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
R. Kumarasamy et al., 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, Pharmaceutical research, 11(3), 1994, pp. 365-371
HPLC analyses of GM-CSF in solution mixtures containing both GM-CSF an
d HSA showed losses of GM-CSF which could not be accounted for using c
onventional electrophoretic and/or RP-HPLC techniques. Further investi
gation of these mixtures by immunoblotting and by immunoaffinity chrom
atography demonstrated the presence of high molecular weight (>67,000)
GM-CSF related species. No such species was detectable in solutions o
f 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 c
onceived which could react only with this type of hybrid molecule. A s
andwich 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 c
onfirming its existence by ELISA, a GM-CSF/HSA adduct was isolated fro
m the solution mixture containing both GM-CSF and HSA. This isolate se
rved as a primary reference standard in the ELISA assay. The immunoass
ay 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 verif
ication, conjugates of GM-CSF/HSA were synthesized using a cross-linki
ng reagent. These covalent conjugates reacted positively in the ELISA
and are employed as a convenient alternative reference standard.