The use of T bag synthesis with paper discs as the solid phase in epitope mapping studies.
Epitope mapping with synthetic peptides bound to derivatized paper discs has been investigated using the discs both as a solid-phase matrix for peptide synthesis as well as the solid phase in immunologic testing procedures without detachment of the peptides from the paper discs. Using the T bag (tea bag) method the simultaneous synthesis and subsequent immunologic testing of large numbers of peptides was demonstrated for the feline major allergen Fel d I. A total of 15,000 paper disc-bound peptides, comprising the 146 nonapeptides overlapping by eight amino acid residues on both chains, were synthesized simultaneously with 100 paper discs per T bag. Using these paper disc-bound peptides as the solid phase in radioimmunoassays the binding sites found coincided with those detected in the PEPSCAN with the commercially available epitope mapping kit and with the binding sites that had been found with Sepharose-coupled peptides. The signal to background-ratio in the paper disc-RIA was comparable to that in the PEPSCAN and the reproducibility was good. The bound antibodies could be eluted from the paper disc-bound peptides, permitting regeneration and repeated use of the paper discs for immunologic testing. This method was shown to be a useful alternative to the PEPSCAN and to have the major advantage that large numbers of antibodies could be tested with large numbers of peptides simultaneously.
van 't Hof
W
W
Central Laboratory, The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
van den Berg
M
M
Aalberse
R C
RC
D000485:Allergens / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002415:Cats; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D006023:Glycoproteins; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood; D009211:Myoglobin / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology*; D011863:Radioimmunoassay
D000485:Allergens; D000911:Antibodies, Monoclonal; D000939:Epitopes; D006023:Glycoproteins; D009211:Myoglobin; D010446:Peptide Fragments; D007073:Immunoglobulin E; C059296:Fel d 1 protein, Felis domesticus