1000ml distilled water) the dilution C-HBeAg of pH9.650mM to debita spissitudo, be coated on solid phase carrier;
B) sealing
Can first wash with physiological saline for some solid phase carriers.The preparation confining liquid uses 0.2gNaH
2PO
42H
2O, 2.9g Na
2HPO
412H
2O, 20g caseinhydrolysate and 0.5ml Tween-20 are dissolved in 1000ml distilled water.The pH value of described confining liquid is 7.2 left and right.Then this confining liquid is carried on solid phase carrier;
In said method, preferably, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
According to the present invention, preferably, described step 2) HBeAg that will recombinate carries out enzyme labeling and adopts the MBS method, comprises the following steps:
A) 8mg horseradish peroxidase (HRP) is dissolved in PB (pH7.0) 1ml of 0.1M;
B) with 0.1ml dimethylformamide dissolving 8mg MBS, join in HRP solution room temperature reaction 1 hour;
C) after centrifugal removal precipitation, remove excessive MBS with the G25 gel filtration chromatography, damping fluid is pH5.0 0.05M acetate buffer;
D) the enzyme liquid that will process is concentrated;
E) 10mg HBeAg monomer is joined in enzyme liquid room temperature reaction 1 hour;
F) reactant liquor is crossed gel chromatography column, conjugate is separated with conjugate not, collect conjugate;
G) coupling liquid is crossed coupling monoclonal anti-HBc antibody sepharose-4B affinity column, collected and pass the peak.
H) will pass the peak concentrated, add equivalent glycerine, freezing preservation after being concentrated to proper volume.
In said method, preferably, described enzyme is horseradish peroxidase or alkaline phosphatase.
According to the present invention, preferably, described step 3) be the chromogenic substrate liquid that preparation is applied to ELISA, comprise the following steps:
A) preparation of nitrite ion A (100ml, PH 4.7):
Citric acid 0.86g and trisodium citrate 1.735g are dissolved in deionized water, are settled to 100ml, add H
2O
250 μ L mixings;
B) preparation of nitrite ion B (100ml, PH 4.7):
1. citric acid 0.8256g, trisodium citrate 1.6656g and sucrose 6g are dissolved in deionized water, are settled to 96ml, and 2. TMB 0.026g is dissolved in 4ml DMSO, will be 1. and 2. mixing;
Preferably, described TMB is 3,3,5,5-tetramethyl benzidine, 3,3,5,5-tetramethyl benzidine sulfate or 3,3,5,5-tetramethyl biphenyl amine hydrochlorate.It is more preferably 3,3,5,5-tetramethyl benzidine.
According to the present invention, preferably, described step 3) be the enzyme-catalyzed chemical luminescence substrate liquid that preparation is applied to CLIA, comprise the following steps:
A) preparation of reactant liquor A:
12.1g Tris, the 2.95ml concentrated hydrochloric acid, the 5g luminol, 1g phenylpropyl alcohol fluoranthene, 0.5mlTween20, constant volume is to 1000ml.
B) preparation of reactant liquor B:
7.3g trisodium citrate, the 4.4g citric acid, the hydrogen peroxide of 1.2ml 30%, constant volume is to 1000ml.
Particularly, the mentioned reagent box can comprise microwell plate, enzyme labeling HBeAg, nitrite ion substrate solution or enzyme-catalyzed chemical luminescence liquid, stop buffer, the washing lotion of coated HBeAg.The microwell plate of coated HBeAg is the micropore lath in 24,48 or 96 holes; The enzyme of mark HBeAg is horseradish peroxidase, and labeling method is the MBS method; The substrate of nitrite ion B is TMB, or the luminous agent of enzyme-catalyzed chemical luminescence liquid A is luminol and derivant and benzofluoranthrene; Washing lotion is Tris-HCl.
