Immunological analysis of food proteins using high-performance thin-layer chromatography-immunostaining.

Area of Science:

• Analytical Chemistry
• Immunochemistry
• Food Science

Background:

• Chromatographic analysis of intact proteins, particularly for antigenicity, presents significant challenges.
• Traditional methods like ELISA and Western blot have limitations in molecular weight range and information loss during sample preparation.
• Investigating antibody cross-reactivity often requires methods with extensive sample processing, impacting structural and conformational data.

Purpose of the Study:

• To explore high-performance thin-layer chromatography (HPTLC) as a promising alternative for protein and peptide analysis.
• To develop and apply an HPTLC-based immunoassay for the immunological investigation of milk proteins, focusing on allergens.
• To demonstrate the applicability of HPTLC for semi-quantitative estimation of antigenic proteins and analysis of protein fragments.

Main Methods:

• High-performance thin-layer chromatography (HPTLC) with various stationary and mobile phases.
• Development of a novel HPTLC-based immunoassay for antigen detection.
• Application of HPTLC-immunostaining for analyzing intact food allergens and their peptides.

Main Results:

• HPTLC demonstrated high variability in chromatographic systems and detection possibilities for protein analysis.
• The developed HPTLC immunoassay successfully detected antigenic proteins in milk.
• The method proved applicable on different stationary phases and allowed for semi-quantitative estimation of antigens.

Conclusions:

• HPTLC is a valuable and versatile technique for the chromatographic separation and immunological detection of intact proteins and peptides.
• The HPTLC immunoassay provides a robust approach for identifying food allergens and assessing allergenic potential.
• This method facilitates the analysis of both intact allergens and their derived peptides, offering comprehensive insights.