Preparation Of Monoclonal Antibodies Against VP1 Protein Of Foot-and-mouth Disease Type A And Establishment Of A Monoclonal Antibody Based Competitive ELISA

Foot-and-mouth disease(FMD),which was also called as "aphtha",is an infectious disease affecting cloven-hoofed animals,in particular cattle,sheep,pigs,goats and deer.It causes fever,followed by the development of blister and erosion,chiefly in the mouth and feet.FMD is a severe plague for animal husbandry and the international trade of animals,since it is highly infectious and spread fast.In recent years,the foot-and-mouth disease type A transmitted from Southeast Asia to East Asian countries,which could be a threat to husbandry of China.Therefore,It is necessary to development a simple,specific detection method for monitoring antibodies to foot-and-mouth disease type A.VPl gene of Foot-and-mouth disease virus type A was amplified by specific primers,then expression plasmid PET-3 2a-VP 1 and pGEX-6p-l-VPl were constructed and transformed into E.coli BL21.Fusion proteins were expressed in recombinant strains,mainly existed in the form of inclusion body by the analysis of protein solution.The SDS-PAGE showed that fusion proteins had a high purity through HIS and GST column chromatography purification.Western-blot analysis illustrate that the purified protein could react with polyclonal anti-serum of guinea pig against FMD type A,but not with type O and Asial.These results indicated that the fusion proteins had a good specificity and antigenicity.Six-week-old female BALB/c mice were immunized with purified fusion protein PET-32a-VP1.Cell fusion could not be done untill the antibody titer were above 1:12800 after three times.The positive hybridism cells were screened by an indirect ELISA coated with purified fusion protein pGEX-6p-l-VP1.Four hybridoma cell lines,which secreted McAbs against foot-and-mouth disease type A VP1 protein,were obtained after 3-4 times cells subcloned,named as ID 10,3D 12,4E12 and 5G7,respectively,these monoclonal antibody were all IgM and kappa light chain.The McAbs titers of 1D10,3D12,4E12 and 5G7 in supernatant were 1:64,1:16,1:2048 and 1:512,while the titers in ascites were 1:2560,1:20480,1:81920 and 1:1280,respectively.The saturation value of 1D10,3D12,4E12 and 5G7 were 1:20,1:40,1:40 and 1:40,while the relative affinity were 1:600,1:600,1:3000 and 1:300,respectively.The AI%of additive ELISA were all less than 50%,which demonstrated that McAbs might recognize the same antigen epitope.Blocking ELISA revealed that all the four McAbs could be blocked by FMDV-A positive serum.Western blot analysis showed that McAb could specifically react with PET-32a-VPl and pGEX-6p-l-VPl，but not with bacteria protein.Competitive ELISA was established based on purified protein pGEX-6p-1-VP1 as coating antigen and 4E12 as competitive antibody.The optimal reactive conditions and cut-off value were determined via test.The results showed that the working concentrations of coating antigen,serum,McAb and HRP-IgG were 0.625μg/mL,1:2,1:400 and 1:5000,respectively.The optimal reaction time of blocking solution,serum sample and McAb were 60min,60min and 45min.The cut-off PI%was more than 30%as positive sample.The agreement ratio was 84.8%between the competitive ELISA and a liquid phase blocking ELISA test.In summary,a competitive ELISA was established for detection Foot-and-mouth disease virus type A antibodies.The test has advantages in specificity and stability.It is a simple and rapid test and provided technical platform for immune antibody detection,monitoring and epidemiological investigation.