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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 7 3401-3408
Copyright © 2003 by The Endocrine Society

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

Abel López-Bermejo, Javad Khosravi, Christopher L. Corless, Radha G. Krishna, Anastasia Diamandi, Umesh Bodani, Eric M. Kofoed, Donna L. Graham, Vivian Hwa and Ron G. Rosenfeld

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.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IGF SYSTEM comprises two ligands, IGF-I and IGF-II, six IGF-binding proteins (IGFBPs), IGFBP-1 to -6, and two receptors, type 1 and type 2 IGF receptors (1). Recently the IGFBP family has been expanded to include the IGFBP-related proteins (IGFBP-rPs), which share significant structural similarities with the IGFBPs (2, 3). Thus, the IGFBP superfamily includes the six conventional IGFBPs, which have high affinity for IGFs, and at least 10 IGFBP-rPs, which not only share the conserved amino-terminal domain of the IGFBPs but also show some degree of affinity for IGFs and insulin in several, but not all, assay systems (4, 5, 6, 7).

The IGFBP-rPs are a group of cysteine-rich proteins that control diverse cellular functions, such as cellular growth, cellular adhesion and migration, and synthesis of extracellular matrix. In addition, these proteins are involved in biological processes that include development and differentiation, reproduction, angiogenesis, wound repair, inflammation, fibrosis, and tumorigenesis (3).

IGFBP-rP1 was initially identified as a gene differentially expressed in normal leptomeningeal and mammary epithelial cells, compared with their counterpart tumor cells, and named meningioma-associated cDNA (MAC25) (8). The expressed protein was independently purified as a tumor-derived adhesion factor (later renamed angiomodulin) (5, 9) and as a prostacyclin-stimulating factor (10). It has additionally been reported as T1A12, a gene down-regulated in breast carcinomas (11).

Although the biological roles of IGFBP-rP1 have not been clearly established, there is a growing body of evidence that suggests that it may act as a tumor suppressor gene. IGFBP-rP1 is preferentially expressed in normal (vs. neoplastic) meningeal, mammary, and prostatic cells (8, 12, 13); it is up-regulated during senescence of mammary and prostatic cells (14, 15); loss of heterozygosity of the IGFBP-rP1 locus has been observed in 50% of cancerous breast tissues in one study (11); and IGFBP-rP1 shows growth inhibitory effects when overexpressed in prostate cancer cells (16, 17) or breast cancer cells (18).

In addition, IGFBP-rP1 may have an important role in vascular biology. It has been detected in tube-like structures in vitro (19) and high endothelial cells (20, 21) and is preferentially localized in the basement membrane of neocapillaries, like those seen in tumor tissues (5, 22, 23). Furthermore, analysis of genes differentially expressed in endothelial cells indicates that IGFBP-rP1 expression is up-regulated in tumor-derived endothelium (24). A role for IGFBP-rP1 in vascular biology emerges from demonstrations that IGFBP-rP1 is capable of stimulating the synthesis of the vasodilator prostacyclin in cultured endothelial cells and may, therefore, be involved in maintaining the permeability of newly synthesized capillaries (10).

We recently generated and characterized a polyclonal anti-IGFBP-rP1 antibody and identified IGFBP-rP1 in conditioned media from cultured human mammary and prostatic cells and in human biological fluids (12, 25). In this report, we further characterize the presence of IGFBP-rP1 in human biological fluids using anti-IGFBP-rP1 monoclonal antibodies. In addition, we provide quantitative data on serum IGFBP-rP1 using a newly developed IGFBP-rP1 immunoassay and examine the distribution of IGFBP-rP1 protein in human tissues.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

HPLC-purified IGFBP-1 from human amniotic fluid was kindly provided by Dr. D. R. Powell (Baylor College of Medicine, Houston, TX); rhIGFBP-2, -4, -5, and -6 were purchased from Austral Biologicals (San Ramon, CA); rhIGFBP-3, a nonglycosylated 29-kDa core protein expressed in Escherichia coli was a generous gift from Celtrix, Inc. (Santa Clara, CA). C-terminallyFLAG-tagged rhIGFBP-rP1, CTGF, and NovH were expressed in a baculovirus system as previously reported (2, 4, 26). Baculovirus-generated nontagged rhIGFBP-rP1 protein was purified over SP Sepharose (Sigma Chemical Co., St. Louis, MO) column equilibrated in MES buffer [50 mM N-morpholino-ethenesulfonic acid (pH 6.0), 2 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride]. IGFBP-rP1 protein retained on the column was eluted with an NaCl gradient [0.2–1.0 M in 2-(N-morpholine) ethane sulfonic acid buffer]. Fractions containing recombinant human (rh)IGFBP-rP1 were pooled and dialyzed against PBS. Analysis for protein purity and quantitation was as previously described (4). 125I-IGF-I and 125I-IGF-II were gifts from Diagnostic Systems Laboratories (Diagnostic Systems Laboratories, Webster, TX).

