Development Of ELISA Mehods And Kits For Rapid Detection And A Colloidal Gold Immunochromategraphic Srip For Detection Of Sulfonamides

4-aminobenzoate （PBPA） acetylamino-benzenesulfonyl chloride（ASC） as raw material, afternucleophilic substitution, ester hydrolysis reaction synthetic sulfa drugs mother nucleus structurebenzoic aminobenzene sulfonamide （SH）, he application of mass spectrometry（ESI-MS） and protonnuclear magnetic resonance spectroscopy（¹H-NMR） identification of SH. Mass spectrometry resultsshow that the [M-]=291.03766with the theoretical value of the [M]=292match, indicating the successof SH synthesis; NMR （¹H-NMR） data match the structure of SH compound.Using the mixed anhydride method, the laboratory synthesis of sulphur amine medicine mothernucleus structure （SH） was conneted with ovalbumin （OVA） and bovine serum albumin （BSA）,synthetic coating antigen OVA-SH and immunogen BSA-SH. Ultraviolet scanning （UV） andSDS-PAGE methods were established. Immune Balb/C mice by BSA-SH, indirect ELISA and indirectcompetitive ELISA method for screening of cell fusion rat by cell fusion standby; hybridomatechnology to establish the SH cell line. In Balb/C mice’ vivo induced ascites prepared SH Mab. Theresults showed that BSA-SH artificial antigen was synthesized successfully, SH and BSA conjugationratio of about11.6:1.5G₁,5G₂and8D₂3strains sensitive to specific hybridoma cell line wassuccessfully screened. Their cell titer of the supernatant solution were1:6.40×10²,1:1.28×10₃and1:3.20×10², ascites titer were1:2.56×10₅,1:5.12×10₅and1.28×10₅. The Mab of5G₂showed goodsensitivity with an IC₅₀of82ng/mL to SH. The crossreactive rates of the Mab after purified toSulfamonomethoxine （SMM）, Sulfametoxydiazine（SMD）, Sulfamerazine（SM）, Sulfadimethoxine（SDM）, Sulfacetamide （SCT）, Sulfaclozine sodium monohydrate（SCZ）, Sulfathiazole（ST）,Sulfamethoxazole（SMZ）, Sulfaphenazole（SPH）, Sulfamethazine（SM₂）, Sulfasulfonamide（SN） andSulfaquinoxaline （SQ） were108%,86%,10₅%,85.6%,72%,26%,32%,54.6%,43%,4.6%,0.5%and76%. SH Mab of high-titer, sensitivity and specificity has been generated.The egg albumin （OVA） and the nucleus of sulfanilamide artificial hapten （SH） by mixed anhydridemethod, connecting the feed ratio of1:2of the package prepared by the former （SH-OVA）, afterhorseradish peroxidase （HRP） and SH-OVA using sodium iodide method by connecting the feed ratioof2:1, that was prepared Enzyme labeled antigen （SH-OVA-HRP）. Direct competitive ELISA methodis determined by the drug competition test and establish the standard curve of Fang Chengcheng in0-1000ng/mL concentration range of direct competitive ELISA was y=-0.4016x+1.2187, correlationcoefficient R²=0.97, half inhibitory concentration (IC₅₀) of61.60ng/mL. SH ELISA Kit preliminarilydeveloped prototype. Every2months the stability of random sampling,4months after the stability isgood.A step strip test descirbed in this study was developed by means of a competitive immunoassay inwhich the detector reagent consisted of colloidal gold particles coated with purified monoclonalantibodies. Colloidal gold partical with a diameter of20nm was used in production of colloidal goldprobe under a pH value of7.8. The capture reagent in the assay was a SH-OVA conjugate which wasimmobilised on the lateral flow membrane of the strip. The control line reagent was Goat anti-mouseIgM. Then the materials and methods were optimized to obtain the testing strip.