1. Mutational analysis of major, sequential IgE-binding epitopes in αs1-casein, a major cow's milk allergen 
Renata R. Cocco, MD, Kirsi-Marjut Järvinen, MD, PhD, Hugh A. Sampson, MD, Kirsten Beyer, MD 
Journal of Allergy and Clinical Immunology 
Volume 112, Issue 2, Pages (August 2003)
DOI: /mai
Copyright © 2003 Mosby, Inc. Terms and Conditions

2. Fig. 1
Selected part of the SPOT membrane, showing 1 of the synthesized, wild-type (WT ) peptides (AA ) and the corresponding mutated peptides with single-AA substitution. Shown is the IgE labeling with pooled sera from 15 CMA patients. Single-AA changes at AA 113, AA 114, and AA 117 resulted in loss of IgE binding to this epitope.
Journal of Allergy and Clinical Immunology  , DOI: ( /mai )
Copyright © 2003 Mosby, Inc. Terms and Conditions

3. Fig. 2
Identification of the critical AA within the major αs1-casein epitopes. Shown is IgE binding of the pooled sera from 15 CMA patients to each individual wild-type and mutated peptide determined by densi-tometry. The arrows mark the AAs that resulted in total abrogation of IgE binding, and the stars indicate the AAs that were chosen for double substitution.
Journal of Allergy and Clinical Immunology  , DOI: ( /mai )
Copyright © 2003 Mosby, Inc. Terms and Conditions

4. Fig. 3
Native and mutated peptides with single- (eg, P114A) or double- (eg, F23A/F24A) AA substitutions were synthesized on cellulose membranes. IgE binding to these membrane-bound peptides was measured by optical densitometry. In A, the results are shown for pooled sera from 15 CMA patients. The black bars represent binding to the native peptides and the gray bars , binding to the mutated ones. In B, IgE binding from 8 individual patient sera, randomly selected from the pool, is shown. Each color represents the OD measurement of an individual patient, resulting in a cumulative OD for all patients.
Journal of Allergy and Clinical Immunology  , DOI: ( /mai )
Copyright © 2003 Mosby, Inc. Terms and Conditions

5. Fig. 4
Peptides AAs in length, representing the major IgE-binding epitopes of αs1-casein, and “engineered” peptides with single-AA changes at each position were synthesized on a cellulose membrane. IgE binding to the native and mutated peptides was determined with pooled and individual patient sera. The results for 2 IgE-binding epitopes (AA and AA ) are presented in this figure. Each column represents a mutated peptide in which an AA change has been made at the indicated position. IgE binding to each modified peptide was measured by OD and is shown for the pooled patient sera in row 1 and for the individual patients in the following rows. The results are expressed as the percentage of IgE-antibody binding to the mutated peptides relative to the wild-type peptides, ranging from no reduction in binding, represented in black squares , to total abrogation of binding, represented in white squares . Although no single-AA substitution resulted in abrogation of IgE-binding in every patient, substitutions highlighted in red showed at least a clear reduction in the majority of patients. Therefore, these AAs were substituted simultaneously, and the results are shown in the last column.
Journal of Allergy and Clinical Immunology  , DOI: ( /mai )
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6. Fig. 5
Selected part of the SPOT membrane, showing the synthesized, wild-type (WT ) peptides for AA and the corresponding mutated peptides with multisided AA substitution. Shown is IgE labeling with pooled sera from 15 CMA patients.
Journal of Allergy and Clinical Immunology  , DOI: ( /mai )
Copyright © 2003 Mosby, Inc. Terms and Conditions

7. Fig. 6
Synthesized peptides (underlined) representing the major IgE-binding epitopes on αs1-casein are shown. The numbers on the top indicate the position of the AA relative to the first AA encoded by this sequence. All hydrophobic AA residues are marked in red . The squares indicate the AAs critical for IgE binding.
Journal of Allergy and Clinical Immunology  , DOI: ( /mai )
Copyright © 2003 Mosby, Inc. Terms and Conditions