A procedure for total protein determination with special application to allergenic extract standardization

Area of Science:

• Biochemistry
• Immunology
• Analytical Chemistry

Background:

• Accurate total protein determination is crucial for standardizing allergenic extracts.
• Existing methods for protein analysis in allergen extracts can yield variable results.

Purpose of the Study:

• To develop and validate a robust method for total protein quantification in allergenic extracts.
• To assess the reliability and applicability of the developed assay for routine use.

Main Methods:

• Hydrolysis of allergenic extract samples using 5 M NaOH.
• Colorimetric determination of released amino acids with ninhydrin, using bovine serum albumin as a standard.
• Assay validation using pollen and mite extracts, with comparison to amino acid analysis and other protein assays.

Main Results:

• The developed method demonstrated good agreement with amino acid analyses for pollen extracts.
• Common excipients in allergenic extracts (glycerol, phenol, mannitol) did not interfere with the assay.
• The assay provided consistent total protein values across 53 different lots of allergenic extracts.
• Standardized mite extracts showed a strong correlation between total protein content and biological activity.

Conclusions:

• A reliable and reproducible method for total protein determination in allergenic extracts has been established.
• This assay is suitable for quality control and standardization of allergenic products.
• The method's accuracy and correlation with biological activity support its use in the manufacturing of allergenic extracts.