CN102175867A - Enzyme-linked immunoassay kit for detecting rabies virus antibody - Google Patents
Enzyme-linked immunoassay kit for detecting rabies virus antibody Download PDFInfo
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- CN102175867A CN102175867A CN2010106206777A CN201010620677A CN102175867A CN 102175867 A CN102175867 A CN 102175867A CN 2010106206777 A CN2010106206777 A CN 2010106206777A CN 201010620677 A CN201010620677 A CN 201010620677A CN 102175867 A CN102175867 A CN 102175867A
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Abstract
The invention provides an enzyme-linked immunoassay kit for detecting a rabies virus antibody, and a preparation method and a detection method of the enzyme-linked immunoassay kit. A recombinant rabies virus G protein serving as an antigen is coated on the enzyme-linked plate and a horse radish peroxidase (HRP)-labeled mouse anti-human IgG Fc fragment monoclonal antibody is used as an enzyme marker in the kit. The kit further comprises a developer A, a developer B, a stop solution, a sample diluent, normal human serum serving as a negative control and rabies-virus-antibody-containing serum serving as a positive control. The kit is high in sensibility and accuracy, and safe, rapid and convenient in operation.
Description
Technical field
The present invention relates to a kind of enzyme linked immunological kit that detects rabies virus antibodies.
Background technology
Rabies have another name called hydrophobia, are a kind of acute viral infectious diseases of encroaching on central nervous system, how to be got by ill animal bites people, in case death rate of the onset is up to 100%.Antirabic main means are to exposed population group's injecting pstudorabies vaccine both at home and abroad at present.World Health Organization's suggestion, the NAT of rabies viruses has protection when reaching 0.5IU/ml.For the evaluation of anti-rabies virus antibody titre in the human serum of vaccinate, can be used as the index of this vaccine validity of clinical assessment.
The detection method of detection rabies virus antibodies commonly used has fluorescence antibody virus neutralization tests (FAVN), rapid fluorescence spot inhibition test (RFFIT), mouse neutralization test (MNT) and enzyme linked immunosorbent assay (ELISA) at present.Wherein the enzyme linked immunosorbent assay handling safety is highly sensitive, and the result is stable, and accuracy is strong, obtains general approval.
Summary of the invention
The purpose of this invention is to provide a kind of enzyme linked immunological kit that detects rabies virus antibodies.
In order to realize the object of the invention, a kind of enzyme linked immunological kit that detects rabies virus antibodies of the present invention comprises:
1) is coated with the ELISA Plate of 0.05 μ g/ml～0.1 μ g/ml recombinant rabies virus G albumen;
2) sample diluting liquid: the phosphate buffer that contains 0.5 ‰～1 ‰ casein sodium salts;
3) cleansing solution: the phosphate buffer that contains 0.5 ‰～1 ‰ tweens;
4) enzyme labeling thing: with the mouse-anti human IgG Fc fragment monoclonal antibody of horseradish peroxidase (HRP) mark;
5) developer A: the citrate buffer that contains 0.4 ‰～0.6 ‰ carbamide peroxides;
6) developer B: the citrate buffer that contains 0.2 ‰～0.25 ‰ tetramethyl benzidines;
7) stop buffer: 1.5mol/L～2mol/L sulfuric acid solution;
8) negative control: normal human serum;
9) positive control: the human serum that contains rabies virus antibodies.
Aforesaid kit, described recombinant rabies virus G albumen prepares according to following method, comprises step:
1) with the base sequence of coding hydrophobin G albumen as target gene fragment: go up data and parent/hydrophobicity program software of announcing according to NCBI and analyze, choose hydrophobin G albumen complete sequence from totally 354 pairing base sequences of amino acid residue of 149 502 as target gene fragment, the synthetic target gene fragment of totally 1062 bases (bp);
2) this target gene fragment is imported host cell by expression vector, screening obtains expressing the engineering cell of hydrophobin G albumen;
3) the culturing engineering cell obtains inclusion body;
4) smudge cells, purifying obtains recombinant rabies virus G albumen.
