Anti-AChR ELISA Assay Kit

Additional Information

Assay Background

Autoantibodies to the acetylcholine receptor (AChR) are responsible for failure of the neuromuscular junction in myasthenia gravis, a neuromuscular disease leading to fluctuating muscle weakness and fatigability. Measurement of these antibodies can be of considerable value in disease diagnosis and management.

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