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Technical Briefs |
1
Department of Pharmacokinetics and Drug Metabolism, Amgen, Inc., One Amgen Center Dr., Thousand Oaks, CA 91320
a author for correspondence: fax 805-499-4953, e-mail
dchen{at}amgen.com
Osteoprotegerin (OPG), also known as osteoclast inhibitory factor, is a soluble receptor of the tumor necrosis factor receptor superfamily. The protein is secreted as a covalent, disulfide-linked homodimer, which is the predominant extracellular form (1), and is expressed in multiple tissues (1)(2)(3). OPG-mediated pathways might have a role in osteoporosis (3)(4)(5)(6) because estrogen increases OPG gene expression (4)(5). OPG maintains the structure of healthy bone and inhibits osteoclast activation and differentiation (3)(7). In the vascular system, OPG inhibits pathological calcification in the media intima (3). OPG has been proposed for therapy of osteopenic disorders, such as postmenopausal osteoporosis, Paget disease, rheumatoid arthritis, hypercalcemia, and lytic bone metastases (8).
Initially, we developed an antibody-based ELISA method with an anti-human OPG monoclonal antibody for capture and an anti-human OPG polyclonal antibody for detection. Yano et al. (9) raised the concern for us that we may not detect the active dimeric OPG with antibody capture because they reported that serum OPG increased with age and that the monomer was the predominant form of OPG in human serum. Although they used a different antibody-dependent ELISA method (monoclonal capture and detection), the results reported by Yano et al. (9) do not correspond with the work performed at Amgen (1)(3)(4)(5)(7)(8)(10)(11)(12). We reasoned that OPG ligand (OPGL) (2)(7)(8)(10)(11)(12)(13)(14)(15)(16), also known as osteoclast differentiation factor, is a potential alternative capture protein for an OPG assay. In an attempt to develop an assay that would measure all bioactive form(s) of OPG, we developed an ELISA assay that uses OPGL as the capture protein. To avoid problems posed by batch-to-batch variability of human serum pools for use as assay diluent, assay development was used to define a serum substitute.
Assay development was performed with AMGN-0007, a modified OPG. Calibrators and quality-control (QC) materials were prepared in human serum or serum substitute. Whereas calibrators were serially diluted, QC materials were prepared individually. Calibration curves were prepared using calibrators containing 0.020–500 µg/L AMGN-0007. Each calibration curve contained at least nine points, including the zero calibrator.
OPGL, AMGN-0007, and murine monoclonal antibody were purified essentially as described previously (1)(7). OPGL was coated onto 96-well microtiter plates (Costar). Plates were blocked with 2 mL/L I-Block (Tropix) and 5 mL/L Tween 20 (Pierce) in phosphate-buffered saline (PBS). Assay buffer, calibrators, and QC materials were added to the wells. After all unbound substances were removed by washing, murine anti-human OPG monoclonal antibody was added to the wells. After another wash, goat anti-mouse IgG conjugated with horseradish peroxidase (IgG-HRP; Zymed) was added to the wells. After the final wash, KPL TMB Microwell Peroxidase Substrate (Kirkegaard & Perry Laboratories) was added to the wells. The colorimetric reaction was stopped with 0.812 mol/L phosphoric acid. The color intensity was measured at 450–650 nm with a ThermoMax Microtiter Plate Reader (Molecular Devices).
The full-length OPG homodimer (OPG-FLD) and the full-length OPG monomer (OPG-FLM) were purified from conditioned medium with Sepharose columns and concentrated into PBS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to confirm size and purity (95%) of the monomer and dimer. Calibrators and QC materials were prepared for each OPG analog: AMGN-0007, OPG-FLD, and OPG-FLM. The assay was performed as described above, except that we used HRP-conjugated murine anti-human OPG monoclonal antibody.
Prepared in PBS, human serum substitute buffers contained 30 mL/L human serum albumin (HSA; Bioreclamation, Inc.) and various concentrations (0–500 mL/L) of fetal bovine serum (FBS; Sigma Chemical Co.). Calibrators and QC materials were prepared and assayed as described above.
OPGL was immobilized on the surface of the microtiter plate. AMGN-0007 was then added to the plate at concentrations of 0.244–31.25 µg/L, the calibration curve range. The analyte was detected with murine anti-human OPG monoclonal antibody plus goat anti-mouse IgG-HRP. Defined as two times the zero calibrator signal, the detection limit was 0.244 µg/L. Other assay configurations, such as monoclonal capture with polyclonal detection and ligand capture with polyclonal detection, were studied and demonstrated low signal-to-noise ratios throughout the calibration curve. For the dynamic range of interest, OPGL capture followed by monoclonal detection produced the best signal-to-noise ratio throughout the calibration curve; most likely, the result was attributable to the specificity of both the ligand and the monoclonal antibody for AMGN-0007.
