Protocol ELISA NF-light®


Step 1 Add Sample

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Step 2 Add tracer

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Step 3 Add conjugate

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Step 4 Add substrate

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  1. Dilute the CSF samples with equal amount (1+1) of sample diluent to a total minimumvolume of 210 μL. The standards reconstituted and diluted according to the standard dilution table are ready to use (i.e. no further dilution should be made).
  2. Wash the wells to be used with wash buffer (3×300 μL). The wash buffer added could be either aspirated or removed by knocking the plate against absorbing material immediately before next washing cycle.
  3. Add 100 μL of each standard and sample in duplicate. Incubate 1 hour at room temperature (20-25°C) with agitation (800 rpm).
  4. Wash the wells with wash buffer (3×300 μL), see point 2.
  5. Add 100 μL of freshly diluted tracer (biotin anti-NF-L) antibody to each well. Incubate 45 minutes at room temperature (20-25°C) with agitation (800 rpm).
  6. Wash the wells with wash buffer (3×300 μL), see point 2.
  7. Add 100 μL of newly diluted conjugate (streptavidin-HRP) to each well. Incubate 30 minutes at room temperature (20-25°C) with agitation (800 rpm).
  8. Wash the wells with wash buffer (3×300 μL), see point 2.
  9. Add 100 μL of TMB to each well. Incubate 15 minutes at room temperature (20-25°C) with agitation (800 rpm).
  10. Add 50 μL of stop reagent to each well and read the absorbance at 450 nm (reference wavelength 620-650 nm).