The Preparation Of Anti-sulfadiazine Monoclonal Antibody And The Establishment Of Chemiluminescen Enzyme Immunoassay

Sulfadimidine(SM2,Sulfadimidine)is a kind of synthetic antibacterial sulphonamide,whose antibacterial effect is quite obvious when used together with antibacterial synergist.Because of its low cost,it is used as additive drugs in livestock and poultry disease treatment,often by increasing the dose.Yet,this also leads to sulfadimidine residue in animal foods and people often suffer fromacute allergy and food poisoning after eating such foods.In view of this,the government stiputlates that the maximum residue limit for sulfadimidine in animal foods is 100 ?g/kg.So it is necessary to develop a rapid,sensitive and low-cost detection method.Featured in sensitivity,rapidity and low interference,chemiluminescence,a new kind of detection technology,has demonstrated its distinct advantage in the field of testing.In this study,synthetic antigen of sulfadimidine(SM2-BSA)was used toimmunize five mice with the dose gradient,five times in all.After the fifth time,serum titers in the mice were determined by indirect ELISA method.The serum titers reached 1:4000 and above,among which serum titer of the fifth mouse,with a dose of 100 ?g,reached as high as 1:8000.Indirect competitive ELISA method was developed for the determination of the serum and its IC50 values were between 10 and 100 ug/L,so the fifth mouse was,in the end,picked to undertake cell fusion.PEG 4000 as fusion agents,the fifth mouse's skin cells were fused with the Sp2/0 cell to conduct cell fusion;the fusion rate was 71.4%.Out of the fused cells two strains of positive cell lines were obtained after 3 times of cloning,the culture of one of which was then expanded and named 1E7.Subclass identification was done on the antibody of the cultured strain,and the result was IgG2b;the supernatant titer was 1:512.Ascites was prepared by in vivo method,and after a week it was collected and purified.The titer,affinity,sensitivity and specificity of the purified antibody were then detected.Its affinity constant was 0.12xl07 mol/L;the rates of cross reaction between the antibody and sulfaguanidine,sulphadiazine,sulfanilamide five a pyrimidine,penicillin,ofloxacin,chloramphenicol and diethylstilbestrol were lower than 0.01%.The chemiluminescent enzyme immunoassay method was developed from the purified antibody to detect SM2,and optimization was done on the condition of package,the time of package,sealing material,sealing time,dilution multiple and competition reaction time of the first antibody,dilution multiple and competition reaction time of the second antibody.Finally,the optimized conditions were used to establish the regression equation,which was y=-22.13x+63.338,R2=0.9904,and IC5o=4.006 ?g/L.The curve displayed good linear relationship between 0.1 and 1000 ?g/L,and the method detection limit was 0.17 ug/L.with recovery rates ranging from 94.42%to 102.73%.This studied method,when compared with the national standard and the kits on market,displayed obvious advantages in detection limit,sensitivity,detection range and recovery.Then,36 of the samples were picked to compare the studied method with the ELISA kits on sale in detection.The comparison demonstrated that two methods had the same number of positive and negative results,and statistical analysis of the results was P=0.684>0.05,which meant the differences between the results from the two methods were small.Detections of the 141 collected samples with this method showed that 91.4%of them were up to standard.In this study two hybridoma cell lines were successfully screened through cell fusion technology,which could secrete antibodies against sulfadimidine.Through in vivo induction one was used to prepare ascites,and the ascites were then purified by applying affinity chromatography methods,after which the obtained antibody was used to establish chemiluminescent enzyme immunoassay method for the detection of sulfadimidine.Research results show that this studied method is obviously superior in detection limit,detection range and recovery to the national standard and the kits on the market.It can actually meet the national requirements in detecting sulfadimidine residue in animal foods.This study lays a foundation for developing reagent kit in the future.