Preparation And Application Of Monoclonal Antibodies Against Non-structural Protein 3ABC Of FMDV

Foot and Mouth Disease is a drastic infectious disease, which deeply thread the cultivation industry. Except Oceania, North America, it is epidemic in the whole world. OIE and FAO set the Food and Mouse Disease as the first one of class-A infectious diseases.In developed country, killing all infected and exposed animal could be the first choice, but in developing country, vaccination is more acceptable. Thus the way of distinguishing the natural infected animals and vaccinated animals is crucial. It is also an evidence to judge the existence of FMD for OIE.Now, lots of paper support the idea that non-structure protein can discriminate the vaccinated animals and infected animals. Now scientists believe that 3ABC-antibody detection is the most reliable method. It is a trust worthy evidence about FMDV infection in any case. In the whole animal group, the infection condition of vaccinated animals can be detected by 3ABC-antibody detection. The routine indirect ELISA usually has enormous false positive induced by the E. coli protein remnant. In this study, 3ABC-antibody detection method by competitive ELISA and indirect-trapping ELISA is established by making monoclonal antibody. Examined by clinical experiment, it has high sensitivity and specialty.1. Generate the monoclonal antibody of FMDV 3ABC proteinUsing purified 3ABC protein as antigen to immune alb/C rates, then spleen cell and myeloma cell SP2/0 were fused. The indirect ELISA method was employed to screen the results. The positive cells were cloned by Limited dilution. After 3 times confluences, 3 lines of Hybridoma cell line which can constant secret antibody of 3ABC, named 1D 1, 1E2, 2E3. They were proved to be Hybridoma cells by Chromosome analysis .The highest titer in all three was employed and all the monoclonal antibodies were proved to be specific with none consensual reaction against vector protein.2. Generate the indirect-trapping ELISA and the prime applicationThe neutralizing monoclonal antibody (Mab) to 3ABC of FMDV was used as trapping antibody in a Mab-linked indirect ELISA. All the items which may influence the reaction were perfected, including temperature in each steps and final concentration of each solution. At last the best way of this method was found, which provide the foundation of next step---promote the ELISA kit.3. Generate the competitive ELISA and the prime application The competitive ELISA method used to detect the antibody of 3ABC was established by using 3ABC protein as antigen and the monoclonal antibody as second antibody. All the items which may influence the reaction were perfected, including temperature in each steps and final concentration of each solution. At last the best way of this method was found, which provide the foundation of next step---promote the ELISA kit.