In research process of the present invention, at first the present inventor has carried out screening experiment and Quality Identification to starting material used, comprises that coated purity with HBeAg and active, mark are with the RZ value of the adsorptive power of the purity of HBeAg and activity, solid phase carrier (as water white microwell plate) and the coefficient of variation, horseradish peroxidase and enzymatic activity etc.Then the method for coating of HBeAg is studied, tests with the concentration of different HBeAg, different coated damping fluids, different coated time and temperature, different sealings.Select optimal coated damping fluid, confining liquid, coated temperature and time, and the protein concentration of most suitable HBeAg.For HBeAg monomer and HRP coupling method, tried out multiple coupling method, finally determined the MBS method; To the method again through repeatedly exploring and the contrast experiment has determined that finally productive rate is high, cost is low, removed the interference of HBcAg activity and the tagging scheme of reliable in quality; For this kind of enzyme mark HBeAg, also grope the formula of most suitable enzyme labeling dilution simultaneously, this enzyme labeling thing and dilution have been carried out long-term investigation test, made the dilution that can make enzyme labeling HBeAg keep for a long time active and thermal stability.At last, determined the optimal proportions of enzyme labeling HBeAg and immobilised HBeAg by chessboard square formation titration experiments repeatedly.
Utilize kit of the present invention to test, highly sensitive, specificity good, result is accurate, clinical applicability is strong, can reflect exactly the amount of E antibody in the hepatitis B patient body.Having or not or quantity of the E antibody that goes out according to this kit measurement, can reflect accurately whether in vivo active situation of the present hepatitis type B virus of determined patient, the therapeutic scheme that adopts have played the effect of virus and the prognosis situation that is in convalescent patient of suppressing.
And detection system of the present invention is enzyme linked immunosorbent assay, and most of hospitals all have this determination techniques and platform at home.And the usage comparison of this method is extensive, to environment require lowly, can carry out in general check laboratory.What is more important, in and the hepatitis B E antibody diagnostic reagent of competition law used at home more than 20 year, all application units that carried out this project can directly use this reagent, need not to increase any new condition.
Embodiment
The preferred embodiments of the invention are described below, comprise the best mode of the present invention of realizing known for inventor.Should be appreciated that these embodiments are only exemplary, should not be used for limiting the scope of the invention.
Embodiment 1 preparation hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent case of the present invention (ELISA method)
One, the preparation of microwell plate
(1) coated
0.05M pH be 9.6 carbonate buffer solution for coated dilution, mix back loading on microwell plate with HBeAg.
Particularly, described method for coating comprises:
Na
2CO
3 1.6g
NaHCO
3 2.9g
Distilled water 1000ml
After damping fluid dissolves mixing fully, add the HBeAg mixing of 2mg, then add the dilution 0.1ml of HBeAg to each hole of microwell plate.Placed 12 hours for 37 ℃.
(2) wash plate
Use physiological saline to be cleansing solution.After HBeAg dilution in microwell plate is got rid of, with physiological saline washing 2 times.Drain at last microwell plate.
(3) sealing
0.01M pH is 7.2 phosphate buffer (PB), contains the solution of caseinhydrolysate and surfactant, loads on microwell plate.
Particularly, described enclosure method comprises:
NaH
2PO
4·2H
2O 0.2g
Na
2HPO
4·12H
2O 2.9g
Caseinhydrolysate 20g
Tween-20 0.5ml
Distilled water 1000ml
After they were mixed to dissolving fully, each hole on microwell plate added 0.11ml, in 37 ℃ of placements 2 hours, afterwards, the solution in the hole is got rid of, and drains, dries.
(4) packing
The microwell plate that will dry is packed in Fresco Bag, after adding an anti-blushing agent, encapsulates with vacuum packing machine.Putting into 4-8 ℃ after labeling deposits.
