Anti-AChR (Acetylcholine Receptor) ELISA Assay Kit

$1,350.00

The Eagle Biosciences Anti-AChR (Acetylcholine Receptor) ELISA Assay kit is intended for the quantitative determination of Anti-AChR in serum by enzyme linked immunoassay (ELISA). The Anti-AChR ELISA Assay kit is for research use only and not to be used in diagnostic procedures.

SKU: ACR31-K01 Categories: , ,

Anti-AChR (Acetylcholine Receptor) ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: 0.23 nmol/l
Dynamic Range: 0.2 – 20 nmol/l
Incubation Time: Overnight
Sample Type: Serum
Sample Size: 100 µl

Controls Included

Additional Information

Assay Principle

The Eagle Biosciences Anti-AChR (Acetylcholine Receptor) ELISA Assay Kit is an enzyme immunoassay for the quantitative determination of autoantibodies to acetylcholine receptor (AChR Abs) in human serum.

The assay system depends on the ability of AChR Abs in human serum to bind to similar sites on the AChR as various monoclonal antibodies such as MAb1 (coated on ELISA plate wells) and/or MAb2 and/or MAb3 (which are labeled with biotin). In the absence of AChR Abs a complex is formed between MAb1 coated on the plate wells, the AChR and MAb2 and MAb3 biotin. MAb2 and MAb3 biotin bound are then detected by addition of streptavidin peroxidase (SA-POD), substrate (TMB) and stop solution. In the presence of AChR Abs the formation of the MAb1-AChR-MAb2/MAb3 biotin complex is inhibited, resulting in less SA-POD being bound and a reduction in final absorbance at 450nm. The higher the concentration of AChR Abs in the test serum the greater the inhibition of MAb biotin binding.

Assay Procedure

  1. Pipette 100 µl of negative control (CI), calibrators (1 – 4), positive controls (CII, CIII) and test sera into individual 1.5 ml Eppendorf tubes, labelled accordingly.
  2. Pipette 25 µl of fetal and adult type AChR mix (K+L) into each Eppendorf tube (from step 1) and seal the tubes. Make sure that all liquid is in the bottom of each tube (if in doubt centrifuge the tubes in a microfuge for 10 seconds at 10-15,000g). Vortex gently and incubate overnight (16 – 20 hrs) at 2 – 8°C.
  3. Gently mix each tube of sample-AChR mixture from step 2 using a vortex mixer. Pipette duplicate 50 µl of each sample-AChR mixture into the corresponding wells (A) according to assay scheme.
  4. Cover the plate and incubate for 60 min at room temperature (18 – 25 °C) while shaking >500 rpm.
  5. Aspirate the plate wells by use of a microplate washer or discard by briskly inverting the frame of stripwells over a suitable receptacle.
  6. Wash the wells 3 times with 300 µl washing solution (diluted from B). Tap the inverted wells gently on a clean dry absorbent surface to remove excess wash.
  7. Add 50 µl of reconstituted MAb-Biotin solution (prepared from H and J) to each well.
  8. Cover the plate and incubate for 60 min at room temperature (18 – 25 °C) while shaking >500 rpm.
  9. Aspirate the plate wells by use of a microplate washer or discard by briskly inverting the frame of stripwells over a suitable receptacle.
  10. Wash the wells 3 times with 300 µl washing solution (diluted from B). Tap the inverted wells gently on a clean dry absorbent surface to remove excess wash.
  11. Add 100 µl reconstituted SA-POD (prepared from D and G) to each well.
  12. Cover the plate and incubate for 30 min at room temperature (18 – 25 °C) while shaking > 500 rpm.
  13. Aspirate the plate wells by use of a microplate washer or discard by briskly inverting the frame of stripwells over a suitable receptacle.
  14. Wash the wells 3 times with 300 µl washing solution (diluted from B), in the case of washing manually, use an additional final wash step with pure water to remove any foam. Tap the inverted wells gently on a clean dry absorbent surface to remove excess wash.
  15. Add 100 µl substrate solution (E) to each well and shake shortly.
  16. Incubate for 30 min in the dark at room temperature.
  17. Add 50 µl stop solution (F) to each well. Shake the plates for 5 seconds.
  18. Read the optical density at 450 nm versus 620 or 690 nm within 5 min after adding the stop solution.

Typical Standard Curve

Anti-AChR (Acetylcholine Receptor) ELISA Assay Kit Standard Curve

Manual

Product Manual


Publications

Recent Citations

Zhang, CJ;Gong, Y;Zhu, W;Qi, Y;Yang, CS;Fu, Y;Chang, G;Li, Y;Shi, S;Wood, K;Ladha, S;Shi, FD;Liu, Q;Yan, Y;, (2016). Augmentation of Circulating Follicular Helper T Cells and Their Impact on Autoreactive B Cells in Myasthenia Gravis. J. Immunol., 197(7), 2610-2617. doi:10.4049/jimmunol.1500725