Anti-CaSR IgG ELISA Assay Kit

$750.00

The Eagle Biosciences Human Anti-CaSR IgG ELISA Assay Kit (enzyme-linked immunoassay kit) kit is produced for the quantitative determination of human anti-CaSR IgG (calcium sensing receptor) autoantibody levels in serum, plasma, tissue extract or other liquid samples. The Human Anti-CaSR ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

SKU: CSR31-K01 Categories: , ,

Anti-CaSR IgG ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: 2.5 U/mL
Dynamic Range: 10 – 270 U/mL
Incubation Time: 2.0 hours
Sample Type: Serum, Plasma, Tissue extract
Sample Size: 100 µL
Controls Included

Additional Information

Assay Background

The human calcium-sensing receptor (CaSR) is a 1078 amino acid cell surface protein, which is predominantly expressed in the parathyroid glands and kidney. It is a member of the family of G protein-coupled receptors. The CaSR allows regulation of parathyroid hormone (PTH) secretion and renal tubular calcium reabsorption in response to alterations in extracellular calcium concentrations. Abnormalities of the CaSR are associated with both hypercalcaemic and hypocalcaemic disorders.

The human CaSR gene is located on chromosome 3q21.1 and loss-of-function CaSR mutations have been reported in the hypercalcaemic disorders of familial benign hypocalciuric-hypercalcaemia (FHH, FBH or FBHH) and neonatal severe primary hyperparathyroidism (NSHPT).

CaSR auto-antibodies have been found in FHH patients who did not have loss-of-function CaSR mutations, and in patients with an acquired form (i.e. autoimmune) of hypoparathyroidism. Autoimmune hypoparathyroidism can occur as an isolated clinical abnormality, as part of autoimmune polyendocrinopathy syndrome (APS)-1 or as part of APS-2. APS-1 most commonly comprises mucocutaneous candidiasis, hypoparathyroidism, and Addison’s disease. APS-2 includes two or more of the following: Addison’s disease, Graves’ disease, autoimmune thyroiditis, type 1 diabetes mellitus, primary hypogonadism, myasthenia gravis, or celiac sprue. Studies have demonstrated that CaSR autoantibody is present in about one third of the patients with isolated acquired hypoparathyroidism. On the other hand, it is also reported that some clinical primary hypoparathyroidism can harbor autoantibodies to human CaSR. Therefore, there is a great clinical value of detecting this autoantibdy to assess the autoimmune origin of the disease.

Assay Principle

The Eagle Biosciences Human Anti-CaSR IgG ELISA Assay Kit designed, developed and produced for the quantitative measurement of human anti-CaSR autoantibody (IgG type) in test samples. The assay utilizes the enzyme linked immunoabsorbent technique with selected immunogenic extracellular CaSR antigen and HRP labeled human IgG specific detection antibody.

Assay standards, controls and prediluted patient samples are added to microtiter wells of a microplate which is coated with a highly purified human CaSR extracellular antigen. After the first incubation period, the CaSR antigen on the wall of microtiter well absorbs or captures human anti-CaSR autoantibody in the sample and unbound proteins in each microtiter well are washed away. Then a HRP conjugated polyclonal anti-human IgG antibody is added to each microtiter well and a link of “CaSR antigen – human anti-CaSR autoantibody – HRP conjugated detection antibody” is formed. The unbound detection antibody is removed in the subsequent washing step. HRP conjugated detection antibody bound to the well is then incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the detection antibody bound to the human anti-CaSR autoantibody on the wall of the microtiter well is directly proportional to the amount of this autoantibody in the sample. A standard curve is generated by plotting the absorbance versus the respective autoantibody concentration for each standard on point-to-point or cubical scales. The concentration of human anti-CaSR autoantibody in test samples is determined directly from this standard curve.

Place a sufficient number of human CaSR microwell strips in a holder to run human Anti-CaSR standards, controls and unknown samples in duplicate.

Test Configuration for Anti-CaSR ELISA Assay Kit:

Assay Procedure

  1. Add 100 µL of standards, controls and 1:100 diluted patient samples into the designated microwell.
  2. Mix gently and cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light.
  3. Incubate plate at room temperature for 60 minutes.
  4. Prepare working Tracer Antibody Working Solution by 1:21 fold dilution of the Human CaSR Detection Antibody with the Tracer Antibody Diluent. For each strip, it is required to mix 1 mL of Tracer Antibody Diluent with 50 µL of the detection antibody in a clean test tube.
  5. Remove the aluminum foil and plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
  6. Add 100 µL of above diluted detection antibody working solution to each of the wells.
  7. Cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light.
  8. Incubate plate at room temperature for 30 minutes.
  9. Remove the aluminum foil and plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
  10. Add 100 µL of ELISA HRP Substrate into each of the wells.
  11. Cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light.
  12. Incubate plate at room temperature for 20 minutes.
  13. Remove the aluminum foil and plate sealer. Add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
  14. Read the absorbance at 450 nm within 10 minutes in a microplate reader

Typical Standard Curve

Manual

Product Manual