Acetylcholine Receptor Ab ELISA Kit

Sample

Serum

Species Reactivity

Human

Detection Method

cELISA

Intended Use

The CD Acetylcholine Receptor Autoantibody (AChRAb) ELISA kit is intended for use by professional persons only, for the quantitative determination of AChRAb in human serum. Autoantibodies to the acetylcholine receptor (AChR) are responsible for failure of the neuromuscular junction in myasthenia gravis. Measurement of these antibodies can be of considerable value in disease diagnosis and management.

Contents of Kit

No.	Components	Size	Storage Conditions
1	AChR MAb1 Coated Wells	12 × 8 wells (96 intotal)	2-8°C
2	Foetal Type AChR, Lyophilised	3 vials	2-8°C
3	Adult Type AChR, Lyophilised	3 vials	2-8°C
4	Reconstitution Buffer for AChR	5 mL	2-8°C
5	AChR MAb - Biotin (MAb2+MAb3), Lyophilised	3 vials	2-8°C
6	Reconstitution Buffer for MAb - Biotin	15 mL	2-8°C
7	Streptavidin Peroxidase (SA - POD) Concentrated	0.7 mL	2-8°C
8	Diluent for SA - POD	15 mL	2-8°C
9	Peroxidase Substrate (TMB)	15 mL	2-8°C
10	Stop Solution	10 mL	2-8°C
11	Concentrated Wash Solution	100 mL	2-8°C
12	Calibrators, 0.5, 1.0, 6.5 and 20 nmol/L	4 ×0.7 mL	2-8°C
13	Negative Control	3 mL	2-8°C

Storage

2-8°C

Precision

Detection Limit

The negative control was assayed 20 times and the mean and standard deviation calculated. The lower detection limit at 2 standard deviations was 0.25 nmol/L.

Sensitivity

Sera from 83 patients diagnosed with myasthenia gravis were assayed in the AChRAb ELISA. 76 (92%) were identified as being positive for AChRAb.
Clinical Accuracy
Analysis of 107 sera from patients with autoimmune diseases other than myasthenia gravis indicated no interference from autoantibodies to thyroglobulin (n=10), thyroid peroxidase (n=11), dsDNA (n=9), TSH receptor (n=40), glutamic acid decarboxylase (n=10), 21-hydroxylase (n=10), or from rheumatoid factor (n=27). Two other samples gave values of 28% (0.74 nmol/L) and 44% (1.5 nmol/L) inhibition and were from a patient with Graves' disease (TRAb positive) and a patient with Systemic Lupus Erythematosus (dsDNA Ab positive) respectively. These samples were assayed in the CD AChRAb RIA kit and were positive (values of 1.3 and 1.5 nmol/L respectively). In addition two samples from patients with rheumatoid arthritis (rheumatoid factor positive) were positive in the CD AChRAb ELISA and gave values of 24% (0.77 nmol/L) and 19% (0.61 nmol/L) inhibition. The first of these samples was also positive in the CD AChRAb RIA (5.3 nmol/L).

Standard Curve

Results can also be expressed as inhibition (%I) of AChR binding calculated using the formula:
100 × [1-(test samples absorbance at 450)/(negative control absorbance at 450)]
This % inhibition value can then be converted to nmol/L toxin bound using the formula;
0.2 × 2^{(0.067 x % inhibition of test sample)}
This formula has been established empirically using a comparison of AChRAb measurements by the CD ELISA and RIA methods. Close agreement between nmol/L values obtained in the AChRAb ELISA using the calibration curve and using this formula should not be expected in the case of all individual sera.
TYPICAL RESULTS USING % INHIBITION

ASSAY CUT OFF:
Negative < 0.45 nmol/L
Positive ≧ 0.45 nmol/L