The abilities of OPG analogs to bind to solid-phase-bound OPGL were
compared (Fig. 1
). The signal at 50 µg/L OPG analog for OPG-FLD (2.352
absorbance units) was very similar to that of AMGN-0007 (2.411
absorbance units), whereas OPG-FLM had signal strength of 1.693
absorbance units. Compared with AMGN-0007 and OPG-FLD, OPG-FLM
demonstrated a curve shift to the right and a lower signal strength
throughout the calibration curve. Although monomeric OPG did bind to
OPGL, the ligand appeared to have greater affinity for dimeric OPG.
Results from a previous study using pulse-chase labeling and
immunoprecipitation of extracts from Chinese hamster ovary cells
transfected with OPG suggested that the primary extracellular form of
OPG is the homodimer (1). Tomoyasu et al. (17)
also determined that dimeric OPG was secreted in greater amounts than
monomeric OPG in mammalian cell systems and was biologically more
active than monomeric OPG. In addition, they suggested that dimeric OPG
might bind to heparin and be transported to the target sites more rapid
than monomeric OPG (17); this may explain the observations
of Yano et al. (9).
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Plapp et al. (18) suggested that QC materials be composed of
the same matrix as the specimens. With human serum as diluent, problems
may arise for the following reasons: (a) the presence of
infectious agents that cannot be screened; (b)
batch-to-batch variability attributable to endogenous factors
(19); and (c) a finite shelf life that may fall
short of the time spans of clinical studies. To minimize the
variability for pharmacokinetic profile studies, a buffer that could be
substituted for human serum as an assay diluent was sought. HSA makes
up 30–55 g/L of serum (20)(21). Therefore,
development was focused on the normal concentration of albumin to
maintain a total protein content similar to the low end of the HSA
reference interval. Weber et al. (19) suggested that
the addition of bovine serum might eliminate interference of
heterophilic antibodies. After comparing the absorbance and slope
obtained for bovine serum with those obtained for human serum, we chose
30 mL/L HSA with 100 mL/L FBS as the serum substitute and diluent for
AMGN-0007 assays. Differences in the assay signals obtained for the
serum substitute and human serum were tested for statistical
significance using one-way ANOVA (Table 1
). No significant differences (P >0.05) were
observed among the signals obtained at different concentrations of
human serum in substitute diluent, suggesting that the human serum
substitute was an adequate substitute for human serum as assay diluent.
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In conclusion, we developed an ELISA AMGN-0007, with OPGL for analyte capture and a monoclonal antibody specific for detection, and found that an albumin-based diluent can be used. The ELISA will be useful for analyzing samples from clinical trials as well as for monitoring therapeutic efforts for osteopenic diseases. Finally, this tool may enable the development of an assay to measure endogenous OPG concentrations.
Acknowledgments
We wish to thank D. Chang, P. Campbell, and D. Yanagihara for providing antibodies. W.J. Boyle, M. Kelley, and E. Davy were instrumental in providing OPGL and support. We also thank C.R. Dunstan for guidance.
References
The following articles in journals at HighWire Press have cited this article:
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  A. D Anastasilakis, D. G Goulis, S. A Polyzos, S. Gerou, V. Pavlidou, G. Koukoulis, and A. Avramidis Acute changes in serum osteoprotegerin and receptor activator for nuclear factor-{kappa}B ligand levels in women with established osteoporosis treated with teriparatide Eur. J. Endocrinol., March 1, 2008; 158(3): 411 - 415. [Abstract] [Full Text] [PDF]
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 A. Rogers and R. Eastell Circulating Osteoprotegerin and Receptor Activator for Nuclear Factor {kappa}B Ligand: Clinical Utility in Metabolic Bone Disease Assessment J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 6323 - 6331. [Abstract] [Full Text] [PDF]
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K. E. Naylor, A. Rogers, R. B. Fraser, V. Hall, R. Eastell, and A. Blumsohn Serum Osteoprotegerin as a Determinant of Bone Metabolism in a Longitudinal Study of Human Pregnancy and Lactation J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5361 - 5365. [Abstract] [Full Text] [PDF]
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 A. Dovio, M. L. Sartori, and A. Angeli Correspondence re: A. Lipton et al., Serum Osteoprotegerin Levels in Healthy Controls and Cancer Patients. Clin. Cancer Res., 8: 2306-2310, 2002. Clin. Cancer Res., June 1, 2003; 9(6): 2384 - 2385. [Full Text] [PDF]
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A. Rogers, G. Saleh, R. A. Hannon, D. Greenfield, and R. Eastell Circulating Estradiol and Osteoprotegerin as Determinants of Bone Turnover and Bone Density in Postmenopausal Women J. Clin. Endocrinol. Metab., October 1, 2002; 87(10): 4470 - 4475. [Abstract] [Full Text] [PDF]
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 K. Jung, M. Lein, K. von Hosslin, B. Brux, D. Schnorr, S. A. Loening, and P. Sinha Osteoprotegerin in Serum as a Novel Marker of Bone Metastatic Spread in Prostate Cancer Clin. Chem., November 1, 2001; 47(11): 2061 - 2063. [Full Text] [PDF]
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), OPG-FLD (
), and OPG-FLM
(
)] were prepared for analysis by ELISA. Each compound was serially
diluted to 0.049–50 µg/L in human serum.