Highly Broad-Specific and Sensitive Enzyme-Linked Immunosorbent Assay for Screening Sulfonamides: Assay Optimization and Application to Milk Samples
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The optimum 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, and solvent) were investigated to develop a broad-specific and sensitive immunoassay for detection of sulfonamides in milk samples. 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 ELISA (ciELISA). In addition, six hapten-HRP conjugates and the two MAbs were evaluated in a competitive direct ELISA. After optimization, a highly broad-specific and sensitive ciELISA for screening for sulfonamides was obtained based on MAb 4D11 and the BS-BSA heterologous-coating antigen, demonstrating a 50 % specific binding (IC50) for 22 sulfonamides at concentrations below 100 ng mL−1. This is the first report of an immunoassay that is capable of detecting more than 20 sulfonamides based on MAbs. The optimized ciELISA was used to quantify the five sulfonamides, SMZ, SDM, SQX, SMM, and SMX in spiked milk samples. Recoveries of 89–104.6 % and coefficients of variation of 11.9–19.1 % demonstrated the potential of the ciELISA to simultaneously monitor multiple sulfonamides in diluted milk samples without further purification steps.
KeywordsBroad-specificity ELISA Milk Optimization Sulfonamides
This work was supported by grants from the National Natural Science Funds (31372475), Trans-Century Training Programme Foundation for the Talents by the Ministry of Education (NCET–12–0529), Key Projects in the National Science & Technology Pillar Program during the twelfth Five-year Plan Period (2012BAK17B16), and the Sino-Russian International Scientific and Technological Cooperation (2011DFR30470).
Conflict of interest
Xiao Lianga has no conflict of interest. Hengjia Nia has no conflict of interest. Ross C. Beier has no conflict of interest. Yanni Dong has no conflict of interest. Jingya Li has no conflict of interest. Xiangshu Luo has no conflict of interest. Suxia Zhang has no conflict of interest. Jianzhong Shen has no conflict of interest. Zhanhui Wang has no conflict of interest. This article does not contain any studies with human or animal subjects
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