Aforesaid kit, described expression vector are pET series plasmid (pET-5a).
Aforesaid kit, described host cell are E.coli BL21 (DE3).
The present invention also provides the using method of the enzyme linked immunological kit of above-mentioned detection rabies virus antibodies, comprises the steps:
1) application of sample: get bag by good elisa plate, blank hole, negative serum control wells, positive serum control wells and sample well are established in each experiment, add sample dilution 100 μ l in each hole earlier, in each corresponding aperture, add each 10 μ l of negative serum, positive serum and sample more successively, mixing.The blank hole only adds sample dilution 100 μ l.Stick the adhesive sticker bar, place 37 ℃ of incubation 20min.
2) wash plate: back-off falls the liquid in the elisa plate, and adds cleansing solution 300 μ l to each hole, leave standstill 30s after, deduct the liquid in the elisa plate, cyclic washing 5 times, last is less important to pat dry liquid.
3) adding two resists: add two antienzyme labels, 100 μ l in each hole, the blank hole does not add, and sticks the adhesive sticker bar, places 37 ℃ of incubation 20min.
4) wash plate, with step 2).
5) colour developing: add developer A and each 50 μ l of developer B to each hole successively, mixing places 37 ℃ of incubation 15min.
6) stop: add stop buffer 50 μ l, mixing to each hole successively.
7) measure: with enzyme connection instrument blank well is returned to zero, measure the absorbance value (OD value) in each hole.
8) result of determination: according to the ratio in judgement of measuring OD value and critical value (cut-off value).If ratio is greater than 1, then this serum is positive, illustrates wherein to contain rabies virus antibodies, and vaccine has protection and renders a service; If ratio is less than 1, then this serum is negative, illustrates wherein not contain antibody.
Determining of critical value:
With the human serum of injecting hydrophobia mouse is carried out neutralization test in the brain, and measure NAT, the human serum of not injecting rabies vaccine is diluted to 0.5IU/ml.To 1000 parts of above-mentioned serum according to step 1)-7) carry out enzyme linked immunosorbent assay, the OD value that records asks average
And standard deviation (SD),
The enzyme linked immunological kit of detection rabies virus antibodies provided by the invention is used for after injecting hydrophobia, detects in the serum whether have corresponding antibodies, to estimate immune effect.The present invention is the kit that utilizes indirect enzyme-linked immunosorbent assay detection rabies virus antibodies, and the inside quality controlled serum measured value that with the antibody titer is 0.5IU/ml has high sensitivity and specificity as critical value (cut-off value).
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment detects the preparation of the enzyme linked immunological kit of rabies virus antibodies
The enzyme linked immunological kit of detection rabies virus antibodies of the present invention comprises:
1) is coated with the ELISA Plate of 0.1 μ g/ml recombinant rabies virus G albumen;
2) sample diluting liquid: the phosphate buffer that contains 1 ‰ casein sodium salts;
3) cleansing solution: the phosphate buffer that contains 1 ‰ tweens;
4) enzyme labeling thing: with the mouse-anti human IgG Fc fragment monoclonal antibody of horseradish peroxidase (HRP) mark;
5) developer A: the citrate buffer that contains 0.6 ‰ carbamide peroxides;
6) developer B: the citrate buffer that contains 0.25 ‰ tetramethyl benzidine;
7) stop buffer: 2mol/L sulfuric acid solution;
8) negative control: normal human serum;
9) positive control: the human serum that contains rabies virus antibodies.
Wherein, recombinant rabies virus G albumen prepares according to following method, comprises step:
1) acquisition of genes of interest: choose hydrophobin G albumen complete sequence from totally 354 pairing base sequences of amino acid residue of 149 502 as target gene fragment, the synthetic target gene fragment of totally 1062 bases (bp).
