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Separation-free amperometric enzyme immunoassay

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Abstract

A new technique for conducting a separation-free amperometric enzyme immunoassay is described using DNP-aminocaproic acid as the analyte. The technique is based on the combined use of a recently described separation-free enzyme immunoassay (19) and an electrode system that senses H2O2. Oxidation of glucose to gluconate and H2O2 by the enzyme reconstituted from DNP-conjugate apoglucose oxidase (DPN-CAGO) and FAD was continuously measured amperometrically. The reconstitution was inhibited by preincubation with anti-DNP antibody before adding FAD. This antibody-induced inhibition of the reconstituting of the holoenzyme was reversed by adding DNP-amino caproic acid to DNP-CAGO before adding the antibody to DNP-CAGO. Based on (a) the antibody-induced inhibition of holoenzyme reconstitution, (b) a specific ligand-induced reversal of the inhibition, and (c) an electrochemical system that measures H2O2, we developed a separation-free (homogeneous) amperometric enzyme immunoassay.

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Authors and Affiliations

  1. Department of Developmental and Cell Biology, University of California, 92717, Irvine, CA, USA

    T. T. Ngo, J. H. Bovaird & H. M. Lenhoff

Authors
  1. T. T. Ngo
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  2. J. H. Bovaird
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  3. H. M. Lenhoff
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Ngo, T.T., Bovaird, J.H. & Lenhoff, H.M. Separation-free amperometric enzyme immunoassay. Appl Biochem Biotechnol 11, 63–70 (1985). https://doi.org/10.1007/BF02824312

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  • DOI: https://doi.org/10.1007/BF02824312

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