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

  • Letizia Abbracciavento
  • Ruggiero Fumarulo
  • Marilina Tampoia
Articolo originale



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.


Antibodies against the acetylcholine receptor Enzyme-linked immunosorbent assay Clinical and Laboratory Standards Institute Validation 


Conflict of interest

The authors Letizia Abbracciavento, Ruggiero Fumarulo, Marilina Tampoia, declare that they have no financial and no-financial competing interests.

Human and animal rights

The article does not contain any study performed on humans and animals by the authors.


  1. 1.
    Leite MI, Waters P, Vincent A (2010) Diagnostic use of autoantibodies in myasthenia gravis. Autoimmunity 43:371–379 CrossRefPubMedGoogle Scholar
  2. 2.
    Hewer R, Matthews I, Chen S et al. (2006) A sensitive non-isotopic assay for acetylcholine receptor autoantibodies. Clin Chim Acta 364:159–166 CrossRefPubMedGoogle Scholar
  3. 3.
    CLSI (2005) User verification of performance for precision and trueness; Approved Guideline, 2nd edn. CLSI document EP15-A2. Wayne, PA, USA:CLSI Google Scholar
  4. 4.
    Vincent A, Newsom-Davis J (1985) Acetylcholine receptor antibody as a diagnostic test for myasthenia gravis: results in 153 validated cases and 2967 diagnostic assays. J Neurol Neurosurg Psychiatry 48:1246–1252 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Lindstrom J (1977) An assay for antibodies to human acetylcholine receptor in serum from patients with myasthenia gravis. Clin Immunol Immunopathol 7:36–43 CrossRefPubMedGoogle Scholar
  6. 6.
    Oosterhuis HJ, Limburg PC, Hummel-Tappel E et al. (1983) Anti-acetylcholine receptor antibodies in myasthenia gravis. Part 2. Clinical and serological follow-up of individual patients. J Neurol Sci 58:371–385 CrossRefPubMedGoogle Scholar
  7. 7.
    Andreetta F, Rinaldi E, Bartoccioni E et al. (2017) Diagnostics of Myasthenic Syndromes: detection of anti-AChR and anti-MuSK antibodies. Neurol Sci 38(Suppl 2):S253–S257 CrossRefGoogle Scholar
  8. 8.
    Bizzaro N, Bossuyt X, Haapala AM et al. (2017) Accreditation in autoimmune diagnostic laboratories. A position paper of the European Autoimmunity Standardisation Initiative (EASI). Autoimmun Rev 16:81–86 CrossRefPubMedGoogle Scholar
  9. 9.
    Tozzoli R, Bizzaro N, Tonutti E et al. (2002) Immunoassay of anti-thyroid autoantibodies: high analytical variability in second generation methods. Clin Chem Lab Med 40:568–573 CrossRefPubMedGoogle Scholar
  10. 10.
    Bizzaro N, Pregnolato M, van Boekel MA et al. (2012) Preliminary evaluation of the first international reference preparation for anti-citrullinated peptide antibodies. Ann Rheum Dis 71:1388–1392 CrossRefPubMedGoogle Scholar

Copyright information

© Società Italiana di Patologia Clinica e Medicina di Laboratorio 2018

Authors and Affiliations

  • Letizia Abbracciavento
    • 1
  • Ruggiero Fumarulo
    • 1
  • Marilina Tampoia
    • 1
  1. 1.Autoimmunology Section, Universitary Clinical Pathology Unit, Polyclinic of Bari, Department of Biomedical Sciences and Human OncologyUniversity of Bari Medical SchoolBariItaly

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