CN104090101A - Human immunodeficiency virus (HIV) antibody detection kit and preparation method thereof - Google Patents

Human immunodeficiency virus (HIV) antibody detection kit and preparation method thereof Download PDF

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Publication number
CN104090101A
CN104090101A CN201410346920.9A CN201410346920A CN104090101A CN 104090101 A CN104090101 A CN 104090101A CN 201410346920 A CN201410346920 A CN 201410346920A CN 104090101 A CN104090101 A CN 104090101A
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hiv
antibody
magnetic particle
immunodeficiency virus
human immunodeficiency
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姚继承
李晓燕
饶志明
闻雯
刘海光
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Weihai Weigao Biotech Co Ltd
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Weihai Weigao Biotech Co Ltd
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/571Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326Magnetic particles

Abstract

The invention belongs to the technical field of immunologic diagnosis and particularly relates to a human immunodeficiency virus (HIV) antibody detection kit using a microparticle chemiluminiscence method and a preparation method of the kit. The kit is composed of magnetic microparticles for detecting an HIV antibody, a tracing conjugate for detecting the HIV antibody, a negative control, a I type positive control, a II type positive control and an analyzing buffering solution. The invention further discloses the preparation method of the detection kit, which adopts a particle chemiluminiscence immunoassay technology; compared with ELISA (Enzyme-Linked Immunosorbent Assay), the technology has higher sensitivity and specificity and is suitable for the clinical auxiliary diagnosis of HIV.

Description

Human immune defect virus antibody detection kit and preparation method thereof
Technical field
The present invention relates to a kind of immune diagnostic technique, specifically a kind of magnetic particle is as human immune defect virus antibody detection kit of carrier and preparation method thereof.
Background technology
Human immunodeficiency virus (Human Immunodeficiency Virus, HIV) is a kind of slow virus that infects human immunity system cells, belongs to the one of retroviruse.Generally believe, the infection of human immunodeficiency virus causes acquired immune deficiency syndrome (AIDS) (AIDS, Acquired Immune Deficiency Syndrome, acquired immunity lacks syndrome, or translations " AIDS "), acquired immune deficiency syndrome (AIDS) is that posteriority cellular immune function occurs defect and causes serious random infection and/or secondary tumor fatal a kind of disease.Acquired immune deficiency syndrome (AIDS) was identified and developed into the whole world from 1981 in the U.S. and is very popular, and to the end of the year 2003, totally caused two over thousands of ten thousand people's death.Human immunodeficiency virus is also commonly called as conventionally as " AIDS virus " or " AIDS virus ".
Spherical in shape or the oval particle of HIV virus, diameter 100-140nm, is to be with tunicary RNA retrovirus, in classification, belongs to the lentiviridae in Retroviridae.HIV virus single copy gene group RNA is about 9.2-9.7Kb.Find that there is at present HIV-1 and HIV-2 amphitypy, these two types very similar on biological characteristics, and their genome has the homology of 40%-50%, and wherein the core protein of two-strain has the cross reaction of height.HIV has three main structural gene: env, gag, pol, and they encode respectively and produce the protein of difference in functionality is antigen, produces corresponding antibody thereby antigen can cause immune response.
HIV invades after body, generally produces immune response at about 6 weeks, produces antibody.In serum, first there is HIV precursor p55 antibody and core protein p24; Then there is outer membrane protein gp120 antibody, transmembrane glycoprotein gp41 antibody, other as the antibody of p66 and p32 also successively occur.Through some months, after several years, when virus appears in blood again, p24, p55 antibody horizontal in serum decline, and other antibody sustainable existences or level rise.Whether diagnose infected by HIV by detecting these different antibodies.China popular be mainly HIV-1 type.
AIDS virus is propagated through exchange body fluid, particularly seminal fluid and blood.The modal routes of infection are: carry out vagina or coitus in ano, share the syringe having stain, the mother who is infected by the virus propagates to baby.In addition, also there are more and more cases to show, infected viral mother and can virus have been passed to baby through breast nursing.
If do not treated, according to HIV different subtype, after aids infection poison the accounts show a surplus of live time average out to 9 to 11 years, and after being diagnosed as AIDS, if, shown according to different research cause treating in the situation that resource-constrained, mean survival time is between 6 to 19 months.And in the area of medical resource abundance, with the effective treatment means treatment HIV the infected of conduct and the AIDS patient of Effective Anti reverse transcription medicine (HAART), can allow mortality ratio reduce 80%, and can be by minimum the HIV the infected's who newly diagnoses out life 30 years.
