Two different acetylcholine receptor (AChR) preparations derived from amputated human muscle (AChRAMP) and from the human rhabdomyosarcoma cell line TE671 (AChRTE67,) were compared in radio-immunoprecipitation assays for the detection of AChR auto-antibodies in serum specimens from 20 patients with proven myasthenia gravis. Tests performed with the AChRTE67, and AChRAMP antigen preparations were positive in all the patients and in 19/20 respectively. A high degree of correlation (r = 0,94) was evident between the two auto-antigen preparations. Assays based on the use of TE671-derived antigen represent a useful alternative to the conventional assay using AChRAMP  for the detection of AChR autoantibodies

Anderson, R.

Duim, W.

Malfeld, S. C. K.

Myer, M. S.

Steenkamp, K. J.

English

AJOL - African Journals Online

Measurement of anti-acetylcholine receptorauto-antibodies inmyasthenia gravisK. J. Steenkamp, W. Duim, M. s. Myer,S. C. K. Malfeld, R. AndersonTwo different acetylcholine receptor (AChR) preparationsderived from amputated human muscle (AChRAMP) and fromthe human rhabdomyosarcoma cell line TE671 (AChRTE67,)were compared in radio-immunoprecipitation assays forthe detection of AChR auto-antibodies in serumspecimens from 20 patients with proven myastheniagravis. Tests performed with the AChRTE67, and AChRAMPantigen preparations were positive in all the patients andin 19/20 respectively. A high degree of correlation(r = 0,94) was evident between the two auto-antigenpreparations. Assays based on the use of TE671-derivedantigen represent a useful alternative to the conventionalassay using AChRAMP for the detection of AChR auto-antibodies.S Atr Med J 1995; 85: 1293-1294.The demonstration of acetylcholine receptor (AChR)antibodies is considered to be essential in the diagnosis ofsuspected myasthenia gravis (MG).' These auto-antibodiesare routinely assayed in serum specimens by radio-immunoassay (RIA) with AChR preparations from humanskeletal muscle radiolabelled by snake toxins, usuallya-bungarotoxin.' The absence of false-positive results inother auto-immune or neuromuscular diseasesdemonstrates the high specificity of this assay,' while thesensitivity is reported to vary between 70%' and 90%2A modified anti-AChR antibody assay using AChRprepared from the human rhabdomyosarcoma cell lineTE671 has recently been described.'-6 This AChRpreparation has the advantages of improved accessibilityand homogeneity, while eliminating the requirements forprocurement and processing of potentially infectious clinicalmateriaL 7 This study compared these two different AChRantigen preparations in the standard radio-immunoprecipitation assay for the detection of AChR auto-antibodies.Departments of Immunology and Neurology. University of PretoriaK. J. Steenkamp. N.H.D. (IMMUNOL)W. Duim. M.B. B.CH.M. S. Myer, PHD.S. C. K. Malfeld, N.D. (!MMUNOL.)R. Anderson, PH.D.SAMJARTICLESMaterials and methodsPatientsSera from 19 patients with generalised MG and 1 withocular disease (patient 4) were analysed for AChRantibodies. Of these, 15 satisfied the electrophysiologicalcriteria (electro-decremental response on repeated nervestimulation and/or increased jitter as measured with singlefibre electromyography) for the diagnosis of MG.s·9 Of theremaining 5 patients, 3 had a positive edrophobium test(patients 5, 6 and 20), while patients 11 and 12 werediagnosed with MG on clinical features (fatigueability andresponse to treatment) alone. Fifteen were women and 5men with average ages of 58 and 54 years respectively. The3 patients younger than 40 years were all female (patients 2,3 and 7). Patients 1, 2 and 3 were newly diagnosed anduntreated, while the others had been diagnosed previouslyand treated with acetylcholine-esterase inhibitors