A kind of universal foot-and-mouth disease virus structural protein antibody and its blocking ELISA detection kit
A technology of foot-and-mouth disease virus and structural protein, which is applied in the field of foot-and-mouth disease virus structural protein antibody and its blocking ELISA detection kit, which can solve the problems of FMDV antibody detection method obstacles, complex antigen structure, and scarcity of monoclonal antibodies, and eliminate non-specificity Reaction, good display of antigen structure, simple effect of reagent composition
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[0030] In the present invention, the preparation method of the monoclonal antibody E32 preferably includes the following steps: combining the coding sequences of the heavy chain variable region and the light chain variable region of the antibody E32 with the bovine IgG2 heavy chain and light chain respectively After the constant region sequences were ligated, they were respectively inserted into the pcDNA3.4 eukaryotic expression vector, and the heavy chain and light chain expression plasmids were mixed in proportion to transfect CHO suspension culture cells, and the complete IgG2 subtype antibody was expressed, and the antibody was purified by affinity chromatography. For the preparation method of the antibody E32, please refer to the description in the patent document application number CN201810929067.1.
[0031]The present invention provides a general monoclonal antibody composition of foot-and-mouth disease virus structural protein, comprising said monoclonal antibody E32 a...
Embodiment 1
[0056] 1. Materials
[0057] Carbonate buffer (Na 2 CO 3 / NaHCO 3 ) and gelatin were purchased from SIGMA.
[0058] BCA protein concentration assay kit was purchased from Beyotime Company.
[0059] EZ-Link Plus Activated Peroxidase Kit was purchased from Thermo Company.
[0060] Horseradish peroxidase-labeled avidin was purchased as a commercial reagent (Genscript product).
[0061] Both FMDV broad-spectrum monoclonal antibodies F104 and E32 were developed and stored in our laboratory using the single B cell antibody preparation technology. The antibody preparation method is described in the patent document CN201810929067.1 (obtained patent number ZL201810929067.1).
[0062] The control serum and the inactivated antigens of type O and A viruses were prepared and preserved by the National Reference Laboratory for Foot-and-Mouth Disease.
[0063] The Asia1 type inactivated antigen of foot-and-mouth disease virus is the reserve inactivated antigen produced in 2015.
[0064...
Embodiment 2
[0093] 1. Serum samples
[0094] Clinically healthy animal serum samples include: 127 healthy bovine serum samples and 30 healthy pig serum samples from clinically healthy cattle, tested negative for O, A, Asia1 type structural protein antibodies and non-structural protein antibodies; 87 sheep serum samples The samples and 88 swine serum samples were from non-immune healthy sheep and pigs in epidemic-free areas. Serum samples from infected animals included: 121 bovine serum samples from 3 to 230 days after infection with type O or A FMDV challenge; 79 sheep serum samples from 7 days to 1 year after challenge with type O FMDV; After 7 days to 6 months, 80 pig serum samples were collected. Quality control sera include: O, A, Asia1 FMDV-infected pigs, cattle, and sheep sera, each of which were tested positive for non-structural protein antibodies.
[0095] 2. Determination of the optimal reaction conditions of the FMD virus structural protein antibody blocking ELISA detection m...
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