development, characterization and application of monoclonal antibodies against fish nervous necrosis virus

• Main Authors: Shieh Jing-Ru, 謝淨如
• Other Authors: 齊肖琪
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/08287812324566429217

碩士 === 國立臺灣大學 === 動物學研究所 === 90 === Abstract Monoclonal antibodies (MAbs) against grouper nervous necrosis virus (GNNV) were established, characterized and applied for diagnosis system and analyzing the antigenic relationship of fish nodaviruses. Five MAbs (2E, 9B, 9D, 10G, 12E) with high 50% neutralizing dose (ND50) were cloned and characterized. Two of them belong to IgG1 and three of them belong to IgM. In western immunoblot assay, two MAbs (9D,10G) can react with the denatured capsid protein derived from GNNV-infected GF-1 cells, while the other three MAbs (2E, 9B, 12E) can not. MAb 9D and 10G were suggested to react with the carboxyl group of viral capsid protein due to the negative result of enzyme-linked immunosorbent assay (ELISA) which coating antigen was recombinant viral capsid protein expressed by E coli. MAb 9D accompanied with GNNV-specific rabbit antiserum were applied for developing a double-antibody sandwich ELISA system. This system can detect 2.5-5 ng / 0.1ml purified viral protein or 5x103 TCID50 / 0.1ml GNNV supernatant. All Taiwan NNV isolates derived from different species of cultured fish were proved to be RGNNV genotype by comparing the nucleotide sequence of capsid protein with four NNV genotypes, and were significantly neutralized by the five MAbs except the isolate from Chinese catfish. Moreover, MAbs 2E and 10G could neutralize four NNV genotypes, but MAbs 9B, 9D and 12E could neutralize three (RGNNV, BFNNV, TPNNV). According to the results of neutralization test and the alignment of deduced amino acids of the NNV isolates, the neutralizing epitopes recognized by MAb 2E was suggested to contain the valine at location of 124th a.a.. The neutralizing epitopes recognized by MAb 9D, 10G was suggested to be carboxyl groups of capsid protein.