1. Determinant analysis of IgE and IgG4 antibodies and T cells specific for bovine αs1- casein from the same patients allergic to cow's milk: Existence of αs1-casein–specific B cells and T cells characteristic in cow's-milk allergy 
Haruyo Nakajima-Adachi, PhDa, Satoshi Hachimura, PhDa, Wataru Isea, Kiri Honma, MD, PhDb, Shinya Nishiwakia, Maiko Hirotaa, Naoki Shimojo, MD, PhDb, Toshiyuki Katsuki, MD, PhDb, Akio Ametani, PhDa, Yoichi Kohno, MD, PhDb, Shuichi Kaminogawa, PhDa 
Journal of Allergy and Clinical Immunology 
Volume 101, Issue 5, Pages (May 1998)
DOI: /S (98)
Copyright © 1998 Mosby, Inc. Terms and Conditions

2. Fig. 1
Screening of allergic patients having high titers of anti-αs1-casein IgE antibodies in their sera. One hundred and twenty-nine allergic infants from the diagnostic department of Chiba University Hospital who manifested severe atopic dermatitis and had positive RAST scores for cow's milk were studied. Binding of their IgE to αs1-casein was analyzed by ELISA, and nine of them (C1 to C3 and C5 to C10) whose sera were available in sufficient quantities for further study were selected.
Journal of Allergy and Clinical Immunology  , DOI: ( /S (98) )
Copyright © 1998 Mosby, Inc. Terms and Conditions

3. Fig. 2
Amino acid sequence of αs1-casein (A) and scheme of overlapping synthetic peptides and CNBr-digested fragments (B). Circled p, phosphoserine; filled bar, αs1-casein; hatched bars, synthetic peptides corresponding to regions of αs1-casein (P1 to P13); open bars, CNBr-digested fragments corresponding to regions of αs1-casein (F1 to F4).
Journal of Allergy and Clinical Immunology  , DOI: ( /S (98) )
Copyright © 1998 Mosby, Inc. Terms and Conditions

4. Fig. 3
Mapping of epitopes on αs1-casein recognized by anti-αs1-casein IgE in sera obtained from normal donors (A) and patients with CMA (B) by using synthetic peptides. Synthetic peptides with sequences located throughout an entire αs1-casein molecule are shown as P1 to P13. A405 (absorbance at 405 nm) obtained at the last step of ELISA indicates binding of serum IgE to each of these synthetic peptides. Values shown indicate titers obtained according to the following formula: Absorbance of each of the peptide-coated wells – Absorbance of uncoated wells in the case of serum from each patient. Binding of serum from donor C1 was assessed in another experiment.
Journal of Allergy and Clinical Immunology  , DOI: ( /S (98) )
Copyright © 1998 Mosby, Inc. Terms and Conditions

5. Fig. 4
Schematic representation of binding of IgE from patients with CMA. Positive binding was evaluated by formula described in Methods section. Black squares, positive binding.
Journal of Allergy and Clinical Immunology  , DOI: ( /S (98) )
Copyright © 1998 Mosby, Inc. Terms and Conditions

6. Fig. 5
Inhibition assay of binding of IgE from patients with CMA to αs1-casein. Sera from two patients with CMA, C1 (A) and C3 (B), were preincubated with various concentrations of αs1-casein (filled circles), P13 (the common binding peptide for IgE from nine selected patients with CMA) (open circles), or P3 (the peptide with which no sera reacted (open triangles). These sera were then tested for their binding to plate-bound αs1-casein.
Journal of Allergy and Clinical Immunology  , DOI: ( /S (98) )
Copyright © 1998 Mosby, Inc. Terms and Conditions

7. Fig. 6
Binding of anti-αs1-casein IgE from sera of normal donors and patients with CMA to CNBr-digested fragments of αs1-casein. Sera from normal donors interacted with none of four fragments, although we observed a slight reaction to αs1-casein (A). Sera from patients with CMA exhibited significant interaction with αs1-casein and F3 (amino acid residues ) compared with other fragments (B).
Journal of Allergy and Clinical Immunology  , DOI: ( /S (98) )
Copyright © 1998 Mosby, Inc. Terms and Conditions

8. Fig. 7
Mapping of epitopes recognized by anti-αs1-casein IgG4 in sera obtained from normal donors (A) and patients with CMA (B) by using synthetic peptides. A405 (absorbance at 405 nm) obtained at last step of ELISA indicates binding of serum IgE to each synthetic peptide. ELISA values shown indicate titers obtained by correcting for nonspecific binding of biotin-labeled anti-human IgG4 in each patient according to following formula: Absorbance of the antigen-coated wells in which the sera diluted with PBS-TG were added – Absorbance of the antigen-coated wells in which PBS-TG only in the place of the sera were added. Positive reactions of anti-αs1-casein IgG4 binding were further evaluated according to the method described in the Methods section.
Journal of Allergy and Clinical Immunology  , DOI: ( /S (98) )
Copyright © 1998 Mosby, Inc. Terms and Conditions

9. Fig. 8
Determinants recognized by αs1-casein–specific TCLs from two patients with CMA. A, Proliferation of TCLs 5A7 and 1A3 established from donor C1 in response to synthetic peptides. B, Proliferation of TCLs 1G1, 2C1 and 1C7 established from donor C2 in response to synthetic peptides and that of 3C3 and 4E11 to CNBr-digested fragments of αs1-casein. TCLs (1 to 2 × 104) were incubated in presence of EB-B cells (1.5 to 3 × 104) as APCs and the optimal concentration of antigen (at 4.0 μmol/L of either αs1-casein or the synthetic peptides) for 3 days. After this incubation, tritiated-thymidine was added, and its uptake was analyzed.
Journal of Allergy and Clinical Immunology  , DOI: ( /S (98) )
Copyright © 1998 Mosby, Inc. Terms and Conditions

10. Fig. 9
Common structures found in peptides recognized by αs1-casein–specific TCLs from each patient with CMA. Although no TCLs from either patient reacted with same peptide, peptides recognized by these T cells had common structure: -E-(X)7-L- in C1 and -E-(X)6-K- in C2. -E-(X)7-L- was found in three different regions, and -E-(X)6-K- was found in four different regions within amino acid sequence of αs1-casein.
Journal of Allergy and Clinical Immunology  , DOI: ( /S (98) )
Copyright © 1998 Mosby, Inc. Terms and Conditions