| | Re: Increasing Sensitivity of ELISA Sandwich Assay
Leucomalachite green (LMG) in peroxidase reaction system is an acceptor, while hydrogen peroxide a substrate. In this reaction the temperature coefficient of peroxidase acting on leucomalachite green is > 25 °C and hydrogen peroxide concentration is about 2.5 mg per litre. A stock solution of LMG reducing substrate was prepared by saturating N/20 acetic acid with the leuco-base in vacuo. The resulting solution contained approximately 10 mg of leucomalachite green per 100 ml, and remained colourless for several weeks if kept in vacuo. The reaction mixture was made up of 10 ml of this solution, 0.2 ml of 0.166 N sodium acetate (0.05 M) which adjusted the reaction to pH 4.1, containing 1.3x10-6 M of hydrogen peroxide. The reaction was carried out in a water-bath at 30-37 °C for a time varying between 2 and 8 minutes, at the conclusion of which the enzyme was destroyed by the addition of 1 ml of N sulphuric acid, which after 30 seconds was neutralised with an equivalent amount of sodium carbonate solution, and the mixture was shaken to remove carbon dioxide, and the malachite green produced (oxidized blue-green color) by the reaction is estimated in a colorimeter at 620 nm against a standard solution prepared by dissolving 10 mg of malachite green in a litre of N/20 acetic acid. The acetic acid inhibits the hydrolysis of the dye and thus prevents fading of the standard solution. LMG can be dissolved in acetone and then diluted with five volumes of methanol, and, in suitable proportion, mixed, just to the use, with sodium acetate buffer 0.05 M at pH 4.1 containing 1.3x10-6 M of hydrogen peroxide. In my foregoing research on human hemoglobin electrophoresis on agarose gel, the support has been incubated for 10 min in a 1:1 dH2O:LMG-peroxidase buffer (0.2 g leucomalachite green ad 0.02 g EDTA in 25 ml 40% [v/v] acetic acid with 0.06% [v/v] H2O2) with good black bands detection. Formulation and application of this reagent has been developed for different purposes and, although Leucomalachite green not true peroxidase test is, high sensitivity "nonspecific" oxidizing system showed. At any rate, is needed to be revalidated to ensure that the LLOQ and the QC samples in the actual concentration range to be used for sample analysis perform accurately and precisely.
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