Location: Food and Feed Safety ResearchTitle: Highly broad-specific and sensitive enzyme-linked immunosorbent assay for screening sulfonamides: Assay optimization and application to milk samples Author
Submitted to: Journal of Food Analytical Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/11/2014
Publication Date: 3/26/2014
Publication URL: http://handle.nal.usda.gov/10113/59791
Citation: Liang, X., Ni, H., Beier, R.C., Dong, Y., Li, J., Luo, X., Zhang, S., Shen, J., Wang, Z. 2014. Highly broad-specific and sensitive enzyme-linked immunosorbent assay for screening sulfonamides: Assay optimization and application to milk samples. Journal of Food Analytical Methods. 7:1992-2002. Interpretive Summary: A broad-specific and sensitive immunoassay for the detection of sulfonamides (antibiotics used in food animal production) was developed by optimizing the conditions of an enzyme-linked immunosorbent assay (ELISA). An ELISA is an easy-to-use test and is similar to commonly used over-the-ounter pregnancy tests. Some of the different conditions tested and optimized were how the ELISA was run (format), different types of reagents required in the ELISA (immunoreagents), and the type of antibody used. Antibodies are substances that are produced by the immune system in response to foreign substances which enter the body. Once the antibodies to a foreign substance are isolated, they can be used in a method to detect the presence of that foreign substance. After optimization, a highly broad-specific and sensitive ELISA for sulfonamides based on a single antibody was obtained showing 50% specific binding (IC50) for 22 sulfonamides at concentrations below 100 ng/mL. The optimized ELISA was used to quantify five sulfonamides in spiked milk samples. The analysis results demonstrated the potential of the ELISA to simultaneously monitor multiple sulfonamides in diluted milk samples without further purification steps. This is important because antibiotics in food may cause additional antibiotic resistance in intestinal bacteria and diminish the effectiveness of important human antibiotics.
Technical Abstract: A broad-specific and sensitive immunoassay for the detection of sulfonamides was developed by optimizing the conditions of an enzyme-linked immunosorbent assay (ELISA) in regard to different monoclonal antibodies (MAbs), assay format, immunoreagents, and several physicochemical factors (pH, salt, detergent, solvent). Two previously produced broad-specific MAbs, 4D11 and 4C7, and eight structurally different haptens conjugated with bovine serum albumin (BSA) were used as coating antigens in a competitive indirect immunoassay (ciELISA). In addition, six hapten-HRP conjugates and the two MAbs were used in a competitive direct immunoassay. After optimization, a highly broad-specific and sensitive ciELISA for sulfonamides based on MAb 4D11 and the heterologous coating antigen (BS-BSA) was obtained, showing 50% specific binding (IC50) of 22 sulfonamides at concentrations below 100 ng mL–1. The optimized ciELISA was used to quantify the five sulfonamides, SMZ, SDM, SQX, SMM, and SMX, in spiked milk samples. The recoveries and coefficients of variation achieved demonstrated the potential of the ciELISA to simultaneously monitor multiple sulfonamides in diluted milk samples without further purification steps.