Nitrocellulose and electrophoresis reagents were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Polyclonal antibodies against IGFBP-rP1FLAG, CTGFFLAG, and NovHFLAG were generated in rabbits, as previously described (2, 25, 26). The IgG fractions were purified by a protein A affinity column (Amersham, Arlington Heights, IL). Monoclonal antibodies against IGFBP-rP1 (designated no. 1 through 11) were produced against the baculovirus-generated rhIGFBP-rP1FLAG described above. Horseradish peroxidase (HRP)-linked donkey antirabbit and sheep antimouse IgG antibodies and enhanced chemiluminescence detection reagents were purchased from Amersham.

Human biological fluids were obtained as anonymous samples from the Oregon Health Sciences University Central Laboratory and were residuals from routine clinical test samples. They were from apparently healthy adult subjects (i.e. with no known acute or chronic diseases); for cerebrospinal fluid (CSF), samples from both apparently healthy adult and pediatric subjects were analyzed. Additional serum and urine samples were collected from apparently healthy adult volunteers. The protocol was approved by the hospital Institutional Review Board, and informed consent was obtained from the healthy adult volunteers.

Adult serum samples included in the analysis were from 18 women, aged 25–76 yr and 19 men, aged 25–67 yr. On collection, blood samples were allowed to clot, then separated; after clinical testing, the residuals were used for these studies within 48 h of collection. Spot urine samples were from 20 women, aged 18–78 yr, and 10 men, aged 28–81 yr, collected after the first morning void. Urinalysis abnormalities excluded samples to be further tested for IGFBP-rP1. Samples were stored at -80 C after centrifugation to discard the cellular pellet. Residuals samples of amniotic fluid were from normal pregnancies between 15 and 20 wk gestation and were stored, after centrifugation, at -80 C until use. Residual samples from normal CSF (two women, four men, two boys, age range for the whole group: 4–88 yr) were also stored at -80 C until use. Urine, amniotic fluid (AF), and CSF were screened for IGFBP-rP1 within 2 months of collection.

Generation of monoclonal anti-IGFBP-rP1 antibodies

Five 10-wk-old Balb/c mice (Charles River, NC) were immunized with 50 µg/ml baculovirus-generated rhIGFBP-rP1FLAG protein with Freund’s complete adjuvant (ICN Biomedicals Inc., Aurora, OH). Three boosters (30 µg rhIGFBP-rP1FLAG with Freund’s incomplete adjuvant) were given at 30-d intervals. Two of the mice with high titers of antibodies against rhIGFBP-rP1FLAG were identified and were subsequently used for hybridoma generation. A third booster (75 µg rhIGFBP-rP1FLAG) was administered to the selected mice, and fusion was performed according to Lane (27).

The resulting hybridomas were screened as follows: IgG from hybridomal cell culture supernatants was captured on goat antimouse IgG-coated plates (DSL). After 2 h, the plates were washed three times with PBS-Tween 20 (0.01 M), and further incubated with 100 µl biotinylated IGFBP-rP1FLAG protein (200 ng/ml in PBS-Tween with 1% BSA and 1% goat serum). After washing with PBS-Tween, the plates were incubated with avidin-HRPO (Zymed Laboratories Inc., South San Francisco, CA) for 30 min, and the signal was developed with TMB (Life Technologies Inc., Grand Island, NY). To obtain pure clones, limiting dilution to 1 cell/well was performed for all positive hybridoma colonies and the resultant monoclonals confirmed by plate assays. The 11 clones are designated no. 1 to 11 in this report and represent the following isolated clones: 1, 1B4A; 2, 1B4B; 3, 1B4C; 4, 1D1A; 5, 1D1B; 6, 2A2A; 7, 2A2B; 8, 2C4A; 9, 2C4B; 10, 2C4C; and 11, 2C5.

