Diagnostics of dysimmune peripheral neuropathies

Abstract

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

Appendix

• 1.0 Preanalytical procedures

Refer to the document on ‘Diagnostics of autoimmune encephalitis associated with antibodies against neuronal surface antigens’

• 2.0 Analytical Procedures

• 2.1 Enzyme linked immunosorbent assay (ELISA) for anti-GM1 IgG/IgM and anti-GQ1b IgG.

• 2.1.1 Materials and reagents

• 2.1.1.1 Microplates for ELISA.

• 2.1.1.2 Antigens for microplate coating: monosialoganglioside GM1 (Sigma) and tetrasialoganglioside GQ1b (Calbiochem).

• 2.1.1.3 Buffers and antisera: bovine serum albumin (BSA), NaCl, NaH₂PO₄, Na₂HPO₄, 3% H₂O₂, citric acid, o-phenylendiamine (OPD, 10 mg tabs), H₂SO₄, distilled H₂O, peroxidase-conjugated rabbit anti-human IgG/IgM (Dako), methanol, 100% ethanol.

• 2.1.2 Reagent preparation

• 2.1.2.1 Antigens for microplate coating: reconstitute lyophils with methanol: GM1, concentration of 1 mg/mL; GQ1b, concentration of 0.1 mg/mL (store at −20 °C for 6 months).

• 2.1.2.2 Non-specific binding site blocking solution: for 200 mL, dissolve in distilled H₂O: 4 g BSA; 1.2 g NaH₂PO₄; 2.4 g NaCl (pH 7.4).

• 2.1.2.3 Washing solution: for 500 mL, dissolve in distilled H₂O: 1.0 g BSA; 3.0 g NaH₂PO₄; 6.0 g NaCl (pH 7.4).

• 2.1.2.4 Stock solutions: Na₂HPO₄ 0.2 M: dissolve 2.85 g Na₂HPO₄ in 100 ml of distilled H₂O (mildly heat to favour salt dissolving). Citric acid 0.1 M: dissolve 2.1 g citric acid in 100 ml of distilled H₂O (store at 4 °C for 1 month).

• 2.1.2.5 Staining solutions: use stock solutions; for 25 mL, mix: 6.4 mL, Na₂HPO₄ 0.2 M; 12.5 mL, H₂O; 6.1 mL, citric acid 0.1 M (pH 5.09). Add an OPD Table 10 min before using; keep it in the dark). Warning! OPD is carcinogenic: use gloves and chemical hood. Add 100 μL of 3% H₂O₂ immediately before using the solution.

• 2.1.2.6 Blocking solutions: H₂SO₄ 0.1 M: dilute 556 μL of H₂SO₄ 18 M in 100 mL of distilled H₂O.

• 2.1.3 Samples and controls

• 2.1.3.1 Dilute serum samples and positive and negative controls at 1:640 (for anti-GM1), or 1:1280 (for anti-GQ1b) using the blocking solution.

• 2.1.4 Procedure

• 2.1.4.1 Divide 96-microwell plates into two areas, and coat half of the microwells with 1 μg GM1 or GQ1b in 100 μL of 100% ethanol/well, the other half of the microwells with 100 μL of 100% ethanol/well.

• 2.1.4.2 Let microwells dry at 4 °C overnight.

• 2.1.4.3 Add 200 μL/well of blocking solution; incubate at 4 °C for 4 hours.

• 2.1.4.4 Remove blocking solution by aspiration; wash with 200 μL/well of washing solution.

• 2.1.4.5 Add 100 μL/well of samples and controls, each in quadruplicate (in duplicate in antigen-coated wells, and in duplicate in non-antigen-coated wells); add 100 μL/well of blocking solution to two antigen-coated wells, and to two non-antigen-coated for blank readings; incubate at 4 °C overnight.

• 2.1.4.6 Remove samples and controls by aspiration; wash with 200 μL/well of washing solution (5 times).

• 2.1.4.7 Add 100 μL/well of anti-human IgG/IgM, previously 1:500 diluted in blocking solution; incubate at 4 °C for 1 hour.

• 2.1.4.8 Wash (point 2.1.4.6).

• 2.1.4.9 Add 100 μL/well of staining solution; incubate at room temperature for 1 hour.

• 2.1.4.10 Block with 50 μL/well of staining blocking solution.

• 2.1.4.11 Read with spectrophotometers at 492 nm, using the appropriate wells as blanks.

• 2.1.5 Readings and result interpretation

Calculate results by subtracting optical density (OD) mean values of non-antigen-coated wells to those of the corresponding antigen-coated wells; OD <0.1 indicate negative samples, OD between 0.1–0.5 positive samples (titer corresponding to the starting dilution); samples with OD >0.5 should be further titered.

• 2.2 Commercial ELISAs and dot-line blots for anti-ganglioside antibody detection.

Certified commercial kits for the contemporary detection of a wide array of anti-ganglioside antibody can be used for routine diagnostics, following the manufacturer’s instructions.

• 3.0 Quality control and sample storage

Refer to the document on ‘Diagnostics of the neuromyelitis optica spectrum disorders (NMOSD)’

• 4.0 Report

• 4.1 Qualitative results (positive/negative/low positive) should be reported.

• 4.2 Positive samples on ELISA should be titered (end-point dilution); titering on immuno-line/dot blots is expensive and thus optional.

• 4.3 Samples with OD between 0.05–0.10 on the herein-reported ELISA could be considered as low positive (no diagnostic meaning).

• 4.4 Reports should contain the following information:

• i) Type of method: in-house ELISA; commercial ELISA or immuno-line/dot blot with the manufacturer.

• ii) Reference values correspond to the testing dilutions: <1:640 (anti-GM1), <1:1280 (anti-GQ1b) for the herein-reported ELISA, or those entailed by commercial kits.

• iii) Comments: refer to the document on ‘Cerebrospinal fluid analysis and the determination of oligoclonal bands’.