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Less IgM spot in human IgG/IgM FluoroSpot kit

Guest Nisa

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I would like to ask some questions about the problem I had during my trial experiment with human IgG/IgM FluoroSpot kit. In this experiment, I used cryopreserved patient PBMC. I wanted to see if there are any IgG/IgM-secreting B cells 3 days after infection. I coated the plate myself using 5 ug/ml of antigen, and I expected to see both IgG and IgM spots since the patients were infected only 3 days ago. I got  IgG spot on my plate; however, only a few IgM spots were found in some well but in general they barely have the spot. So I have a few questions:

1. What could be the problem of not having IgM spots? Is it because the subjects themselves do not have IgM-secreting B cells, or is there any technical problem? However, I see that in the positive control well (stimulated healthy donor PBMC in total IgG/IgM), the result looks good.

2. I would also like to know why the spots of IgG seem to be really fuzzy in some wells.

Please find the result of my experiment in the attached file. It would be very helpful if you could provide me with some of the ideas you have from your experiences.

Best Regards,


B cell FluoroSpot.pptx

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1. When looking at your results it might be that your "red spots" in the Cy3 filter (or LED550 as it is called on IRIS) is in fact IgM spots. Not IgG spots. Please check the reagents you purchased! We do have ability to detect IgM in green spots and IgG in red spots, but the other way around is actually more common. If this is the case, then your results make more sense! IgM have tendency to give false positive spots in PBS coated wells, simply because a few IgM are so sticky. By contrast, this is very uncommon for IgG!

2. IgM spots tend to be more fuzzy than IgG. 

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