Immunoadsorption of IGFBP-rP1 polyclonal antibody

Aliquots of the IGFBP-rP1 polyclonal antiserum were incubated overnight at 4 C with either rhIGFBP-rP1FLAG at a ratio of 15 µg/µl of antiserum (subsequently designated preadsorbed fraction) or buffer alone (immune fraction). To remove the rhIGFBP-rP1/anti-IGFBP-rP1 immune complexes, equal volumes of anti-FLAG M2 agarose beads (Sigma) were added to the aliquots above, and samples were incubated for another hour at 4 C. Samples were pelleted (5 min, at 12,000 g) and supernatants collected and frozen for subsequent Western immunoblot (WIB) studies.

WIB studies

For the initial characterization of the IGFBP-rP1 monoclonal antibodies, conditioned medium from normal human prostate epithelial cells, which are known to secrete large amounts of IGFBP-rP1 protein (12), was used as the source of endogenous IGFBP-rP1. Equal amounts of total protein per lane were dissolved in nondenaturing SDS sample buffer [0.5 mol/liter Tris (pH 6.8), 1% SDS, 10% glycerol, and bromphenol blue] and boiled for 5 min. Samples were electrophoresed on 15% SDS-polyacrylamide gels, electroblotted onto nitrocellulose, and membranes blocked with 4% milk-TBS-T [Tris-buffered saline-Tween-20 (0.1%)] for 1 h at 22 C. Western blots were incubated with IGFBP-rP1 polyclonal antiserum (IgG fraction, 6 µg/µl) at a 1:3000 dilution or with IGFBP-rP1 monoclonal antibodies (IgG fraction, 1 µg/µl) at a dilution of 1:2000 in TBS-T overnight at 4 C. Membranes were washed with TBS-T and incubated for 1 h at 22 C with a 1:3000 dilution of HRP-linked antirabbit or antimouse IgG secondary antibodies. Proteins of interest were detected with enhanced chemiluminescence reagents, according to the manufacturer’s protocol.

For the characterization of IGFBP-rP1 in human biological fluids, representative samples from healthy human subjects were prepared similarly and resolved on 15% SDS-polyacrylamide gels. Normal human serum was concentrated 10-fold using a heparin affinity column (Amersham) before these studies. Immunoblotting of nitrocellulose membranes involved: 1) both the immune and the preadsorbed fractions of the IGFBP-rP1 antiserum, and 2) anti-IGFBP-rP1 monoclonal antibody no. 5 and/or 10.

Western ligand blotting

Equimolar amounts of IGFBPs and IGFBP-rPs were resuspended in SDS sample buffer and resolved on 15% SDS-polyacrylamide gels. Separated proteins were electroblotted onto nitrocellulose membranes. Membranes were rinsed in 3% IGEPAL (Sigma) in TBS-T for 30 min at 22 C, blocked with 1% BSA IGEPAL (Sigma) in TBS-T for 1 h at 22 C and incubated with 2 x 106 cpm of a mixture of 125I-IGF and 125I-IGF-II in 1% BSA/TBS-T overnight at 4 C. Membranes were washed, dried, and exposed to Biomax film (Eastman Kodak Co., Rochester, NY).

IGFBP-rP1 ELISA development

Anti-IGFBP-rP1 monoclonal antibodies were employed to construct a noncompetitive sandwich-type immunoassay (see below). The antibody selection was based on extensive pair-wise evaluations in both one-step (equilibrium) and two-step (sequential) immunoreaction formats. Using this protocol, combinations of the 11 different anti-IGFBP-rP1 monoclonal antibodies were analyzed. The protocol optimization was based on the initial evaluation of a number of factors that could potentially affect detection limit, dynamic range, precision, and delayed sample addition (28). Antibody combinations demonstrating favorable analytical performances were further assessed for accuracy and comparative IGFBP-rP1 determinations. The sources of the raw materials and composition of the various buffers employed have been previously described (29, 30).

IGFBP-rP1 antibody coating to microtiter wells was performed at a concentration of 0.25–20 mg/liter by using previously published methods (29, 30). The IGFBP-rP1 detection antibodies were coupled to HRP as previously described (29). IGFBP-rP1 calibrators were prepared by appropriately diluting the recombinant IGFBP-rP1 in a protein-based buffer matrix [0.05 mol/liter sodium phosphate (pH 7.4), 9 g/liter NaCl, 6 g/liter BSA, and 0.5% Proclin 300]. The preparation was stable for at least 5 d at 4 C and more than 6 months at -70 C.

