Quantification of insulin-like growth factor I (IGF-I) without interference by IGF binding proteins.
A chemiluminescent dot blot assay has been developed by our laboratory for rapid determinations of IGF-I in serum-free conditioned media (CM) collected from cultured cells. In contrast to IGF-I radioimmunoassays (RIAs), the IGF binding proteins (IGFBPs) did not interfere with the dot blot assay and did not require the laborious (and sometimes ineffective) removal of IGFBPs. Although all six IGFBPs were shown to bind to 125I IGF-I, none interfered with IGF-I detection on nitrocellulose dot blots. In contrast, an RIA using the same Oncogene monoclonal antibody (clone 82-9A) showed interference by IGFBP-1, IGFBP-2, and IGFBP-4. The IGF-I dot blot assay was sensitive (0.125-8.0 ng IGF-I), specific (assay crossreactivity with IGF-II is less than 1%), and reproducible (intra-assay variance < or = 6%; inter-assay variance < 12%) when chemiluminescence was quantified by phosphorimager and Molecular Analyst software (BioRad). The apparent sensitivity of the enhanced chemiluminescence (ECL) reagent to serum, precludes the use of this assay for IGF-I determination in serum or in serum-containing media.
De León
D D
DD
Department of Physiology, Loma Linda University School of Medicine, CA 92350, USA.
Asmerom
Y
Y
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D017077:Culture Media, Conditioned / Q000737:chemistry*; D006801:Humans; D007163:Immunosorbent Techniques; D018969:Insulin-Like Growth Factor Binding Proteins / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000032:analysis* / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000032:analysis; D008163:Luminescent Measurements; D011786:Quality Control; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II