Preparation And Identification Of McAbs Against Ciguatoxin

Ciguatoxins (CTXs) are kinds of marine toxins exist in coral fishes. A trace residual might lead to ciguatera poisoning. Establishment of an effective method to detect CTXs rapidly and sensitively becomes necessary.Monensin, an analogue of CTXs, was used to prepare CTXs artificial antigen as a substituent. Hybridomas were obtained by the fusion of spleen cells and SP2/0 cells. Large amout of monoclonal antibodies (McAbs) were purified from mice ascites to establish the immunoassays.Mixed anhydride and EDC methods were used for coupling monensin with BSA/OVA to prepare immunogen/coating antigen of CTXs respectively. Spleen cells from Balb/c mice immuned with immunogen were fused with SP2/0 cells to produe hybridomas. Four times of subcloning were carried out by limited dilution and cell lines secret antibodies specific to CTXs were piceked out. McAbs were purified by Octylic acid-ammonium sulfate precipitation method from the ascites induced by hybridomas in vivo. Then a rapid method, indirect competitive ELISA, was established with CTXs McAbs.Results:(1) The immunogen and coating antigen were synthesized successfully and identified by TLC and SDS-PAGE, and the coupling ration of monensin to BSA and OVA were 18.5:1 and 7:1 respectively. (2) Three hybridoma cell lines secret McAbs against ciguatoxin named 1D5,2G11 and 3G11 were obtained. Subtypes and light chain types of all the three cell lines were IgGl andκchain. (3) Ascites McAbs after purification were separated to two chains by SDS-PAGE. Molecular weight of the heavy chain and light chain were about 55KD and 28KD. The content of antibodies from the ascites was about 4.127mg/mL. (4) Conditions for competitive ELISA were as followed:concentration of coating antigen was 5μg/mL for 2 h; 3% defatted milk powder buffer for 1 h; McAbs titer was 1:6400; methanol concentration was 10%; dilution of HRP was 1:10000; coloring for 15 minutes. (5) The linear regression line equation was y= 10.722x+25.399, correlation coefficient was R2=0.9939. The ELISA experiment also indicated that the linear detecting the range of calibration curve for CTXs was 0.12ng/mL～15.6ng/mL.The detection limitation was 0.12ng/mL. (5) Specificity of the McAbs were proved by the across reactivities with Monensin, Maduramicin and Hainanmycin. and there were merely cross-reaction with Maduramicin and Hainanmycin, except for Monensin.This research established a meaningful idea for immunoassays of CTXs.