bioactive SCLEROSTIN ELISA
|Method:||Sandwich ELISA, HRP/TMB, 12x8-well strips|
|Sample type:||Serum, plasma (EDTA, citrate), protocols for cell culture supernatant and urine samples available|
|Standard range:||0-320 pmol/l (7 serum based standards)|
|Standard points:||0/10/20/40/80/160/320 pmol/l|
|Control:||2 serum based controls|
|Sample size:||20 µl / well|
|Incubation time:||2 h / 1 h / 30 min|
|Unit conversion:||1 pg/ml = 0.046 pmol/l (MW: 21.8 kDa)|
LOD: 1.9 pmol/l (0 pmol/l + 3 SD); LLOQ: 1.3 pmol/l
Intra-assay (n=3) ≤ 1%, Inter-assay (n=7) ≤ 5%
The mean recovery of recombinant Sclerostin (spike: 26 pmol/l and 110 pmol/l) in human samples is:
serum (n=5): 93% / 86%
EDTA plasma (n=5): 94% / 93%
citrate plasma (n=1): 104% / 99%
Expected average dilution of recombinant Sclerostin in human samples for 1+1, 1+3 and 1+7 dilution is:
serum (n=6): 98% / 86% / 89%
EDTA plasma (n=6): 102% / 99% / 91%
citrate plasma (n=1): 119% / 132% / 103%
Expected average dilution of endogenous Sclerostin in human samples for 1+1, 1+3 and 1+7 dilution is:
serum (n=7): 100% / 103% / 106%
EDTA plasma (n=6): 105% / 108% / 123%
citrate plasma (n=2): 91% / 91% / 103%
The assay does not detect Noggin.
The assay does not detect Wise (SOSTDC1).
Serum values from apparently healthy individuals:
Median (serum, n=32): 61.5. pmol/l
Median (EDTA plasma, n=24): 87 pmol/l
Median (citrate plasma, n=24): 61.5. pmol/l
It is recommended to establish the normal range for each laboratory.
Principle of the assay:
Manual ELISAs - easily adaptable for automation!
INSTRUCTIONS FOR USE
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BI-20472 bioactive Sclerostin ELISA Validation Data (S/R, dilution linearity, precision, ...)
The human bioactive Sclerostin immunoassay is a 3.5 hour, 96-well, sandwich ELISA for the quantitative determination of bioactive Sclerostin in human serum, EDTA plasma, and citrate plasma. The assay employs human based serum standards to ensure biological reliable data. The antibody characterization of both antibodies utilized in this assay comprises epitope mapping, binding kinetics, and determination of antibody purity (for further information on antibody characterization please see Validation Data).
Sclerostin is a 22.5 kDa secreted glycoprotein that functions as a potent inhibitor of Wnt signaling. It acts by binding to the Wnt-coreceptor LRP5/6 thus inhibiting bone formation by regulating osteoblast function and promoting osteoblast apoptosis (1,2). The Sclerostin protein consists of two flexible N- and C-terminal arms and a cystine-knot with three loops, whereas the second loop binds to the LRP5/6 complex (3,4). Sclerostin is classically considered to be a monomeric protein, but data from Hernandez and colleagues (5) postulate that circulating sclerostin has a dimeric configuration. In addition, it is not yet well documented if also Sclerostin fragments circulate, but the comparison of different Sclerostin ELISAs suggest that fragments exist as well (6,7).
As the epitope of the monoclonal capture antibody utilized in the bioactive Sclerostin ELISA is located in loop 2, the binding region to the LRP 5/6 complex, all Sclerostin molecules (including potential fragments) containing this receptor binding region can be detected.
Sclerostin is nearly exclusively produced in osteocytes (8). Mutations in the Sclerostin (SOST) gene can cause sclerosteosis and van Buchem disease which are bone dysplasia disorders characterized by progressive skeletal overgrowth (9,10). Sclerostin levels are altered in response to hormonal stimuli or due to pathophysiological conditions. Sclerostin concentrations are increased in disorders such as hypoparathyroidism (11), Paget’s disease (12), multiple myeloma (13) and in cancer induced bone diseases (12). Sclerostin levels are decreased in primary hyperparathyroidism (14), as well as by the mechanical stimulation of bone (15). Several studies have found a positive association between sclerostin and bone mineral density (16,17). Sclerostin levels in chronic kidney disease (CKD) patients are up to 4-fold increased compared to patients without CKD and increase with CKD stage and declining kidney function (18,19). In CKD patients, renal elimination of sclerostin increases with decreasing renal function (20). In dialysis patients, sclerostin is an independent predictor of bone loss (21). Numerous studies have shown that serum sclerostin levels are also associated with cardiovascular events (22,23). A monoclonal sclerostin antibody for the treatment of osteoporosis is currently undergoing clinical trials (24). For reviews please see references (25,26).
