An enzyme-linked immunosorbent assay (ELISA) for marograstim (KW-2228) was established. This ELISA proved to be highly sensitive with the detection limit of 0.01 ng/ml (about 0.5 fmol/ml) of KW-2228 and the assay range between 0.01 and 2 ng/ml. When 0.02 to 2 ng/ml of KW-2228 added to human plasma was determined, the variation coefficiencies of intra-day and inter-day assays were 0.4 to 7.6% and 5.2 to 15.8%, respectively, with good recoveries. These results indicate that this ELISA will be applicable to pharmacokinetic studies on KW-2228. With respect to the specificity, recombinant human granulocyte colony-stimulating factor (rhGCSF) produced in Esherichia coli as well as KW-2228 which does not have sugar chains in its structure showed slightly less immunoreactivity toward the antibody raised against KW-2228. The rhGCSF produced in Chinese hamster ovary cells (CHO) having sugar chains showed the lower immunoreactivity. The antigenic domains of KW-2228 were evaluated using a number of variants of hG-CSF. The variants having different amino acids from KW-2228 in the 1st to 5th residue of the N-terminus showed almost equal immunoreactivities to KW-2228. The immunoreactivities of the variants lacking 7 to 18 amino acids of N-terminus were less than 50% of that of KW-2228. No immunoreactivity was observed for the variants deleted in the area of 70th to 130th amino acids from the N-terminus. In addition, the immunoreactivity of a variant lacking the 10 amino acids from the C-terminus was 20% of that of KW-2228. From these findings, it was assumed that there are multiple antigenic domains in the sequence of KW-2228 except for a small region of the N-terminus.