Incorporation of Different Bridge Length Linkers in Enzyme and Its Use in the Preparation of Enzyme Conjugates for Immunoassay

Abstract

An enzyme horseradish peroxidase (HRP), as a starting material, has been used to introduce different bridge length linkers, and its use in the preparation of enzyme conjugates for immunoassay is described. HRP was conjugated to adipic acid dihydrazide (ADH), gamma amino butyric acid (GABA), followed by ADH and 6‐amino caproic acid (6ACA) followed by ADH. The different bridge length linkers‐incorporated enzyme was coupled to a carboxylic derivative of cortisol. Four enzyme conjugates with different bridge length were prepared, such as cortisol‐21‐hemisuccinate–HRP (cortisol‐21‐HS–HRP), cortisol‐21‐HS–ADH–HRP, cortisol‐21‐HS–ADH–GABA–HRP, and cortisol‐21‐HS–ADH–6ACA–HRP. The influence of linker on sensitivity and specificity of the cortisol assay was studied. The study revealed that incorporation of a linker between hapten and enzyme increases the sensitivity and specificity of the assay.