Highly sensitive Immuno-PCR assay for the detection of the circulating antigens in trichinellosis / 中国人兽共患病学报

ABSTRACT

A highly sensitive immuno-PCR assay based on sandwich ELISA and PCR was developed to detect the circulating antigen in trichinellosis. Antigens were purified from the muscle larvae of Trichinella spiralis, and the myeloma cells were fused with spenocytes immunized with T. spiralis antigens to product the specific monoclonal antibodies. Indirect ELISA was used to select the antibody-secreting hybrodoma cells. By this method of procedure, monoclonal antibody F4C6 against the T. spiralis ES antigen was obtained, which was used as the indicator antibody, while the rabbit polyclonal antibodies against T. spiralis were to be used as capturing antibodies. The plasmid Bluecript Ⅱ KS was amplified by PCR with biotin-labeled primer M13-20, and thus the biotin-labeled DNA was obtained. Both the second antibody and DNA labeled with biotin were to be linked with 100 ng/ml avidin. The whole procedures of assay consisted of two steps, in which the circulating antigens were captured by monoclonal antibody through sandwich ELISA in the first step, and the DNA linked by monoclonal antibody was amplified by PCR in the second step. The sensitivity of this method was compared with that of the ELISA assay. It was found that the measuring ranges to detect the circulating antigens in trichinellosis were 50 pg/L to 0.005 pg/L for the immuno-PCR assay, and 5 μg/L to 0.05 μg/L for ELISA assay, the former was quite higher than that of the latter. It is evident that this method is highly sensitive for the detection of circulating antigens in trichinellosis.