Measurement of an analog of insulin-like growth factor-I in blood plasma using a novel enzyme-linked immunosorbent assay.
Long-Arg3-IGF-I (LR3IGF-I) is a synthetic analog of IGF-I that has much lower affinity for IGF-binding proteins than do native IGFs-I and -II. Comparisons of the effects of LR3IGF-I with those of IGFs-I and-II in in vitro and in vivo studies have proved useful in defining the functions of IGF-binding proteins. We have developed a sensitive noncompetitive nonisotopic assay of LR3IGF-I. Mouse IgG 1A7-F5-E5 binds an epitope that contains the substituted arginine3 in LR3IGF-I and was used as the solid phase antibody. The solution phase antibody was a rabbit immunoglobulin Nelson which binds to an epitope that is common to IGF-I and LR3IGF-I. The ELISA system was able to detect as little as 50 pg LR3IGF-I in 100 microliters and the native peptides IGFs-I and -II have less than 0.01% activity. Blood plasma from animals treated with pharmacologically active doses of this growth factor analog could be diluted 33.3-fold before assay, at which concentrations plasma had no significant effect on the assay. The ELISA response to LR3IGF-I was unaffected by the presence of IGF-binding proteins. The intra-assay and interassay coefficients of variation are 2.8 and 7.3% respectively. Recovery of LR3IGF-I added to blood plasma was approximately 90%. The ELISA was used to measure LR3IGF-I concentrations in plasma of cows treated with a pharmacologically active dose of this peptide and the results were compared with those obtained by a previously established LR3IGF-I RIA that requires size exclusion chromatography of plasma under acidic conditions to eliminate IGF-binding protein artefacts from the RIA. There was a positive correlation between results obtained by the two assays. The LR3IGF-I ELISA permits discrimination between the exogenous synthetic IGF-I analog and the endogenous native IGFs-I and -II in animals treated with this growth factor without the need for radioiodination of LR3IGF-I and elimination of the requirement for extraction of plasma before assay.
Gajanandana
O
O
Department of Biochemistry, University of Adelaide, Australia.
Irvine
K
K
Grant
P A
PA
Francis
G L
GL
Knowles
S E
SE
Wrin
J
J
Wallace
J C
JC
Owens
P C
PC
D000818:Animals; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006728:Hormones / Q000097:blood*; D018969:Insulin-Like Growth Factor Binding Proteins / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000031:analogs & derivatives* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011817:Rabbits; D011863:Radioimmunoassay; D012680:Sensitivity and Specificity
D006728:Hormones; D018969:Insulin-Like Growth Factor Binding Proteins; C085700:long R(3)-insulin-like growth factor-I; D007334:Insulin-Like Growth Factor I