Preparation And Characterization Of Monoclonal Antibodies Against PRRSV HH08Strain And Establishment Of Antigen-capture ELISA

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent ofporcine reproductive and respiratory syndrome (PRRS), which leads to reproductive failure insows and respiratory problems in piglets, and spreads rapidly. Sensitive and effective ELISA testmethod was developed for detecting PRRSV and the method has vital significance and applicationvalue for the PRRS research on diagnostics and immunology.The BALB/c mice were immunized with purified PRRSV HH08strain antigen, once in twoweeks. Until the serum antibody titer up to1:10000or more, the spleen cells from immunizedmice were fused with SP2/0myeloma cell after final boost. Five hybridoma cell lines wereobtained by the indirect ELISA detection and limited dilution cloning, and named as A7, B4, B8,B12, G12. Then ascites of A7, B4, B8, B12were prepared, and ascites of A7and B8were purified by caprylic acid-saturate ammoniumsulphate method, and SDS-PAGE analysis indicated that therewere H chain and L chain obviously without other visible proteins. The ELISA titers of McAbs ofascites, were higher than those of culture supernatants of hybridoma cells, which were1:6400~1:51200versus1:256~1:2048respectively. The specific band between97.2~66.4kDa wasshowed and this indicated the McAbs reacted with PRRSV but not with Marc-145cells by Westemblot analysis，the same positive results of IFA demonstrated that the McAbs specifically recognizedPRRSV. The subtype of A7, B8, B12were IgG2b, and B4and G12were IgM. The averagechromosome number of the five hybridoma cells were84~100, which was much more than that ofSP2/0cell. Specificity analysis revealed that the McAbs reacted only with PRRSV, not with otherreference swine-originated pathogens.Rabbits were immunized with PRRSV to generate rabbit-anti-PRRV polyclonal antibodies.An antigen-capture ELISA (AC-ELISA) was established for the detection of PRRSV, in whichpolyclonal antibody was used as capturing antibody and mouse-anti-PRRSV McAb of B8asdetecting antibody. The results showed that the optimal coating concentration of rabbit-anti-PRRSV was40μg/mL, the optimal working concentration of B8McAb was1.25μg/mL, the reaction time of sample was90min, and the optimal dilution of HRP-labeledgoat-anti-mouse IgG was1:5000. The cutoff value was0.175(OD490nm). The coefficient ofvariation of reproducibility was less than10%, and at least1735TCID50PRRSV could bedetectable. The result of ELISA showed no cross-reactions with Marc-145cells and other referenceswine-derived virus. The agreement rate from eleven clinical infected samples detected by theELISA and RT-PCR was high. The results revealed that the AC-ELISA possessed good specificityand higher sensitivity, indicating a suitable method for rapid detection of PRRSV.