Evaluation by SDS-PAGE and immunoblotting of residual antigenicity in hydrolysed protein formulas


Dr P. Restani, Institute of Pharmacological Sciences, via Balzaretti 9, 20133 Milano, Italy.


Background Extensively hydrolysed protein formulas are widely used as an alternative diet for children with cow's milk allergy. Partially hydrolysed protein formulas have been noted in some studies as useful in the prevention of allergy in infants at high risk of atopy. Although normally well tolerated, these ‘hypoallergenic’ products have been reported to cause serious immunological reactions in very sensitive subjects.

Objective Starting from these considerations, we studied some commercial hydrolysed formulas in search of biological data supporting the observed clinical reactions.

Methods We set up an electrophoretic method sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which allowed us to study the molecular weight of peptides contained in hydrolysed products. Then, using the immunoblotting technique we evaluated the reactivity of circulating IgE (from serum of children allergic to cow's milk proteins) with the residual intact proteins and with the peptides present in these formulas.

Results Both group of milk proteins (caseins and whey proteins) were important allergens for children included in this study. The presence of high-molecular polypeptides was shown in partial hydrolysed formulas as such and in extensive hydrolysed products after protein enrichment by trichloroacetic acid (TCA) precipitation. Intact residual proteins were mainly responsible for the formation of FgE-antigen complexes observed in immunoblotting. More rarely, polypeptides of partial hydrolysed formulas were involved in immunological responses.

Conclusions Both partial and extensive hydrolysed formulas could induce clinical reactions in very sensitive subjects. These responses are mainly associated with allergy to the small amounts of residual intact proteins.