CN101598730A - Hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent case and preparation method - Google Patents
Hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent case and preparation method Download PDFInfo
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Abstract
The invention provides a kind of hepatitis B virus E antibody through dual-antigen sandwich method external diagnosis reagent case (ELISA method), it comprises: 1) wrapped by in the reorganization HBeAg of solid phase carrier; 2) carry out the reorganization 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.In addition, the invention provides the method for preparing the mentioned reagent box, it may further comprise the steps: the HBeAg that 1) will recombinate bag quilt is in solid phase carrier; 2) reorganization HBeAg is carried out enzyme labeling; 3) preparation substrate solution; With 4) be assembled into the finished product kit.Kit of the present invention is highly sensitive, specificity good, the result is accurate, and clinical applicability is strong, can reflect the amount of E antibody in the hepatitis B patient body exactly.
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 one of deadly virus the most widely at present in the world.According to statistics, the whole world has the crowd above 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 the district occurred frequently and the popular district 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 arranged approximately.And, 1,000,000 people are arranged in the world approximately because of infecting HBV dead [Parkin D.M., etal., Cancer Surv, 1999,33:5～33] every year.Still do not have at present and cure the chronically infected effective ways of hepatitis type B virus, so vaccine inoculation and early diagnosis be the important means of prevention, to the detection of dynamic of the infection of virus 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 the antibody of corresponding generation at these three kinds of antigens, and they are respectively hepatitis B surface antibody, hepatitis B core antibody and hepatitis B E antibody, and justacrine is (blood is that main antibody exists the place) in body fluid.Because the cAg of hepatitis B can't be secreted in the 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 two double ".
The genome of hepatitis B be one small and exquisite and only there is 3.2kb in system efficiently, only have 4 main open reading frames, but but bearing expression task [GalibertF., Mandrat E., the et al. of nearly ten kinds of albumen, Nature, 1979,281:646～650; Mandrat E., Kay A., J.Virol., 1984,49:782～792].Its genome generally is divided into: parts such as preceding S district, S district, CORE district (pre-c/c) and corresponding promoter, wherein the CORE district is responsible for expressing two kinds of albumen: hepatitis B virus core albumen and hepatitis B E albumen.That is to say the shared genomic same section of HBeAg and HBcAg.Studies have shown that more and more HBeAg is the product of HBcAg after proteinase decomposes.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 that the appearance of anti-HBe is the sign that hbv replication weakens or stops in patient's body, indicating patient's self-healing preferably or result of treatment [will Fang Junfu, clinical patient, 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 two double explored secrets (three)].Therefore the detection of anti-HBe is during " hepatitis B two double " 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, chief editors such as Hu Zonghan, the virus hepatitis laboratory diagnosis, first published, the People's Health Publisher, 1986], radiommunoassay finally (RIA) based on immunoadsorption principle (IA), enzyme linked immunosorbent assay (ELISA) (ELISA), gold test strip is measured (GIA), the method of chemiluminescent immunoassay(CLIA) (CLIA) and time-resolved fluoroimmunoassay determining adsorption high sensitives such as (TRFIA).Present routine clinical assay method all is based on the method for IA, because the use of RIA method is complicated and the radioactivity risk is arranged, is eliminated basically.The ELISA method is to use maximum methods because of easy to use and low price; The gold test strip method is because it is easy-to-use and quick, 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 subjected to clinical welcome.