Two, the preparation of enzyme-labelled antigen
(1) enzyme labeling of HBeAg
The mark of HBeAg adopts the MBS method.The enzyme that the method is used is horseradish peroxidase (HRP).Particularly, described method comprises:
A) 8mg HRP is dissolved in PB (pH7.0) 1ml of 0.1M;
B) with 0.1ml dimethylformamide dissolving 8mg MBS, join in HRP solution room temperature reaction 1 hour;
C) after centrifugal removal precipitation, remove excessive MBS with the G25 gel filtration chromatography, damping fluid is pH5.0 0.05M acetate buffer;
D) the enzyme liquid that will process is concentrated;
E) the 10mgHBeAg monomer is joined in enzyme liquid room temperature reaction 1 hour;
F) reactant liquor is crossed gel chromatography column, conjugate is separated with conjugate not, collect conjugate;
G) coupling liquid is crossed coupling monoclonal hepatitis B E antibody sepharose-4B affinity column, collected and pass the peak.
H) will pass the peak concentrated, add equivalent glycerine, freezing preservation after being concentrated to proper volume.
(2) preparation of enzyme dilution
0.01M the phosphate buffer of pH7.2 contains the solution of calf serum, antiseptic and surfactant.
Particularly, described compound method comprises:
NaH
2PO
4·2H
2O 0.2g
Na
2HPO4·12H
2O 2.9g
Calf serum 200ml
Tween-20 0.5ml
Proclin300 0.1ml
Distilled water 800ml
They are mixed to dissolving fully.
(3) dilution of enzyme labeling HBeAg
Get 2ml enzyme labeling HBeAg, join the enzyme dilution of 50ml, the concussion mixing.And then it is joined in the enzyme dilution of 950ml, convolution was rocked reagent bottle more than 20 minutes.After measuring mixing, taking sample determination, qualified after, put into 4-8 ℃ after labeling and deposit.
(4) packing
According to the standard of each Packaging Bottle 6ml, accurately diluting good enzyme labeling HBeAg packing.Putting into 4-8 ℃ after labeling deposits.
Three, the preparation of nitrite ion
It is substrate that this method adopts TMB.Nitrite ion is divided into A liquid and the preparation of B liquid, deposits respectively, and equal proportion is mixed in use.Concrete grammar comprises:
1) preparation of nitrite ion A (100ml, PH 4.7):
Citric acid 0.86g
Trisodium citrate 1.735g
H
2O
2 50μL
Distilled water 100ml
They are mixed to dissolving fully.
2) preparation of nitrite ion B (100ml, PH 4.7):
Citric acid 0.8256g
Trisodium citrate 1.6656g
Sucrose 6g
Distilled water 96ml
TMB 0.026g
DMSO 4ml。
They are mixed to dissolving fully.
3) get nitrite ion A and the B sample is measured, rear packing conforms to quality requirements.Standard packing according to each Packaging Bottle 6ml.After packing was completed, labeling was put into the lucifuge 4-8 of place ℃ and deposits.
Four, the preparation of stop buffer
Stop buffer is 2M sulfuric acid.Method comprises particularly:
Concentrated sulphuric acid 200ml
Distilled water 800ml
After solution mixes, packing.Standard packing according to each Packaging Bottle 6ml.After packing was completed, labeling was deposited in room temperature.
Five, the preparation of cleansing solution
Tris 24g
NaCl 160g
KCl 4g
HCl 15ml
Distilled water 1000ml
After solution mixes, measure the pH value, should be 7.4.Qualified rear packing.Standard packing according to each Packaging Bottle 20ml.After packing was completed, labeling was deposited in room temperature.
Six, the assembling of semi-manufacture and finished product
The packing of above-mentioned steps products obtained therefrom is semi-manufacture.Extract three parts out through accuracy, specificity, sensitivity and Detection of Stability is qualified just can be assembled into kit.Be assembled into and also need inspect qualified just can dispatch from the factory afterwards after kit by random samples.
Embodiment 2～4 preparation hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent cases of the present invention (ELISA method)
Except respectively take plastic bead, plastic tube or magnetic-particle as carrier, all the other all prepare the hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent case with the method identical with embodiment 1.
Embodiment 5 preparation hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent cases of the present invention (ELISA method)
Be outside the marker enzyme of HBeAg divided by alkaline phosphatase, all the other all prepare the hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent case with the method identical with embodiment 1.
