Monoclonal and polyclonal antibodies against casein components of cow milk for evaluation of residual antigenic activity in hypoallergenic infant formulas

Background: Hydrolysed casein and whey protein formulas have been developed with the aim of preventing sensitization in infants at high risk of cow milk allergy. Subsequently these products have also been used for treatment of children with cow milk allergy. However, severe reactions have occurred in some allergic infants fed with these formulas raising doubts about their absolute safety and suggest the need for developing in vitro techniques for detection of eventual residual allergenic activity in such preparations. Objectives: Our purpose was to evaluate the usefulness of monoclonal and polyclonal antibodies against casein components (α, β and κ casein) as reagents for the detection of the residual antigenic activity of casein components in several hydrolysed formulas. Methods: The monoclonal and polyclonal antibodies were produced according to standard procedures by immunizing female Balb/c mice with casein fraction (a mixture of α, β and κ casein). ELISA assays were developed to test the specificity of the antibodies and to detect and evaluate the amount of residual antigenic activity of the casein components in hydrolysed formulas. Results: Use of polyclonal antiserum specific for casein allowed detection of residual antigenic activity of casein components in all partial hydrolysates and in the two extensive whey protein hydrolysates in the amounts ranging from 0.05 to 0.67% of total protein. No such activity was detectable in either the two extensive casein hydrolysates tested or the aminoacid based formula. The polyclonal antiserum proved to be more suitable than monoclonals for detecting residual antigenic activity in the hydrolysates. The monoclonal antibodies were directed against epitopes expressed on different casein components. Conclusions: In this study the ELISA inhibition assay with polyclonal antibodies specific for casein components of cow milk proved to be a sensitive method for estimating residual antigenicity in the hydrolysed formulas commercially available for infants with cow milk allergy suggesting their potential application for the quality control of hypoallergenic infant formulas.