ABSTRACT
Some N-alkyl phenothiazines with different ionic groups were studied as enhancers of chemiluminescence catalyzed by soybean peroxidase. Phenothiazines carrying positive charged groups had no an enhancement ability, whereas phenothiazine derivatives with negative charged groups increased significantly an intensity of chemiluminescence. Correlation between the enhancement activity ofphenothiazines and their ability to enzymatically oxidize by hydrogen peroxide was found. The discovery of new enhancers open good perspectives for an improvement of sensitivity of analytes determination by chemiluminescent enzyme immunoassay.
Subject(s)
Hydrogen Peroxide/chemistry , Immunoenzyme Techniques/methods , Luminescence , Phenothiazines/chemistry , Horseradish Peroxidase/chemistry , Luminescent Measurements , Luminol/chemistry , Oxidation-Reduction , Glycine max/enzymologyABSTRACT
An indirect competitive enzyme-linked immunosorbent assay (ELISA) of hexestrol (HES), an antibiotic forbidden for use in livestock farming, has been developed. Conditions of ELISA have been optimized by varying the concentrations of the coating conjugate (HES-ovalbumin), anti-HES antiserum, casein, and Tween 20. In the absence of Tween 20 in the reaction mixture, the detection limit (IC10) equaled 0.01 ng/ml, IC50 equaled 0.17 ng/ml, and the working range (IC20-IC80) equaled 0.03-0.86 ng/ml, while, in the presence of 0.05% Tween 20, these values equaled 0.05 ng/ml, 2.9 ng/ml, and 0.26-32.0 ng/ml, respectively. Standard deviation of the analysis results did not exceed 5.4%. If ELISA was performed in the absence of detergents, the recovery value upon HES determination in spiked beef samples ranged from 74 to 147%.
Subject(s)
Anti-Bacterial Agents/analysis , Enzyme-Linked Immunosorbent Assay/methods , Hexestrol/analysis , Animals , Antibodies/metabolism , Binding, Competitive , Caseins/chemistry , Cattle , Food Contamination/analysis , Haptens/chemistry , Haptens/metabolism , Immune Sera/chemistry , Inhibitory Concentration 50 , Limit of Detection , Meat/analysis , Ovalbumin/chemistry , Ovalbumin/metabolism , Polysorbates/chemistryABSTRACT
Conditions of luminol oxidation by hydrogen peroxide in the presence of peroxygenase from the mushroom Agrocybe aegerita V.Brig have been optimized. The pH value (8.8) at which fungal peroxygenase produces a maximum chemiluminescent signal has been shown to be similar to the pH optimum value of horseradish peroxidase. Luminescence intensity changed when the concentration of Tris buffer was varied; maximum intensity of chemiluminescence was observed in 40 mM solution. It has been shown that enhancer (p-iodophenol) addition to the substrate mixture containing A. aegerita peroxygenase exerted almost no influence on the intensity of the chemiluminescent signal, similarly to soybean, palm, and sweet potato peroxidases. Enzyme detection limit in the reaction of luminol oxidation by hydrogen peroxide was 0.8 pM. High stability combined with high sensitivity make this enzyme a promising analytical reagent.
