Mutational analysis of immunoglobulin E-binding epitopes of beta-casein and beta-lactoglobulin showed a heterogeneous pattern of critical amino acids between individual patients and pooled sera.
BACKGROUND
For immunotherapeutic approaches, 'critical' amino acids (AAs) within allergenic epitopes are replaced with alternate AAs to eliminate IgE antibody binding.


OBJECTIVE
To determine the critical AAs for IgE binding in beta-casein and beta-lactoglobulin (BLG).


METHODS
Peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single AA substitutions (alanine or glycine) at each position. Membranes were incubated with a pool of sera from 15 cow's milk-allergic patients and individual sera from six of the 15 patients. In cases where no decrease in binding occurred with a single AA substitution, peptides with two AA substitutions were generated and labelled.


RESULTS
Using pooled patient sera, single AA substitutions led to complete elimination of binding to six of 11 peptides for beta-casein and to all six peptides for BLG. Substituting two AAs led to an elimination of binding to four of the remaining five beta-casein epitopes. However, in three of the 11 modified beta-casein peptides and five of the six BLG peptides, no decrease in IgE binding occurred in at least one individual patient. For these patients, critical AAs other than those defined by the patient serum pool were identified, indicating a heterogeneous pattern of IgE recognition.


CONCLUSIONS
These results indicate that AAs critical for IgE binding are more heterogeneous than initially defined by pooled milk-allergic patient sera. For future immunotherapeutic interventions with mutated peptides, critical AAs should also be identified with individual patient sera to account for heterogeneity of IgE binding between patients.
Cocco
R R
RR
Division of Pediatric Allergy & Immunology and Jaffe Institute for Food Allergy, The Mount Sinai School of Medicine, New York, NY 10029-6574, USA.
Järvinen
K-M
KM
Han
N
N
Beyer
K
K
Sampson
H A
HA
D000293:Adolescent; D019943:Amino Acid Substitution / Q000276:immunology*; D000818:Animals; D002364:Caseins / Q000235:genetics / Q000276:immunology*; D002417:Cattle; D002648:Child; D002675:Child, Preschool; D004252:DNA Mutational Analysis; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D005260:Female; D014644:Genetic Variation; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D007167:Immunotherapy; D007782:Lactoglobulins / Q000235:genetics / Q000276:immunology*; D008297:Male; D008892:Milk / Q000009:adverse effects / Q000276:immunology*; D016269:Milk Hypersensitivity / Q000097:blood / Q000235:genetics / Q000276:immunology* / Q000628:therapy; D010455:Peptides / Q000235:genetics / Q000276:immunology
D002364:Caseins; D000939:Epitopes; D007782:Lactoglobulins; D010455:Peptides; D007073:Immunoglobulin E