The Preparation And Characteristics Analysis Of Polyclonal Antibody And Monoclonal Antibody Of Vibrio Anguillarum

Vibrio anguillarum,which could attack eel,weever,trout,sweetfish,turbot,halibut,rainbow trout and prawn,causes the extensive conern of researchers.V.anguillarum is characterized by spreading widely,high attack rate and high mortality,and brings huge economic losses to fishery.V anguillarum(SMW5)was used as antigen to immune the rabbit in order to get polyclonal antibody(pAb).The titer of the pAb was 1:320000,while its sensitivity to SMW5 was 1 × 104 CFU/mL.There were weak cross-reactions between the pAb and V.parahaemolyticus(ATC17802),Aeromonas hydrophila(JS01-1)and Plesiomonas shigelloides(LCCi190625).Western-bolt analysis showed that the pAb could bind with the proteins with molecular weight as 40 kDa and 25-35 kDa.Vanguillarum(SMW5)inactivated by formaldehyde was used to immune Balb/C mice.Using monoclonal antibody technology by cell fusion,screening and limiting dilution cloning,3 mAbs anti-Vanguillarum were prepared.The cell fusion rate was 94.6%and the positive rate was 62.4%.These mAbs were named 1H9,1C12 and 6F8,whose titer of the supernatant were 1:6400,1:3200 and 1:800.Their subtype were IgG2a?IgM?IgG2b.The sensitivity of 1H9,1C12and 6F8 was 4×103cells/mL,2×103 cells/mL and 8x 103 cells/mL.The titer of the mAb named 1H9 in ascite was1:640000.Acorrding the corss-reaction,the mAb named IC12 cross-reacted with the strains like V.Alginolyticus(Js60517NA1),V vulnificus(FJ04-L1),A.hydrophila(JS01-1).The mAb named 6F8cross-reacted with the strains like V.parahaemolyticus(ATC17802),V.harveyi(03152),V cholerae(96-2).The mAb named 1H9 didn't have cross-reaction with the strains like V.alginolyticus(Js60517NAl),V.vulnificus(FJ04-Ll),V.parahaemolyticus(ATC17802),V harveyi(03152),V.flurialis,A.hydrophila(ML316),A.hydrophila(JS01-1),A.sobria,V.cholerae(96-2),P.shigelloides(LCCi190625),Streptococus agalactiae,Escherichia coli.The results showed that the mAb named 1H9 had strong specificity.Stability test showed that these 3 mAbs all had had a good stability.The mAb named 1H9 with high titer,well stability,high sensitivity and strong specificity could be applied to the development of ELISA and gold labeled method to identify V.anguillarum.