SMALL SYNTHETIC PEPTIDES OF HUMAN GM-CSF REQUIRE DIFFERENT CONDITIONSFOR IMMOBILIZATION, EPITOPE DENSITY AND PRESENTATION IN ELISA

Different conditions influencing hapten immobilization and presentatio n of five small synthetic peptides (amino acid residues 4-16, 14-24, 4 -24, 30-41 and 79-91) of the human granulocyte-macrophage colony stimu lating factor (hGM-CSF) molecule, were studied by measurement of antib ody binding using antiprotein and antipeptide antibodies, by ELISA. Ei ther peptides covalently coupled to another protein carrier than the o ne used for immunization or free peptides were used as the coating tar get antigen. Directional coupling of synthetic peptides was assured vi a a cysteine residue, incorporated at the peptide C-terminus, through a maleimide containing hetero-bifunctional crosslinker. Using rhGM-CSF or peptide-protein conjugates as the coat, maximum O.D.-readings occu red with carbonate buffer at pH 9.6. With antipeptide antibodies, it w as necessary in some cases to use free peptides in order to exclude th e influence of anti-linker antibodies. We found that different conditi ons with regard to pH and ionic strength of the coating buffer, had to be determined for each peptide because some of them lacked sensitivit y due to poor adsorption to the solid phase or improper presentation. To improve epitope density and presentation, multiple copies of the sa me peptide were synthesized on a branched polylysine core. The data sh owed that multiple branched peptides (i.e. 4-or 8-copies) bound antibo dies more effectively than monomeric or dimeric peptides.