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Guest Hari B

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Guest Hari B

Dear Mabtech,

  I am optimizing an IgG ELISpot assay for Hep B vaccinated subject. I stimulate my PBMCs from a recently hep B vaccinated subject with R848 and IL2 for 5 days. Wash my cells 1X and resuspend in fresh 10%FCS RPMI before plating 2e5 cells/well. My negative control is PBS coated well (Image B5), positive control is Total IgG (15ug/mL) (image C5) and antigen-specific cells are identified with HBsAg coated (10ug/mL)(image F5) or IgG coated well followed by a biotinylated HBsAg (5ug/mL)(D5).  I noticed that the background in my PBS coated well as high. Could you please advise?

Thank you.

B5.JPG

C5.JPG

F5.JPG

D5.JPG

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Dear Hari B,

Welcome to the forum. Here are my intital reactions/questions:

1. After 5 days stimulation with R848+IL-2, the amount of IgG in the cell cultures is massive. Washing 1x is on the small side. I would recommend doing 3x or 2x and carefully removing all supernatant all the way down to the cell pellet. In this way, free IgG in solution will be reduced which can ofcourse cause high backgorund.

2. The 200,000 cells/well for Total IgG is too high. A better option would be 50,000 cells/well. For your antigen specific wells 200,000 cells/well is ofcourse good. 

3. When coating with only PBS, do you include a blocking step in your assay with cell culture medium containing 10% FBS? Blocking of the PVDF membrane is needed. 

4. The well names of you uploaded images is not shown to me. My guess is that the really dark ELISpot membrane above is the PBS control, correct?

 

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