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.
Kumarasamy
R
R
Schering-Plough Research Institute, Kenilworth, New Jersey 07033.
Bausch
J
J
Kopcha
D
D
Patel
S
S
McGonigle
E
E
D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000032:analysis*; D006801:Humans; D015151:Immunoblotting; D008970:Molecular Weight; D012709:Serum Albumin / Q000032:analysis*; D012996:Solutions; D013696:Temperature
D003432:Cross-Linking Reagents; D012709:Serum Albumin; D012996:Solutions; D016178:Granulocyte-Macrophage Colony-Stimulating Factor