So far also do not succeed in developing the vaccine that can prevent AIDS, control AIDS only has in early days and diagnoses, could treat in early days and prevent and treat the further deterioration of the state of an illness, otherwise will be transmitted to other people and make self aggravation, affect healthy and pass to offspring.Therefore be necessary to develop a kind of diagnostic method of better performances, can be effectively in early days just by this medical diagnosis on disease out, treatment in time, reduces the health brought because of AIDS and the loss of property.
The traditional detection method of human immune defect virus antibody comprises euzymelinked immunosorbent assay (ELISA), colloidal gold method and chemiluminescence detecting method, although these methods have lot of advantages, need further raising at aspects such as the sensitivity detecting, specificity, stability.Full-automatic microparticle chemiluminescence immunoassay is on EIA enzyme immunoassay basis, to combine highly sensitive chemical luminescent detecting technology and magnetic particle isolation technics, compared with additive method, this method has the advantage of many uniquenesses, first it uses paramagnetic particles as solid phase carrier, because particle volume is little, surface area is large, has expanded reaction area, has greatly improved detection sensitivity; Secondly owing to using full-automatic instrument and matched reagent, human factor is minimized, improved the stability of method and the repeatability of result, also make batch interior difference and differences between batches all less simultaneously.Compared with radioimmunology, particulate chemistry luminescence method is except having the advantages such as high sensitivity, pinpoint accuracy, high reliability, and also tool has the following advantages: a. no radioactivity pollute, good stability; B. specificity is high; C. reagent can ready access upon use, and it is rapidly convenient to measure, and can be used as emergency treatment test item.According to a large amount of experimental results and clinical practice data, from practicality, stability, accuracy and development prospect thereof, the method becomes the first-selection that replaces radioimmunoassay and EIA enzyme immunoassay gradually.
Summary of the invention
Technical matters to be solved by this invention is to overcome above-mentioned the deficiencies in the prior art, provide one to there is high sensitivity and specificity, be suitable for particulate chemistry luminescence method HIV antibody assay kit of the auxiliary diagnosis of clinical human immunodeficiency virus and preparation method thereof.
The technical scheme that the present invention solves the problems of the technologies described above employing is: a kind of human immune defect virus antibody detection kit, it comprises surveys HIV antibody magnetic particle, survey HIV antibody spike bond, negative control, I type positive control, II type positive control, analysis buffer, and described survey HIV antibody magnetic particle is for being marked with the magnetic particle of HIV antigen (gp41, gp36); Survey HIV antibody spike bond for being marked with the different luminol of HIV antigen (gp41, gp36); I type positive control is the human serum/blood plasma that contains HIV-1 antibody; II type positive control is the rabbit anteserum/blood plasma that contains HIV-2 antibody; Analysis buffer is the phosphate buffer containing bovine serum albumin(BSA).
The present invention also provides the preparation method of above-mentioned human immune defect virus antibody detection kit, it is characterized in that: concrete steps are as follows:
(1) preparation is surveyed HIV antibody magnetic particle and will be with carboxyl magnetic particle and EDC 1: 2 in mass ratio, the ratio of magnetic particle and gp41 is the gp41 of every milligram of magnetic particle mark 20 μ g, the ratio of magnetic particle and gp36 is the gp36 of every milligram of magnetic particle mark 25 μ g, under 22 ~ 26 DEG C of conditions, mix mark, 1 hour mark time; After mark, adopt glycocoll to seal unnecessary site, make it concentration and reach 25mM, react 0.5 hour, wash three times, add magnetic particle to preserve liquid (containing the 0.01M PBS buffer system of 1% bovine serum albumin(BSA)), making it final concentration reaches every 20 μ L and surveys in HIV antibody magnetic particles and contain respectively the magnetic particle mark gp 41 Antigens of 60 μ g and the magnetic particle mark gp36 antigen of 50 μ g, 2 ~ 8 DEG C of preservations.
