Objective: CASPR1 antibodies screening in Guillain-Barré patients after SARS-CoV-2 infection.
Samples: 21-2-0768, 21-2-0769, 21-2-0770, 21-2-0771, 21-2-0772, 21-2-0773, 21-2-0774, 21-2-0775, 21-2-0776.
Materials:
- Maxisorb 96-well ELISA plates (Thermo Fisher Scientific, Massachusetts, US). NUNC ref: 442404.
- Coating buffer: carbonate/bicarbonate buffer (pH 9.6)
- Recombinant Human protein: CASPR1 (2418-CR, R&D Systems).
- RAH-HRP IgG secondary antibody: P0214, Agilent
- Non-fat dry milk.
- PBS-Tween 0.1%.
- H2SO4 25%.
- Substrate: TMB solution (BioLegend).
Procedure:
- Coat ELISA plate with protein: dilute the CASPR1 protein at 3 µg/ml with carbonate/bicarbonate buffer (50µL/well) and incubate overnight at 4ºC rocking.
- The day after, wash plate 3 times with PBS-Tween 0.1% and block with non-fat milk 5% in PBS-Tween for 1 hour (200µL /well).
- Remove blocking solution and add patients’ sera diluted 1/100 in non-fat milk 5% in PBS-Tween (100µL/well) and incubate 1 hour at RT.
- Wash 3 times PBS-Tween 0.1%.
- Add secondary antibody RAH IgG diluted 1/3000 in 5% in PBS-Tween (100µL/well) and incubate 1 hour at RT.
- Wash 3 times with PBS-Tween 0.1%
- Add TMB solution (100µL/well) an incubate 5 minutes at RT.
- Stop reaction with 25% H2SO4 with 50µL/well.
- Lecture at Multiscan ELISA Reader (filter 450 nm).
Results: All samples were negative.
