AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) FOR QUANTITATION OF ADDUCTS OF GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR (GM-CSF) AND HUMAN SERUM-ALBUMIN (HSA) IN STRESSED SOLUTION MIXTURES

HPLC analyses of GM-CSF in solution mixtures containing both GM-CSF and HSA showed losses of GM-CSF which could not be accounted for using conventional electrophoretic and/or RP-HPLC techniques. Further investigation of these mixtures by immunoblotting and by immunoaffinity chromatography demonstrated the presence of high molecular weight (>67,000) GM-CSF related species. No such species was detectable in solutions of GM-CSF alone. This experiment pointed to the formation of an adduct between GM-CSF and HSA in the solution mixtures. To probe further the hypothesis of a GM-CSF/HSA adduct, an immunologically based test was conceived which could react only with this type of hybrid molecule. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed using two antibodies, anti-GM-CSF (capture antibody) and anti-HSA (detection antibody), as part of the quantitation of GM-CSF/HSA adducts. After confirming its existence by ELISA, a GM-CSF/HSA adduct was isolated from the solution mixture containing both GM-CSF and HSA. This isolate served as a primary reference standard in the ELISA assay. The immunoassay has a subnanogram sensitivity and is highly specific for GM-CSF/HSA adducts in the presence of either free GM-CSF or free HSA. As a verification, conjugates of GM-CSF/HSA were synthesized using a cross-linking reagent. These covalent conjugates reacted positively in the ELISA and are employed as a convenient alternative reference standard.