(2) mark of HIV antibody spike bond HIV antigen (gp41, gp36) and different luminol is surveyed in preparation, reaction system is: glutaraldehyde working concentration is 1.0%-2.0%, its best working concentration is 1.25%, the mass ratio of gp41 and different luminol is 1: 5,22 ~ 26 DEG C of reactions 1.5 hours, dialyse with the 0.01M PBS of pH7.2-7.4, after dialysis, add equal-volume glycerine-20 DEG C to deposit; Glutaraldehyde working concentration is 1.0%-2.0%, and its best working concentration is 1.25%, the mass ratio of gp36 and different luminol is 1: 5,22 ~ 26 DEG C of reactions 1.5 hours, dialyse with the 0.01M PBS of pH7.2-7.4, after dialysis, add equal-volume glycerine-20 DEG C to deposit; Be the dilution in 1: 4000 of different luminol mark gp 41 Antigens by dilution ratio respectively by different luminol mark HIV spike bond dilution for antigen (containing the 0.01M PBS buffer system of 20% calf serum), different luminol mark gp36 antigen 1: 3000 dilutions, final composition is surveyed HIV antibody spike bond.
(3) prepare positive and negative contrast and select the negative human serum of more than 5 parts HIV antibody test or blood plasma to mix, after 60 DEG C, 1 hour processes, aseptic filtration, in 2~8 DEG C of preservations, as negative control; Select the positive human serum of more than 5 parts HIV-1 antibody test or blood plasma to mix, after 60 DEG C, 1 hour processes, aseptic filtration, in 2~8 DEG C of preservations, as I type positive control; Select after HIV-2 antibody test positive rabbit anteserum or diluted plasma, after 60 DEG C, 1 hour processes, aseptic filtration, in 2~8 DEG C of preservations, as II type positive control.
(4) prepare analysis buffer sodium dihydrogen phosphate 0.39g/L, sodium hydrogen phosphate 2.68g/L, sodium chloride 8.50g/L, bovine serum albumin(BSA) 10g/L, thimerosal 1.0g/L, by above-mentioned formulated dilution.
Principle of the present invention is to utilize microparticle chemiluminescence immunoassay technology, adopt dual-antigen sandwich method, HIV antigen on magnetic particle solid phase labelling (gp41, gp36), add sample to be tested, reaction forms magnetic particle labelled antigen-antibody conjugates, after incubation, add the HIV antigen of different luminol mark, after reaction, form magnetic particle labelled antigen-antibody-different luminol labelled antigen compound, set up immune response and chemiluminescent contact.Add the different luminol of exciting liquid catalytic label antigen to send the light of 425nm, in sample, the content of HIV antibody and luminous value (RLU) are proportionate, and according to S/CO value, pattern detection result are judged.
The advantage of kit of the present invention is to have adopted microparticle chemiluminescence immunoassay technology, has higher sensitivity and better specificity than ELISA.
Embodiment
Kit of the present invention adopts microparticle chemiluminescence immunoassay technology, detects in serum or blood plasma whether have HIV antibody.Specifically describe HIV antibody assay kit and preparation method thereof below.
A kind of human immune defect virus antibody detection kit, it comprises surveys HIV antibody magnetic particle, survey HIV antibody spike bond, negative control, I type positive control, II type positive control, analysis buffer.Described survey HIV antibody magnetic particle is for being marked with the magnetic particle of HIV antigen (gp41, gp36); Survey HIV antibody spike bond for being marked with the different luminol of HIV antigen (gp41, gp36); I type positive control is the human serum/blood plasma that contains HIV-1 antibody; II type positive control is the rabbit anteserum/blood plasma that contains HIV-2 antibody; Analysis buffer is the phosphate buffer containing bovine serum albumin(BSA).
The preparation method of the above-mentioned HIV antibody assay kit of the present invention, its concrete steps are as follows:
(1) preparation is surveyed HIV antibody magnetic particle and will be with carboxyl magnetic particle and EDC 1: 2 in mass ratio, the ratio of magnetic particle and gp41 is the gp41 of every milligram of magnetic particle mark 20 μ g, the ratio of magnetic particle and gp36 is the gp36 of every milligram of magnetic particle mark 25 μ g, under 22 ~ 26 DEG C of conditions, mix mark, 1 hour mark time; After mark, adopt glycocoll to seal unnecessary site, make it concentration and reach 25mM, react 0.5 hour, wash three times, add magnetic particle to preserve liquid (containing the 0.01M PBS buffer system of 1% bovine serum albumin(BSA)), making it final concentration reaches every 20 μ L and surveys in HIV antibody magnetic particles and contain respectively the magnetic particle mark gp 41 Antigens of 60 μ g and the magnetic particle mark gp36 antigen of 50 μ g, 2 ~ 8 DEG C of preservations.
