Molecular basis of IgE cross-reactivity between human b-casein and bovine b-casein, a major allergen of milk H. Bernard a, *, L. Negroni a , J.M. Chatel a , G. Clement a , K. Adel-Patient a , G. Peltre c , C. Creminon b , J.M. Wal a a INRA-Laboratoire d'Immuno-Allergie Alimentaire, Ba Ãtiment 136, Saclay, 91191 Gif Sur Yvette, France b CEA, Service de Pharmacologie et d'Immunologie, CEA-Saclay, 91191 Gif Sur Yvette, France c Unite  d'Immuno-Allergie, Institut Pasteur, Paris, France Abstract Twenty patients allergic to cow's milk proteins and with high levels of speci®c IgE directed against bovine whole casein were selected to evaluate reactivity of their IgE antibodies with human b-casein. Highly puri®ed human and bovine b-caseins were prepared by selective precipitations and FPLC separation. Their identity and purity were assessed by HPLC, analysis of amino acid composition, sequencing of the ®ve N-terminal amino acid residues and immunochemical tests. Direct and indirect ELISAs were performed using human and bovine b-casein coated into microtiter plates and monoclonal anti-human IgE antibody AChE labelled for revelation. Seven sera contained speci®c IgE directed against human b-casein. Inhibition studies using native human and bovine b-caseins as well as bovine b-casein-derived peptides demonstrated that, depending on the sera, one or several common epitopes located in dierent parts of the molecule were shared by the two homologous proteins. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Cow's milk allergy; b-casein; Human milk; IgE; Cross-reactivity 1. Introduction Whole casein accounts for 80% of the total protein in cow's milk (27 g/l) and acts as a major allergen in cow's milk allergy (StoÈger and WuÈtrich, 1993; Docena et al., 1996; Bernard et al., 1998). It comprises four dierent caseins, aS1-, aS2-, b-, and k-casein, with b- casein representing 38% of the whole casein fraction (approximately 10 g/l). In human milk, the proportion of total protein accounted for by the casein fraction depends on the stage of lactation (Kunz and LoÈnnerdal, 1990a, 1990b, 1992; Sanchez-Hidalgo et al., 1998) and increases from early lactation (casein vs. whey proteins ratio: 10:90), to mature milk (40:60), and to late lactation (50:50) (Kunz and LoÈnnerdal, 1990a, 1990b, 1992). The casein fraction is essentially composed of b-casein, at an aver- age concentration of 5 g/l of milk (Chtourou et al., 1985; Kroening et al., 1998). The amino acid sequence of human b-casein has been established by protein sequencing using recurring Edman degradation and by deduction from the nucleo- tide sequence (Greenberg et al., 1984; LoÈnnerdal et al., 1990). Comparison of the sequences of bovine and human b-caseins requires alignment of the N-terminal extremity of the human protein with residue 10 of bovine b-casein. Marked homology is then observed between the two proteins, which share 47% of identi- cal residues. The allergenicity of bovine b-casein has already been demonstrated since 92% of sera from patients allergic Molecular Immunology 37 (2000) 161±167 0161-5890/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S0161-5890(00)00029-8 www.elsevier.com/locate/molimm * Corresponding author. Tel.: +33-1-69-08-92-25; fax: +33-1-69- 08-59-07. E-mail address: bernardh@dsvidf2.cea.fr (H. Bernard).