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Technical Briefs |
1
Division of Laboratory Diagnosis and
2
Department of Clinical Laboratory Medicine, Sapporo Medical University School of Medicine, Sapporo 060-8543, Japan
3
Research Institute of Life Science, Snow Brand Milk Products Co., Ltd., 519, Shimoishibashi, Ishibashi-machi, Shimotsuga-gun, Tochigi 329-0512, Japan
aAddress correspondence to this author at: Department of Clinical Laboratory Medicine, Sapporo Medical University School of Medicine, South-1, West-16, Chuo-ku, Sapporo 060-8543, Japan; fax 81-11-622-7502, e-mail watanabn{at}sapmed.ac.jp
Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor is a recently cloned cytokine that specifically inhibits differentiation and maturation of osteoclasts (1)(2)(3)(4). OPG is present as a monomer (60 kDa) and a homodimer (120 kDa) in conditioned media from human fibroblasts and from CHO cells producing human OPG (3)(5). Notably, the homodimeric form of recombinant OPG exerted more potent biologic activity than the monomeric form in vivo (5)(6)(7). Which form of OPG predominates in human serum has not been definitively determined. Determining the amount of each form of OPG in patient serum may be useful in the diagnosis and treatment of metabolic bone diseases associated with abnormal osteoclast recruitment and function, such as osteopetrosis, osteoporosis, metastatic bone disease, Paget disease, rheumatoid arthritis, and periodontal disease.
Yano et al. (8) have described ELISAs with tetramethylbenzidine for total OPG and the homodimeric form of OPG (detection range for both ELISAs, 65–500 ng/L). The homodimeric form of OPG, however, cannot be detected in human serum by conventional ELISAs because the concentrations are below the detection limit. Additionally, the monomeric form of OPG cannot be measured directly at the present time because no specific antibody for the monomeric form is available. We therefore devised a highly sensitive immuno-PCR assay to measure the homodimeric form of OPG in sera from healthy individuals and patients with osteoporosis.
Blood samples were obtained from 11 healthy volunteers and 22 patients with osteoporosis. The immuno-PCR assay consisted of the following steps: an ELISA for antigen detection, followed by PCR for signal amplification. The ELISA for human OPG (hOPG) was performed according to a method described previously (8). Monomeric and homodimeric forms of recombinant hOPG (rhOPG), monoclonal antibodies OI-4 and OI-19 (specific for both forms of rhOPG), and monoclonal antibody OI-26 (specific for the homodimeric form) were prepared as reported previously (8). Briefly, an ELISA for total hOPG was set up with OI-19 as the immobilized and capture antibody and OI-4 as the biotinylated second antibody. In an ELISA for the homodimeric form, OI-26 was used in place of OI-19 as the immobilized and capture antibody.
To determine the lower detection limit of the ELISA, diluted solutions of rhOPG and substrate solution (50 µL of 0.1 mol/L sodium phosphate-citrate buffer containing 0.7 g/L o-phenylenediamine and 0.3 mL/L H2O2, pH 5.0) were used for signal detection, and the absorbance at 492 nm was measured (relative to the absorbance at 650 nm). When the minimum detection limit was determined as the lowest concentration of rhOPG that produced a signal equal to the mean + 2 SD of the blank, the mean detection limit of both ELISA systems was 125 ng/L. The mean ± SD absorbance value at 492 nm with 125 ng/L rhOPG did not overlap that obtained with 62.5 ng/L. The assay was performed in triplicate. The total OPG concentration measured by ELISA in our subjects with osteoporosis was 1147 ± 448 ng/L, 
1.5-fold higher than in control subjects (760 ± 176 ng/L; Table 1
). However, our ELISA with the OPD colorimetric method did not detect the homodimeric form in samples from either controls or patients, because the concentrations of OPG were below the detection limit (125 ng/L). Our results confirmed that the homodimeric form can not be detected by conventional ELISA. We therefore selected an immuno-PCR assay from among several sensitive methods, such as chemiluminescent assays.
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We next performed immuno-PCR for the homodimeric form of hOPG, according to the method described previously (9). The procedures up to the addition of biotinylated OI-4 were identical to those for the ELISA mentioned above, except that we used 0.1 g/L salmon sperm DNA (Sigma) in 500 mL/L BlockAce instead of the blocking reagent alone. After the biotinylated OI-4 had reacted and the wells were washed six times with wash buffer to remove unbound biotinylated OI-4, free streptavidin (Chemicon International) at 1 mg/L in phosphate-buffered saline containing 10 g/L bovine serum albumin was added, and the wells were incubated at room temperature for 1 h. The wells were washed six times with wash buffer, after which biotinylated DNA was added at a concentration of 1 ng/L in phosphate-buffered saline containing 1.0 g/L bovine serum albumin.
