We propose a new approach of an indirect enzyme-linked immunosorbent assay (ELISA) for determination of D-glutamic acid (D-Glu) using a monoclonal antibody against D-glutamic acid (D-Glu-MAb), which recognizes D-Glu-glutaraldehyde (GA) molecule but not D-Glu molecule. Human serum albumin (HSA) was coated on an immunoplate and reacted with D-Glu via GA to produce D-Glu-GA-HSA conjugates in situ in the well to be recognized by D-Glu-MAb, which enabled the development of an indirect ELISA for the determination of free D-Glu. In this indirect ELISA, D-Glu can be specifically detected with limit of detection of 7.81 μ g/mL. Since anti-conjugate antibodies are often produced, even though anti-hapten antibodies are desired, this new approach could be very useful as an application of anti-conjugate antibodies to the development of quantitative analysis for detecting hapten.
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.
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