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Fluorospot: total Ig vs. protein coating

  • GL
    Guest Laura2023-07-14T10:00:50Z

    Hello!

    We have performed IgG fluorospot using a cell line that was engineered to express a particular antibody, which is an IgG. Therefore we thought that if we coated the plate with anti-Ig or with the protein recognized by the antibody we should get the same amount of spots. But as you can see in the pdf attached, this is not the case. We get about double amount of spots when using anti-Ig for coating. Increasing the amount of protein used for coating did not change the results.

    What could be the reason for this discrepancy?

    Thanks in advance!

    Best,

    Laura

    20230714 Fluorospot.pdf

  • a
    admin2023-07-14T10:25:35Z
    Admin

    Hi Laura,

    Interesting experiment!

    I have a few comments:

    1. What count settings are you using for the analysis of these wells in LED550? The anti-IgG spots are stronger compared to the antigen specific ones but my eyes tell me that spot forming units are fairly similar. Around 85-90% of spots in total IgG. Sure there are more spots in total IgG but not much more.  If you use default settings of 10, I would recommend lowering it something like 5 and see if this does not result in more similar spot counts.

    2. When using a cell line engineered to secrete IgG of a particular type I would not be surprised if some of the cells will generate IgGs that have not folded correctly. Something that makes them assemble incorrectly inside of the cells before they are secreted. When using plasmids there will be some cells that simply generate these incorrect IgG. If this happens it might lead to the IgG loosing its ability to bind its antigen effeciently but still get detected as secreted IgG.

  • GL
    Guest Laura2023-07-14T13:48:08Z

    Hi,

    Thank you very much for your response!

    1. We used the default settings of 10. We are going to try and read the plate using lower settings.

    2. That could be indeed an explanation for this result

  • a
    admin2023-07-14T16:28:51Z
    Admin

    Hi again Lara,

    You do not need to re-read the plate in order to use another count-setting.

    Just open up the plate in Apex and change the default preset intensity to 5 (instead of 10). Spotnumbers will update immeditatly and you can see the difference.