A Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for Determination of Homoharringtonine

Abstract

Homoharringtonine (HHT), also known as omacetaxine, is a natural compound found in the genus and is a promising pharmaceutical drug used for the treatment of chronic or accelerated phase chronic myeloid leukemia. As a tool for the quantitative determination of HHT, a specific monoclonal antibody against HHT (MAb 6A1) was generated by conjugates prepared via sodium periodate-mediated oxidation. The developed indirect competitive enzyme-linked immunosorbent assay (icELISA) using MAb 6A1 was found to be highly specific and sensitive with a limit of detection for HHT of 48.8 ng/mL. Validation assays to evaluate precision and accuracy of the method were conducted by the use of intra- and inter-assay analysis, recovery test, and comparison analysis between the amounts of HHT determined by ELISA and high-performance liquid chromatography. These results revealed that the established icELISA using MAb 6A1 is specific, sensitive, and reliable enough to be applied to the qualitative analysis for HHT. Furthermore, the results of this study support the usefulness of sodium periodate as a reagent for the conjugation between alkaloids and proteins for producing specific antibodies.

Sensitive quantitative analysis of the bitter glycoside amarogentin by specific monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay

“…Considering our recent reports about specic MAbs prepared using BSA conjugates containing only two target molecules, [20][21][22] very low hapten numbers may be sufficient for producing highly specic antibodies although some study concluded between 8 and 25 hapten number were optimal.…”

“…Approximately 40 years ago, the appropriate hapten numbers had already been investigated by Erlanger for the production of antihapten antibodies using BSA conjugates and he concluded that between 8 and 25 molecules were optimal. 19 However, in our recent reports, highly specic and highly sensitive antibodies were successfully produced against harringtonine, 20 homoharringtonine, 21 and monocrotaline, 22 despite only having hapten numbers of 2 per BSA molecule. Thus, AG-BSA conjugates obtained herein were used as immunogens for further antibody production.…”