20181210-11: ELISA NF155 in GBS from IGOS

Objective: To test IgG positivity towards NF155 in GBS samples from the spain cohort of IGOS + confirmation of positivity of sample 18-860.

SAMPLES: 0-42, 18-860, CTRL – (1 to 5) and CTRL + (LAC)

MATERIALS:

  • ELISA plates NUNC ref: 442404 Thermoscientific
  • Coating buffer:  sodium carbonate (pH 9.6)
  • Recombinant Human NF155 protein 
  • RAH-HRP IgG secondary antibody, Dako P0214
  • TMB solution (BioLegend)
  • Non-fat dry milk
  • H2SO4
  • PBS-Tween 0.1%
  • PROCEDURE:

    • Coat ELISA plate with NF155 protein (1ugr/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.
    • Wash 3xPBS-Tween 0.1%
    • Add secondary antibody using RAH IgG 1/5000 diluted in milk 5% in PBS-Tween and incubate 1h at RT.
    • Wash 3xPBS-Tween
    • Add TMB solution 10′
    • Stop reaction with H2SO4
    • Lecture at 450-620 nm.

    RESULTS:

    The figure of ELISA results is here.

    All samples were negative except 18-860.

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