1. Sclerostin and Wnt signaling-the pathway to bone strength. Ott SM, J Clin Endocrinol Metab, 2005; 90(12):6392-6395.
2. SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor. Semenov M et al., J Biol Chem, 2005; 280(29):26770-26775.
3. Characterization of the structural features and interactions of sclerostin: molecular insight into a key regulator of Wnt-mediated bone formation. Veverka V et al., J Biol Chem, 2009; 284:10890-10900.
4. Characterization of the Interaction of Sclerostin with the Low Density Lipoprotein Receptor-related Protein (LRP) Family of Wnt Co-receptors. Holdsworth G et al., J Biol Chem, 2012; 284(16), 287(32): 26464-26477.
5. New insights into the location and form of sclerostin. Hernandez P et al., Biochem Biophys Res Commun, 2014; 446 (4):1108-1113.
6. Association of circulating sclerostin with bone mineral mass, microstructure, and turnover biochemical markers in healthy elderly men and women. Durosier C et al., J Clin Endocrinol Metab, 2013; 98 (9):3873-3883.
7. Circulating sclerostin levels are decreased in patients with endogenous hypercortisolism and increase after treatment. van Lierop AH et al., J Clin Endocrinol Metab, 2012; 97:E1953-E1957.
8. The Osteocyte: An Endocrine Cell … and More. Dallas SL et al., Endocrine Rev, 2013; 34:658-690.
9. Sclerostin in mineralized matrices and van Buchem disease. van Bezooijen RL et al., J Dent Res, 2009; 88(6):569-574.
10. Patients with Van Buchem disease, an osteosclerotic genetic disease, have elevated bone formation markers, higher bone density, and greater derived polar moment of inertia than normal. Wergedal, JE et al., J Clin Endocrinol Metab, 2003; 88:5778.
11. Circulating sclerostin in disorders of parathyroid gland function. Costa AG et al., J Clin Endocrinol Metab, 2011; 96: 3804-3810.
12. Serum sclerostin levels in Paget’s disease and prostate cancer with bone metastases with a wide range of bone turnover. Yavropoulou MP et al., Bone, 2012; 51:153-157.
13. Elevated circulating sclerostin correlates with advanced disease features and abnormal bone remodeling in symptomatic myeloma: reduction post-bortezomib. Monotherapy. Terpos E et al., Int J Cancer, 2012; 131:1466-1471.
14. Patients with primary hyperparathyroidism have lower circulating sclerostin levels than euparathyroid controls. Van Lierop AH et al., Eur J Endocrinol, 2010; 163:833-837.
15. Mechanical stimulation in vivo reduces osteocyte expression of sclerostin. Robling AG et al., J Musculoskelet Neuronal Interact, 2006; 6(4):354.
16. Association of serum sclerostin with bone mineral density, bone turnover, steroid and parathyroid hormones, and fracture risk in postmenopausal women: the OFELY study. Garnero P et al., Osteoporos Int, 2013; 13; 24(2):489-494.
17. Sclerostin and its association with physical activity, age, gender, body composition, and bone mineral content in healthy adults. Amrein K et al., J Clin Endocrinol Metab, 2012; 97:148-154.
18. Sclerostin and Dickkopf-1 in renal osteodystrophy. Cejka D et al., Clin J am Soc Nephrol, 2012; 6: 877-882.
19. The Relation between Renal Function and Serum Sclerostin in Adult Patients with CKD. Pelletier S et al., Clin J am Soc Nephrol, 2013; 8 (5): 819-823.
20. Renal elimination of Sclerostin increases with declining kidney function. Cejka D et al., J Clin Endocrinol Metab, 2014; 99(1):248-255.
21. Bone mineral density and serum biochemical predictors of bone loss in patients with CKD on dialysis. Malluche HH et al., Clin J Am Soc Nephrol, 2014; 9:1254-1262.
22. Serum sclerostin and adverse outcomes in nondialyzed chronic kidney disease patients. Kanbay M et al., J. Clin. Endocrinol Metab, 2014; 99:E1854-E1861.
23. Sclerostin: Another bone-related protein related to all-cause mortality in haemodialysis? Viaene L et al., Nephrol Dial Transplant, 2013; 28:3024-3030.
24. Clinical utility of anti-sclerostin antobodies. McClung MR, Bone, 2017; 96:3-7.
25. Sclerostin measurement in human disease: Validity and current limitations. Costa AG et al.,; Bone, 2017; 96:24-28.
26. Hormonal and systemic regulation of sclerostin. MT Drake and S Khosla, Bone, 2017; 96:8-17.