Bioactive Sclerostin ELISA Assay Kit

Additional Information

Assay Background

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. 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.

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. Mutations in the Sclerostin (SOST) gene can cause sclerosteosis and van Buchem disease which are bone dysplasia disorders characterized by progressive skeletal overgrowth. Sclerostin levels are altered in response to hormonal stimuli or due to pathophysiological conditions. Sclerostin concentrations are increased in disorders such as hypoparathyroidism, Paget’s disease, multiple myeloma and in cancer induced bone diseases. Sclerostin levels are decreased in primary hyperparathyroidism, as well as by the mechanical stimulation of bone. Several studies have found a positive association between sclerostin and bone mineral density. 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. In CKD patients, renal elimination of sclerostin increases with decreasing renal function. In dialysis patients, sclerostin is an independent predictor of bone loss. Numerous studies have shown that serum sclerostin levels are also associated with cardiovascular events. A monoclonal sclerostin antibody for the treatment of osteoporosis is currently undergoing clinical trials. For reviews please see references.

Assay Principle

This kit is a sandwich enzyme immunoassay for the quantitative determination of bioactive sclerostin in human serum and plasma samples (EDTA, citrate). In a first step, assay buffer is pipetted into the wells of the microtiter strips. Thereafter, STD/sample/CTRL are pipetted into the wells, which are pre-coated with the recombinant human monoclonal Sclerostin antibody. Any bioactive Sclerostin present in the STD/sample/CTRL binds to the pre-coated antibody in the well. After incubation, a washing step is applied where all non-specific unbound material is removed. In a next step, the conjugate (anti sclerostin-HRPO) is pipetted into the wells and reacts with bioactive Sclerostin present in the sample, forming a sandwich. After another washing step, the substrate (TMB Tetramethylbenzidine) is pipetted into the wells. The enzyme catalysed colour change of the substrate is directly proportional to the amount of bioactive sclerostin. This colour change is detectable with a standard microtiter plate ELISA reader. A dose response curve of the absorbance (optical density, OD at 450 nm) versus standard concentration is generated, using the values obtained from the standards. The concentration of bioactive sclerostin in the sample is determined directly from the dose response curve.

Assay Procedure

1. Pipette 100 μl ASYBUF (Assay buffer, red cap) into each well.
2. Add 20 μl STD/SAMPLE/CTRL (Standard/Sample/Control) in duplicate into respective well. Swirl gently.
3. Cover tightly and incubate for 2 hours at room temperature (18-26°C).
4. Aspirate and wash wells 5x with 300 μl diluted WASHBUF (Wash buffer). After final wash, remove remaining WASHBUF by strongly tapping plate against paper towel.
5. Add 100 μl CONJ (Conjugate, amber cap) into each well. Swirl gently.
6. Cover tightly and incubate for 1 hour at room temperature (18-26°C) in the dark.
7. Aspirate and wash wells 5x with 300 μl diluted WASHBUF (Wash buffer). After final wash, remove remaining WASHBUF by strongly tapping plate against paper towel.
8. Add 100 μl SUB (Substrate, blue cap) into each well. Swirl gently.
9. Incubate for 30 min at room temperature (18-26°C) in the dark.
10. Add 50 μl STOP (Stop solution, white cap) into each well. Swirl gently.
11. Measure absorbance immediately at 450 nm with reference 630 nm, if available.

Expected Values

Values from apparently healthy individuals:
Median Serum (n=411): 24.14 pmol/l
This value lies between calibration point 2 and 3 of the standard curve. It is recommended to establish the normal range for each laboratory.