Immunoassay of insulin-like growth factor-binding protein-3 (IGFBP-3): new means to quantifying IGFBP-3 proteolysis.
Posttranslational modifications, particularly proteolysis, may play a significant role in the regulation of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) physiology, and thus, measurement of modified variants of IGFBP-3 and/or their combination ratios may have important research and diagnostic relevance. Based on evaluation of a panel of monoclonal and polyclonal IGFBP-3 antibodies, we constructed three new enzyme-linked immunosorbent assays (ELISAs) using a common capture and polyclonal (ELISA-3) or monoclonal (ELISA-1 and -2) detection antibodies and evaluated them in a two-step colorimetric procedure. Evaluation of ELISA-1-3 demonstrated detection limit, dynamic range, overall precision, and recovery of the added IGFBP-3 to be generally less than 0.04 microg/L, 2-100 microg/L, less than 10%, and 91-113%, respectively. IGF-I and -II, and IGFBP-1, -2, -4, -5, and -6 did not interfere. In normal adult sera (n = 26), seminal plasma (n = 14), pregnancy sera (n = 30), and amniotic fluid (n = 30), ELISA-1-3 detected significantly different IGFBP-3 levels (by up to 6-fold, on the average), whereas levels in seminal plasma determined by ELISA-1 were undetectable. Comparison of the values obtained vs. corresponding levels by an established method (Diagnostic Systems Laboratories, Inc., active IGFBP-3 ELISA) were similarly sample dependent and, on the average, varied by up to 19-fold. Only ELISA-3 compared well with the Diagnostic Systems Laboratories, Inc., IGFBP-3 ELISA when samples from normal adults were analyzed. The observed variability could not be totally explained by 50% lower reactivity of ELISA-1-3 for glycosylated IGFBP-3 vs. the nonglycosylated form, and changes in phosphorylation had no effect on immunoreactivity. Evaluation of IGFBP-3 after proteolysis by seminal plasma, plasmin, or thrombin suggested recognition of intact IGFBP-3 by ELISA-1, whereas ELISA-3 appeared to measure intact and proteolyzed IGFBP-3 (total IGFBP-3) with similar potency. In contrast, levels determined by ELISA-2 increased severalfold, indicating preferential recognition of IGFBP-3 fragments. We propose that immunoassay capable of differential determination of IGFBP-3 variants may help better define the physiological importance and potential clinical value of IGFBP-3 measurements.
Diamandi
A
A
Diagnostics Systems Laboratories, Inc., Toronto, Ontario, Canada.
Mistry
J
J
Krishna
R G
RG
Khosravi
J
J
D000328:Adult; D000653:Amniotic Fluid / Q000737:chemistry*; D000906:Antibodies; D000911:Antibodies, Monoclonal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D005341:Fibrinolysin / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000032:analysis* / Q000097:blood*; D007700:Kinetics; D008297:Male; D008875:Middle Aged; D011247:Pregnancy; D015203:Reproducibility of Results; D012661:Semen / Q000737:chemistry; D012680:Sensitivity and Specificity
D000906:Antibodies; D000911:Antibodies, Monoclonal; D018972:Insulin-Like Growth Factor Binding Protein 3; D005341:Fibrinolysin