2) reorganization:, and utilize the T4 ligase that target gene fragment is reconstituted in plasmid with described target gene fragment and plasmid pET-5a restriction enzyme Bgl II and BamH I double digestion.
3) transform: adopt lime chloride-glycerine method that plasmid is converted into E.coli BL21, and thalline is recovered.
4) verify: transformant is coated on the nutrient culture media that contains 25 μ g/ml～50 μ g/ml ammonia benzyl mycins cultivated 16～18 hours.Picking white colony upgrading grain carries out single endonuclease digestion and double digestion to plasmid, and determines according to the analysis contrast of SDS-PAGE electrophoretic band whether plasmid recombinates.
5) express: the clone of 1 recombinant plasmid of picking is cultured to OD in fluid nutrient medium
600Value is 0.3～0.5 o'clock adding 0.05mmol/L～0.1mmol/LIPTG, expressing quantity maximum after about 3.5 hours.
6) purifying: bacterium liquid is carried out the ultrasonication thalline, and 4000rpm is centrifugal, gets supernatant, with 4B gel chromatography column purifying and measure protein content, determines protein concentration.
Experimental example detects the application and the detection effect of the enzyme linked immunological kit of rabies virus antibodies
The using method of the enzyme linked immunological kit that the foregoing description makes comprises the steps:
1) application of sample: get bag by good elisa plate, blank hole, negative serum control wells, positive serum control wells and sample well are established in each experiment, add sample dilution 100 μ l in each hole earlier, in each corresponding aperture, add each 10 μ l of negative serum, positive serum and sample more successively, mixing.The blank hole only adds sample dilution 100 μ l.Stick the adhesive sticker bar, place 37 ℃ of incubation 20min.
2) wash plate: back-off falls the liquid in the elisa plate, and adds cleansing solution 300 μ l to each hole, leave standstill 30s after, deduct the liquid in the elisa plate, cyclic washing 5 times, last is less important to pat dry liquid.
3) adding two resists: add two antienzyme labels, 100 μ l in each hole, the blank hole does not add, and sticks the adhesive sticker bar, places 37 ℃ of incubation 20min.
4) wash plate, with step 2).
5) colour developing: add developer A and each 50 μ l of developer B to each hole successively, mixing places 37 ℃ of incubation 15min.
6) stop: add stop buffer 50 μ l, mixing to each hole successively.
7) measure: with enzyme connection instrument blank well is returned to zero, measure the absorbance value (OD value) in each hole.
8) result of determination: according to the ratio in judgement of measuring OD value and critical value (cut-off value).If ratio is greater than 1, then this serum is positive, illustrates wherein to contain rabies virus antibodies, and vaccine has protection and renders a service; If ratio is less than 1, then this serum is negative, illustrates wherein not contain antibody.
Determining of critical value:
With the human serum of injecting hydrophobia mouse is carried out neutralization test in the brain, and measure NAT, the human serum of not injecting rabies vaccine is diluted to 0.5IU/ml.To 1000 parts of above-mentioned serum according to step 1)-7) carry out enzyme linked immunosorbent assay, the OD value that records asks average
And standard deviation (SD),
The kit (the accurate word S2006008 of the title of a reigning dynasty) of kit of the present invention and the production of Ningbo Tian Run biological products company limited is carried out the rabies virus antibodies detection to crowd, immunity and non-immune crowd after import hydrophobia and the homemade hydrophobia immunity respectively, and the result as shown in Table 1 and Table 2.
The omnidistance immunity of table 1 import seedling and homemade seedling back antibody male rotary situation relatively
The testing result of the omnidistance immunity of contrast import seedling and homemade seedling back antibody male rotary rate shows that kit of the present invention detects effect and is significantly higher than and a day profit kit (P=0.012).
