Purpose | This immunoassay kit allows for the in vitro quantitative determination of human OPG concentrations in serum, plasma and other biological fluids. |
Sample Type | Serum, Plasma, Biological Fluids |
Analytical Method | Quantitative |
Detection Method | Colorimetric |
Specificity | This assay recognizes recombinant and natural human OPG. |
Cross-Reactivity (Details) | No significant cross-reactivity or interference was observed. |
Characteristics | Homo sapiens,Human,Tumor necrosis factor receptor superfamily member 11B,Osteoclastogenesis inhibitory factor,Osteoprotegerin,TNFRSF11B,OCIF,OPG |
Components |
Reagent (Quantity):
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Material not included | Microplate reader. Pipettes and pipette tips. EP tube Deionized or distilled water. |
Alternative Name | TNFRSF11B (TNFRSF11B ELISA Kit Abstract) |
Background | Osteoprotegerin, also known as osteoclastogenesis inhibitory factor (OCIF), is a cytokine and a member of the tumor necrosis factor (TNF) receptor superfamily. It is a basic glycoprotein comprising 401 amino acid residues arranged into 7 structural domains. It is found as either a 60 kDa monomer or 120 kDa dimer linked by disulfide bonds.Osteoprotegerin inhibits the differentiation of macrophages into osteoclasts and also regulates the resorption of osteoclasts in vitro and in vivo. Osteoprotegerin is a RANK homolog, and works by binding to RANK ligand on osteoblast/stromal cells, thus blocking the RANK-RANK ligand interaction between osteoblast/stromal cells and osteoclast precursors. This has the effect of inhibiting the differentiation of the osteoclast precursor into a mature osteoclast. Recombinant human osteoprotegerin specifically acts on bone, increasing bone mineral density and bone volume. Osteoprotegerin has been used experimentally to decrease bone resorption in women with postmenopausal osteoporosis and in patients with lytic bone metastases. Osteoprotegerin production is stimulated in vivo by the female sex hormone estrogen,as well as the experimental osteoporosis drug, strontium ranelate. |
Sample Volume | 100 μL |
Plate | Pre-coated |
Protocol | The microtiter plate provided in this kit has been pre-coated with an antibody specific to OPG. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for OPG. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain OPG, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm 2 nm. The concentration of OPG in the samples is then determined by comparing the O.D. of the samples to the standard curve. |
Reagent Preparation |
Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard. The Sample Diluent serves as the zero standard (0 ng/ml). |
Sample Collection | Serum - Use a serum separator tube and allow samples to clot for 30 minutes before centrifugation for 20 minutes at approximately 1000 g. Remove serum and assay immediately or aliquot and store samples at -20 or -80 . Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 g at 2 - 8 within 30 minutes of collection. Store samples at -20 or -80 . Avoid repeated freeze-thaw cycles. Other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20 or -80 . Avoid repeated freeze-thaw cycles. Note: Serum and plasma to be used within 7 days may be stored at 2-8 , otherwise samples must stored at -20 ( 1 month) or -80 ( 2 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature. |
Assay Procedure |
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37 °C directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4 °C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments. |
Calculation of Results |
Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the SAA concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 13.0. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. |
Restrictions | For Research Use only |
Handling Advice |
1. The kit should not be used beyond the expiration date on the kit label. 2. Do not mix or substitute reagents with those from other lots or sources. 3. If samples generate values higher than the highest standard, further dilute the samples and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding. 4. This assay is designed to eliminate interference by soluble receptors, ligands, binding 3 proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded. 5. Limited by the current condition and scientific technology, we can't completely conduct the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit. |
Storage | 4 °C/-20 °C |
Storage Comment | The Assay Plate, Standard, Detection Reagent A and Detection Reagent B should be stored at -20°C upon being received. After receiving the kit , Substrate should be always stored at 4°C. |
Supplier Images |
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