Janckila Anthony J, Simons Ruth M, Yam Lung T
Special Hematology Laboratory, Department of Veterans Affairs Medical Center, Louisville, KY 40206, USA.
Clin Chim Acta. 2004 Sep;347(1-2):157-67. doi: 10.1016/j.cccn.2004.04.021.
Our purpose was to develop a specific immunoassay for tartrate-resistant acid phosphatase (TRACP) 5b using naphthol ASBI phosphate (N-ASBI-P) as a selective substrate for isoform 5b and heparin as a selective inhibitor of isoform 5a.
Serum TRACP 5a and 5b and recombinant TRACP 5a were used to optimize and standardize the immunoassay for specificity, linearity, analytical recovery, sensitivity and reproducibility. Serum N-telopeptide cross-links (NTX) and bone alkaline phosphatase (bone ALP) were also measured. The clinical sensitivity and specificity were assessed in healthy control subjects and patients with osteoarthritis (OA), rheumatoid arthritis (RA) and endstage renal disease (ESRD).
TRACP 5b specificity was achieved at pH 6.3 with 2.5 mmol/l substrate and 25 U/ml heparin. Isoform 5b specificity was increased over our original immunoassay using 4-nitrophenyl phosphate (4-NPP) without heparin. The alternative immunoassay was linear with 110% analytical recovery and no serum matrix effects. The average intra-assay error was 10.65%; the average inter-assay error was 10.11% for values of 1-3 U/l and 6.5% for values of 7-11 U/l. Mean serum TRACP 5b in OA and RA were not significantly different from control using either immunoassay. Mean serum TRACP 5b was significantly increased in ESRD with both immunoassays. Serum TRACP 5b levels correlated significantly with NTX and bone ALP in the disease groups, but not always in the control group.
This alternative immunoassay for TRACP 5b activity is highly specific. It should have applications in evaluating patients with bone disease and will improve our understanding of the biological significance of TRACP 5b expression in health and disease.
我们的目的是开发一种针对抗酒石酸酸性磷酸酶（TRACP）5b的特异性免疫测定法，使用萘酚ASBI磷酸盐（N - ASBI - P）作为5b同工型的选择性底物，肝素作为5a同工型的选择性抑制剂。
使用血清TRACP 5a和5b以及重组TRACP 5a来优化和标准化该免疫测定法，以评估其特异性、线性、分析回收率、灵敏度和重现性。还测定了血清N - 端肽交联物（NTX）和骨碱性磷酸酶（骨ALP）。在健康对照受试者以及骨关节炎（OA）、类风湿关节炎（RA）和终末期肾病（ESRD）患者中评估临床敏感性和特异性。
在pH 6.3、2.5 mmol/l底物和25 U/ml肝素的条件下实现了TRACP 5b的特异性。与我们最初使用无肝素的对硝基苯磷酸酯（4 - NPP）的免疫测定法相比，5b同工型的特异性有所提高。替代免疫测定法呈线性，分析回收率为110%，且无血清基质效应。平均批内误差为10.65%；对于1 - 3 U/l的值，平均批间误差为10.11%，对于7 - 11 U/l的值，平均批间误差为6.5%。使用任何一种免疫测定法，OA和RA患者的血清TRACP 5b平均值与对照组无显著差异。两种免疫测定法均显示ESRD患者的血清TRACP 5b平均值显著升高。在疾病组中，血清TRACP 5b水平与NTX和骨ALP显著相关，但在对照组中并非总是如此。
这种针对TRACP 5b活性的替代免疫测定法具有高度特异性。它在评估骨病患者方面应具有应用价值，并将增进我们对TRACP 5b在健康和疾病中表达的生物学意义的理解。