According to the principle of immunoadsorption (IA) method, because of the relation of test event and used raw material quality is divided into multiple assay methods such as sandwich method, indirect method, competition law (in comprising and competition law) and prize law again.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 big and result's inconvenience interpretation of differences between batches, and the result is inaccurate etc.These shortcomings cause the problems in the clinical use.Such as: because sensitivity is low, in patient's body, produced hepatitis B E antibody, in and the competition law kit but survey to come out, this provide correctly information timely just can not for the doctor when judging result of treatment and formulation successive treatment scheme; Because poor specificity often has false-positive result, thereby cause the detection positive rate to be higher than actual positive rate [ChengBing Li, the problem of anti--HBe detection method is inquired into, 302 hospital's epi chambers]; Because the used CUTOFF value of interpretation as a result of this method can arbitrarily be adjusted by the technician, cause the official written reply difference of product big, and different manufacturers product measured result is also inconsistent.Equally, in and competition law also measured the influence of operating habit, when the mistiming that sample adds and labelled antibody adds is inconsistent, since hepatitis B E antibody in the sample and the hepatitis B E antibody in the 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; ]; Because the inconvenient interpretation of result, the result is inaccurate, and causing reviewer's a sample that can't intuitively judge rightly is the feminine gender or the positive, 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 the blood in advance than competition law; The 2nd, antibody in the sample and labelled antigen are not competed envelope antigen, thereby it is inconsistent the results that cause because of 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 different batches product or the different manufacturers product comparability to same pattern detection result.The 4th, the colour developing 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 used a kind of most important material hepatitis B E antigen 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 two double " kit is developed success, and is applied to clinical examination and uses.At that time, the Antigens raw material that is used for these kit productions all was that blood or the liver from hepatitis B patient proposes, and the source is rare, and the quality instability.Along with the demand to " hepatitis B two double " 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 made up the HBeAg and the HBcAg[Zhu Qiu duckweed of reorganization, 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 the researchist finds that the quality of the HBeAg of reorganization 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 folding with three-dimensional structure accurately to HBcAg, this has just caused the active component that always exists HBcAg among the 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 that 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 use the double antigens sandwich law technology to prepare the hepatitis B E antibody diagnostic kit, 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 development of technology with according to the characteristics of used albumen and enzyme, a lot of methods have been developed, as: chemical coupling method of glutaraldehyde method, sodium periodate method, 1,4-benzoquinone method, adjacent phenylenedimaleimide method or the like 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 the coupling.Practice for many years thinks, during to the enzyme that contains glycosyl and big molecule coupling, the periodic acid method be effect better and the technology simpler method, 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 also is 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 on one's body.For HBeAg, molecular weight is less, and antigenic determinant also has two, so the space structure of its molecule is huge to the activity performance influence of HBeAg.When adopting the sodium periodate method with HBeAg and enzyme coupling, its probability that causes the interior amino of enzyme and the determinant scope of HBeAg to combine with enzyme is bigger.When the enzyme molecule with after HBeAg combines, on the one hand, enzyme can cause the inactivation of this antigenic determinant when amino on the HBeAg antigenic determinant combines; On the other hand,, thereby produce sterically hinderedly, cause most of inactivation of HBeAg 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.Therefore,, will cause the inactivation of HBeAg if adopt inappropriate coupling mode to make HBeAg and enzyme coupling, reflect on apparent seemingly HBeAg can't with the enzyme coupling.
In addition, owing to be mixed with the activity of HBcAg among the prepared now reorganization 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 the solid phase, thereby causes the sample of the hepatitis B core antibody positive is produced cross reaction.
Summary of the invention
The invention solves the mark of HBeAg and HBcAg activity the difficult problem that influences, thereby prepare hepatitis B E antibody external diagnosis reagent case based on the dual-antigen sandwich method principle to kit.
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, has only exposed [the Michael Nassal and Andrea Rieger outside of a 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 space steric effect simultaneously as much as possible.
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 in the future the cross reaction to hepatitis B core antibody as much as possible, 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 extensive use clinically.
Hepatitis B E antibody external diagnosis reagent case based on dual-antigen sandwich method provided by the invention comprises: 1) wrapped by in the reorganization HBeAg of solid phase carrier; 2) carry out the reorganization 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 the method based on the enzyme immunoadsorption such as enzyme linked immunosorbent assay (ELISA) and enzyme-catalyzed chemical luminescence method (CLIA).
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:
Colour developing liquid A: citric acid 0.86g, trisodium citrate 1.735g is dissolved in the deionized water, is settled to 100ml, adds H
2O
250 μ L mixings;
Colour developing liquid B: 1. citric acid 0.8256g, trisodium citrate 1.6656g and sucrose 6g are dissolved in the 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) will recombinate HBeAg bag quilt 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, will recombinate HBeAg bag of described step 1) be may further comprise the steps in solid phase carrier:
A) bag quilt
Carbonate buffer solution (1.6g sodium carbonate and 2.9g sodium bicarbonate are dissolved in the 1000ml distilled water) the dilution C-HBeAg that adopts pH9.6 50mM is to debita spissitudo, and bag is by on solid phase carrier;
B) sealing
Can wash with physiological saline earlier 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 the 1000ml distilled water.The pH value of described confining liquid is about 7.2.Then this confining liquid is carried on the 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, may further comprise the steps:
A) 8mg horseradish peroxidase (HRP) is dissolved among PB (pH7.0) 1ml of 0.1M;
B) with 0.1ml dimethylformamide dissolving 8mg MBS, join in the HRP solution room temperature reaction 1 hour;
C) centrifugal removal post precipitation is removed excessive MBS with the G25 gel filtration chromatography, and damping fluid is a pH5.0 0.05M acetate buffer;
D) the enzyme liquid that will handle concentrates;
E) 10mg HBeAg monomer is joined in the 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 and concentrate, 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) is the chromogenic substrate liquid that preparation is applied to ELISA, may further comprise the steps:
A) preparation of colour developing liquid A (100ml, PH 4.7):
Citric acid 0.86g and trisodium citrate 1.735g are dissolved in the deionized water, are settled to 100ml, add H
2O
250 μ L mixings;
B) preparation of colour developing liquid B (100ml, PH 4.7):
1. citric acid 0.8256g, trisodium citrate 1.6656g and sucrose 6g are dissolved in the 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.More preferably be 3,3,5, the 5-tetramethyl benzidine.
