Anti-AChR antibodies detected by ELISA method using an automated system (SkyLAB 752): evaluation of the analytical performance according to the Clinical and Laboratory Standards Institute (CLSI) EP15-A

Abstract

Antibodies against the acetylcholine receptor (AChRAb) are currently detected in the clinical Laboratory by using the radioimmunoassay which uses receptors extracted from human muscles, identified and quantified by means of labelling with 125I- $\alpha$ -bungarotoxin. For the first time, Hewer et al. developed a new enzyme-linked immunosorbent assay (ELISA) based on the use of purified foetal and adult AchR. The aim of the study was to validate the ELISA test and define a protocol for its implementation on an automatic instrumentation, according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. Two commercial control sera, high positive (C1, 7.5 nmol/L) and low positive (C2, 1.6 nmol/L), provided by the manufacturer were assayed in triplicate, in five independent runs for within-run, between-day and within-laboratory precision. The four calibrators at different concentration level included in the diagnostic kit (E1, 0.5 nmol/L; E2, 1.0 nmol/L; E3, 6.5 nmol/L; E4, 20 nmol/L) were tested in duplicate in five consecutive days for the trueness study. The commercial controls, tested in the course of precision test, at the mean concentration measured of 11.2 nmol/L (control C1) showed a within-run CV value of 7.9% (95% CI, 0.61 to 1.52), a between-day CV of 8.4% (95% CI, 0.00 to 2.98) and a within-laboratory CV of 11.5% (95% CI, 0.92 to 3.12). At the mean concentration measured of 3.2 nmol/L (control C2) showed a within-run CV of 5.6% (95% CI, 0.13 to 0.32), a between-day CV of 1.9% (95% CI, 0.00 to 0.33) and a within-laboratory CV of 6.0% (95% CI, 0.15 to 0.39). The total repeatability, expressed as Standard Deviation (SD) (within laboratory), resulted 1.27 for C1 and 0.19 for C2, more less than the verification value (1.37 and 0.49, respectively) obtained on the basis of the repeatability declared by the manufacturer [SD (C1) = 0.95874 and SD (C2) = 0.3589]. The analytical performance obtained in “routine activities” has confirmed the specifications produced in the experimental stage and has shown that the reliability of the test persists even when the assay is performed in automation. The good precision and accuracy of the test and its availability on an automated system featuring ease of use and rapid response lead us to conclude that the test is satisfactory for routine use and it is potentially suitable for the long-term monitoring of AChRAb.