(2) mark of HIV antibody spike bond HIV antigen (gp41, gp36) and different luminol is surveyed in preparation, reaction system is: glutaraldehyde working concentration is 1.0%-2.0%, its best working concentration is 1.25%, the mass ratio of gp41 and different luminol is 1: 5,22 ~ 26 DEG C of reactions 1.5 hours, dialyse with the 0.01M PBS of pH7.2-7.4, after dialysis, add equal-volume glycerine-20 DEG C to deposit; Glutaraldehyde working concentration is 1.0%-2.0%, and its best working concentration is 1.25%, the mass ratio of gp36 and different luminol is 1: 5,22 ~ 26 DEG C of reactions 1.5 hours, dialyse with the 0.01M PBS of pH7.2-7.4, after dialysis, add equal-volume glycerine-20 DEG C to deposit; Be the dilution in 1: 4000 of different luminol mark gp 41 Antigens by dilution ratio respectively by different luminol mark HIV spike bond dilution for antigen (containing the 0.01M PBS buffer system of 20% calf serum), different luminol mark gp36 antigen 1: 3000 dilutions, final composition is surveyed HIV antibody spike bond.
(3) prepare positive and negative contrast and select the negative human serum of more than 5 parts HIV antibody test or blood plasma to mix, after 60 DEG C, 1 hour processes, aseptic filtration, in 2~8 DEG C of preservations, as negative control; Select the positive human serum of more than 5 parts HIV-1 antibody test or blood plasma to mix, after 60 DEG C, 1 hour processes, aseptic filtration, in 2~8 DEG C of preservations, as I type positive control; Select after HIV-2 antibody test positive rabbit anteserum or diluted plasma, after 60 DEG C, 1 hour processes, aseptic filtration, in 2~8 DEG C of preservations, as II type positive control.
(4) prepare analysis buffer sodium dihydrogen phosphate 0.39g/L, sodium hydrogen phosphate 2.68g/L, sodium chloride 8.50g/L, bovine serum albumin(BSA) 10g/L, thimerosal 1.0g/L, by above-mentioned formulated dilution.
The above-mentioned various raw-material selections of the present invention require as follows:
1, mark is with the selection of HIV antigen first with regard to outward appearance, concentration, the purity of antigen with tire and study, and antigen should be the liquid of clear, containing foreign matter, without shaking not loose precipitation; Detect its protein content with ultraviolet absorption method and should be not less than 1mg/mL; SDS-PAGE detects purity and answers master tape clear, without the band of obviously mixing; Tire should be not less than to indicate and tire and be not less than 1: 10000, result of study shows that HIV antigen can be used for the preparation of kit of the present invention completely.
2, the outward appearance to magnetic particle is passed through in the selection of magnetic particle, the ratio of labelled protein, and magnetic responsiveness, the aspects such as magnetic particle absorption consistance are analyzed, and through repeatedly analysis and research, magnetic particle are mixed, and under light, observe, easily disperse, without gathering, foreign; Adopt diverse ways to carry out mark in albumen, mark rate should be greater than 90%; Magnetic particle is put on the magnet of 370-380 tesla, observed the aggregation velocity of magnetic particle, finely dispersed magnetic particle is assembled completely within 10 seconds; Magnetic particle absorption consistance CV≤10%.Result of study shows that diameter is the magnetic particle of 0.90-1.10 μ m, contains carboxylic group, and mark rate is the highest, can be used for the preparation of diagnostic kit of the present invention.
3, the selection of different luminol by different for luminol DMSO (dimethyl sulfoxide (DMSO)) dissolve, be diluted to 1.2 × 10 by purified water -5the amount of M, adds the different luminol liquid of 10 μ L, respectively adds 200 μ L exciting liquids, measures its luminous value, and luminous value answers>=160000, and through research, satisfactory different luminol can be used as luminous raw material.
The detection method that HIV antibody assay kit of the present invention detects HIV antibody in sample is: first take out concentrated washing lotion, dilute by purified water according to multiple.Then take out and excite A liquid and B liquid, place the suitable position of Full-automatic chemiluminescence analyzer.Exciting A liquid is the damping fluid that contains 4%NaOH, excites B liquid for containing 0.12%H 2o 2damping fluid.Then after kit being taken out from refrigerator, be put in after instrument reagent district at least mixes 30 minutes and can use, and carry out application of sample and incubation in strict accordance with the program of setting.
Specifically add quadrat method as follows: 50 μ L blood serum/blood plasma samples, 100 μ L analysis buffer and 20 μ L survey HIV antibody magnetic particle and under the condition of 37 DEG C, react 20 minutes, add 150 μ L to survey HIV antibody spike bond, 37 DEG C are reacted 20 minutes, again with the washing lotion washing after dilution 3 times, finally add respectively 200 μ L to excite A liquid and excite B liquid, measure luminous value, according to S/CO value, pattern detection result is judged.