According to the present invention, preferably, described step 3) is the enzyme-catalyzed chemical luminescence substrate liquid that preparation is applied to CLIA, may further comprise the 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 and having wrapped by the microwell plate of HBeAg, enzyme labeling HBeAg, colour developing liquid substrate solution or enzyme-catalyzed chemical luminescence liquid, stop buffer, washing lotion.Wrapped by the microwell plate of HBeAg is the micropore lath in 24,48 or 96 holes; The enzyme of mark HBeAg is a horseradish peroxidase, and labeling method is the MBS method; The substrate of colour developing liquid 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, the present inventor has at first carried out screening experiment and Quality Identification to used starting material, comprise by with the purity of HBeAg and active, mark 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 or the like.Then the method for coating of HBeAg is studied, with the concentration of different HBeAg, different bags be cushioned liquid, different bags was tested with temperature, different sealings by the time.Select optimal bag and be cushioned liquid, confining liquid, bag by temperature and time, and the protein concentration of only HBeAg.For HBeAg monomer and HRP coupling method, tried out multiple coupling method, finally determined the MBS method; To this method again through interference and the reliable quality tagging scheme explored repeatedly and the contrast experiment has determined that finally productive rate height, cost are low, removed the HBcAg activity; While has also been groped the prescription of only enzyme labeling dilution at this kind of enzyme mark HBeAg, and this enzyme labeling thing and dilution have been carried out long-term investigation test, has made to make enzyme labeling HBeAg keep the dilution of active and thermal stability for a long time.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, the result is accurate, clinical applicability is strong, can reflect the amount of E antibody in the hepatitis B patient body exactly.The E antibody that goes out according to this kit measurement have or not or quantity what, can reflect accurately whether in vivo active situation of the present hepatitis type B virus of determined patient, the therapeutic scheme that is adopted 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 an enzyme linked immunosorbent assay, and most of at home hospitals all have this determination techniques and platform.And the use of this method is more extensive, and is low to environment requirement, 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 realization known for inventor best mode of the present invention.Should be appreciated that these embodiments only are exemplary, should not be used to limit 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) bag quilt
0.05M pH be 9.6 carbonate buffer solution for the diluted liquid of bag, 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, add the dilution 0.1ml of HBeAg then 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 the microwell plate got rid of, with physiological saline washing 2 times.Drain microwell plate at last.
(3) sealing
0.01M pH is 7.2 phosphate buffer (PB), contains the solution of caseinhydrolysate and surfactant, loads on the 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 fully dissolving, each hole on microwell plate added 0.11ml, placed 2 hours in 37 ℃, afterwards, the solution in the hole was got rid of, and drained, dried.
(4) packing
The microwell plate that will dry is packed in the Fresco Bag, add an anti-blushing agent after, encapsulate with vacuum packing machine.Putting into 4-8 ℃ after the 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 this method is used is horseradish peroxidase (HRP).Particularly, described method comprises:
A) 8mg HRP is dissolved among PB (pH7.0) 1ml of 0.1M;
B) with 0.1ml dimethylformamide dissolving 8mg MBS, join in the HRP solution room temperature reaction 1 hour;
C) centrifugal removal post precipitation is removed excessive MBS with the G25 gel filtration chromatography, and damping fluid is a pH5.0 0.05M acetate buffer;
D) the enzyme liquid that will handle concentrates;
E) the 10mgHBeAg monomer is joined in the 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 and concentrate, 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, circle round and rock reagent bottle more than 20 minutes.After measuring mixing, taking sample determination, qualified after, put into 4-8 ℃ after the 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 the labeling deposits.
Three, the preparation of colour developing liquid
It is substrate that this method adopts TMB.Colour developing liquid 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 colour developing liquid 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 colour developing liquid 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 colour developing liquid A and B sample and measure, the back packing conforms to quality requirements.Standard packing according to each Packaging Bottle 6ml.After packing was finished, labeling was put into the lucifuge place and deposits for 4-8 ℃.
Four, the preparation of stop buffer
Stop buffer is a 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 finished, 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 back packing.Standard packing according to each Packaging Bottle 20ml.After packing was finished, 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 also need inspect by random samples behind the kit and just can dispatch from the factory after qualified.