IGFBP-rP1 ELISA protocol

Calibrators or samples (0.020 ml) were added in duplicate to the precoated wells, followed by addition (0.1 ml) of the detection antibody-HRP conjugate (diluted in the assay buffer to approximately 0.1–0.25 mg/liter) and 4 h of incubation at room temperature with continuous shaking. The wells were washed five times and incubated with 0.1 ml/well of the TMB/H2O2 substrate solution for 10 min. Stopping solution (0.1 ml) was then added and absorbance measured by dual-wavelength measurement at 450 nm with background wavelength correction set at 620 nm. Absorbance measurements and ELISA data analysis were performed with the Labsystems Multiskan Multisoft microplate reader (Labsystems, Helsinki, Finland).

The best performances were obtained with a coating antibody concentration of 10 mg/liter (1000 ng/0.1 ml/well), a detection antibody concentration of approximately 0.1–0.25 mg/liter (10–25 ng/0.1 ml per well), a sample size of 0.02 ml, and a 4-h one-step (equilibrium) ELISA configuration. With this protocol, the differences in assay results caused by 1- to 20-min delay between addition of the same samples into the coated wells was less than 10%.

IGFBP-rP1 ELISA validation procedures

The lower limit of detection (sensitivity) was determined by interpolating the mean plus 2 SD of 12 replicate measurements of the zero calibrator. The intraassay coefficients of variation were determined by replicate analysis (n = 12) of four samples at IGFBP-rP1 concentrations of approximately 10–50 µg/liter in one run and interassay coefficients of variation by duplicate measurement of the samples in 12 separate assays. Recovery was assessed by adding 25 µl recombinant IGFBP-rP1 diluted in the standard matrix to 225 µl of three sera and analyzing the spiked and unspiked samples. Percent recovery was determined by comparison of the amount of added IGFBP-rP1 with the amount measured after subtracting the endogenous IGFBP-rP1 levels. Linearity was tested by analyzing three serum samples serially diluted (2- to 8-fold) in the zero calibrator of the assay.

The standard range and performance characteristics of IGFBP-rP1 are summarized in Table 1Go. Analysis of IGFBP-1, IGFBP-2, IGFBP-4–6 (up to 500 µg/liter) and IGFBP-3 (up to 5 mg/liter) did not show any cross-reactivity or interference. There was no cross-reactivity with IGF-I or IGF-II (up to 600 µg/liter) added to the assay zero standard followed by IGFBP-rP-1 analysis (data not shown).


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  		TABLE 1. IGFBP-rP1 ELISA validation data

 
Immunohistochemistry

Anonymous samples of normal and neoplastic human tissues were provided by the Cancer Pathology Shared Resource of the Oregon Cancer Center. These samples had been collected in the fresh state shortly after surgical resection and either snap frozen in optimal cutting temperature embedding compound, using a dry-ice/pentane slurry, or fixed in 10% buffered formalin. The fixed tissues were processed and embedded in paraffin using standard techniques.

Five-micrometer sections of the frozen samples were prepared in a cryostat, placed on Fisherbrand Plus slides (Fisher Scientific, Pittsburgh, PA) and allowed to air dry for 15 min at room temperature. Dried slides were wrapped in cellophane and stored at -80 C. The cryostat sections were allowed to warm to room temperature and then fixed for 10 min in freshly prepared 1% paraformaldehyde/PBS. Five-micrometer sections of the paraffin-embedded tissues were cut on a microtome and placed on Fisherbrand Plus slides. The slides were deparaffinized through xylenes and alcohol and then placed in TBS buffer for use in immunohistochemistry.

Immunohistochemistry was performed using an automated immunostainer (DAKO Corp., Carpinteria, CA), with all steps carried out at room temperature. Antibodies were diluted in a buffer containing 1% BSA, 0.1% Tween 20, 0.1% sodium azide in PBS. TBS was used for all wash steps. Following a 10-min incubation in dilution buffer, primary antibody (IgG fractions of anti-IGFBP-rP1 polyclonal or the anti-IGFBP-rP1 monoclonal antibodies) was added at a concentration of 1 µg/ml for 45 min, followed by washing and application of the secondary antibody. The secondary antibody (at a 1:400 dilution) was either biotinylated goat antirabbit or biotinylated horse antimouse (Vector Laboratories, Burlingame, CA). After 30-min incubation with the secondary antibody, the samples were treated with quench buffer (methanol/6% H2O2), washed, and bound antibodies were detected using the Vectastain Elite ABC kit (Vector Laboratories) as per the manufacturer’s recommendations. Premixed DAB solution (DAKO) was used in the final reaction (10 min). Slides were counterstained with hematoxylin before dehydration and coverslipping.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of IGFBP-rP1 monoclonal antibodies

