Prepareration Of Monoclonal Antibody And Establishment Of Immunological Rapid Test Method Of Sulfamethazine

Sulfonamides (SAs) are mainly applied to prevent and cure bacterial infection and usually used as feed additive of animals. Their overuse may cause anaphylactic reaction and increase their carcinogenicity. Therefore, more attention was paid to their harm to human healthy and potential pollution to environment due to their residues in animal food. Sulfamethazine (SM2) is one kind of SAs widely used in animal husbandry. It is of great benefit to the safety of animal food to develop kinds of sensitive, fast and simple detection methods of SM2 residue.Based on the analysis of molecular structure and immunogenicity of SM2, the technology of monoclonal antibodies production was applied to assemble rapid ELISA kit for SM2 residual detection. The main contents and results of this study were as follows:1. The artificial immunogen BSA-SM2 and the coating antigen OVA-SM2 were obtained by conjugating SM2 to bovine serum albumin (BSA) and Ovalbumin (OVA) with Diazotization. The conjugation of BSA-SM2 was successfully identified with ultraviolet and SDS-PAGE. The high-titer, sensitive and specific SM2 antiserum identified with indirect ELISA was produced from the Balb/C mice immunized with BSA-SM2.2. One Balb/C mouse was chosen from five Balb/C mice immunized with BSA-SM2 for cell fusion by the identification of indirect ELISA and blocking ELISA. Three hybridoma lines of2H10, 2E2, 2A10and 2G5 that secrete SM2 mAb were screened by indirect ELISA and blocking ELISA. The indirect ELISA titers of them were 1:6.4×102, 1:6.4×102, 1:3.2×102 and 1:3.2×102 in supernatant, 1:1.28×105, 1:6.4×104, 1:3.2×105 and 1:2.56×105 in ascites respectively, The best one of them was2G5, SM2 mAb of 2G5 showed good sensitivity with an IC50 of 5.82 ng/ml to SM2.3. Based on principle of the enzyme-linked immunosorbent assay a blocking enzyme-linked immunosorbent assay (ELISA) kit was developed with SM2 mAb of 2G5 and applied to the screening for SM2 residues in pig urine. The calibration curve of the SM2-Kit with standard SM2 inhibitor was typical sigmoid curve fitted to the four parameter logistic equation with the linear detection of 0.625 to 80.0μg/L(R2=0.9911),the sensitivity of 0.9μg/L,the IC50 of 5.82μg/L and the detection limit of 1μg/L. The recoveries of SM2 spiked in pig feed were 83.4%,in pig urine were 89.5%. The precision and accuracy of the assay as determined by inter-assay and intra-assay coefficient variation was both below 15%. The SM2-Kit generally had 2.08% cross-reactivity towards sulfamerazine and little or no cross-reactivity towards other sulfonamides. The dilution solution of SM2 had no effect on results of SM2-Kit. The validity of SM2-Kit in 4℃was above six months.