Gangliosides ELISA protocol

This protocol can be used for detection of gangliosides.Spicific antibodies to gangliosides are added to cells and allowed to bind to the cell surface. The nonbound antibody is washed away and an enzyme conjugated second antibody is then detected by the addition of an enzyme-spicific substrate.

Procedure

2. Trypsinize confluent flasks
4. pool and count cells
6. Resuspend to 4x100,000 cells/ml medium.
8. Add100ul/cell to 96 well culture plates
10. Incubate 24Hrs at 37c
12. Wash plates twice with PBS
14. add 150ul/well 10% Buffered Formalin
16. Fix for 20 minutes at room temperature
18. Wash three times with PBS
20. Add 250ul/well PBS:2%BSA
22. Incubate 30minutes at room temperature.
24. Wash plates twice with PBS.
26. Add 100ul/well primary antibody diluted in PBS
28. incubate for 2hr at 37c
30. Wash 5 times with PBS.
32. Add 50ul/well secondary antibody:HRP diluted in PBS.
34. Incubate for 1hr at 37c.
36. Wash 5 times with PBS.