20200923-24: CASPR1 and NF155 ELISA titration

Objective:

  • Titrarion of samples included in NF155+ CIDP patients study.
  • Evaluate CASPR1 positivity in samples from Luuk dataset (ICOS_biobank).

SAMPLES:

  • Titration NF155: 24.1, 24.2, 24.3, 407
  • CASPR1: 507, 363, 350, 356, 357, 361, 344, 362, 391, 392, neg control and pos control.

MATERIALS:

  • ELISA plates NUNC ref: 442404 Thermoscientific
  • ELISA plate ref. 1581
  • Coating buffer:  sodium carbonate (pH 9.6)
  • Recombinant Human NF155 protein 
  • Recombinant Human CASPR1 protein
  • MAH IgG4 for NF155 titration, MAH total IgG for CASPR1.
  • TMB solution (BioLegend)
  • Non-fat dry milk
  • H2SO4
  • PBS-Tween 0.1%

PROCEDURE:

  • Coat ELISA plate with NF155 protein (1ug/ml) or CASPR1 protein (5ug/ml) in carbonate/bicarbonate buffer (pH 9.6) and incubate O/N at 4ºC rocking.
  • The day after, wash plate 3xPBS-Tween 0.1% and block with milk 5% in PBS-Tween for 1 hour, 200uL per well).
  • Add patients sera diluted 1/100 (200uL/well)  and incubate 1h at RT. For titration: Make serial dilutions 1:3 of the samples in a ELISA plate ref. 1581 (8 wells). In the first well, add 200uL of the sample (diluted at 1/100), in the other wells add 140uL of milk 5% in PBS-Tween and 70uL of the previous well (serial dilutions). Add the dilutions in the blocked ELISA plate (ref. 442404) and  incubate 1h at RT.
  • Wash 3xPBS-Tween 0.1%
  • Add secondary antibody MAH IgG4 1/1000 or MAH IgG 1/2000 in milk 5% and incubate 1h at RT.
  • Wash 3xPBS-Tween
  • Add TMB solution 10′
  • Stop reaction with H2SO4
  • Lecture at 450-620 nm.

RESULTS: The results of the ELISA were here.

All samples were negative for CASPR1. Sample 407 was negative for NF155 (IgG4 and total IgG).NF155 titration: 1/8100 for 24.1, 24.2 and 24.3.

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