Protein hydrolysates are used in special infant formulas for dietary treatment of allergy to cow's milk protein in infants(Reference Host, Koletzko and Dreborg1). The aim of this study was to characterise two different milk casein hydrolysate (HCN-1 and HCN-2) and one whey protein hydrolysate (HWP), and to evaluate their potential for allergenicity in in vitro studies. The IgE reactivity was evaluated using sera of patients with clinically demonstrated allergy to cow's milk proteins that contains ≥20 KU/l of specific IgE antibodies towards milk proteins, measured using FEIA-CAP System (Pharmacia diagnostics, Uppsala, Sweden).
IgE binding was measured using an indirect ELISA coupled to a signal amplification system (ELAST ELISA amplification System, Perkin Elmer Life Sciences, Waltham, MA, USA(Reference Chicón, López-Fandiño and Alonso2). Each sample was also fractionated into three different fractions using Centriprep columns (Amicon): L-Fraction (>10 kDa), M-Fraction (<10 kDa–3 kDa) and S-Fraction (<3 kDa) and IgE reactivity was measured as previously described. In addition, reactivity against several commercial antibodies anti-casein, anti-β-lactoglobulin and anti-α-lactalbumin was measured in each product.
Hydrolysates presented no reactivity against IgE in serum of allergic patients when they were used at concentrations similar to native protein (0.0025 mg/ml). However, when they were used in concentrated form (1000×), HCN-2 showed the lower reactivity, whereas HCN-1 and HWP showed moderate reactivity (Fig. 1). Tests with protein fractions showed that reactivity in the low molecular weight fractions (<3000 Da) was lower than reactivity in the other two fractions. Again HCN-2 fractions also showed the lower IgE reactivity compared to CN-1 and HWP.
HCN-1 showed reactivity against anti-caseins antibodies and HWP also showed reactivity against antibodies anti-β-lactoglobulin and anti-α-lactalbumin and to anti-caseins, whereas HCN-2 showed very low reactivity against anti-caseins. In conclusion, we have thus identified a casein hydrolysate that shows a very low in vitro reactivity (HCN-2). Even though clinical evidence is still a must, in vitro allergenic approach may help to predict reactivity in allergic individuals.
Fig. 1. IgE reactivity.
