Prepareration Of Monoclone Antibody And Establishment Of Immunology Rapid Test Method Of Sulfadimethoxine

Sulfonamides (SAs) are mainly applied to prevent and cure bacterial infection andusually used as feed additive of animals. Their overuse may cause anaphylacticreaction and increase their carcinogenicity. Therefore, more attention was paid to theirharm to human healthy and potential pollution to environment due to their residues inanimal food. Sulfadlmethdme (SDM) is one kind of SAs widely used in animalhusbandry. It is of great benefit to the safety of animal food to develop kinds ofsensitive, fast and simple detection methods of SDM residue.Based on the analysis of molecular structure and immunogenicity of SDM, thetechnology of monoclonal antibodies production was applied to assemble rapid ELISAkit and strip for SDM residual detection. The main contents and results of this studywere as follows:1. The artificial immunogen BSA-SDM and the coating antigen OVA-SDM wereobtained by conjugating SDM to bovine serum albumin (BSA) and Ovalbumin (OVA)with Diazotization. The conjugation of BSA-SDM was successfully identified withultraviolet and SDS-PAGE. The high-titer, sensitive and specific SDM antiserumidentified with indirect ELISA was produced from the Balb/C mice immunized withBSA-SDM.2. One Balb/C mouse was chosen from five Balb/C mice immunized with BSA-SDM for cell fusion by the identification of indirect ELISA and blocking ELISA.Three hybridoma lines of 1B4, 3D9, and 4E1 that secrete SDM mAb were screened byindirect ELISA and blocking ELISA. The indirect ELISA titers of them were1:3.2×10², 1:5.12¹0² and 1:8.1×10² in supernatant, 1:2.56×10⁵, 1:2.56×10⁵and 1:5.12×10⁵ in ascites respectively, and the affinity constant (Ka) was 2.16×10⁹,3.28×10⁹,1.71×10¹⁰ L/mol respectively. The best one of them was 4E1, SDM mAb of4E1 showed good sensitivity with an IC₅₀ of 16.64 ng/ml to SDM and 5% CR to SMM and no CR to other compounds. SDM mAb obtained can be used to establishimmunoassay of SDM residues in animal food.3. Based on principle of the enzyme-linked immunosorbent assay and goldimmunochromatography assay (GICA), a blocking enzyme-linked immunosorbentassay (ELISA) kit and a lateral flow immunoassay (LFIA) strip were developed withSDM mAb of 4E1 and applied to the screening for SDM residues in pig urine. Thecalibration curve of SDM-Kit with standard SDM inhibitor was typical sigmoid curvefitted to the four parameters logistic equation with the linear determination of 1.0 to128.0 ng/ml. By SDM-kit, the test of SDM residues in swine urine samples could beaccomplished within 60 minutes. By SDM-strip, the test could be accomplished within8 to 10 minutes. The SDM-Kit and SDM Strip were sensitive, specific, simple andreliable, could be used for the rapid determination of SDM residues in animal food.