Embodiment 6 preparation hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent cases of the present invention (CLIA method)
Divided by chemiluminescence reaction liquid replace ELISA in sending out chromogenic substrate liquid and do not have outside stop buffer, all the other all prepare the hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent case with the method identical with embodiment 1
The compound method of chemiluminescence reaction liquid:
This reactant liquor is take luminol and benzofluoranthrene as shiner, is divided into A and B liquid is prepared respectively, separately deposits, and the ratio with 1: 1 before using mixes rear use.Concrete grammar comprises:
1) preparation of reactant liquor A (100ml pH8.5):
Tris 1.21g
Concentrated hydrochloric acid 0.295ml
Luminol 0.5g
Benzofluoranthrene 0.1g
Tween20 0.5ml
Deionized water 100ml
2) preparation of reactant liquor B (100ml pH4.6)
Trisodium citrate 0.73g
Citric acid 0.44g
30% hydrogen peroxide 0.12ml
Deionized water 100ml
3) get nitrite ion A and the B sample is measured, rear packing conforms to quality requirements.Standard packing according to each Packaging Bottle 6ml.After packing was completed, labeling was put into the lucifuge 4-8 of place ℃ and deposits.
The using method 1 of embodiment 7 kits of the present invention
The present embodiment uses the hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent case (ELISA method) of embodiment 1 preparation.
1) application of sample:
Pre-packet response plate (bar), every plate is established positive and negative and is contrasted each 2 holes, and every hole adds 50 μ l.If blank 1 hole (not adding any reagent), all the other each holes add serum 50 μ l to be checked.Every hole adds enzyme conjugates 50 μ l (or 1) again, fully shakes up rearmounted 37 ℃ of insulations 30 minutes;
2) wash plate:
Get rid of each hole liquid, fill it up with each hole with washing lotion, dry after standing 10 seconds, wash so again three times, pat dry at last;
3) colour developing:
Every hole (comprising blank well) adds nitrite ion A 50 μ l (or 1), adds nitrite ion B 50 μ l (or 1) again, after fully shaking up, puts 37 ℃ of colour developings 15 minutes;
4) stop:
Every hole adds stop buffer 50 μ l (or 1) cessation reaction.
5) result is judged:
With wavelength 450nm calibration blank well OD=0, then measure each hole OD value, and press following formula result of calculation:
The positive S/N in S/N 〉=2.1＜2.1 are negative
Negative control OD value＜0.07 o'clock by 0.07 calculating, was calculated by actual OD value in＞0.07 o'clock.
The using method 2 of embodiment 8 kits of the present invention
The present embodiment uses the hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent case (CLIA method) of embodiment 6 preparations.
1) application of sample:
Pre-packet response plate (bar), every plate is established positive and negative and is contrasted each 2 holes, and every hole adds 50 μ l.All the other each holes add serum 50 μ l to be checked.Every hole adds enzyme conjugates 50 μ l (or 1) again, fully shakes up rearmounted 37 ℃ of insulations 30 minutes;
2) wash plate:
Get rid of each hole liquid, fill it up with each hole with washing lotion, dry after standing 10 seconds, wash so again three times, pat dry at last;
3) mixed luminescence reactant liquor:
With reactant liquor A and B, mix with the ratio of 1: 1;
4) luminescence-producing reaction:
Every hole adds mixed luminescence reactant liquor 100 μ l (or 2), puts room temperature lucifuge reaction 5 minutes.
5) measure:
Sequentially measure the luminous intensity (RLU) in each hole on the chemiluminescence measuring instrument, measure duration: 1 second/hole.
6) result is judged:
With wavelength 450nm calibration blank well OD=0, then measure each hole OD value, and press following formula result of calculation:
The positive S/N in S/N 〉=1.0＜1.0 are negative
The methodology Quality Identification of embodiment 9 kits of the present invention
According to the state quality standard of hepatitis B E antibody external diagnosis reagent case, adopting national standard serum dish is standard, detects the kit of preparation in embodiment 1 and 6.The results are shown in following table 1.
