Development Of Fast Detection Kits Against AFTB₁

Aflatoxin B1(AFTB1)has strong toxicity, which was ranked A carcinogen by international cancer organization in 1988. The production of milk and eggs will be decreased when animals intake fodder polluted by AFTB1.AFTB1 is harm to the people,and it also influence the economy. It is necessary to develop a sensitive, specific, quick and reliable detection method in order to monitor the content level of Aflatoxin B1. The aim of our study is to prepare the monoclonal antibodies against AFTB1 and lay a foundation of development ELISA method for detection of AFTB1.This study consulted many documents, improved their methods,and prepared AFTB1 and OVA combination,which was used for clinial in indirect ELISA. Mice used for production of hybridomas were six-week-old BALB/C mice and were immunized as follows.Mice were inoculated feet-pad with 10ug AFTB1-BSA antigen in complete Freund's Adjuvant and 4 weeks later with AFTB1-BSA in incomplete Freund's adjuvant.At least 3 weeks after the last injection the mice were injected intravenously or intraperitoneally(IP) and 3-4 days later the spleens were harvested.Test method of hybridomas must be established before immunity programme completed.The study definited some reaction conditions through chess-board test,such as,coating antigen 1:100,mouse anti-AFTB1 serum 1:5000 as PC,mouse normal serum 1:100 as NC,horseradish peroxidase comjugated goat anti-mouse IgG 1:400 .Using the fusion of murine splenocytes with SP2/0 plasmacytoma cells to obtain monoclonal mouse antibody(McAb) against AFTB1, indirect ELISA to filtrate masculine cells. We couldn't obtain stably masculine cells through 8 fusions and many times filtrate, and we had to prepare pdyclonal antibody(pAb).AFTB1-BSA used for immunization of rabbits,collected serum. The pAb was obtained by purify the ascitic fluid with sediment method. The characters of pAb were detected as follows. The pAb was specificity against AFTB1 by western blotting.The titers of pAb is1:2.56×105 measured by indirect ELISA.Through indirect competition method,we got the least detection concentration of AFTB1 , 0.05μg/ml.