andvarious combinations of immunosuppressive agents,thymectomy or plasma exchange.Control test sera were obtained from 10 healthyindividuals (1 man and 9 women with average ages of 24and 27 years respectively).Sources of AChRFor the preparation of amputated human muscle AChR(AChRAMP) we used human gastrocnemius muscle obtainedfrom the amputated leg of a diabetic patient. The tissue wasprocessed as previously described' and the AChR-enricheddetergent extract aliquoted and stored at -72°C until used.Immediately prior to use in assays of AChR auto-antibodies,this antigen preparation was labelled for 4 hours at 4°C with'25I-labelled a-bungarotoxin (Amersham International,Amersham, Buckinghamshire, UK, > 200 Ci/mmol) aspreviously described.'Detergent-solubilised AChR derived from the 'TE671 cellline (AChRTE671 ) were obtained from a commercial source(RSR Ltd, Pentwyn, Cardiff, UK). These preparations werepre-Iabelled with '251-labelled a-bungarotoxin.Measurement of AChR auto-antibodiesThis was accomplished using a standard radio-immunoprecipitation assay.' Briefly, when AChRAMP was usedas the source of auto-antigen, 50 ~I of '25Ia-bungarotoxin-labelled AChRAMP were co-incubated with 5 ~I serumovernight at 4°C (the quantities of radiolabelled AChRAMP andserum were pre-determined in standardisation experiments),followed by the addition of 50 ~I of goat anti-human IgG(RSR Ltd) and incubation for an additional 4-hour period at4°C. Thereafter, 1 ml of washing solution (phosphate-buffered saline containing 0,05% Triton-x 100 and 0,01 Msodium azide) was added to the tubes, which werecentrifuged at 1 500 x g for 30 minutes. The supernatantswere then aspirated and the pellets resuspended and thewashing procedure repeated. Thereafter the radioactivity inthe pellet was measured using a LKB 1261 Multigamma-counter (LKB, Turku, Finland).The method was almost identical when the AChRTE671 wasused in the RIA procedure, except that shorter incubationSAMJ Volllme 85 No. 12 December 1995 1293periods were used (2 hours at room temperature duringincubation of AChRTE671 (50 1-l1) with serum (5 1-l1) and a further2 hours at 4°C following addition of goat anti-human IgG).With both types of AChR preparation the auto-antibodyconcentrations in serum specimens are expressed asnanomoles/litre (nmoles/I) toxin bound.'ResultsComparison of AChRAMP and AChRTE671The antibody concentrations in the sera of the patients withMG are shown in Table I. Although antibody titres weregenerally higher in assays using the AChRAMP preparation, ahigh degree of correlation (r = 0,94) between the AChRAMPand AChRTE671 antibody titres was observed. Elevated levelsof anti-AChR antibodies were found in the sera of 20/20 and19/20 MG patients with the AChRTE671 and AChRAM? auto-antigen preparations respectively; those of the 3 untreatedpatients were somewhat higher than those of the 17 treatedpatients (15,7 nmoles/l v. 8,3 nmoles/l for untreated andtreated patients respectively using AChRAMP; thecorresponding values with AChRTE6]1 were 12,4 nmoles/I and6,3 nmoles/l).Table I. Serum AChR auto-antibody titres in patients withmyasthenia gravisPatient Sex Age (yrs) AChRTE671 AChRAMP1 F 43 7,159 5,7732 F 39 13,025 18,0963 F 13 16,925 23,2474 F 63 11,105 12,4475 M 73 2,950 3,5656 F 52 11,040 34,6307 F 39 14,380 25,3108 F 63 4,978 4,2179 F 79 14,990 9,10010 F 53 8,540 8,76011 F 61 1,611 0,89112 F 53 2,011 1,63613 M 60 3,971 4,09114 M 55 0,939 0,50415 M 50 0,791 0,49216 M 51 2,700 4,67017 F 49 0,390 0,28018 F 77 12,653 16,79319 F 59 4,564 4,09320 F 51 8,927 9,000Resu~s are expressed as nmolesll toxin bound with cut-off points of 0,28 and 0,32 forthe AChATE671 and AChR....o.tP preparations respectively.The mean values ± the standard deviation of the 10control sera were 0,16 ± 0,16 