Free soluble RANKL ELISA

$900.00

The Free soluble RANKL ELISA Assay Kit is an enzyme immunoassay for the quantitative determination of free, soluble, uncomplexed human RANKL in serum or heparin plasma. The Eagle Biosciences Free soluble RANKL ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

SKU: BI-20462 Categories: , ,

Free soluble RANKL ELISA

Free soluble RANKL ELISA Developed and Manufactured in Austria by Biomedica

Size: 1×96 wells
Sensitivity: (0 pmol/l + 3 SD): 0.01 pmol/l / 0.008pmol/l
Dynamic Range: 0 to 2 pmol/l
Incubation Time: 3.5 hours
Sample Type: Human serum, Heparin Plasma
Sample Size: 150 µL
Alternative Names: Receptor Activator of Nuclear Factor Kappa B Ligand
For Research Use Only
Controls Included


Unit conversion: 1 pg/ml = 0.05 pmol/l (MW: 20 kD, monomer)


Assay Principle

This kit is a sandwich enzyme immunoassay for the quantitative determination of free soluble RANKL in human serum and heparın plasma samples. In a first step, wells which are pre-coated with recombinant Osteoprotegerin (OPG), RANKL’s interaction partner, are prewashed to ensure optimal sensitivity. Thereafter, assay buffer is pipetted into the wells of the microtiter strips followed by the addition of standard/control/sample. Soluble RANKL present in the standard/control/sample binds to the pre-coated OPG in the well. After incubation, a the plate is washed to remove all non-specifically bound material. In a next step, biotinylated detection antibody (polyclonal goat anti-human sRANKL) is pipetted into the wells and reacts with the sRANKL present in the sample, forming a sandwich. Next, all unbound antibody is removed during another washing cycle. In the following step, the conjugate (streptavidin-polyHRP) is added and reacts with the detection antibody. After a final washing step, the substrate (TMB, tetramethylbenzidine) is pipetted into the wells. The enzyme-catalyzed color change of the substrate is directly proportional to the amount of soluble RANKL present in the sample. This color change is detectable with a standard microtiter plate 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 soluble RANKL in the sample is determined directly from the dose response curve.


Related Products

Osteoprotegerin ELISA Assay Kit
Periostin ELISA Assay Kit
Sclerostin ELISA Assay Kit

Additional Information

Assay Background


RANKL, the receptor activator of nuclear factor kappa B ligand, a member of the tumor necrosis factor (TNF) family (https://www.uniprot.org/uniprot/O14788), is the main stimulatory factor for the formation of mature osteoclasts and is essential for their survival. RANKL activates its specific receptor RANK, located on osteoclasts and dendritic cells. The effects are counteracted by OPG which acts as an endogenous soluble receptor antagonist (see: BI-20403 – OPG ELISA). The major source of RANKL are osteocytes, former osteoblasts that become embedded within the mineralized bone matrix. RANKL is a ~35 kD type II transmembrane-type protein and is cleaved to release a soluble biologically active product that forms a homotrimer.

RANKL and its specific receptor RANK are not only key regulators of bone remodeling but also play an essential role in immunobiology, e.g. lymph node formation, establishment of the thymic microenviroment, mammary gland development during pregnancy, bone metastasis in cancer and sex-hormone, progestin-driven breast cancer, thermoregulation, and finally in the development of type 2 diabetes mellitus.

Assay Procedure


  1. Prewash wells with 300 µl diluted WASHBUF (wash buffer, natural cap) five times. Remove remaining WASHBUF by tapping plate against paper towel after the last wash.
  2. Add 50 µl ASYBUF (assay buffer, red cap) into each well. Pipette additional 150 µl ASYBUF into well marked as blank.
  3. Pipette 150 µl STD/SAMPLE/CTRL (Standard/Sample/Control) in duplicate into respective wells, except blank.
  4. Cover tightly and incubate at room temperature (18-24°C) for 2 hours.
  5. Aspirate and wash wells with 300 µl diluted WASHBUF (wash buffer, natural cap) five times. Remove remaining WASHBUF by tapping plate against paper towel after the last wash.
  6. Add 200 µl AB (biotinylated anti sRANKL antibody, green cap) into each well, except blank. Swirl gently.
  7. Pipette additional 200 µl ASYBUF (assay buffer, red cap) into well marked as blank.
  8. Cover tightly and incubate at 4°C (2-8°C) over night (18-24 hours).
  9. Aspirate and wash wells with 300 µl diluted WASHBUF (wash buffer, natural cap) fives times. Remove remaining WASHBUF by tapping plate against paper towel after the last wash.
  10. Add 200 µl CONJ (conjugate, amber cap) into each well.
  11. Cover tightly and incubate at room temperature (18-24°C) for 1 hour in the dark.
  12. Aspirate and wash wells with 300 µl diluted WASHBUF (wash buffer, natural cap) fives times. Remove remaining WASHBUF by tapping plate against paper towel after the last wash.
  13. Add 200 µl SUB (substrate, blue cap) into each well.
  14. Incubate at room temperature (18-24°C) for 30 min in the dark.
  15. Add 50 µl STOP (stop solution, white cap) into each well.
  16. Measure absorbance immediately at 450 nm with reference 630 nm if available.

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Typical Standard Curve


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Documents

Product Documents


Publications

Citations


  • Immunology and bone. Danks L and Takayanagi H, J Biochem 2013; 154: 29-39.
    Physiology and pathophysiology of the RANKL/RANK system. Hanada R et al., Biol Chem 2010; 391(12): 1365-1370.
  • Evidence for osteocyte regulation of bone homeostasis through RANKL expression. Nakashima T et al., Nat Med 2011; 17(10): 1231-1234.
  • RANKL Employs Distinct Binding Modes to Engage RANK and the Osteoprotegerin Decoy Receptor. Nelson CA et al., Structure, Nov 2012; 20(11): 1971-1982.
  • Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer. Schramek D et al., Nature 2010; 468(7320): 98-102.
  • RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis. Gonzalez-Suarez E et al., Nature 2010; 468: 103-107.
  • Central regulation of body temperature by RANKL/RANK pathway. Hanada R and Penninger JM, Clin Calcium 2011; 21(8): 1201-1208.
  • Blockade of receptor activator of nuclear factor-κB (RANKL) signaling improves hepatic insulin resistance and prevents the development of diabetes mellitus. Kiechl S et al., Nature Medicine 2013; 19(3):358-363.