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Generation of Anti-Insulin-Like Growth Factor-Binding Protein-Related Protein 1 (IGFBP-rP1/MAC25) Monoclonal Antibodies and Immunoassay: Quantification of IGFBP-rP1 in Human Serum and Distribution in Human Fluids and Tissues

Departments of Pediatrics (A.L.-B., E.M.K., D.L.G., V.H., R.G.R.) and Pathology (C.L.C.), Oregon Health Sciences University, Portland, Oregon 97201; Department of Laboratory Medicine and Pathobiology (J.K.), Mount Sinai Hospital, Toronto M5G IX5, Canada; Diagnostic System Laboratories (J.K., A.D.), Toronto, Canada; and Diagnostic System Laboratories (R.G.K., U.B.), Webster, Texas 77598

Address all correspondence and requests for reprints to: Ron G. Rosenfeld, M.D., Department of Pediatrics-CDRCP, Oregon Health Sciences University, 707 SW Gaines Road, Portland, Oregon 97201. E-mail: rosenfer{at}ohsu.edu.

The IGF-binding protein (IGFBP)-related proteins (rPs) are a group of recently described cysteine-rich proteins that share significant amino-terminal structural similarity with the conventional IGFBPs. IGFBP-rP1 (also known as MAC25/angiomodulin/prostacyclin-stimulating factor and T1A12), regulates cellular proliferation, adhesion, and angiogenesis and stimulates prostacyclin synthesis. We characterized new monoclonal antibodies generated against IGFBP-rP1 and have used them to study the distribution of IGFBP-rP1 in human biological fluids and tissues. Additionally, we have developed a noncompetitive sandwich-type immunoassay to quantitate the concentrations of IGFBP-rP1 in human serum. IGFBP-rP1 was readily detectable in serum, urine, amniotic fluid, and cerebrospinal fluid by immunoblot analysis. Evaluation of the newly developed immunoassay demonstrated acceptable analytical performance, with a detection limit of 0.7 µg/liter, a dynamic range of 3.1–100 µg/liter, and intra- and interassay coefficients of variation of 2.5–6.8% and 3.1–6.4% at approximately 24–85 ng/ml IGFBP-rP-1, respectively. No significant cross-reactivity with IGFBP-1–6 was observed. In random normal human adult sera (n = 37), the median IGFBP-rP1 was 21.0 µg/liter, and values did not correlate with levels of IGF-I (r = 0.085, P = 0.61), IGF-II (r = 0.051, P = 0.75), or IGFBP-3 (r = 0.061, P = 0.74). The monoclonal anti-IGFBP-rP1 antibodies also readily detected IGFBP-rP1 expression in human tissue sections, with preferential expression of IGFBP-rP1 in the microvascular endothelium associated with tumorigenesis. In summary, using newly developed IGFBP-rP1 monoclonal antibodies, we confirm the presence of IGFBP-rP1 in the major human body fluids, provide quantitative normative data on the concentrations of IGFBP-rP1 in human serum, and show preferential expression of IGFBP-rP1 in the microvascular endothelium associated with tumorigenesis. The use of these novel IGFBP-rP1 detection tools should prove useful in the elucidation of the biological role(s) of this protein.

This work was supported by NIH Grant CA58110 and DMD17-00-1-0042. A.L.-B. is supported by a Fellow Research Funding Grant from Eli Lilly & Co. and is also a recipient of Grants 97/5309 and 98/9198 from the Fondo de Investigacion Sanitaria, Spain.

Abbreviations: AF, Amniotic fluid; CCN, connective tissue growth factor/Cyr61/Nov; CSF, cerebrospinal fluid; CTGF, connective tissue growth factor; HRP, horseradish peroxidase; IGFBP, IGF-binding protein; IGFBP-rP, IGF-binding protein-related protein; rh, recombinant human; WIB, Western immunoblot.

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