Preparation Of The Monoclonal Antibodies Against CA16 VP1 And The VP1 Epitope Analysis

Coxsackievirus A16?CA16?can cause outbreaks and epidemics of hand-foot-mouth disease?HFMD?in infants and preschool children worldwide.Up to now,there is no pretty effective vaccine or antiviral drugs for anti-CA16 related diseases,and its multiple and complex genotypes also pose challenges to the clinical diagnosis and detection of CA16.This paper is dedicated to the preparation of monoclonal antibodies against CA16 VP1,which not only lays a foundation for the development of preventive or therapeutic anti-CA16virus drugs,but also provides an important basis for the specific methods to detecting CA16.In this paper,the reaction conditions of experimental steps were optimized,including the concentration of CA16 virus antigen and serum which will to be tested,the type and concentration of the blocking reagent in ELISA,the temperature and time of the blocking,the reacting time of DON-HRP-conjugated antibodies,the reacting time of the H₂O₂substrate solution,so as to establish a specific indirect ELISA detection method for the preparation of CA16 VP1 monoclonal antibodies.Monoclonal antibodies of CA16 VP1 were prepared by immunizing mice with pGEX-CA16-VP1 recombinant protein antigen and hybridoma technique,as a result,3 ideal monoclonal cell lines were obtained:CA16 VP1-1A4?CA16 VP1-1C8?CA16 VP1-4E3.The full length of 891bp CA16 VP1 gene was obtained by RT-nPCR and sequenced successfully.At the same time,the antigen epitopes and other biological characteristics of CA16 VP1 were analyzed by using bioinformatics softwares.EMBOSS combined with Protean predicted 11 B cells antigen epitopes of CA16VP1,and SYFPEITHI predicted that the VP1 has six peptides,contianing KMTDPPAQV,RVYMRIKHV,NLIETRCVL,MMGTFSIRT,WQTATNPSV and FDAEFTFVV,which may be the optimal MHC epitopes of CA16 VP1.In this experiment,the self-made monoclonal antibody of CA16 VP1 was purified by gel chromatography and labeled with fluorescein.The titer was reach to 1.28×10⁴ which determined by ELISA method,and the state of purified fluorescence monoclonal antibodies was good.At the same time,according to the gradient dilution method,1:32 was determined as the optimal dilution ratio of fluorescence antibodies.Through direct immunofluorescence,it was verified that fluorescent monoclonal antibody did not react cross with EV71 and CB3,indicating that it had good specificity.According to the experimental results,the indirect ELISA method established in this study could effectively detect the CA16 VP1 antibodies,and the prepared monoclonal antibodies of CA16 VP1 had good quality,high titer and obvious specificity,which could be applied to the rapid detection of the viruses,furthermore,to the separation and identification of viruses.