Development of Epitopephore-Based Rational Hapten Design Strategy: A Combination of Theoretical Evidence and Experimental Validation

Abstract

Antibody is a key biomolecule that governing sensitivity and specificity of an immunoassay for chemical compound, also named hapten molecule. Obviously, predication of hapten effectiveness before chemical synthesis is beneficial to boost success, save cost and improve controllability for antibody production. Here, we proposed and evaluated an epitopephore based rational hapten design (ERHD) to assist antibody production, combining theoretical evidence and then experimental validation by using dinitrocarbanilide (DNC) as a model analyte. To prove and clarify the usability of the ERHD, two retrieved full haptens (HFU), one non-retrieved HFU hapten and three non-retrieved fragment haptens (HFR) were all selected to produce monoclonal antibodies (mAbs) for comparison purpose. A maximal 6000-fold increased affinity of mAb from retrieved HFU than HFR was observed, while, non-retrieved HFU failed to produce antibody to DNC. More importantly, mAbs from HFU haptens provided highly specificity to DNC, while, mAbs from HFR haptens could recognize 15 others analogues. We then constructed antibody structure and investigated molecular recognition of the mAbs to DNC, well supporting the rationality of the ERHD. Lastly, an icELISA was developed for DNC with an IC50 value as low as 0.19 ng/mL with high specificity, which has never achieved before.

Keywords: Hapten rational design, epitopephore, antibody production, virtual screening, recognition patterns