Sorting polyclonal antibodies into functionally distinct fractions using peptide phage display: 'a library on top of a library'.
A general approach for sorting antibodies (Abs) to a restricted protein domain was developed using phage-displayed peptide libraries. The method is demonstrated by fractionating polyclonal antibodies (pAbs), raised against a short peptide derived from the extracellular, juxtamembrane region of fibroblast growth factor receptor 1 (FGFR1) into fractions with distinct chemical and biological characteristics. Screening two combinatorial peptide libraries, with the pAb, several sequences, homologous to different regions within the original peptide, were identified. Four of the corresponding peptides were synthesized and used as peptide-conjugated affinity columns for the fractionation of the pAbs. The fractions obtained were unique in their recognition patterns and in their capacity to immunoprecipitate and immunoblot, as well as to modulate the activity of FGFR1. This technique is, therefore, highly sufficient in separating pAbs to monospecific fractions and may also be used for fine mapping of different, even overlapping, sequences within a restricted peptide or protein domain.
Moshitch-Moshkovitz
S
S
Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel.
Heldman
Y
Y
Yayon
A
A
Katchalski-Katzir
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification*; D002460:Cell Line; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000276:immunology; D011817:Rabbits; D020794:Receptor Protein-Tyrosine Kinases / Q000276:immunology*; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017468:Receptors, Fibroblast Growth Factor / Q000276:immunology*
D000906:Antibodies; D019151:Peptide Library; D010455:Peptides; D017468:Receptors, Fibroblast Growth Factor; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1