Anti-IGFBP-rP1 monoclonal antibodies (IgG fractions) were initially screened by WIB for their ability to recognize rhIGFBP-rP1FLAG protein. As shown in Fig. 1AGo, baculovirus-generated rhIGFBP-rP1FLAG protein was detected by both the panel of monoclonal antibodies (no. 1 through 10) and the polyclonal anti-IGFBP-rP1 antibody. Anti-IGFBP-rP1 monoclonal antibody no. 11 was significantly less potent in recognizing the recombinant protein. We next examined the ability of these antibodies to identify human nonrecombinant (endogenous) IGFBP-rP1. As shown in Fig. 1BGo, all 11 anti-IGFBP-rP1 monoclonal antibodies and the polyclonal anti-IGFBP-rP1 antibody recognized the secreted IGFBP-rP1 in conditioned medium from normal human prostate epithelial cells (15). Although the sensitivity of the monoclonal antibodies for endogenous IGFBP-rP1 appeared to be lower than that of the polyclonal antibody, the sensitivity of the antibodies for human endogenous IGFBP-rP1 was comparable when electrophoresis was performed under reducing conditions (Fig. 1CGo).



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  		FIG. 1. Characterization of anti-IGFBP-rP1 monoclonal antibodies. Immunoreactivity of 11 anti-IGFBP-rP1 monoclonal antibodies was compared with our polyclonal anti-IGFBP-rP1 antibody (25 ). A, WIB using rhIGFBP-rP1FLAG and the IgG fractions of these antibodies. B, WIB using conditioned media from normal prostate epithelial cells, which are known to secrete large amounts of IGFBP-rP1 (15 ). C, Same as in B, except a reducing agent (dithiothreitol) was added to the conditioned media before gel electrophoresis.

 
IGFBP-rP1 is structurally related to the conventional IGFBPs as well as to other members of the IGFBP superfamily (3), like connective tissue growth factor (CTGF) (31) and NovH (32), both members of the CTGF/Cyr61/Nov (CCN) family. To confirm the specificity of the anti-IGFBP-rP1 monoclonal antibodies, equimolar amounts of the six IGFBPs (IGFBP-1 through 6), rhIGFBP-rP1FLAG, CTGFFLAG (2) and NovHFLAG (26) were electrophoresed and immunoblotted with two representative anti-IGFBP-rP1 monoclonal antibodies (no. 5 and 10). Neither of these antibodies cross-reacted significantly with CTGFFLAG, NovHFLAG, or any of the IGFBPs (Fig. 2AGo). The presence of these proteins was demonstrated by immunoblotting the same membranes with anti-CTGF and anti-NovH antibodies (data not shown) and by Western ligand blotting with 125I-IGF-I and 125I-IGF-II for the IGFBPs (Fig. 2BGo).



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  		FIG. 2. Specificity of anti-IGFBP-rP1 monoclonal antibodies. Monoclonal anti-IGFBP-rP1 antibody (no. 5) was tested against equimolar amounts of IGFBPs (IGFBP-1 through 6) and rhIGFBP-rP1FLAG, CTGFFLAG (2 ), NovHFLAG (26 ). Note that rhIGFBP-3 is Escherichia coli expressed and nonglycosylated, with a molecular mass of 29 kDa. Identical results were obtained with monoclonal anti-IGFBP-rP1 no. 10 (data not shown). A, WIB. B, Western ligand blot. The WIB in A was incubated with [125]IGF-I and [125]IGF-II (ligand blot) to confirm the presence of the six IGFBPs.

 
Characterization of IGFBP-rP1 in normal human biological fluids by WIB

Once the sensitivity and specificity of the anti-IGFBP-rP1 monoclonal antibodies were demonstrated, we investigated the presence of IGFBP-rP1 in the major human body fluids, such as serum, urine, AF, and CSF using these antibodies. In pooled normal human serum from healthy adults, both the polyclonal anti-IGFBP-rP1 antibody and the panel of monoclonal antibodies recognized an approximately 31-kDa protein that ran at slightly higher molecular mass than the baculovirus-generated rhIGFBP-rP1FLAG. Furthermore, the specificity of this band was demonstrated by immunoblotting with an rhIGFBP-rP1FLAG preadsorbed fraction of the polyclonal antibody (Fig. 3AGo). Similarly, distinct and specific IGFBP-rP1 bands were detected by both the polyclonal anti-IGFBP-rP1 antibody and monoclonal anti-IGFBP-rP1 antibodies in pooled normal human urine, AF, and CSF from healthy adults (Fig. 3BGo). The lower sensitivity of the monoclonals can be attributed to the fact that nonreducing conditions were employed for these WIB analyses because, as demonstrated above (see Fig. 1Go), reducing conditions enhance the affinity of the monoclonals for endogenous IGFBP-rP1.



