Determining factor of enzyme conjugates, bridge heterology and analytical variables of immunogens in prednisolone ELISA

In this study, adipic acid dihydrazide (ADH), carbohydrazide (CH), ethylenediamine (EDA), and urea (U) were used as spacer molecules, covalently linking prednisolone (PSL) to carrier proteins for immunogen preparation, and PSL to enzymes for enzyme conjugate preparation using N-hydroxysuccinimide (NHS)-mediated carbodiimide reactions. The resulting immunogens were used to generate antiserum in New Zealand white rabbits. Antibodies produced against immunogens with various spacers were tested for immunoreactivity with enzyme conjugates, both with and without spacers, in a total of twenty different combinations. All combinations demonstrated binding and were subsequently evaluated through displacement studies. Sensitivity and specificity tests revealed that the combination of PSL-21-HS-U-BSA-antibody with PSL-21-HS-HRP enzyme conjugate exhibited the best sensitivity (0.032 ng/mL) and limited cross-reactivity with other steroids. This combination was further examined for analytical parameters, showing recovery rates of 93.87–101.19 % for PSL from spiked human serum samples, with intra- and inter-assay CVs of <8.88 %. The serum PSL values obtained by this method showed strong correlation with a commercially available ELISA kit (r² = 0.97, n = 78).