CN102445548A - Detection kit for indirect ELISA of FAVI antibody based on penton protein - Google Patents
Detection kit for indirect ELISA of FAVI antibody based on penton protein Download PDFInfo
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- CN102445548A CN102445548A CN2011103610068A CN201110361006A CN102445548A CN 102445548 A CN102445548 A CN 102445548A CN 2011103610068 A CN2011103610068 A CN 2011103610068A CN 201110361006 A CN201110361006 A CN 201110361006A CN 102445548 A CN102445548 A CN 102445548A
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Abstract
The invention discloses a detection kit for indirect ELISA (Enzyme-Linked Immuno Sorbent Assay) of an FAVI (Fowl Adenovirus Group I) based on a penton protein, wherein a fowl adenovirus group I penton recombinant protein is used as the coating antigen of the coated enzyme label plate of the detection kit. The detection kit for indirect ELISA of the FAVI antibody based on the penton protein is obtained by taking the epitope of the penton recombinant protein as the coating antigen according to the characteristics of the fowl adenovirus group I penton recombinant protein, and by taking goat anti-chicken IgG labeled by a horse radish peroxidase as an enzyme-labeled secondary antibody. The kit has the advantages of simple antigen preparation process, high biological safety, low cost, easy production and application and the like. With the detection kit provided in the invention and with chicken serum as a detection sample, an indirect ELISA detection method with the penton recombinant protein as the coating antigen is established; and the method is good in specificity, high in sensitivity, good in repeatability, and suitable for large-batch detection, and thereby can be applied to the clinic detection, epidemiological survey and active immune effect detection of the fowl adenovirus group I antibody.
Description
Technical field
The present invention relates to FAVI antibody indirect ELISA detection kit, especially a kind of FAVI antibody indirect ELISA detection kit based on penton albumen.
Background technology
I crowd's aviadenovirus (fowl adenovirus group I; FAVI) belong to the Adenoviridae Aviadenovirus; Have common group antigen, can be divided into 5 kinds (FAV A-E), can be divided into 12 serotypes (FAV1-12) based on the cross neutralization test result based on the difference of molecular structure.This group virus comprises conventional aviadenovirus (representative strains is the deadly orphan of chicken embryo), worldwide extensively exists, and more is present in the bird body, and does not fall ill.Though FAVI is not the important cause of disease that causes morbidity, can with other cause of disease actings in conjunction in poultry, have Secondary cases.In recent years, the rotten to the corn infection of the inclusion body hepatitis that is caused by FAVI, hydropericardium syndrome and gizzard is in rising trend, has at home and abroad caused widely and has paid close attention to.Research shows that this disease is relevant with infectious bursa of Fabricius and infectious anemia.In addition, this disease both can be polluted the chicken embryo through the ovum vertical transmission, also can be through the excreta horizontal transmission, and this has brought huge difficulty to this disease of prevention and control.
At present, adopt the regular-PCR method to detect the FAVI clinical sample, time and effort consuming can't fast processing and a large amount of samples of diagnosis; Utilize the serology detection method,, can not definitely diagnose, and need in vivo to carry out at animal, chicken embryo or tissue culture cells etc. owing to there is cross reaction like neutralization test; The agar gel immunodiffusion is because the precipitin diffusion need be hatched 2 days, and it is low again to have delayed Diagnostic Time susceptibility.Therefore, quick, accurate, easy diagnosis is the key of this disease of control, particularly seeks a kind of dominant antigen its diagnosis is had considerable meaning, also can reliable assurance be provided for FAVI antibody detection and epidemiology survey.
Penton, six adjacent bodies and fibrin are three primary structure albumen of FAVI, and they constitute virus nucleocapsid jointly.Wherein, each penton by substrate and stretch out the surface an end the fibrous of heading arranged, help virus to attach to cell surface; Penton also can combine with the virus receptor of cell surface, in the virus infected cell process, plays important effect.Research shows, when adenoviral fiber protein too in short-term, penton mediates the intrusion and the internalization of adenovirus through the integrin effect of its Arg-Gly-Asp (RGD) sequence with cell surface.Simultaneously, the penton conservative property is better, can stimulate body to produce in the antibody and virion.Therefore, penton albumen is significant at pathogenic, diagnosis, prevention and the aspect such as control of FAVI.
Summary of the invention
The technical matters that the present invention will solve provides a kind of quick, accurate, easy, FAVI antibody indirect ELISA detection kit based on penton albumen of being suitable for mass detection, so that the Clinical detection of I crowd's aviadenovirus antibody, epidemiology survey and active immunity effect detection.
Adopt following technical scheme for solving the problems of the technologies described above the present invention:
FAVI antibody indirect ELISA detection kit based on penton albumen; Comprise coated elisa plate, negative standard serum, positive criteria serum, goat-anti chicken IgG ELIAS secondary antibody, cleansing solution, dilution, substrate solution and stop buffer, coated elisa plate with I crowd's aviadenovirus penton recombinant protein as envelope antigen.