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  		FIG. 3. WIB studies of IGFBP-rP1 in human body fluids. A, Two-microliter aliquots of 10-fold heparin affinity column-concentrated normal human serum were electrophoresed in triplicate and immunoblotted with polyclonal anti-IGFBP-rP1 antibody, rhIGFBP-rP1FLAG-preadsorbed fraction of anti-IGFBP-rP1 polyclonal antibody, or a representative monoclonal antibody (no. 5 and/or 10). Baculovirus-generated rhIGFBP-rP1 (100 ng) was run as control ("C"). B, Similar WIB studies were carried out with human urine (50 µl), AF (20 µl), CSF (50 µl), and rhIGFBP-rP1 (100 ng) as control ("C").

 
Quantification of IGFBP-rP1 in normal human serum by ELISA

An IGFBP-rP1 noncompetitive sandwich-type immunoassay (see Materials and Methods) was developed to analyze individual human serum samples. The IGFBP-rP1 ELISA standard curve for the assay is shown in Fig. 4Go.



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  		FIG. 4. IGFBP-rP1 ELISA standard curve. Linearity of the curve is as indicated (R2).

 
In normal human adult sera (n = 37), the median IGFBP-rP1 was 21.0 µg/liter (Table 2Go). The values did not correlate with 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) levels. A sexual dimorphism in circulating IGFBP-rP1 was evident, with higher IGFBP-rP1 concentrations in male subjects (26.3 ± 6.8, n = 19 vs. 19.8 ± 9.2, n = 18; P = 0.01). In matched samples from normal adult males and females (n = 13), median IGFBP-rP1 levels were similar in serum, plasma-EDTA, and plasma-heparin (ANOVA P = 0.768) and were 20.8, 19.9, and 22.0 ng/ml, respectively. The anti-IGFBP-rP1 antibodies also immunodetected circulating IGFBP-rP1 in other mammals (e.g. pigs and cows) but not rodents (data not shown). IGFBP-rP1 in the matched samples, in fetal bovine serum, as well as the recombinant (nontagged) form, was stable at 4 C for at least 4 d.


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  		TABLE 2. IGFBP-rP1 levels in human body fluids

 
Distribution of IGFBP-rP1 in human tissues

Immunostaining of human tissues with IGFBP-rP1 antibodies generated against a decapeptide in the C terminus of IGFBP-rP1 has been reported by Akaogi et al. (5) and more recently by Degeorges et al. (33). With our panel of characterized antibodies, the tissue distribution of IGFBP-rP1 in cryostat sections of normal and malignant human tissues was analyzed.

To ascertain the specificity of the immunostaining, parallel tissue sections (Fig. 5Go, A and B, lung squamous carcinoma tissue) were initially prepared with both the polyclonal anti-IGFBP-rP1 antibody and antibody preadsorbed with rhIGFBP-rP1FLAG. Figure 5AGo shows typical positive immunostaining (arrows), which was reduced to background with the preadsorbed fraction of the polyclonal antibody (Fig. 5BGo, arrows). Identical patterns of staining were observed in these sections with the monoclonal anti-IGFBP-rP1 antibodies (Fig. 5CGo, lung squamous carcinoma tissue).



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  		FIG. 5. Immunohistochemical analyses of IGFBP-rP1 in normal and malignant human tissues. Cryostat sections of various human tissues were fixed on slides and immunohistochemistry analysis performed using either the polyclonal or representative monoclonal antibodies (no. 5 and/or 10). Representative sections are as shown. Arrows indicate regions of specific immunostaining. A, Lung squamous carcinoma immunostained with the IgG fraction of anti-IGFBP-rP1 polyclonal antibody ({alpha}IGFBP-rP1 polyclonal). B, Parallel section of A immunostained with the preadsorbed fraction of {alpha}IGFBP-rP1 polyclonal antibody. C, Lung squamous carcinoma immunostained with the IgG fraction of anti-IGFBP-rP1 monoclonal antibody no. 10. Identical results were obtained with monoclonal antibody no. 5 (data not shown). D, Normal prostatic tissue immunostained with {alpha}IGFBP-rP1 polyclonal. E, Prostate cancer stained with {alpha}IGFBP-rP1 monoclonal no. 10. F, Colon cancer stained with polyclonal {alpha}IGFBP-rP1 antibody. Magnification, x200 (A–D) and x400 (E and F).

