Comparison of Classical Lowry, Modified Lowry and a Dye-Binding Assay for the Estimation of Protein in Allergen Extracts and Influence of Different Parameters on the Modified Lowry Assay

Abstract

Protein values of dialysed allergen extracts determined by Lowry, modified Lowry (trichloroacetic acid precipitation of the proteins) and dye-binding assay were compared. The influence of different parameters on the modified Lowry was examined. The reproducibility of the modified Lowry was checked with three independent measurements. For the examination of recovery a constant amount of 6-grass pollen allergen proteins was added to the samples of the standardized human serum albumin prepared for the calibration curve. The samples were measured by modified Lowry. The mean of the ratio between the protein values of the dialysed allergen extracts obtained by modified Lowry and those obtained by classical Lowry was 3.59 (coefficient of variation Vv=45%). The mean of the ratio between the protein values of the allergen extracts obtained by modified Lowry and dye-binding assay was 1:0.71 (Cv=31%). Phenol interfered with the modified Lowry. Phenolic allergen extracts showed higher "protein values" than non-phenolic allergen extracts. This influence could be reduced by a second precipitation of the dissolved precipitate. The precipitation of non-phenolic dialysed aqueous allergen extracts was complete after the first trichloroacetic acid precipitation. By incubating samples with the Folin-Ciocalteu''s reagent at 55.degree. C in a waterbath, the time necessary for developing the colour could be reduced from 45 min to 5 min. Protein measurements by modified Lowry of a 6-grass pollen allergen extract in three different laboratories showed good reproducibility. For these extract 785 .mu.g protein/ml (Cv=4%) could be measured. The addition of a constant amount of allergen proteins to the samples prepared for the human serum albumin calibration curve produced a curve parallel to this calibration curve.