Quality control requirement of the present invention is: the positive and negative contrast in kit is for process of the test control.Negative control testing result (S/CO) must not be higher than 0.8, and positive control testing result (S/CO) must not be lower than 10.
Kit of the present invention detects HIV antibody National reference, and the result of detection shows: every quality index such as the positive and negative reference material coincidence rate of this kit, minimum detectability, precision, stability all meet national requirements.Negative match-rate: 20 parts of negative reference material coincidence rates 20/20; Positive coincidence rate: 20 parts of positive reference material coincidence rates 20/20, and P12 > P11; Minimum detectability detects: S1, S2 are all negative, and S3, S4, S5 and S6 are all positive; Precision: the coefficient of variation (CV)≤15% is detected in 10 holes; Stability: the each component of reagent was in 37 DEG C of placements 6 days, and verification result reaches standard.
Kit of the present invention carries out clinical examination, result by Henan College Of Traditional Chinese Medicine the second affiliated hospital, Henan College Of Traditional Chinese Medicine the 3rd affiliated hospital, No. 153 Central Hospital, PLA, Chinese People's Liberation Army's acquired immune deficiency syndrome (AIDS) detection Confirmatory laboratory, five clinical detection mechanisms of Beijing Disease Prevention and Control Centre:
(1) Henan College Of Traditional Chinese Medicine the second affiliated hospital examines 307 routine negative sample and 3 routine positive sample, and kit negative sample 307 examples of the present invention all detect, and positive sample 3 examples all detect.The negative recall rate of this kit is 100%, and positive rate is 100%.
(2) Henan College Of Traditional Chinese Medicine the 3rd affiliated hospital examines 312 routine negative sample and 3 routine positive sample, and kit negative sample 312 examples of the present invention all detect, and positive sample 3 examples all detect.The negative recall rate of this kit is 100%, and positive rate is 100%.
(3) No. 153 Central Hospital, PLA examines 315 routine negative sample and 2 routine positive sample, and kit negative sample 315 examples of the present invention all detect, and positive sample 2 examples all detect.The negative recall rate of this kit is 100%, and positive rate is 100%.
(4) Chinese People's Liberation Army's acquired immune deficiency syndrome (AIDS) detects Confirmatory laboratory and examines 89 routine negative sample and 355 routine positive sample, and kit negative sample 89 examples of the present invention all detect, and positive sample 355 examples all detect.The negative recall rate of this kit is 100%, and positive rate is 100%.
(5) Beijing Disease Prevention and Control Centre examines 32 routine negative sample and 64 routine positive sample, and kit negative sample 32 examples of the present invention all detect, and positive sample 64 examples all detect.The negative recall rate of this kit is 100%, and positive rate is 100%.
The total sensitivity of this detection kit is 100%, and specificity is 100%.
Human immune defect virus antibody detection kit (particulate chemistry luminescence method) sensitivity is 100%, and specificity is 100%, is suitable for the auxiliary diagnosis of Clinical HIV poison.

Claims (3)

1. a human immune defect virus antibody detection kit, it comprises surveys HIV antibody magnetic particle, survey HIV antibody spike bond, negative control, I type positive control, II type positive control, analysis buffer, it is characterized in that: described survey HIV antibody magnetic particle is the magnetic particle potpourri that is marked with respectively Human Immunodeficiency Virus antigen gp41 and Human Immunodeficiency Virus antigen gp36; Surveying HIV antibody spike bond is the different luminol potpourri that is marked with respectively Human Immunodeficiency Virus antigen gp41 and Human Immunodeficiency Virus antigen gp36; I type positive control is the human serum/blood plasma that contains HIV-1 antibody; II type positive control is the rabbit anteserum/blood plasma that contains HIV-2 antibody; Analysis buffer is the phosphate buffer containing bovine serum albumin(BSA).