 
In normal tissues (prostate, breast, and colon), immunoreactivity was weak and limited to small blood vessels (Fig. 5DGo, normal prostate). Diffuse staining of the stroma was also observed. The immunostaining was, however, notably enhanced in the endothelial cells of the microvasculature of most of the human cancer tissues examined (lung, prostate, colon), using either the polyclonal or the monoclonal anti-IGFBP-rP1 antibodies (Figs. 5Go, A, C, E, and F).

Immunohistochemical analysis of IGFBP-rP1 expression was extended to include paraffin-embedded tissue sections (data not shown). Unlike frozen tissue sections, only the monoclonal anti-IGFBP-rP1 antibodies were capable of detecting IGFBP-rP1 expression, but the overall signal was less robust than that detected in frozen tissue sections. The pattern of staining, however, remained the same, with immunostaining predominantly identified in vascular endothelium, particularly in the microvasculature of tumor tissues.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The biological roles of IGFBP-rP1 have been evaluated in numerous studies and include diverse actions, such as tumor suppression (8, 11, 12, 14, 16, 34), stimulation of prostacyclin synthesis (10), and involvement in angiogenesis (5, 19, 24).

We previously characterized a polyclonal anti-IGFBP-rP1 antibody and identified IGFBP-rP1 in conditioned media from cultured human cells as well as in human biological fluids (25). Here we extend these studies with the characterization of new anti-IGFBP-rP1 monoclonal antibodies to study the distribution of IGFBP-rP1 in human biological fluids and tissues. These monoclonal antibodies, like the polyclonal antibody, do not cross-react with the six conventional IGFBP proteins or proteins of the CCN family. All 11 of the monoclonal antibodies specifically recognize the approximately 31-kDa secreted IGFBP-rP1 protein in conditioned medium from cultured human cells.

Here we also show for the first time quantitative data on serum IGFBP-rP1 in humans and indicate a dimorphic distribution of circulating IGFBP-rP1, with higher concentrations of IGFBP-rP1 in males. Because IGFBP-rP1 could be down-regulated by estrogens, according to one report (8), a plausible mechanism for the lower concentrations of circulating IGFBP-rP1 in females could be its tonic inhibition by estrogens. Although the assay was not developed to quantitate IGFBP-rP1 in other body fluids, the assay readily detected IGFBP-rP1 in human urine, AF, and CSF (data not shown), consistent with the findings obtained by immunoblot analysis. Further investigations are warranted to evaluate the clinical significance of these observations.

Both the polyclonal and the panel of monoclonal anti-IGFBP-rP1 antibodies were capable of detecting IGFBP-rP1 in major human body fluids, such as serum, urine, AF, and CSF. Interestingly, the IGFBP-rP1 protein detected in most of these samples (with the exception of amniotic fluid) appeared to be of a slightly higher molecular mass than the rhIGFBP-rP1FLAG protein (Fig. 3Go). This observation may be accounted for by differences in glycosylation or other posttranslational modifications between the baculovirus-generated recombinant protein and human IGFBP-rP1 detected in body fluids. These observations are consistent with the molecular weight of IGFBP-rP1 protein detected in the conditioned media of mammalian cells (12, 15, 25).

To further characterize IGFBP-rP1 protein expression in vivo, we used the panel of anti-IGFBP-rP1 antibodies to evaluate IGFBP-rP1 distribution in tissues. For both the polyclonal and panel of monoclonal antibodies, frozen tissue sections were superior to paraffin-embedded sections for immunodetection. Identical patterns of staining in the frozen tissues were observed with polyclonal and monoclonal antibodies, and the staining appears to be specific because preclearing of the relevant IgG fraction with rhIGFBP-rP1FLAG protein abrogated the signals. Most striking was that the distribution of IGFBP-rP1 was predominantly among endothelial cells of tumor tissues, with reduced, but detectable, signals in endothelial cells of normal tissues and in the stroma of all tissues examined. Similar observations were made with the paraffin-embedded tissue sections.

