Enzyme linked immunosorbent assay (ELISA) kit of foot-and-mouth disease virus antibody and preparation method thereof
A technology of foot-and-mouth disease virus and kit, applied in the field of biotechnology detection, can solve the problems of high non-specific background reading value, low sensitivity of peptide ELISA, affecting the combination of peptide and target antibody, etc., achieves wide application prospects, is economical and convenient, reduces non-specific Effect of idiosyncratic background readings
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[0040] The preparation method of the coupling polypeptide adopted in the present invention is as follows:
[0041] 1) Screening of reactogenic peptides of foot-and-mouth disease virus
[0042] For the selection and determination of effective peptides, the antigenic epitopes of porcine foot-and-mouth disease virus proteins are predicted by DNAStar, Genebank, ProtScale and other analysis software, the amino acid sequence of the natural antigen is determined, and then the epitopes targeted by the peptides are searched , use ELISA to detect the reaction of different polypeptides and porcine foot-and-mouth disease virus antiserum, finally select the foot-and-mouth disease virus reactogenic polypeptide with good response, adopt the following polypeptides screened out as follows in the present invention:
[0043] P1: Lys Tyr Ser Asp Ala Arg Val Ser Asn Val Arg Gly Asp Leu Arg Val Leu AlaGln Lys Ala Glu Arg Ala Leu Pro Thr Ser Ser Asn Tyr;
[0044] P2: Lys Tyr Ser Asp Ala Arg Ala Ser...
Embodiment 1
[0064] An ELISA kit for foot-and-mouth disease virus antibody, prepared by the following method:
[0065] 1) Select any one of the above-mentioned conjugated polypeptide antigens and dilute it with a coating solution at a concentration of 5 μg / ml (the coating solution is 1.59g Na 2 CO 3 , 2.93 g NaHCO 3 Dissolve in 800mL double-distilled water, adjust the pH value to 9.6, and make up to 1000mL), then coat the diluted conjugated polypeptide antigen on a 96-well plate, 100μL per well, coat overnight at 4°C, and wash with PBST the next day Wash with 250 μl / well for 3 times and pat dry, block with 150 μl / well of blocking solution at 37°C for 1 hour, wash with 250 μl / well of washing solution PBST for 3 times, pat dry, put in a vacuum bag, add desiccant, vacuumize, 4°C save;
[0066]2) Preparation of Antibody Diluent (AD): Weigh 1.0g BSA (0.1%), add PBS to 1000ml, store at 4°C;
[0067] 3) Preparation of substrate liquid chromogenic solution: 4ml TMB (50mg TMB dissolved in 10mL ...
Embodiment 2
[0073] An ELISA detection kit for foot-and-mouth disease virus antibody, prepared by the following method:
[0074] 1) Select any of the above-mentioned conjugated polypeptide antigens and dilute it with a coating solution at a concentration of 1 μg / ml (the coating solution is 1.59g Na 2 CO 3 , 2.93 g NaHCO 3 Dissolve in 800mL double-distilled water, adjust the pH value to 9.6, and make up to 1000mL), then coat the diluted conjugated polypeptide antigen on a 96-well plate, 100μL per well, coat overnight at 4°C, and wash with PBST the next day Wash 2 times with 250 μl / well and pat dry, block with 150 μl / well of blocking solution at 37°C for 1 hour, wash 4 times with 250 μl / well of washing solution PBST, pat dry, put in a vacuum bag, add desiccant, vacuumize, 4°C save;
[0075] 2) Preparation of Antibody Diluent (AD): Weigh 1.0g BSA (0.1%), add PBS to 1000ml, store at 4°C;
[0076] 3) Preparation of substrate liquid chromogenic solution: 4ml TMB (50mg TMB dissolved in 10mL D...
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