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

Abstract

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 TIA12), 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.