Influence of Hapten–protein Conjugate Composition on Competitive Elisa Performance: A Case Study of Bisphenol A Determination

Upon immunochemical determination of low-molecular-weight compounds capable of binding only to one antibody molecule (haptens), competitive interactions of the hapten in the sample and the hapten-carrier conjugate (usually, a hapten-protein conjugate) with the antibodies are conducted. The characteristics of such analytical systems largely depend on the properties of the conjugates used. However, the relationships between the conjugate composition and the achieved limit of detection (LOD) are described fragmentarily and are not systematized. In this study, a panel of nine conjugates of bisphenol A, a relevant toxic environmental pollutant, with soybean trypsin inhibitor (STI) and bovine serum albumin is considered. The composition of the conjugates synthesized by the carbodiimide-succinimide method was determined using fluorescamine. Concentration dependences of binding between antibodies and conjugates immobilized in microplates and competitive interaction with free bisphenol A were studied. The enzyme-linked immunoassay (ELISA) implemented using various conjugates was characterized by LODs and working ranges. It was demonstrated that the minimum LOD of 1.9 ng/mL was achieved using the bisphenol A–STI conjugate with a 5 : 1 composition. The developed ELISA was successfully tested for the detection of bisphenol A in natural and drinking water.