A novel affinity purification method to isolate peptide specific antibodies.
Site-specific, high affinity polyclonal antisera are effectively and successfully produced by immunizing rabbits with synthetic peptides. The use of these antisera in subsequent immune analysis is often limited because of non-specific binding. We describe a new and simple method to effectively affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact, antigenic protein in immunoblot analyses. The sequence-specific nature of the eluted antibodies was confirmed since binding to the antigenic proteins could be displaced by the immunizing but not by unrelated peptides.
Karlsen
A
A
Hagedorn Research Laboratory, Niels Steensensvej, Gentofte, Denmark.
Lernmark
A
A
Kofod
H
H
Dyrberg
T
T
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006683:HLA-DQ Antigens / Q000138:chemical synthesis / Q000276:immunology; D015151:Immunoblotting; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology*; D011817:Rabbits; D051381:Rats; D011986:Receptors, Somatotropin / Q000138:chemical synthesis / Q000276:immunology
D000906:Antibodies; D006683:HLA-DQ Antigens; D010455:Peptides; D011986:Receptors, Somatotropin