Detection and identification of a soy protein component that cross-reacts with caseins from cow's milk

Abstract

Soy-based formulas are the most employed cow's milk substitutes in the treatment of cow's milk allergy in our country. Since adverse reactions have been reported in allergic patients as a consequence of exposure to soy proteins, we have investigated the possible cross-reactivity between components from soybean and cow's milk. A cow's milk specific polyclonal antiserum and casein specific monoclonal antibodies were used in immunoblotting and competitive ELISA studies to identify a 30-kD component from soybean that cross-reacts with cow's milk caseins. Its IgE binding capacity was tested by EAST, employing sera from cow's milk allergic patients, not previously exposed to soy proteins. The 30 kD protein was isolated and partially sequenced. It is constituted by two polypeptides (A5 and B3) linked by a disulphide bond. The protein's capacity to bind to the different antibodies relies on the B3 poly-peptide. These results indicate that soy-based formula, which contains the A5-B3 glycinin molecule, could be involved in allergic reactions observed in cow's milk allergic patients exposed to soy-containing foods.

Fig. 1

Competitive inhibition ELISA tests Increasing concentrations of CMP (▪) or soybean proteins (•) were preincubated with specific CM polyclonal antiserum in order to inhibit the binding of antibodies to immobilized CMP (a) or soybean proteins (b).

Fig. 2

SDS-PAGE analysis of protein extracts. MW: molecular weight markers; 1: CMPextract (under reducing conditions); 2: total casein components (under reducing conditions), 3: soy extract (under non reducing conditions). Gels were stained with Coomasie Blue R 250 (15–25 μg of protein were loaded per lane).

Fig. 3

Immunoblottings of the different protein extracts Lanes 1: CMPextract (under reducing conditions); lanes 2: soybean extract, lanes 3: S30 component (under non reducing conditions). Immunoblottings were revealed with rabbit polyclonal antiserum (A) and the monoclonal antibodies 1D5-I (B), 2A1-I (C) or 3B5-I (D).

Fig. 4

Comparison of the amino acid sequences of soybean glycinins A5 and B3, and proteolitic peptides obtained from S30. Similarities in amino acid residues are indicated as *.

Fig. 5

Analysis by Tricine SDS-PAGE and immunoblotting of the S30 component. MW 1 and 2: different molecular weight markers, lanes 1 and 2: Tricine SDS-PAGE electrophoresis of the S30 protein under nonreducing (lane 1) or reducing conditions (lane 2), stained with silver staining (2 μg of protein per lane), lane 3–7: immunoblots of the S30 component under nonreducing (lane 3) or reducing conditions (lanes 4–7), revealed with the polyclonal antiserum (lanes 3 and 4) or the monoclonal antibodies ID5-I (lane 5), 2A1-I (lane 6) and 3B5-I (lane 7).

Fig. 6

IgE immunoblots revealed with a pooled serum obtained from CMA patients. Lane 1: CM immunoblotting under reducing conditions, lane 2: soybean immunoblotting (under non reducing conditions), lane 3: S30 immunoblotting under nonreducing conditions, lane 4: S30 immunoblotting under reducing conditions.