Table 1: the testing result of national standard serum dish
Above result shows that sensitivity, specificity, accuracy and the stability of " hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent case " all comply with the national standard requirements fully.
Contrast with the competition law kit serum sample that detects clinically on embodiment 10 use kits of the present invention and market.
Use kit of the present invention (ELISA method) and ABBOTT Murex in and the competition law kit detect simultaneously 76 parts of clinical random samples, the comparative result of two kinds of kits sees the following form 2.
For 6 parts of serum that this kit and ABBOTT kit are not inconsistent, by consulting source patient's case history archive, find that they are all the hepatitis B patients that are in the treatment different times, decline has all appearred in their hepatitis B E antigen quantity.This conforms to clinical result for the treatment of: the sign that takes a turn for the better has all appearred in these six patients' treatment, and namely hepatitis B E antibody occurs, and shows that beginning " turns out cloudy ".This explanation kit of the present invention can than in and the kit of competition law detect in advance hepatitis B E antibody in patient body.
Table 2: the kit comparing result of the Murex of this kit and ABBOTT
In sum, " hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent case (ELISA method) " of the present invention with used in compare with the kit of competition law, sensitivity is higher, specificity is better, can earlier detect E antibody.
Claims (10)
1. hepatitis B E antibody external diagnosis reagent case based on the dual-antigen sandwich method principle, it comprises: the restructuring HBeAg that 1) has been coated in solid phase carrier; 2) carry out the restructuring HBeAg of enzyme labeling; 3) substrate solution; The described restructuring HbeAg that carries out enzyme labeling is that enzyme passes through the coupling of MBS method with restructuring HBeAg.
2. according to claim 1 kit, wherein said solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
3. according to claim 1 kit, wherein said enzyme is horseradish peroxidase or alkaline phosphatase.
4. according to claim 1 kit, wherein said substrate solution is the chromogenic substrate liquid that is applied to ELISA, comprises:
Nitrite ion A: citric acid 0.86g, trisodium citrate 1.735g is dissolved in deionized water, is settled to 100ml, adds H
2O
250 μ L mixings;
Nitrite ion B: 1. citric acid 0.8256g, trisodium citrate 1.6656g and sucrose 6g are dissolved in deionized water, are settled to 96ml, and 2. TMB 0.026g is dissolved in 4ml DMSO, will be 1. and 2. mixing.
5. according to claim 4 kit, wherein said TMB is 3,3,5,5-tetramethyl benzidine, 3,3,5,5-tetramethyl benzidine sulfate or 3,3,5,5-tetramethyl biphenyl amine hydrochlorate.
6. according to claim 1 kit, wherein said substrate solution is the enzyme-catalyzed chemical luminescence substrate liquid that is applied to CLIA, comprises:
Reactant liquor A:12.1g Tris, the 2.95ml concentrated hydrochloric acid, the 5g luminol, 1g phenylpropyl alcohol fluoranthene, 0.5ml Tween20, constant volume is to 1000ml.
Reactant liquor B:7.3g trisodium citrate, the 4.4g citric acid, the hydrogen peroxide of 1.2ml 30%, constant volume is to 1000ml.
7. method for preparing the hepatitis B E antibody external diagnosis reagent case, it comprises:
1) HBeAg is coated in solid phase carrier;
2) HBeAg that will recombinate carries out enzyme labeling;
3) preparation of substrate solution;
4) be assembled into the finished product kit; The described restructuring HBeAg that carries out enzyme labeling is that enzyme passes through the coupling of MBS method with restructuring HBeAg.
8. according to claim 7 method, wherein said step 2) comprise the following steps:
A) MBS is dissolved with dimethylformamide, join in HRP solution, room temperature reaction 1 hour;
B) remove excessive MBS with the G25 gel filtration chromatography;
C) the enzyme liquid that will process is concentrated;
D) HBeAg is joined in enzyme liquid room temperature reaction 1 hour;
E) reactant liquor is crossed gel chromatography column conjugate is separated with conjugate not, collect conjugate;
F) conjugate that has the HBeAg activity with the absorption of hepatitis B core antibody affinity column.
