Epitope mapping of the cat (Felis domesticus) major allergen Fel d I by overlapping synthetic peptides and monoclonal antibodies against native and denatured Fel d I.
The major cat allergen Fel d I is a homodimer of which each monomer consists of two disulfide-linked polypeptide chains: chain 1 (70 amino acid residues) and chain 2 (92 amino acid residues). Twenty-one synthetic peptides of 14 amino acid residues length, overlapping by seven residues and spanning the entire sequence of both chains, were synthesized. These peptides were coupled to CNBr-activated Sepharose-4B and used as solid-phase antigens in epitope-mapping studies with monoclonal antibodies against native and reduced/alkylated Fel d I. Two monoclonal antibodies directed against reduced/alkylated chain 1 bound to the overlapping peptides 53-66 and 60-70 of chain 1. The monoclonal antibody directed against reduced/alkylated chain 2 bound to the overlapping peptides 36-49 and 43-56 of chain 2. Binding specificity was demonstrated by inhibition by reduced/alkylated Fel d I for all three monoclonal antibodies. Another monoclonal antibody against reduced/alkylated Fel d I had been found to bind predominantly to reduced/alkylated chain 2 on immunoblot in previous studies. It bound to peptides 1-16 and 60-70 of chain 1 and peptides 1-14 and 50-63 of chain 2; it is therefore probably directed against a conformational epitope formed by these four regions. Possibly because of low affinity of this monoclonal antibody, specificity of its binding could not be verified by inhibition studies. A panel of monoclonal antibodies directed against native Fel d I bound to peptides 1-16 and 60-70 of chain 1 and peptides 1-14 and 43-56 of chain 2.(ABSTRACT TRUNCATED AT 250 WORDS)
van 't Hof
W
W
Central Laboratory, The Netherlands Red Cross Blood Transfusion Service, University of Amsterdam.
van Milligen
F J
FJ
van den Berg
M
M
Lombardero
M
M
Chapman
M D
MD
Aalberse
R C
RC
D000478:Alkylation; D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D002415:Cats; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D010449:Peptide Mapping; D011487:Protein Conformation; D011489:Protein Denaturation
D000485:Allergens; D000911:Antibodies, Monoclonal; D000939:Epitopes