I crowd's aviadenovirus penton albumen is by the penton gene code of the brachymemma of sequence table SEQ .ID.No.1.
I crowd's aviadenovirus penton recombinant protein is from escherichia coli prokaryotic expression.
Coated elisa plate is with coating buffer envelope antigen to be diluted to 1～30 μ g/mL, adds 96 hole ELISA Plates, 100 μ L/ holes, place 37 ℃ of incubation 1h after, 4 ℃ are spent the night; Wash plate 3 times with PBST, clap and do, with confining liquid sealase target, 37 ℃ of incubation 1h wash plate 3 times with PBST, clap drying getting; Confining liquid is 5% skimmed milk; Coating buffer is the carbonate buffer solution of pH 9.6.
Goat-anti chicken IgG ELIAS secondary antibody is the goat-anti chicken IgG with horseradish peroxidase-labeled.
Negative standard serum is the serum that SPF chicken embryo is hatched 2～5 Japanese instar chicklings that produce;
Positive criteria serum is the SPF chicken serum behind twice of mode collunarium, eye droppings that I crowd's aviadenovirus simulating nature infects;
Cleansing solution is the phosphate buffer that contains 0.05% Tween-20, and pH is 7.4;
Dilution is 1%BSA;
Substrate solution is TMB-H
2O
2Solution;
Stop buffer is 2M H
2SO
4
Phosphate buffer is by NaCl 8.0g, KCl 0.2g, KH
2PO
40.2g, Na
2HPO
4.12H
2O 2.9g, Tween-20 500uL, adding distil water is settled to 1000mL, transfers pH value to 7.4 to process.
TMB-H
2O
2Solution is by TMB damping fluid 10ml, TMB solution 0.5ml, H
2O
232ul forms; The TMB buffering is by Na
2HPO
418.27g citric acid 4.665g with the 900mL dissolving, transfers PH to 5.5, adds water to 1000mL and processes; TMB solution is processed by 5mL anhydrous alcohol solution TMB 10mg.
According to the characteristic of I crowd's aviadenovirus penton albumen, the present invention utilizes the epitope of penton albumen as envelope antigen, is ELIAS secondary antibody with the goat-anti chicken IgG of horseradish peroxidase-labeled, has processed FAVI antibody indirect ELISA detection kit.This kit has that antigen preparation technology is simple, the bio-safety degree is high, with low cost, be easy to characteristics such as production and application.Use the present invention; With the chicken serum is test sample; Set up and utilize the indirect ELISA detection method of penton recombinant protein as envelope antigen; This method high specificity, highly sensitive, good reproducibility, be suitable for mass detection, can be used for Clinical detection, epidemiology survey and the active immunity effect detection of I crowd's aviadenovirus antibody.
Description of drawings
Fig. 1 is the SDS-PAGE figure of I crowd's aviadenovirus penton recombinant protein, and among the figure: M is a dna molecular amount standard, the 1st, and the empty thalline expression product of pGEX-4T-1,2 and 5 is penton fusion protein expression products, 3 and 4 is that the penton fusion is not induced.
Fig. 2 is a detection method process flow diagram of using the FAVI antibody indirect ELISA detection kit that the present invention is based on penton albumen.
Embodiment
One, the preparation of envelope antigen penton recombinant protein
Used envelope antigen is the epitope that utilizes primer PCR amplification I crowd aviadenovirus penton among the present invention; The penton gene of brachymemma is inserted construction recombination plasmid pGEX-penton among the expression vector pGEX-4T-1, again with the recombinant protein (like Fig. 1) that carries out prokaryotic expression in this recombinant plasmid transformed bacillus coli DH 5 alpha and form.(Deng Xianwen waits the clone and the prokaryotic expression [J] of .I crowd's aviadenovirus penton gene for Luo Sisi, Xie Zhixun. guangdong agricultural science, 2011,38 (7): 154-157).The penton recombinant protein is behind inclusion body purification, as the envelope antigen of coated elisa plate in the kit.
