Diagnostics of myasthenic syndromes: detection of anti-AChR and anti-MuSK antibodies

Abstract

This paper presents the Italian guidelines for autoantibody testing in myasthenia gravis that have been developed following a consensus process built on questionnaire-based surveys, internet contacts and discussions during dedicated workshops of the sponsoring Italian Association of Neuroimmunology (AINI). Essential clinical information on myasthenic syndromes, indications and limits of antibody testing, instructions for result interpretation and an agreed laboratory protocol (Appendix) are reported for the communicative community of neurologists and clinical pathologists.

Appendix

Preanalytical procedures

Refer to the document on “Diagnostics of autoimmune encephalitis associated with antibodies against neuronal surface antigens.”

Recommendation

The use of radioactive materials requires that executors should comply with established radioprotection rules.

Analytical procedures

Assay for the determination of anti-AChR antibody titre

The execution of the radioimmunoassay for anti-AChR antibodies using commercial kits is briefly described.

Several commercially available kits (IBL, Hamburg, Germany; DLD, Hamburg, Germany; RSR, Cardiff, UK) use as antigen the AChR extracted from human muscle, from muscle-derived human cell lines (TE671) that express AChR in the adult form, or from TE671 cell lines expressing both adult and foetal AChR forms (γ/ε TE671 cells).

Materials and reagents (provided by the kit, to be stored at 2–8 °C):

• Antigen: [¹²⁵I]-α-bungarotoxin-labelled AChR (lyophilized)

• Positive control: a serum with known anti-AChR antibody titre, or a series of sera with known titers—for generating the calibration curve

• Negative control: pooled normal sera—as cut-off control

• Normal serum for sample dilution

• Washing buffer

• Buffer for the reconstitution of the labelled AChR

• Anti-human IgG

• Instruments and materials are not provided by the kit

• Polystyrene tubes (5 mL); pipettes and tips; refrigerated centrifuge; vortex; system for liquid aspiration; system for collecting/wasting radioactive materials; gamma-counter

Procedure:

• Reconstitute the labelled AChR

• Incubate sera and controls, in duplicate/triplicate, with the labelled AChR at room temperature for 2 h. The highest analytical sensitivity can be achieved by using 5 μl of undiluted serum, or 20 μl of diluted serum (depending on different kits). Sample can also be incubated overnight at 4 °C

• Add the anti-human IgG antibody and incubate in accordance with kit’s instructions, approximately for further 2 h, at room temperature

• Wash with washing buffer (1 mL) and centrifuge at 1500×g (2–8 °C), 20 min

• Remove supernatants by aspiration (pay attention to the immunoprecipitated pellet). Resuspend the pellet by vortex and repeat the washing procedure

• Remove the supernatants and measure the radioactivity in the pellet with a gamma-counter

• For further details, refer to the instructions provided by the kit manufacturer.

Calculation of results

Radioactivity (cpm) in the pellet is proportional to the labelled-AChR immunocomplexes formed with MG antibodies. Results are expressed as picomole of [¹²⁵I]-αBTX precipitated per millilitre of serum (pmol/ml). To calculate the results, follow the manufacturer’s instructions. Even if the normal range value is indicated for each assay/lot number, the laboratory should calculate the own normal reference range/cut off value by testing an appropriate number of sera from non-myasthenic patients or healthy subjects (at least 20 healthy control samples; guidelines from Lombardia region).

Assay for the determination of anti-MuSK antibody titre

The assay utilizes human recombinant MuSK (aa 1-490) labelled with [¹²⁵I]. The labelled protein is incubated with patient serum and the stable antigen-antibody complexes are immunoprecipitated by anti-human IgG antibody. The amount of specific autoantibodies is proportional to the immunoprecipitated radioactivity.

Materials and reagents (provided by the kit)

• [¹²⁵I]-labelled MuSK (lyophilized)

• Positive control: a serum sample with known amount of anti-MuSK antibodies

• Negative control: pool of normal sera

• Normal serum for sample dilution

• Washing buffer

• Buffer for the reconstitution of labelled receptor

• Anti-human IgG

• Precipitation buffer (enhancer)

• Materials not provided: pipettes and tips; polystyrene tubes; refrigerated centrifuge; vortex; system for liquid aspiration; system for collecting/wasting radioactive materials; gamma-counter.

Procedure:

• The highest analytical sensitivity is reached by using 5 μL of undiluted serum

• Reconstitute and shake the labelled MuSK with the proper buffer 30 min before use (1.5 ml of buffer for 1 vial of lyophilized MuSK)

• Incubate sera (5 μl) and positive and negative controls (50 μl), in duplicate, with 50 μl of labelled MuSK over night at room temperature

• Add 50 μl of anti-human IgG antibody and incubate for further 2 h at room temperature

• Vortex the precipitation buffer (enhancer) and add 25 μl to each sample

• Add washing buffer (1 mL) to each sample and centrifuge at 1500×g (4 °C) for 20 min

• Remove supernatant by aspiration (pay attention to the pellet). Resuspend the pellet by vortex and repeat the washing step

• Remove the supernatant and measure the radioactivity with a gamma-counter—count reading time 5 min.

Calculation of results

The radioactivity is proportional to the immunocomplexes composed of MuSK-labelled and anti-MuSK antibodies. The results are expressed in picomole of [¹²⁵I]-labelled MuSK per millilitre of serum and are calculated according to the following formula:

$$ \mathrm{anti}-\mathrm{MuSK}\kern0.4em \mathrm{Abs}\kern0.4em \left(\mathrm{pmol}/\mathrm{ml}\right)=\frac{\left(\mathrm{sample}\kern0.4em \mathrm{cpm}-\mathrm{negative}\kern0.4em \mathrm{control}\kern0.4em \mathrm{cpm}\right)\times A}{C\times K\times B\times 2.22} $$

A = [¹²⁵I] decay factor at the time of the assay, C = serum volume used (μl), K = specific activity (KCi/mmol) of labelled MuSK and B = gamma counter efficency

Quality control and sample storage

Each analytical run is accepted and validated only when the anti-AChR/MuSK antibody titre of the positive control is in the range established by each manufacturer. If the laboratory routinely utilizes an internal positive serum, its result is plotted on a quality control chart (Shewhart-Levey-Jannings). The correlation between anti-AChR/MuSK antibody titre and counts from immunoprecipitated [¹²⁵I]-αBTX is linear only within the limits of the positive control/calibration curves. Sera with values exceeding these limits should be retested after appropriate dilutions with normal serum.

• In every analytical run, a negative and two positive (one provided by the kit and one from the serum bank of the laboratory to monitor the assay results over time) controls should be used at least

• External quality controls should be performed at least yearly (e.g., AINI external quality control schemes; for anti-AChR antibodies: Euro EQAS (www.immqas.org.uk); IBL ARAb (www.ibl-international.com).

• Storage, see the document on “Cerebrospinal fluid analysis and the determination of oligoclonal bands”

Report

The following information should be reported:

• Type of the test: RIA with manufacturer’s name

• Results (in pmol/mL or nmol/L)

• Normal reference range

• Result interpretation (positive, out of the normal range, negative, in the normal range). It is not advisable to indicate border-line results

• Comments: Refer to point 4.1.9 of the document on “Cerebrospinal fluid analysis and the determination of oligoclonal bands”