Study On The Method Of Olaquindox Immunoassay

A polyclonal antibody capable of specifically recognizing 3-methylquinoxaline-2-car-boxylic acid(MQCA)was prepared and an indirect competitive enzyme linked to the detection of 3-methylquinoxaline-2-carboxylic acid was established.Immunoassay(ELISA)method;a method for colloidal gold 3-methylquinoxaline-2-carboxylic acid immunochrom-atographic test strip was established by using anti-3-methylquinoxaline-2-carboxylic acid monoclonal antibody.Through the design and synthesis of 3-methylquinoxaline-2-carboxylic acid hapten and artificial immunogen,the high titer and specificity of MQCA polyclonal antibody were successfully prepared,and an indirect competition for rapid detection of MQCA was established.Enzyme-linked immunosorbent assay(ELISA),the optimal working conditions of MQCA indirect competition ELISA were determined by optimizing the reaction conditions of the method:the original coating amount was 0.25 ?g/well;the antibody was diluted 2000 times;HRP-goat anti-rabbit IgG enzyme label The secondary antibody was diluted 25,000 times;the MQCA standard and the antibody dilution were pH 7.4 PBS buffer solution;the blocking solution was 0.5%skim milk powder;the MQCA polyclonal antibody and its structural analogs did not cross-react and the specificity was good.The method sensitivity(IC50)was 20.86±0.313 ?g/L;the method detection limit(IC15)was 1.68 ±0.043 ?g/L;the intraplate variation coefficient(CV)of the method was less than 10%,and the inter-plate variation coefficient was less than 15%.Good precision;through the sample matrix interference elimination experiment,it is determined that the dilution of the bovine and pork samples can eliminate the matrix effect by 10 times;at the three spiked levels of 50,200,500 ?g/kg,the recovery rate of the ELISA method is 90%-100%,the confirmation test of the sample by HPLC method,the indirect competition ELISA method and the HPLC method,the correlation coefficient of the detection results of both the bovine and pork samples were 0.9999.The correlation between the two methods was good and the method accuracy was high.The coating was optimized by using the monoclonal antibody of 3-methylquinoxaline-2-carboxylic acid.The colloidal gold solution was prepared and the colloidal gold was used as the labeled MQCA monoclonal antibody to establish colloidal gold 3-methyl.The optimal working conditions of the quinoxaline-2-carboxylic acid immunochromatographic test strip method were as follows:0.2 mol/L K2CO3 solution was added in the solution of colloidal gold solution,and the amount of antibody added was 20 ?L;gold standard antibody reconstitution working solution volume 200 ?L;comparison of MilliporeHF135 and 90 nitrocellulose membrane by experiment,the results show that 90 nitrocellulose membrane is better;determine the gold standard antibody loading 1.2 ?L;loading buffer The solution is pH7.0 PB buffer;the original and secondary antibody dilutions are pH7.0 PB buffer;the dilution ratio of the original and secondary antibodies is 95 times and 15 times respectively;the detection limit of the method is 1 ?g/L;The 10-fold dilution of the bovine and pork sample extracts can eliminate the matrix effect;the detection limit of the method in cattle and pork samples is 10 ?g/L.