Two, the preparation of ELISA detection kit agents useful for same and preparation
(1) the goat-anti chicken IgG ELIAS secondary antibody of horseradish peroxidase-labeled is available from KPL company;
(2) encapsulate the preparation of enzyme mark target: with coating buffer envelope antigen is diluted to 1～30 μ g/mL, adds 96 hole ELISA Plates, 100 μ L/ holes, place 37 ℃ of incubation 1h after, 4 ℃ are spent the night; Wash plate 3 times with PBST, clap and do, every hole adds confining liquid 200 μ L, places 37 ℃ of sealing 1h, washes plate 3 times with PBST, claps drying getting; Coating buffer: carbonate buffer solution: Na
2CO
31.59g, NaHCO
32.93g adding distil water is settled to 1000mL, transfers pH value to 9.6; Confining liquid: 5% skimmed milk, the 5g skimmed milk is settled to 100mL with PBST;
(3) cleansing solution: contain the phosphate buffer of 0.05% Tween-20, i.e. PBST:NaCl 8.0g, KCl 0.2g, KH
2PO
40.2g, Na
2HPO
4.12H
2O 2.9g, Tween-20 500uL, adding distil water is settled to 1000mL, transfers pH value to 7.4;
(4) dilution: 1%BSA:1g bovine serum albumin(BSA) BSA is settled to 100mL with PBST;
(5) substrate solution (TMB-H
2O
2Solution): TMB damping fluid (Na
2HPO
418.27g citric acid 4.665g with the 900mL dissolving, transfers PH to 5.5, adds water to 1000mL) 10mL, TMB solution (TMB 10mg, 5mL anhydrous alcohol solution) 0.5mL, H
2O
232uL;
(6) stop buffer: 2M H
2SO
4: the 21.7mL concentrated sulphuric acid slowly adds in the 178.3mL distilled water.
Three, ELISA method of operating (like Fig. 2)
(1) encapsulate: with coating buffer envelope antigen is diluted to 1～30 μ g/mL, adds 96 hole ELISA Plates, 100 μ L/ holes, place 37 ℃ of incubation 1h after, 4 ℃ are spent the night; Wash plate 3 times with PBST, clap and do;
(2) sealing: every hole adds 5% skimmed milk 200 μ L, places 37 ℃ of sealing 45min, washes plate 3 times with PBST, claps and does;
(3) combine with serum: establish negative gauge orifice and positive criteria hole, all the other holes add sample to be checked, and plate behind 37 ℃ of incubation 1h, is washed 3 times with PBST in 100 μ L/ holes, clap and do;
(4) combine with ELIAS secondary antibody: the goat-anti chicken IgG of horseradish peroxidase-labeled with 1%BSA with times dilution in 1: 1000～1: 8000,100 μ L/ holes, place 37 ℃ of incubation 1h after, wash plate 3 times with PBST, clap to do;
(5) colour developing: every hole adds 100uL substrate colour developing liquid, in 37 ℃ of lucifuge effect 10min;
(6) stop: every hole adds the 50uL stop buffer, and ELIASA detects the OD450 value;
(7) result judges: calculate the OD450 mean value X and the standard deviation SD that detect 50 parts of SPF negative serum samples, if sample OD450 value＞X+3SD is judged to the positive; If sample OD450 value＜X+3SD is judged to feminine gender.Calculating X is 0.182, and SD is 0.051, according to principle of statistics; The X+3SD of negatives is the positive and negative critical value, and promptly the yin and yang attribute critical value of this method is that 0.182+3 * 0.051=0.335 is if sample OD450 value＞0.335 is positive; If sample OD450 value＜0.335 is negative.
Four, the practical application of FAVI antibody indirect ELISA detection kit
1. clinical sample detects
(1) sample collecting: 10 parts of clinical samples of random acquisition, gather live-bird market chicken blood with vein, the serum of separating out is sample to be checked;
(2) detect: concrete grammar is the same;
(3) result and judgement: measure the OD450 value of each sample, according to above-mentioned standard determination, result such as table 1.
Table 1 clinical sample ELISA testing result
| |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| OD450 | 1.375 | 0.208 | 1.029 | 0.270 | 0.301 | 0.233 | 0.978 | 0.211 | 0.197 | 0.154 |
| The result | + | - | + | - | - | - | + | - | - | - |
2. immune effect evaluation
(1) sample collecting: the chicken blood of acquisition test immunity inactivated vaccine respectively before immunity, immunity one week of back, immunity two weeks of back, immunity back three all and chicken blood samples all around, is a detected object with the serum of separating out;
(2) detect: concrete grammar is the same;
(3) result: measure each sample OD450 value, based on above-mentioned standard determination, result such as table 2.
Table 2 immune effect ELISA evaluation result
| Sample | 0 |
1 |
2 |
3 |
4 weeks |
| OD450 | 0.081 | 0.276 | 0.798 | 1.034 | 1.122 |
| The result | - | - | + | + | + |
The above results shows, as sample to be checked, utilizes kit of the present invention can be successfully used to Clinical detection and immune effect evaluation that I crowd's aviadenovirus infects with the chicken serum sample.
Claims (8)
1. FAVI antibody indirect ELISA detection kit based on penton albumen; Comprise coated elisa plate, negative standard serum, positive criteria serum, goat-anti chicken IgG ELIAS secondary antibody, cleansing solution, dilution, substrate solution and stop buffer, it is characterized in that: said coated elisa plate with I crowd's aviadenovirus penton recombinant protein as envelope antigen.
