Osteoprotegerin ELISA Assay Kit

$825.00

The Osteoprotegerin (OPG) ELISA Assay Kit (enzyme-linked immunoassay kit) is intended for the quantitative determination of human osteoprotegerin levels in serum or plasma samples. The Eagle Biosciences Human Osteoprotegerin (OPG) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

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Osteoprotegerin ELISA Assay Kit

Osteoprotegerin ELISA Assay Kit Developed and Manufactured in Austria by Biomedica

Size: 1×96 wells
Sensitivity: LOD: (0 pmol/l + 3 SD): 0.07 pmol/l; LLOQ: 0.08 pmol/l
Dynamic Range: 0 to 20 pmol/l
Incubation Time: 5.5 hours
Sample Type: Serum, plasma (EDTA, heparin, citrate)
Sample Size: 20 µL
Alternative Names: OPG ELISA, Human OPG ELISA, Human Osteoprotegerin ELISA
For Research Use Only
Controls Included

Unit conversion: 1 pg/ml = 0.05 pmol/l (MW: 19.9 kD)
Specificity: The ELISA recognizes human endogenous and recombinant OPG. The OPG ELISA detects monomeric and dimeric OPG as well as OPGRANKL complexes. The assay does not cross-react with rat or mouse samples.

Assay Principle

The Osteoprotegerin ELISA Assay Kit is a sandwich enzyme immunoassay for the determination of OPG in human serum and plasma samples. In a first step, assay buffer, standard/control/sample and detection antibody (biotinylated monoclonal mouse anti-human OPG) are pipetted into the wells of the microtiter strips, which are pre-coated with polyclonal goat anti-human OPG antibody. OPG present in the standard/control/sample binds to the pre-coated antibody in the well and forms a sandwich with the detection antibody. In the washing step all non-specific unbound material is removed. In a second step, the conjugate (streptavidin-HRP) is pipetted into the wells and reacts with the detection antibody. After another 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 OPG. This color change is detectable with a standard microtiter plate reader. A dose response curve of the absorbance (optical density, OD at 450 nm) vs. standard concentration is generated. The concentration of OPG in the sample is determined directly from the dose response curve.

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Additional Information

Assay Background

Osteoprotegerin (OPG) or Osteoclast inhibitory factor (OCIF) is a glycoprotein of the TNF receptor superfamily 11b (gene name TNFRSF11B) https://www.uniprot.org/uniprot/O00300. OPG is synthesized as a monomer of 380 amino acids and is assembled as a homodimer within the cell and then secreted mainly as a disulfide-linked homodimer into the extracellular compartment. OPG is produced by many different tissues and cell types including osteoblasts. OPG is a negative regulator of bone resorption by acting as decoy receptor for RANKL, thus neutralizing its function in osteoclastogenesis. This glycoprotein is also involved in the regulation of vascular calcification.

Assay Procedure

  1. Pipette 150 µl ASYBUF (Assay Buffer, red cap) into each well.
  2. Add 20 µl STD/SAMPLE/CTRL (Standard/Sample/Control) in duplicate into respective wells, swirl gently.
  3. Add 50 µl AB (biotinylated anti OPG antibody, green cap) into each well, swirl gently.
  4. Cover tightly and incubate for 4 hours at room temperature (18-24°C).
  5. 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.
  6. Add 200 µl CONJ (Conjugate, amber cap) into each well.
  7. Cover tightly and incubate for 1 hour at room temperature (18-24°C).
  8. 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.
  9. Add 200 µl SUB (Substrate, blue cap) into each well.
  10. Incubate for 30 min at room temperature (18-24°C) in the dark.
  11. Add 50 µl STOP (Stop solution, white cap) into each well, swirl gently.
  12. Measure absorbance immediately at 450 nm with reference 630 nm, if available.

osteoprotegerin-opg-elisa-assay-kit-1

Typical Standard Curve

OPG ELISA Standard Curve

Expected Values

Values from apparently healthy individuals:

  • Median serum (n=60): 2.7 pmol/l
  • Median EDTA plasma (n=6): 2.2 pmol/l
  • Median heparin plasma (n=7): 2.3 pmol/l
  • Median citrate plasma (n=5): 2.3 pmol/l

It is recommended to establish the normal range for each laboratory.

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