Development of a synthetic peptide-based tartrate-resistant acid phosphatase radioimmunoassay for the measurement of bone resorption in rat serum |
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Authors: | Apurva K Srivastava Cesar Libanati Nancy Lane David J Baylink |
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Institution: | (1) Musculoskeletal Disease Center (151), Jerry L. Pettis VA Medical Center and Loma Linda University, 11201 Benton Street, Loma Linda, CA 92357, USA, US;(2) Division of Rheumatology, University of California at San Francisco, San Francisco, CA, USA, US |
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Abstract: | Selective markers of bone turnover provide a convenient and reproducible alternative to the complex and expensive histochemical
techniques used commonly to study the effect of pharmacological agents and the pathogenesis of bone disease in the ovariectomized
(OVX) rat model. One marker, which has been specifically linked to terminally differentiated osteoclasts and, thus, provides
useful insight at cellular levels, is type-5 tartrate-resistant acid phosphatase (TRACP). We describe the development of a
TRACP radioimmunoassay (RIA), which requires synthetic peptide for antibody development. To develop the RIA, polyclonal antibodies
were generated in goats against a synthetic peptide, DPSVRHQRKCY, corresponding to amino acid residues 267–275 of the rat
type-5 TRACP sequence. In the RIA, 50 μl of rat serum, 100 μl of goat anti-TRACP antibodies, and 100 μl of tracer were incubated
overnight. The antibody-bound fraction was separated, counted, and unknown values were calculated by comparison with the peptide
calibrator. Rat serum shows parallelism with the synthetic peptide calibrator used in the RIA. The sensitivity of the RIA
was 24.7 μg/l, and the measuring range was 19–2476 μg/l. The average intra-assay coefficients of variation for (CV) two controls
were less than 7%. The average dilution and spike recoveries were 107% and 87%, respectively. We applied our peptide-based
RIA to study bone resorption in an OVX rat model. TRACP concentrations in serum in 12-week-old OVX Sprague Dawley rats were
14%–22% (P < 0.05) higher than those in the sham-operated rats, and TRACP concentrations in OVX rats treated with estradiol were 24%–32%
lower (P < 0.01) than those in the vehicle-treated OVX group. Similarly, as compared with those in OVX rats, TRACP concentrations
decreased to those of sham levels in OVX rats receiving 10 μg/kg per day of alendronate for 10 days. In addition, the TRACP
levels determined by RIA showed a significant correlation with serum C-telopeptide (type-I collagen) concentrations (r = 0.56; P < 0.001) measured by an enzyme-linked immunosorbent assay (ELISA) developed earlier for the rat model. In conclusion, we
have developed a TRACP RIA that could be used to monitor the rate of bone resorption in the rat model.
Received: June 18, 2001 / Accepted: October 26, 2001 |
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Keywords: | tartrate-resistant acid phosphatase radioimmunoassay ovariectomy alendronate estradiol |
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