Diagnostics of anti-MAG antibody polyneuropathy

Abstract

This document presents the guidelines for anti-myelin-associated glycoprotein (MAG) antibody testing that have been developed following a consensus process built on questionnaire-based surveys, internet contacts, and discussions at workshops of sponsoring Italian Association of Neuroimmunology (AINI) congresses. The main clinical information on anti-MAG antibody polyneuropathy, indications and limits of anti-MAG antibody testing, instructions for result interpretation, and an agreed laboratory protocol (Appendix) are reported for the communicative community of neurologists and clinical pathologists.

Appendices

Appendix

Preanalytical procedures

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

Analytical procedures

ELISA

The herein described analytical procedures refer to the certified commercial anti-MAG IgM ELISA currently used by the AINI centers (Bühlmann, Schönenbuch, Switzerland).

Reagent preparation

Dilute the concentrated buffer solution with deionized water (1:10) (about 80 mL per strip). Use the diluted buffer cooled (put the beaker on ice).

Reconstitute the four calibrators (anti-MAG antibody concentrations: 70,000, 15,000, 3000, and 1000 BTU). Store in aliquots at −20 °C for up to 2 months.

Controls and samples

Reconstitute the two controls with incubation buffer (1 mL). Store in aliquots at −20 °C for up to 2 months.

Dilute sera with incubation buffer (1:1000). Let the diluted samples rest for 1 h at 18–28 °C, vortexing from time to time. Put the sample tubes on ice for 10 min before dispensing.

Procedure

Take the strips needed to test the samples and immediately seal the remaining ones in the original envelope.

Wash the pre-coated microwells four times with 300 μL/well of the cooled washing solution.

Empty the wells and tap the microplate onto absorbent paper sheets to completely remove the liquid.

Pipet four calibrators, two controls, and samples in duplicate (100 μL/well).

Cover the microplate with a sealing sheet and incubate for 2 h (±5 min) at 2–8 °C.

Empty the wells, and wash and empty them as above described.

Add peroxidase-conjugated anti-human IgM (100 μL/well), cover with a sealing sheet and incubate for 2 h (±5 min) at 2-8°C.

Empty the wells, and wash and empty them as above described.

Important: Let the substrate solution reach the temperature of 18–28 °C.

Add substrate solution (100 μL/well).

Cover the microplate with a sealing sheet, put it onto a plate mixer at 800–1000 rpm, protect from light, and incubate for 30 min (±5 min) at 18–28 °C.

Add stopping solution (100 μL/well). Take care of getting rid of air bubbles. Read the microplate by 30 min.

Read absorbances at 450 nm using a microplate ELISA reader (if available, set the instrument doubling the wavelengths at 450 nm and at 600/620 nm to minimize background).

Result interpretation

Design standard curves by placing absorbance values (OD) on the vertical axis and BTU values on the horizontal axis for each calibrator (mean values). Absorbances are proportional to anti-MAG antibody titers (expressed in arbitrary units, BTU). If the microplate reader cannot read OD >2.0 read at 490/492 nm wavelengths (reference filters, 600/620 nm).

Serum samples with BTU values ≤1000 are “negative,” those with BTU values ≥10,000 “positive,” and those with BTU values between 1000 and 10,000 “weak positive” or “negative” on the basis of what reported in “clinical and laboratory aspects” section of this document.

Quality control and sample storage

Refer to the document on “diagnostics of the neuromyelitis optica spectrum disorders (NMOSD).”

Report

A qualitative result (positive/negative/weak positive) should be reported, along with the BTU value.

The report should contain the following general information:

• i) Type of method (ELISA) and name of the kit manufacturer.

• ii) Reference values: report the cutoff of positivity.

• iii) Comments: refer to the document on “cerebrospinal fluid analysis and the determination of oligoclonal bands.”