2. the FAVI antibody indirect ELISA detection kit based on penton albumen according to claim 1 is characterized in that: said I crowd's aviadenovirus penton albumen is by the penton gene code of the brachymemma of sequence table SEQ .ID.No.1.
3. the FAVI antibody indirect ELISA detection kit based on penton albumen according to claim 1 is characterized in that: said I crowd's aviadenovirus penton recombinant protein is from escherichia coli prokaryotic expression.
4. the FAVI antibody indirect ELISA detection kit based on penton albumen according to claim 1; It is characterized in that: said coated elisa plate is with coating buffer envelope antigen to be diluted to 1～30 μ g/mL; Add 96 hole ELISA Plates; 100 μ L/ holes, place 37 ℃ of incubation 1h after, 4 ℃ are spent the night; Wash plate 3 times with PBST, clap and do, with confining liquid sealase target, 37 ℃ of incubation 1h wash plate 3 times with PBST, clap drying getting; Said confining liquid is 5% skimmed milk; Said coating buffer is the carbonate buffer solution of pH 9.6.
5. the FAVI antibody indirect ELISA detection kit based on penton albumen according to claim 1 is characterized in that: said goat-anti chicken IgG ELIAS secondary antibody is the goat-anti chicken IgG with horseradish peroxidase-labeled.
6. the FAVI antibody indirect ELISA detection kit based on penton albumen according to claim 1 is characterized in that:
Said negative standard serum is the serum that SPF chicken embryo is hatched 2～5 Japanese instar chicklings that produce;
Said positive criteria serum is the SPF chicken serum behind twice of mode collunarium, eye droppings that I crowd's aviadenovirus simulating nature infects;
Said cleansing solution is the phosphate buffer that contains 0.05% Tween-20, and pH is 7.4;
Said dilution is 1%BSA;
Said substrate solution is TMB-H
2O
2Solution;
Said stop buffer is 2M H
2SO
4
7. the FAVI antibody indirect ELISA detection kit based on penton albumen according to claim 4 is characterized in that: said phosphate buffer is by NaCl 8.0g, KCl 0.2g, KH
2PO
40.2g, Na2HPO
4.12H
2O2.9g, Tween-20 500uL, adding distil water is settled to 1000mL, transfers pH value to 7.4 to process.
8. the FAVI antibody indirect ELISA detection kit based on penton albumen according to claim 4 is characterized in that: said TMB-H
2O
2Solution is by TMB damping fluid 10ml, TMB solution 0.5ml, H
2O
232ul forms; Said TMB buffering is by Na
2HPO
418.27g citric acid 4.665g with the 900mL dissolving, transfers PH to 5.5, adds water to 1000mL and processes; Said TMB solution is processed by 5mL anhydrous alcohol solution TMB 10mg.
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| CN106018780A (en) * | 2016-05-17 | 2016-10-12 | 扬州大学 | F1-protein-based indirect immunofluorescence kit for detecting type 4 fowl adenovirus antibody |
| CN106443015A (en) * | 2016-09-21 | 2017-02-22 | 扬州大学 | ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting fowl adenovirus antibody based on hexon protein N-terminal conservative area |
| CN106755106A (en) * | 2017-01-06 | 2017-05-31 | 华中农业大学 | The preparation method and application of Ankara subunit vaccine |
| CN107102138A (en) * | 2017-04-14 | 2017-08-29 | 杨凌职业技术学院 | Detect indirect ELISA reagent kit and its detection method and its application of I group I fowl adenovirus antibody |
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| CN103323591A (en) * | 2013-07-16 | 2013-09-25 | 广西壮族自治区兽医研究所 | Enzyme linked immunosorbent assay kit for detecting Mycobacterium bovis antibody and preparation method thereof |
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| CN107365364A (en) * | 2016-05-12 | 2017-11-21 | 兰州雅华生物技术有限公司 | A kind of quick detection kit of Adenovirus Antigen preparation method and the detection adenovirus antibody prepared using the antigen |
| CN106018780A (en) * | 2016-05-17 | 2016-10-12 | 扬州大学 | F1-protein-based indirect immunofluorescence kit for detecting type 4 fowl adenovirus antibody |
| CN106443015A (en) * | 2016-09-21 | 2017-02-22 | 扬州大学 | ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting fowl adenovirus antibody based on hexon protein N-terminal conservative area |
| CN106755106A (en) * | 2017-01-06 | 2017-05-31 | 华中农业大学 | The preparation method and application of Ankara subunit vaccine |
| CN107102138A (en) * | 2017-04-14 | 2017-08-29 | 杨凌职业技术学院 | Detect indirect ELISA reagent kit and its detection method and its application of I group I fowl adenovirus antibody |
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Application publication date: 20120509 |