Preparation of ganglioside ELISA plates
- Reconstitute the ganglioside vials with 1 ml of chloroform/methanol (1:1), except in the case of GQ1b, which is reconstituted with 0.5 ml. Once reconstituted, they are stored at -20ºC
- GM1: 1mg + 1ml: 1mg/ml
- GD1b: 1mg + 1ml: 1mg/ml
- GQ1b: 0.5mg + 0.5ml: 1mg/ml
- Prepare the methanol+ganglioside solution depending on the plates to be prepared. The final concentration is 2 ug/ml (except GQ1b which is 4 ug/ml).
- For 5 plates (3 columns of each ganglioside/plate)
- GM1: 15 ml methanol + 30 ul reconstituted GM1
- GD1b: 15 ml methanol + 30 ul reconstituted GD1b
- GQ1b: 15 ml methanol + 30 ul reconstituted GQ1b
- Vortex the dilutions.
- Add 100 ul of diluted ganglioside to each well (in the corresponding column). Only methanol is added to the blank wells.
- Let the plates dry at room temperature (preferably in an extractor hood) until the methanol evaporates (minimum 4 hours)
- Freeze plates at -20ºC
ELISA Gangliosides day 1
Samples: all samples are made for IgG and IgM, at 1/100 and 1/500 dilutions (a total of 16 wells will be used for each sample). In each plate we can test 5 samples and the controls.
SAMPLES: 21-2-109, 21-2-110, 21-2-111, 21-2-112, 21-2-113
- Protocol:
- Thaw plates with gangliosides
- Block with PBS-BSA 1% – 200 ul/well, incubate 2h at 4ºC
- Wash 2 times with PBS (in cuvette)
- Prepare serum dilutions (in PBS-BSA 0.1%)
- Dil 1/100 (x8 wells): 1 ml PBS-BSA 0.1% + 10 ul serum
- Dil 1/500 (x8 wells): 0.8 ml of PBS-BSA 0.1% + 0.2 ml of the previous dilution.
- In the case of controls, as we put a serum for IgG and another for IgM, we prepare half of each dilution
- Add 100 ul of serum to each well, incubate overnight at 4ºC
ELISA Gangliosides day 2
- Wash 4 times with PBS
- Prepare secondary Ab: RAH HRP IgG or IgM dil. 1/3000 in PBS-BSA 0.1%. Add 100 ul of the dilution to each well, incubate 2h at 4ºC
- Wash 4 times with PBS
- Prepare the substrate: 1 OPD tablet + 1 urea hydrogen peroxidase tablet + 20 ml distilled water (protect from light with aluminum foil and shake).
- Add 100 ul to each well, incubate 40 min at room temperature
- Stop the reaction by adding 50 ul of 25% H2SO4 to each well.
- Read in ELISA plate reader at 490-630nm
RESULTS: All patients were negative. The results were here.
