Preparation Of Polyclonal Antibodies For The Detection Of T-2 Toxin By Cleia

The trichothecene mycotoxin T-2 is a fungal metabolite known to contaminate stored grain and other agricultural products. T-2 toxin is the most toxic compound in Fusarium graminearum. In this paper, a competitive chemiluminescence immunoassay assay (CLEIA) with a polyclonal antibodie has been developed, including synthesis of the T-2 toxin artificial antigen, preparation of the polyclonal antibodie and establishing the mothod of CLEIA. The main results are as follows:T-2 toxin was first converted to T-2 hemisuccinate (T-2 HS) and then conjugated with bovine serum albumin (BSA) in the presence of water-soluble carbodiimide (EDPC) according to Chu's method. The thin layer chromatography (TLC) proved that T-2 toxin had been conjugated to protein (BSA/OVA). The results showed that the molecular weights of conjugates (T-2 HS-BSA and T-2 HS-OVA) are larger than those of the original proteins. The largest absorption peaks of ultraviolet scanning had shifted, it demonstrated that the preparation of T-2 toxin antigens were successful. The couple rate of T-2 toxin with BSA was 6:66, while with OVA was 10:11 from the ultraviolet scanning. The results showed the complete antigens can be used in animal immunity to further prepare T-2 toxin antibodiesUsing T-2 BSA as a source to immune BALB/c mice, and the titers of antiserum for mouse BALB/c is 1:12800 through reinforcing immunity 3 times, which is the highest. The block experiment was performed by choosing the mice blood serum with higher inhibition rates. The results showed the inhibition rates of 104ng/ml T-2 and 103ng/ml T-2 to T-2 BSA are 90.71% and 3.54%, respectively.Using the blood serum of a mice that has not been immunized as negative control, dilute immune blood serum as the following rates of 1:100,1:200,1:400,1:800,1:1600, 1:3200,1:6400 and 1:12800, respectively. And the method of the indirect competitive ELISA for T-2 was established. The results showed the optimal concentration of the coated antigen and the Balb/c mice serum dilutions in the indirect competitive ELISA were approximately 20μg/ml and 1:4000 respectively.In this paper, a competitive chemiluminescence immunoassay assay (CLEIA) with polyclonal antibodie has been developed for quantitative determination of trichothecene T-2 toxin (T-2), compared with enzyme-linked immunosorbent assay (ELISA), CLEIA have high sensitivity and detectability.