Table 2 immune group and non-immune group crowd's rabies virus antibodies testing result
A kit of the present invention and a day profit kit are carried out the rabies virus antibodies detection to immunity with non-immune crowd respectively, and the result is shown in table 3 and table 4.
Table 3 immune group and non-immune group crowd's rabies virus antibodies testing result
Table 4 immune group and non-immune group crowd's rabies virus antibodies testing result
Draw immune group crowd rabies virus antibodies recall rate by table 2, table 3 and table 4 and be significantly higher than contrast rabies virus antibodies kit (P＜0.05).Non-immune group crowd's rabies virus antibodies and contrast agents box do not have significant difference (P＞0.05).
Above-mentioned comparative result shows that kit of the present invention has stronger specificity, and higher sensitivity meets clinical request for utilization.
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1. enzyme linked immunological kit that detects rabies virus antibodies is characterized in that it comprises:
1) is coated with the ELISA Plate of 0.05 μ g/ml～0.1 μ g/ml recombinant rabies virus G albumen;
2) sample diluting liquid: the phosphate buffer that contains 0.5 ‰～1 ‰ casein sodium salts;
3) cleansing solution: the phosphate buffer that contains 0.5 ‰～1 ‰ tweens;
4) enzyme labeling thing: with the mouse-anti human IgG Fc fragment monoclonal antibody of horseradish peroxidase-labeled;
5) developer A: the citrate buffer that contains 0.4 ‰～0.6 ‰ carbamide peroxides;
6) developer B: the citrate buffer that contains 0.2 ‰～0.25 ‰ tetramethyl benzidines;
7) stop buffer: 1.5mol/L～2mol/L sulfuric acid solution;
8) negative control: normal human serum;
9) positive control: the human serum that contains rabies virus antibodies.
2. kit according to claim 1 is characterized in that, described recombinant rabies virus G albumen prepares according to following method, comprises step:
1) going up data and the parent/hydrophobicity program software announced according to NCBI analyzes, choose hydrophobin G albumen complete sequence from totally 354 pairing base sequences of amino acid residue of 149 502 as target gene fragment, the synthetic target gene fragment of totally 1062 bases;
2) this target gene fragment is imported host cell by expression vector, screening obtains expressing the engineering cell of hydrophobin G albumen;
3) the culturing engineering cell obtains inclusion body;
4) smudge cells, purifying obtains recombinant rabies virus G albumen.
3. kit according to claim 2 is characterized in that, described expression vector is the pET-5a plasmid.
4. according to each described kit of claim 1-3, it is characterized in that described host cell is E.coli BL21.
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102520169A (en) * | 2011-11-29 | 2012-06-27 | 唐山怡安生物工程有限公司 | ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of animal rabies neutralizing antibody and application thereof |
| CN102721816A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Rabies virus diagnostic reagent kit |
| CN102749445A (en) * | 2012-06-29 | 2012-10-24 | 中国人民解放军军事医学科学院军事兽医研究所 | Method for improving rabies neutralizing antibody detection sensitivity in latex agglutination test |
| CN103323588A (en) * | 2012-03-19 | 2013-09-25 | 郑州中道生物技术有限公司 | Method for detecting canine rabies virus antibody and detection kit |
| CN104101711A (en) * | 2013-04-07 | 2014-10-15 | 广州瑞博奥生物科技有限公司 | Improved enzyme-linked immunoassay kit and detection method thereof |
