Development of Sandwich Double-Competitive ELISA for Sulfonamides. Comparative Analytical Characteristics and Matrix Effect Resistance

Sandwich enzyme-linked immunosorbent assay (ELISA) of sulfonamides based on double-competitive interaction between haptenized protein as a captured antigen and analyte for binding to immobilized and enzyme-labeled antibodies was developed. This experimental assay format was examined in analytical properties and matrix effect resistance in comparison with usual ones: indirect, direct antigen-coated and antibody-coated ELISAs. All four assay formats were designed on the basis of interactions between the previously prepared monoclonal antibody and immunizing hapten, 4-(4-(4-aminophenylsulfonamido) phenyl)butanoic acid, providing the uniform output optical signal for correct comparison of each assay characteristic. The sensitivity (IC50) of the developed competitive sandwich assay was rather below 100 ng/ml for 11 sulfonamides which was suitable for their determination in milk, muscles and animal sera at established maximum residue limit concentration. Comparative examination did not reveal changes in assay specificity, and advantages in sensitivity, matrix effect resistance, and procedure duration before commonly used assay formats. Moreover, new design of assay was shown to be three-fold more consumable in antibody reagents in comparison with direct assay formats.