Enzyme-linked immunosorbent assay for human insulin-like growth factor-I using monoclonal and polyclonal antibodies with defined epitope recognition.
Monoclonal antibodies (McAb) and polyclonal antibodies (PcAb) against human insulin-like growth factor-I (somatomedin C; hIGF-I) were produced. Using these two antibodies, an enzyme-linked immunosorbent assay (ELISA) system for hIGF-I was established. The ELISA system was able to detect hIGF-I at a range of 1-25 micrograms/l, compared with the range of 1-50 micrograms/l detected by radioimmunoassay (RIA). Human IGF-II and human insulin could not be recognized in this system. The plasma concentrations of IGF-I found using the ELISA agreed well with those found using RIA after conventional Sep-Pak C18 cartridge pretreatment. Epitopes of hIGF-I to McAb and PcAb were investigated by enzymatic digestion of hIGF-I followed by comparing the affinity of the antibodies to the peptides obtained proteolytically. The epitope to McAb was found to be a peptide containing Leu10-Val11-Asp12 (epitope 2). Five epitopes to PcAb containing the following key fragments were identified: a conformational structure formed by the disulphide bonds between Cys6 and Cys48, and between Cys47 and Cys52 (epitope 1), Leu10-Val11-Asp12 (epitope 2), Val17-Cys18-Gly19-Asp20 (epitope 3), Arg21-Gly22-Phe23-Tyr24 (epitope 4) and Lys68-Ser69-Ala70 (epitope 5). Of these, the peptide containing epitope 5 showed the highest affinity to PcAb. The results indicated that our ELISA system combined recognition by epitope 2 of McAb and recognition by epitope 5 of PcAb to obtain its good specificity.
Product Development Laboratories, Fujisawa Pharmaceutical Co. Ltd, Osaka, Japan.
K
K
Tamura
M
M
Kobayashi
S
S
Suzuki
Y
Y
Ishii
S
S
Koyama
H
H
Yamada
K
K
Hashimoto
M
M
Niwa
F
F
Shibayama
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000032:analysis; D005260:Female; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D013002:Somatomedins / Q000032:analysis*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I