nmoles/l and 0,10 ± 0,17nmoles/l with the AChRAMP and AChRTE671 preparationsrespectively. Cut-off points were taken as the mean valuefor each AChR preparation ± 1 standard deviation.DiscussionIn this study we have demonstrated a close correlation(r = 0,94) between the AChRAMP and AChRTE671 antibodyconcentrations in serum specimens from patients with MG.These observations are in agreement with several previousstudies.',5,] Moreover, we have also demonstrated theusefulness of the recently introduced, commercially availableAChRTE671 -based AChR-antibody RIA kit. This method isclearly of comparable sensitivity and specificity to AChRAMP-based methods but, in addition, has several distinctadvantages, including elimination of procurement oU[ssue,cumbersome antigen processing procedures and antigenradiolabelling steps. With the removal of these potentialdeterrents, it is probable that an increasing number oflaboratories will introduce this important serodiagnostic test.Although a high degree of correlation was obs~ed whenthe two different AChR preparations were used, titresobserved with AChRAMP were generally higher (by about 25%on average). This difference has been reported blothers ]and may be related to TE671's expressing only t~eextrajunctional isoform of AChR, whereas AChRAMP containsboth junctional and extrajunctional AChR.lOIn conclusion, the commercially avanable AChRTE671 -basedauto-antibody detection kit compares remarkably well withconventional AChRAMP-based methods and is a more-than-useful alternative,REFERENCES1. Bentwich Z, Beverley pelf Hammarstrom L, et al. Laboratory investigations inclinical immunology: methods, pitfalls and clinical indications. Clin ImmunolImmunopatho/1988; 49: 478-497, .2. Lindstrom J. Radioimmunoassay of antibodies to acetylcholine receptors. In: RoseNL, Friedman H, eds. Manual of Clinical Immunology. 2nd ed. Washington: AmericanSociety Of Microbiolo9Y, 1980: 386-390.3. Lindstrom JM, Seybold ME, Lennon VA, Withlingham S. Duane DD. Antibody toacetylcholine receptor in myasthenia gravis. Neurology 1976; 26: 1054-10594. Lindstrom J, Criado M, Aatnam M, et al. Using monoclonal antibodies to determinethe structures of acetylcholine receptors from electric organs, muscles and neurons.Ann N Y Acad Sci 1987; 505: 208-225.5. Walker A, Vincent A, Newsom-Davis J. Immunological and pharmacologicalheterogeneity of a-bungarotoxin binding sites extracted from TE671 cells.J Neuroimmuno/1988; 19: 149-157.6. Pachner AA. Anti-acetylcholine receptor antibodies block a.-bungarotoxin binding tonative human acetylcholine receptor on the surface of TE6?1 cells. Neurology 1989;39: 1057-1061,7. Voltz A, Hohlfeld A, Fateh-Moghadam A, et al. Myasthenia gravis: measurement ofanti-AChA autoantibodies using cell line TE6?1. Neurology 1991; 41: 1836-1838.8. Kimura J. Myasthenia gravis and other disorders of neuromuscular transmission. In:Kimura J, ed. EJectrodiagnosis in Diseases of Nerve and Muscle: Principles andPractice. 2nd ed. Philadelphia: FA Davis Company, 1989: 519-534.9. Kimura J. Single-fiber and macroelectromyography. In: Kimura J, eel.EJectrodiagnasis in Diseases af Nerve and Muscle: Principles and Practice. 2nd ed.Philadelphia: FA Davis Company, 1989: 288-304.10. Conroy WG, Saedi MS, Lindstrom J. TE671 cells express an abundance of apartially mature acetylcholine receptor alpha subunit which has characteristics ofassembly intermediate. J Bioi Chem 1990; 165: 21642-21651.Accepted 16 Mar 1994.1294 Volume 85 No. 12 December 1995 SAMJ

Measurement of antiacetylcholine receptor auto-antibodies in myasthenia gravis

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Measurement of antiacetylcholine receptor auto-antibodies in myasthenia gravis

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