9. according to claim 7 method, wherein said step 3) be the chromogenic substrate liquid that preparation is applied to ELISA, comprise the following steps:
A) preparation of nitrite ion A (100ml, PH 4.7):
Citric acid 0.86g and trisodium citrate 1.735g are dissolved in deionized water, are settled to 100ml, add H
2O
250 μ L mixings;
B) preparation of nitrite ion B (100ml, PH 4.7):
1. citric acid 0.8256g, trisodium citrate 1.6656g and sucrose 6g are dissolved in deionized water, are settled to 96ml, and 2. TMB 0.026g is dissolved in 4ml DMSO, will be 1. and 2. mixing.
10. according to claim 7 method, wherein said step 3) be the enzyme-catalyzed chemical luminescence substrate liquid that preparation is applied to CLIA, comprise the following steps:
A) preparation of reactant liquor A:
12.1g Tris, the 2.95ml concentrated hydrochloric acid, the 5g luminol, 1g phenylpropyl alcohol fluoranthene, 0.5mlTween20, constant volume is to 1000ml.
B) preparation of reactant liquor B:
7.3g trisodium citrate, the 4.4g citric acid, the hydrogen peroxide of 1.2ml 30%, constant volume is to 1000ml.
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| CN102998462B (en) * | 2012-11-20 | 2014-12-10 | 博奥赛斯（天津）生物科技有限公司 | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for procollagen III (PC III), and preparation method of kit |
| CN103712982B (en) * | 2013-12-13 | 2016-02-03 | 山东博科生物产业有限公司 | A kind of highly sensitive TMB nitrite ion and preparation method thereof |
| CN108152488A (en) * | 2017-12-22 | 2018-06-12 | 太原瑞盛生物科技有限公司 | A kind of magnetic microparticle chemiluminescence detection kit of human t lymphotropic virus antibody and preparation method thereof |
| CN108267577B (en) * | 2018-01-15 | 2020-10-09 | 广州市妇女儿童医疗中心 | EV71 virus IgA antibody detection test strip |
| CN113358878B (en) * | 2021-06-04 | 2022-11-01 | 深圳市辰纳生物科技有限公司 | Reagent and method for in vitro diagnosis of hepatitis B virus antibody |
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| US5501948A (en) * | 1989-02-06 | 1996-03-26 | Imclone Systems Incorporated | Stabilized hepatitis B e antigen suitable for immunoassays |
| CN1356554A (en) * | 2001-11-23 | 2002-07-03 | 上海晶泰生物技术有限公司 | Protein chip for diagnosing hepatism |
| CN1963512A (en) * | 2006-11-10 | 2007-05-16 | 郑州安图绿科生物工程有限公司 | Chemiluminescence method for qualitative and quantitative detection of hepatitis B virus |
| CN101251540A (en) * | 2008-03-26 | 2008-08-27 | 博阳生物科技（上海）有限公司 | Hepatitis B virus e antigen testing corpuscle, preparation and application thereof |
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2009
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Patent Citations (4)
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|---|---|---|---|---|
| US5501948A (en) * | 1989-02-06 | 1996-03-26 | Imclone Systems Incorporated | Stabilized hepatitis B e antigen suitable for immunoassays |
| CN1356554A (en) * | 2001-11-23 | 2002-07-03 | 上海晶泰生物技术有限公司 | Protein chip for diagnosing hepatism |
| CN1963512A (en) * | 2006-11-10 | 2007-05-16 | 郑州安图绿科生物工程有限公司 | Chemiluminescence method for qualitative and quantitative detection of hepatitis B virus |
| CN101251540A (en) * | 2008-03-26 | 2008-08-27 | 博阳生物科技（上海）有限公司 | Hepatitis B virus e antigen testing corpuscle, preparation and application thereof |
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