Use of synthetic peptides in the production and characterization of antibodies directed against predetermined specificities in rat immunoglobulin E.
Two peptides, P123 and P124, representing amino acid sequences His 542-Lys 557 and Tyr 459-Arg 472, respectively, of the CH4 domain of rat IgE and predicted to be located on accessible regions of the protein were synthesized by a solid-phase procedure. Rabbits were immunized with the peptides conjugated to KLH and their antisera were tested for reactivity with free peptide and rat IgE by inhibition-ELISA. Each animal produced antibodies which reacted specifically with its immunizing peptide (titre greater than 1/62,500), but not with other synthetic peptides of similar chain-length and composition. Antisera directed against peptides P123 and P124 specifically bound purified rat IgE (IR 162) and IgE in whole myeloma serum (greater than 1/6400), but showed no reaction with normal rat serum proteins and only very low binding to purified human IgE. In addition the binding of anti-peptide sera to rat IgE could be completely inhibited with either homologous peptide or purified rat IgE, but not by other peptides or purified human IgE. Heating rat IgE for 1 hr at 56 degrees C enhanced its binding to anti-peptide antibodies by between 4- and 60-fold, but markedly reduced its reactivity with a rabbit anti-rat IgE (Fc) serum. These results suggest that antibodies directed against the synthetic peptides employed recognize and specifically bind to sites within the CH4 domain of rat IgE represented by their respective immunizing peptides. Furthermore, these antibodies are capable of detecting subtle alterations in structural conformation resulting from heating at 56 degrees C. Epitopes represented by peptides P123 and P124 may contribute to the heat-sensitive cytophilic region of rat IgE.
D S
DS
Burt
G Z
GZ
Hastings
D R
DR
Stanworth
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006358:Hot Temperature; D007106:Immune Sera / Q000276:immunology*; D007073:Immunoglobulin E / Q000276:immunology*; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology*; D011817:Rabbits; D051381:Rats
D007106:Immune Sera; D010455:Peptides; D007073:Immunoglobulin E