Abstract
When developing immunochemical test systems, it is necessary to obtain specific antibodies. Their quality depends, among other things, on the immunogen used. When preparing hapten-protein conjugates to obtain antibodies for low-molecular-weight compounds, the key factors are the structure of the hapten itself, the presence of a spacer, the size of the carrier protein and the degree of its modification by hapten molecules. This work shows that one additional factor-the conditions for obtaining the hapten-protein conjugate-is overlooked. In this work, we have synthesized conjugates of bisphenol A derivative 4,4-bis(hydroxyphenyl)valeric acid (BVA), the protein carrier soybean trypsin inhibitor (STI), and bovine serum albumin (BSA) in reaction media combining water with two organic solvents: dimethylformamide (DMF) or dimethyl sulfoxide (DMSO). Namely, BSA(DMF)-BVA, STI(DMF)-BVA, BSA(DMSO)-BVA and STI(DMSO)-BVA conjugates were obtained. Rabbit polyclonal antibodies against the BSA(DMF)-BVA conjugate demonstrated basically different interactions in the developed ELISA systems using either STI(DMF)-BVA or STI(DMSO)-BVA conjugates. The use of the STI(DMF)-BVA conjugate demonstrated the absence of competition in combination with antisera obtained from BSA(DMF)-BVA in an ELISA. A competitive interaction was observed only with the use of the STI(DMSO)-BVA conjugate. Under the selected conditions, the detection limit of bisphenol A was 8.3 ng/mL, and the working range of determined concentrations was 18.5-290.3 ng/mL. The obtained data demonstrate the possibility of achieving sensitive immunoassays by simply varying the reaction media for the hapten-protein conjugation, which could provide an additional tool in the development of immunoassays for other low-molecular-weight compounds.