Purification Of Type O Foot-and-mouth Disease Virus And Preparation Of Monoclonal Antibodies

Foot-and-mouth disease (FMD) is a febrile acute disease caused by Foot-and-mouth disease virus (FMDV). FMDV mainly infects a variety of cloven-hoofed livestock and reduces animal production performance, but also restricts the market and export of livestock products. The outbreak of FMD would bring huge economic losses to the global animal trade, so FMD has always been under an extensive attention of the world. At present, the rapid and accurate diagnosis and effective vaccine play an important role in preventing and controling FMD. Therefore, development of more sensitive, rapid and accurate diagnostic reagents on prevention and control of FMD has a very important practical significance. Monoclonal antibody is high purity, good specificity and good repeatability that can be used to develop new diagnostic reagents and the basic research. This study aims to establish one or several hybridoma cell lines which can stably secrete monoclonal antibodies against type O FMDV, provide experimental material for the development of new diagnostic reagents and genetic engineering vaccines and lay the foundation for etiological study of type O FMDV.Using virus regeneration, cell culture and virus multiplication methods, the cell culture supernatant containing FMDV was obtained. In order to obtain FMDV with high purity, the cell culture supernatant was purified through a series of steps, such as inactivation, PEG precipitation, concentration and sucrose densitygradient centrifugation. Finally, SDS-PAGE and Western-blot analysis indicate that type O FMDV complete virus particle antigen with high purity and good antigenicity was obtained.To establish indirect ELISA, the best antigen concentration for coating was5.0μg/mL which was determined by checkerboard titration method. The BALB/c mice were immunized4times with the purified antigen by routine immunization. Then, the spleen cells of immunized mice were fused with myeloma cell line SP2/0cells by PEG1500to prepare hybridoma. After3times limiting dilution assay, one monoclonal hybridoma cell lines secreting antibodies against type O FMDV were screened by indirect ELISA, named5F10, respectively. In order to obtain abundant monoclonal antibodies,5F10hybridoma cell line was injected in BALB/c mice for in vivo preparation.Monoclonal antibodies from in vivo preparation were purified by bitterness-saturated ammonium sulfate precipitation. SDS-PAGE showed that the molecular weights of the heavy chain and light chain of5F10monoclonal antibody were about50.0kD and25.0kD. The5F10antibody titer in cell culture supernatant was1:512, and1:25600in abdomen liquor. The specificity cross test indicated that5F10monoclonal antibody was not specific reaction with the normal BHK-21cells, A, C or Asia I type of FMDV. Determination of antibody subtype results showed that5F10monoclonal antibody belonged to IgGl subclass.O type FMDV infected BHK-21cells preliminary study indicate that virus infected cells can be detected after2h, and the virus only exists in the cytoplasm, newly formed infectious virions begin to appear at4h after infection.In this study we successfully prepared the monoclonal antibody against type O FMDV, and provide experimental material for the development of new diagnostic reagents and genetic engineering vaccines and lay the foundation for etiological study of type O FMDV.