Addition of reducing agents to the peroxidase-o-phenylenediamine buffer reduces background of enzyme immunoassays.
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 2-3 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.
Coll
J M
JM
Instituto Nacional de Investigaciones Agrarias, Departamento de Sanidad Animal, Madrid, Spain.
D000083:Acetanilides; D002097:C-Reactive Protein / Q000032:analysis*; D002951:Citrates; D004797:Enzyme-Linked Immunosorbent Assay; D006861:Hydrogen Peroxide; D007124:Immunoenzyme Techniques; D008623:Mercaptoethanol; D009571:Nitrilotriacetic Acid; D010655:Phenylenediamines; D011522:Protons
D000083:Acetanilides; D002951:Citrates; D010655:Phenylenediamines; D011522:Protons; D008623:Mercaptoethanol; C034193:1,2-diaminobenzene; D002097:C-Reactive Protein; D006861:Hydrogen Peroxide; D009571:Nitrilotriacetic Acid