| CN104792996A (en) * | 2014-01-20 | 2015-07-22 | 辽宁成大动物药业有限公司 | Rabies virus antibody (IgG) enzyme-linked immunoassay kit and detection method thereof |
| CN108135999A (en) * | 2015-08-13 | 2018-06-08 | 马萨诸塞大学 | For rabic human antibody and application thereof |
| CN109444410A (en) * | 2018-10-26 | 2019-03-08 | 成都普利泰生物科技有限公司 | A kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit |
| CN109828109A (en) * | 2018-12-18 | 2019-05-31 | 武汉生命科技股份有限公司 | For detecting the preparation method and kit of the antigen protein of rabies virus antibodies |
| CN109870570A (en) * | 2018-12-29 | 2019-06-11 | 广东云天抗体生物科技有限公司 | A kind of enzyme linked immunological kit detecting monkey IL-18 |
| CN109870571A (en) * | 2018-12-29 | 2019-06-11 | 广东云天抗体生物科技有限公司 | A kind of enzyme linked immunological kit detecting monkey G-CSF |
| CN110305874A (en) * | 2019-06-19 | 2019-10-08 | 浙江省肿瘤医院 | Meriones unguiculatus Immunoglobulin IgG1, IgG2 recombinant protein, gene and its application |
| CN112798787A (en) * | 2019-11-14 | 2021-05-14 | 安徽智飞龙科马生物制药有限公司 | Rabies vaccine antigen content detection method and reagent or kit |
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Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102520169A (en) * | 2011-11-29 | 2012-06-27 | 唐山怡安生物工程有限公司 | ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit of animal rabies neutralizing antibody and application thereof |
| CN103323588A (en) * | 2012-03-19 | 2013-09-25 | 郑州中道生物技术有限公司 | Method for detecting canine rabies virus antibody and detection kit |
| CN102721816A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Rabies virus diagnostic reagent kit |
| CN102749445A (en) * | 2012-06-29 | 2012-10-24 | 中国人民解放军军事医学科学院军事兽医研究所 | Method for improving rabies neutralizing antibody detection sensitivity in latex agglutination test |
| CN104101711A (en) * | 2013-04-07 | 2014-10-15 | 广州瑞博奥生物科技有限公司 | Improved enzyme-linked immunoassay kit and detection method thereof |
| CN104101711B (en) * | 2013-04-07 | 2016-03-23 | 广州瑞博奥生物科技有限公司 | A kind of ELISA measuring reagent kit of improvement and detection method thereof |
| CN104792996A (en) * | 2014-01-20 | 2015-07-22 | 辽宁成大动物药业有限公司 | Rabies virus antibody (IgG) enzyme-linked immunoassay kit and detection method thereof |
| CN104792996B (en) * | 2014-01-20 | 2017-10-27 | 辽宁成大动物药业有限公司 | A kind of rabies virus antibodies（IgG）Enzyme-linked immunologic detecting kit and its detection method |
| CN108135999A (en) * | 2015-08-13 | 2018-06-08 | 马萨诸塞大学 | For rabic human antibody and application thereof |
| CN108135999B (en) * | 2015-08-13 | 2022-05-31 | 马萨诸塞大学 | Human antibodies to rabies and uses thereof |
| CN109444410A (en) * | 2018-10-26 | 2019-03-08 | 成都普利泰生物科技有限公司 | A kind of veterinary rabies poison neutralizing antibody chemiluminescence detection kit |
| CN109828109A (en) * | 2018-12-18 | 2019-05-31 | 武汉生命科技股份有限公司 | For detecting the preparation method and kit of the antigen protein of rabies virus antibodies |
| CN109870570A (en) * | 2018-12-29 | 2019-06-11 | 广东云天抗体生物科技有限公司 | A kind of enzyme linked immunological kit detecting monkey IL-18 |
| CN109870571A (en) * | 2018-12-29 | 2019-06-11 | 广东云天抗体生物科技有限公司 | A kind of enzyme linked immunological kit detecting monkey G-CSF |
| CN110305874A (en) * | 2019-06-19 | 2019-10-08 | 浙江省肿瘤医院 | Meriones unguiculatus Immunoglobulin IgG1, IgG2 recombinant protein, gene and its application |
| CN112798787A (en) * | 2019-11-14 | 2021-05-14 | 安徽智飞龙科马生物制药有限公司 | Rabies vaccine antigen content detection method and reagent or kit |
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