Validation of SDS-PAGE method for the proteins composition determination in an allergenic extract from soybean
Abstract
Soybean is one of the foods that most frequently cause food allergies. Obtaining allergenic extracts makes possible the specific diagnosis of allergic sensitization through skin tests. Proteins composition is an essential quality requirement for allergenic extracts. Validation of analytical methods evaluates their performance for their use as quality control in the pharmaceutical industry. The aim of this work was to validate SDS-PAGE method for the proteins composition determination in an allergenic extract from soybean. Precision (Repeatability and Intermediate precision) was evaluated in determining the protein relative content of the main bands. The possible interference of glycerol (excipient of the finished product) was determined to evaluate the Specificity of the method. Were also analyzed the Accuracy and Limit of Detection of the method in the determination of the molecular weight of the extract main proteins. The bands of the main protein components were identified by comparing the electrophoresis patterns with the bands identified by IgE/IgG4 Immunoblotting. All pre-established acceptance criteria were met. The coefficient of variation was less than 5% in all cases. There were no significant differences between different analysts, within or between tests (Fischer, p˃0.05). Accuracy was demonstrated in the determination of molecular weights, there were no significant differences between the mean value found for the main bands and the theoretical values of the allergens (p˃0.05). There was no interference from glycerol in the formulation, there were no significant differences in the percentages of the main bands between the lyophilized extract and the glycerinated formulation (p˃0.05). The detection limit of the technique was 0.5 µg of proteins. In conclusion, the SDS-PAGE technique was validated for allergenic soybean extracts and can be used to control the quality of the finished product.
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