2. a preparation method who prepares human immune defect virus antibody detection kit described in claim 1, is characterized in that concrete steps are as follows:
(1) preparation is surveyed HIV antibody magnetic particle and will be with carboxyl magnetic particle and EDC 1: 2 in mass ratio, the ratio of magnetic particle and Human Immunodeficiency Virus antigen gp41 is the Human Immunodeficiency Virus antigen gp41 of every milligram of magnetic particle mark 20 μ g, the ratio of magnetic particle and Human Immunodeficiency Virus antigen gp36 is the Human Immunodeficiency Virus antigen gp36 of every milligram of magnetic particle mark 25 μ g, under 22 ~ 26 DEG C of conditions, mix mark, 1 hour mark time; After mark, adopt glycocoll to seal unnecessary site, make it concentration and reach 25mM, react 0.5 hour, wash three times, add magnetic particle to preserve liquid, it is the 0.01M PBS buffer system containing 1% bovine serum albumin(BSA) that described magnetic particle is preserved liquid, makes it final concentration and reaches every 20 μ L and survey in HIV antibody magnetic particles and contain respectively the magnetic particle mark Human Immunodeficiency Virus antigen gp41 of 60 μ g and the magnetic particle mark Human Immunodeficiency Virus antigen gp36 of 50 μ g, 2 ~ 8 DEG C of preservations;
(2) mark of HIV antibody spike bond Human Immunodeficiency Virus antigen gp41, Human Immunodeficiency Virus antigen gp36 and different luminol is surveyed in preparation, reaction system is: glutaraldehyde concentration is 1.0-2.0%, the mass ratio of Human Immunodeficiency Virus antigen gp41 and different luminol is 1: 5,22 ~ 26 DEG C of reactions 1.5 hours, dialyse with the 0.01M PBS of pH7.2-7.4, after dialysis, add equal-volume glycerine-20 DEG C to deposit; Glutaraldehyde concentration is 1.0-2.0%, and the mass ratio of Human Immunodeficiency Virus antigen gp36 and different luminol is 1: 5,22 ~ 26 DEG C of reactions 1.5 hours, dialyses with the 0.01M PBS of pH7.2-7.4, adds equal-volume glycerine-20 DEG C to deposit after dialysis; By spike bond dilution for different luminol mark HIV antigen, described spike bond dilution is the 0.01M PBS buffer system containing 20% calf serum, be the dilution in 1: 4000 of different luminol mark gp 41 Antigens by dilution ratio respectively, different luminol mark gp36 antigen 1: 3000 dilutions, final composition is surveyed HIV antibody spike bond;
(3) prepare positive and negative contrast and select the negative human serum of more than 5 parts HIV antibody test or blood plasma to mix, after 60 DEG C, 1 hour processes, aseptic filtration, in 2~8 DEG C of preservations, as negative control; Select the positive human serum of more than 5 parts HIV-1 antibody test or blood plasma to mix, after 60 DEG C, 1 hour processes, aseptic filtration, in 2~8 DEG C of preservations, as I type positive control; Select after HIV-2 antibody test positive rabbit anteserum or diluted plasma, after 60 DEG C, 1 hour processes, aseptic filtration, in 2~8 DEG C of preservations, as II type positive control;
(4) prepare analysis buffer sodium dihydrogen phosphate 0.39g/L, sodium hydrogen phosphate 2.68g/L, sodium chloride 8.50g/L, bovine serum albumin(BSA) 10g/L, thimerosal 1.0g/L, by above-mentioned formulated dilution.
3. the preparation method of human immune defect virus antibody detection kit according to claim 2, is characterized in that: in step (2), glutaraldehyde working concentration is 1.25%.
CN201410346920.9A 2014-07-21 2014-07-21 Human immunodeficiency virus (HIV) antibody detection kit and preparation method thereof Pending CN104090101A (en)

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CN105954510A (en) * 2016-06-30 2016-09-21 深圳市亚辉龙生物科技股份有限公司 Chemiluminescence immunodetection kit for anti-beta 2 glycoprotein I antibodies IgG and preparation method of kit
CN106501519A (en) * 2016-06-30 2017-03-15 深圳市亚辉龙生物科技股份有限公司 Human immunodeficiency virus antigen antibody chemical luminescence immunity detection reagent and preparation method thereof
CN106546732A (en) * 2015-09-17 2017-03-29 深圳迈瑞生物医疗电子股份有限公司 Human immunodeficiency virus antigen and antibody combined detection kit and application thereof and detection method
CN107202889A (en) * 2017-07-21 2017-09-26 苏州华益美生物科技有限公司 Four pathogen synchronous detection reagent kits and its application and preparation
CN109541229A (en) * 2018-11-09 2019-03-29 广州源起健康科技有限公司 A kind of+2 type antibody kit of detection human immunodeficiency virus I and preparation method thereof
CN110907645A (en) * 2019-12-17 2020-03-24 郑州安图生物工程股份有限公司 Detection kit for human immunodeficiency virus antibody
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