Biotinylation of DNA was performed according to the method described previously (9). These strips were incubated at room temperature for 1 h. After the strips were washed six times with wash buffer, 50 µL of PCR mixture was added to each well. Each strip was subjected to PCR in a Perkin-Elmer Cetus 9600 cycler. The PCR reaction mixture included 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3.0 mM MgCl2, 0.2 mM each deoxyribonucleotide, 0.2 µM each of the forward primer (5'-AGCGCGCGTAATACGACTC-3') and reverse primer (5'-ACCATGATTACGCCAAGCG-3'), and 2 U of AmpliTaq DNA polymerase (Takara). The number of cycles and temperatures were as follows: initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 1 min, annealing at 58 °C for 1 min, and extension at 72 °C for 1 min. A 10-µL aliquot of the resulting PCR products (196 bp) was electrophoresed in a 3% agarose gel. The amounts of PCR products were determined by a Fluorimager SI (Molecular Dynamics) and indicated in relative fluorescent units.
To determine the detection limit of the immuno-PCR, serial dilutions of different concentrations (0, 1, 2.5, 5, 10, 25, 50, and 100 pg/L) of the homodimeric form of rhOPG were used. The band was detected at rhOPG concentrations down to 5.0 pg/L. The mean ± SD relative fluorescent units at 5.0 pg/L did not overlap those for the other concentrations.
To study specificity, monomeric or homodimeric rhOPG was dissolved at a final concentration of 50 pg/L in diluted human serum (control). Human sera were diluted at 1:105 or 1:106 with 0.2 mol/L Tris-HCl (pH 7.4) containing 400 mL/L BlockAce, 10 mg/L normal mouse IgG, and 1 mL/L Tween 20. The results showed a significant difference between control sera and samples supplemented with the homodimeric form of rhOPG (P <0.01; Fig. 1
). On the other hand, no significant difference was seen between control sera and samples supplemented with the monomeric form of rhOPG (Fig. 1
, monomer). The immuno-PCR assay for the homodimeric form could detect as little as 5.0 pg/L recombinant human homodimer, a detection limit 2.5 x 104 times lower than that of the ELISA. Moreover, the use of a fluorescence dye (SYBR Green) and radioisotopes rather than ethidium bromide may increase the sensitivity.
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Homodimer concentrations measured by this assay in patients with osteoporosis (106 ± 11 ng/L) were 3.3-fold higher than those in healthy controls (32 ± 5 ng/L). These results suggest that in human serum, OPG exists mainly as the monomeric form. Moreover, the ratio of homodimer (measured using immuno-PCR) to total OPG (measured by using ELISA) was 2.2-fold higher in patients with osteoporosis than in controls.
Tomoyasu et al. (5) have suggested that the half-life of the OPG homodimer in the 
phase is significantly shorter than that of the monomer and that the OPG homodimer exerts more potent biologic activity than the OPG monomer in reducing the calcium concentration in rat serum, suggesting that the homodimer may tend to redistribute more rapidly from the circulation to the tissues than the monomer. Yamaguchi et al. (10) suggested that the monomer may represent a degradation product of the homodimer, given that it lacks several amino acids in the COOH-terminal region, including Cys379, which participates in an intermolecular disulfide bond. On the basis of the reports of Tomoyasu et al. (5) and Yamaguchi et al. (10), homodimeric OPG may exert more potent biologic activity than the monomeric form. Accordingly, accurate determination of homodimer concentrations in human serum is particularly important. The immuno-PCR described here can accomplish this and may be useful in diagnosing and monitoring treatment of metabolic bone diseases involving osteoclastic abnormalities.
References
The following articles in journals at HighWire Press have cited this article:
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  D. Vega, N. M. Maalouf, and K. Sakhaee The Role of Receptor Activator of Nuclear Factor-{kappa}B (RANK)/RANK Ligand/Osteoprotegerin: Clinical Implications J. Clin. Endocrinol. Metab., December 1, 2007; 92(12): 4514 - 4521. [Abstract] [Full Text] [PDF]
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 L. A. Schneeweis, D. Willard, and M. E. Milla Functional Dissection of Osteoprotegerin and Its Interaction with Receptor Activator of NF-{kappa}B Ligand J. Biol. Chem., December 16, 2005; 280(50): 41155 - 41164. [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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