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 that being the carrier with plastic bead, plastic tube or magnetic-particle respectively, 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)
Divided by alkaline phosphatase is outside the marker enzyme of HBeAg, and 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 the 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 a shiner with luminol and benzofluoranthrene, is divided into A and B liquid is prepared respectively, separately deposits, and mixes the back with 1: 1 ratio before using and uses.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 colour developing liquid A and B sample and measure, the back packing conforms to quality requirements.Standard packing according to each Packaging Bottle 6ml.After packing was finished, labeling was put into the lucifuge place and deposits for 4-8 ℃.
The using method 1 of embodiment 7 kits of the present invention
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 are established each 2 hole of positive and negative contrast, 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, dry after leaving standstill for 10 seconds, give a baby a bath on the third day after its birth so again time, pat dry at last with washing lotion;
3) colour developing:
Every hole (comprising blank well) adds colour developing liquid A 50 μ l (or 1), adds colour developing liquid 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 judges:
With wavelength 450nm calibration blank well OD=0, measure each hole OD value then, 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,＞0.07 o'clock by actual OD value calculating.
The using method 2 of embodiment 8 kits of the present invention
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 are established each 2 hole of positive and negative contrast, 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, dry after leaving standstill for 10 seconds, give a baby a bath on the third day after its birth so again time, pat dry at last with washing lotion;
3) mixed luminescence reactant liquor:
With reactant liquor A and B, mix with 1: 1 ratio;
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:
On the chemiluminescence measuring instrument, measure the luminous intensity (RLU) in each hole in regular turn, measure duration: 1 second/hole.
6) result judges:
With wavelength 450nm calibration blank well OD=0, measure each hole OD value then, 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.
Embodiment 10 use on kits of the present invention and the market in competition law kit comparison and detection serum sample clinically.
Use kit of the present invention (ELISA method) and ABBOTT Murex in and the competition law kit detect 76 parts of clinical samples at random simultaneously, 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 all are 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 of treatment: the sign that takes a turn for the better has all appearred in these six patients' treatment, and promptly hepatitis B E antibody occurs, and shows that beginning " turns out cloudy ".This illustrate kit of the present invention can than in and the kit of competition law detect hepatitis B E antibody in patient's body in advance.
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 in-vitro diagnosis examination of the present invention Agent box (ELISA method) " with used in compare with the kit of competition law, sensitivity is higher, the spy The opposite sex 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: 1) wrapped by in the reorganization HBeAg of solid phase carrier; 2) carry out the reorganization HBeAg of enzyme labeling; 3) substrate solution.
2. according to the kit of claim 1, wherein said solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
3. according to the kit of claim 1, wherein said enzyme is horseradish peroxidase or alkaline phosphatase.
4. according to the kit of claim 1, wherein said substrate solution is the chromogenic substrate liquid that is applied to ELISA, comprises:
Colour developing liquid A: citric acid 0.86g, trisodium citrate 1.735g is dissolved in the deionized water, is settled to 100ml, adds H
2O
250 μ L mixings;
Colour developing liquid B: 1. citric acid 0.8256g, trisodium citrate 1.6656g and sucrose 6g are dissolved in the 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 the kit of claim 4, 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 the kit of claim 1, 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 wrapped quilt 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.
8. according to the method for claim 7, wherein said step 2) may further comprise the steps:
A) MBS is dissolved with dimethylformamide, join in the HRP solution, room temperature reaction 1 hour;
B) remove excessive MBS with the G25 gel filtration chromatography;
C) the enzyme liquid that will handle concentrates;
D) HBeAg is joined in the 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 HBcAg activity with the absorption of hepatitis B core antibody affinity column.
9. according to the method for claim 7, wherein said step 3) is the chromogenic substrate liquid that preparation is applied to ELISA, may further comprise the steps:
A) preparation of colour developing liquid A (100ml, PH 4.7):
Citric acid 0.86g and trisodium citrate 1.735g are dissolved in the deionized water, are settled to 100ml, add H
2O
250 μ L mixings;
B) preparation of colour developing liquid B (100ml, PH 4.7):
1. citric acid 0.8256g, trisodium citrate 1.6656g and sucrose 6g are dissolved in the 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 the method for claim 7, wherein said step 3) is the enzyme-catalyzed chemical luminescence substrate liquid that preparation is applied to CLIA, may further comprise the 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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| CN103712982A (en) * | 2013-12-13 | 2014-04-09 | 山东博科生物产业有限公司 | High-sensitivity TMB (tetramethylbenzidine) color developing liquid 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 |
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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 |
| CN101251540B (en) * | 2008-03-26 | 2012-12-05 | 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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| 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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