Addition of reducing agents to the peroxidase-o-phenylenediamine buffer reduces background of enzyme immunoassays

J.M. Coll
45

Abstract

The concentrations of o-phenylenediamine (OPD), H2O2, citrate and H+ in a substrate buffer for peroxidase immunoassays were optimized for minimal background.The background was reduced 2-03 fold with 5.5 mM OPD, 3 mM H2O2, 150 mM citric acid/sodium citrate, pH 4.8, and the reproducibility interassay was increased.A further 3-5 fold reduction of the background was obtained by the addition of 1.5 mM acetanilide, 0.14 mM beta-mercaptoethanol and 5 mM nitrilotriacetic acid to the substrate buffer.This low-background substrate buffer allows increased sensitivity and lowers the interassay variation coefficient.It has been used successfully in peroxidase immunoassays of human C-reactive protein, human antiestreptolysin and human rheumatoid factor.

Keywords:
Citrates, Enzyme-Linked Immunosorbent Assay, Hydrogen Peroxide, Phenylenediamines, Acetanilides, C-Reactive Protein/analysis, Immunoenzyme Techniques, Mercaptoethanol, Nitrilotriacetic Acid, Protons, ELISA, Peroxidase-o-phenylcnediamine, Enzyme immunoassay

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J.M. Coll


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