Our findings concur, in part, with other reports of the IGFBP-rP1 distribution in human tissues (5, 11, 33, 35). In these previous studies, the tissue sections examined were all paraffin embedded, and immunohistochemical analysis employed an incompletely characterized monoclonal antibody (5) or a polyclonal antibody (11, 33, 35) generated against a decapeptide in the C terminus of IGFBP-rP1 and, therefore, of uncertain specificity. The latter polyclonal antibody (11) recognized only the reduced approximately 37-kDa form of IGFBP-rP1 protein (33). Because our polyclonal and monoclonal antibodies were generated against intact IGFBP-rP1 protein, the patterns and sensitivity of staining might be expected to differ from that of other laboratories. Nevertheless, Akaogi et al. (5) detected tumor-derived adhesion factor (IGFBP-rP1) immunoreactivity in the vascular membrane of small blood vessels as well as capillaries of diverse cancer tissues but not those associated with normal tissues. Degeorges et al. (33), in contrast, did observe immunostaining of normal endothelial cells, but intense immunoreactivity was associated predominantly with supporting cells of peripheral nerves and stromal cells of numerous tissues.

The implication from this present study (and others) is that not only do endothelial cells express IGFBP-rP1 protein but also expression is considerably higher in endothelial cells associated with cancers. Intriguingly, a recent comparative study profiling gene expression in endothelium derived from normal and tumor tissue supports these observations (24). Analysis by serial analysis of gene expression determined that IGFBP-rP1 was the pan endothelial marker most abundantly expressed in endothelial cells and there was a 2-fold increase in gene expression in malignant tissues (24). These observations support an important role of IGFBP-rP1 in vascular biology and, as first proposed by Akaogi et al. (5), suggest that IGFBP-rP1 may be involved in the process of neoangiogenesis in malignancy. Furthermore, because IGFBP-rP1 appears to induce prostacyclin synthesis in endothelial cells (10), IGFBP-rP1 may also have roles in noncancerous human vascular diseases, such as atherosclerosis and hypertension. The new tools presented here should, therefore, prove useful in dissecting out the involvement of this protein in vascular function of normal and malignant tissues.

Other than endothelial cells, IGFBP-rP1 was detected rarely in epithelial cells in our immunohistochemical analysis of IGFBP-rP1 distribution. This was somewhat unexpected because we previously demonstrated that epithelial cells, at least in vitro, do express IGFBP-rP1 protein, and in situ hybridization studies of normal prostate tissues indicate IGFBP-rP1 mRNA in glandular epithelium that surrounds the lumen (12, 15). The apparent contradiction could be due to the sensitivity of antibodies. Akaogi et al. (5), similarly, did not detect immunostaining of epithelial cells with their antibody. However, the polyclonal antibody raised against the C-terminal region of IGFBP-rP1 was reactive to the luminal epithelial cell of normal lobules and ducts of breast tissues (11); the tumor epithelial cells of prostate tissue (35); and ciliated cells of bronchial, epididymal, and fallopian epithelia (33). Thus, the variations in the immunoreactivity of tissues and cell types in each study clearly depend on the source of antibodies and, most likely, also on the method of tissue preparation and processing.

Existing data on IGFBP-rP1 indicate that its role in cancer remains to be defined. Based on recent serial analysis of gene expression analysis (24) and immunohistochemical studies, including the present report, IGFBP-rP1 is implicated in the neovascularization process so critical for tumor growth. This contrasts with a growing body of evidence that suggests IGFBP-rP1 is a potential tumor suppressor gene. In a number of cancers, such as breast (8, 11, 13, 18), meningiomas (8), prostate cancer (12), and liver tumorigenesis (36), IGFBP-rP1 expression was down-regulated, although expression appeared to be up-regulated in carcinogenesis of colon mucosa (23, 37). Interestingly, IGFBP-rP1 is associated with a 50% loss of heterozygosity (LOH) in breast cancer (11). Furthermore, overexpression of IGFBP-rP1 in a prostate cancer cell line was shown to dramatically reduce tumorigenic potential of the cell line (16, 17), and exogenous addition of the protein to cancer cell lines appeared to inhibit cell growth (38). Taken altogether, these observations suggest that although IGFBP-rP1 is clearly important in tumorigenesis, its specific role(s) is still unclear, and most likely depends on a number of factors, including the tissue and cell type under study, the sensitivity, and specificity of reagents used as well as the techniques employed.

In summary, using a panel of newly developed IGFBP-rP1 monoclonal antibodies and an immunoassay, we demonstrate that IGFBP-rP1 is detectable in serum and other biological fluids and provide quantitative data on the concentrations of IGFBP-rP1 in human serum. We also have shown distribution of IGFBP-rP1 in human normal and cancerous tissues. IGFBP-rP1 appears to be involved in vascular biology, possibly in the process of neoangiogenesis that occurs in tumor tissues. The use of these novel IGFBP-rP1 detection tools should prove useful in the elucidation of the biological role of this IGFBP related protein.


   Footnotes
 
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.

Received August 16, 2002.

Accepted March 24, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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