Development of a synthetic peptide-based tartrate-resistant acid phosphatase radioimmunoassay for the measurement of bone resorption in rat serum.
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 microl of rat serum, 100 microl of goat anti-TRACP antibodies, and 100 microl 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 microg/l, and the measuring range was 19-2476 microg/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 microg/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.
Srivastava
Apurva K
AK
Musculoskeletal Disease Center (151), Jerry L. Pettis VA Medical Center and Loma Linda University, 11201 Benton Street, Loma Linda, CA 92357, USA.
Libanati
Cesar
C
Lane
Nancy
N
Baylink
David J
DJ
D000135:Acid Phosphatase / Q000097:blood* / Q000235:genetics; D019386:Alendronate / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D015415:Biomarkers; D015519:Bone Density; D001862:Bone Resorption; D024042:Collagen Type I / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D010052:Ovariectomy; D010455:Peptides / Q000378:metabolism*; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley
D000906:Antibodies; D015415:Biomarkers; D024042:Collagen Type I; D010455:Peptides; D004958:Estradiol; D000135:Acid Phosphatase; D019386:Alendronate