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Help with antigen-specific ELISpot assay

  • GF
    Guest Federico Ruiz Moreno2024-02-01T16:00:25Z

    Hi everyone

    I am writing to seek clarification regarding the results of an ELISpot assay conducted using your kit. I work with vaccines and recently immunized subjects with a formulation containing OVA as the antigen. After 20 days post-immunization, and following a 6-day pre-sacrifice stimulation, I extracted spleen, inguinal lymph nodes, and bone marrow to analyze cells producing specific antibodies against OVA using the ELISpot assay.

    However, I did not observe any cells producing specific antibodies for OVA in any of the organs, despite detecting these specific cells through flow cytometry. Additionally, ELISA results showed a significant quantity of anti-OVA antibodies. I sensitized the plate with 10ug of OVA per well and then added 1.2 million cells/well for specific analysis, both unstimulated and stimulated with R848/IL-2 from the kit, but observed no spots. Furthermore, an unspecific assay revealed cells producing IgG antibodies.

    I would greatly appreciate your assistance in understanding why I am encountering these issues with the ELISpot assay and any potential modifications that could be made to improve the results.

    Thank you

  • C
    Christian@mabtech.com2024-02-02T08:10:46Z
    Admin

    Hi Frederico,

    OVA generally gives very good responses in mouse IgG ELISpot.

    Can you give me some details on the anti-mouse antibodies you used in this experiment? What code was the kit you used?

    What substrate was used for spot-development?

    I ask these questions because we have a number of anti-mouse IgG antibodies but for the ELISpot we recommend a special pab mix. Furthermore, if wrong substrate is used in ELISpot there are no spots. The substrate needs to be "precipitating".

  • GF
    Guest Federico2024-02-02T18:17:50Z
    10 hours ago, Christian@mabtech.com said:

    Hi Frederico,

    OVA generally gives very good responses in mouse IgG ELISpot.

    Can you give me some details on the anti-mouse antibodies you used in this experiment? What code was the kit you used?

    What substrate was used for spot-development?

    I ask these questions because we have a number of anti-mouse IgG antibodies but for the ELISpot we recommend a special pab mix. Furthermore, if wrong substrate is used in ELISpot there are no spots. The substrate needs to be "precipitating".

    Hi Christian,

    First, thank you for your prompt response.

    I used the ELISpot Flex Mouse IgG (ALP) 3825-2A kit, with BCIP/NBT as the substrate. The cells were stimulated with R848 and IL-2 for 72 hours. Interestingly, in the non-specific determination using the anti-IgG capture antibody, the spots appeared perfect.

  • GF
    Guest Federico2024-02-02T18:18:33Z

    Hi Christian,

    First, thank you for your prompt response.

    I used the ELISpot Flex Mouse IgG (ALP) 3825-2A kit, with BCIP/NBT as the substrate. The cells were stimulated with R848 and IL-2 for 72 hours. Interestingly, in the non-specific determination using the anti-IgG capture antibody, the spots appeared perfect.

  • C
    Christian@mabtech.com2024-02-05T07:25:06Z
    Admin

    Aha, I see. So when testing for total IgG using the mouse Mabtech kit spots did appear correctly.

    For some reason, the antigen-specific OVA wells are not working as they should.

    How many cells/well are you plating in these antigen wells?

    Can you give me details on the coating protocol you use for the OVA into the ELISpot plates? Do you use EtOH pre-activation? What concentration of OVA per well?

    best,

    Christian

  • GF
    Guest Federico2024-02-05T12:29:30Z

    Thank you, Christian.

    For the specific OVA assay, 1.2 million cells were seeded per well. These cells were then stimulated for 72 hours with R848 and IL-2. The assay followed the protocol provided by you on the Mabtech website. The plate was pre-treated with 15 μl/well of 35% EtOH for 1 minute. Subsequently, it was washed four times with PBS, and the plate was sensitized with 100 μl/well of OVA at a concentration of 10 μg/well, incubating the plate overnight at 4-8°C.

  • C
    Christian@mabtech.com2024-02-05T15:38:05Z
    Admin

    This sounds like a good coating protocol. You did the etoh pre-activation and concentration of OVA is good.

    What I am suspecting is that the cell number is simply too high. At 1.2 million there will be plenty of antigen specific B-cells and spots could become confluent. Try titrating the cell number. For strong antigens like OVA I think 50-100K cells/well might be enough, especially for the splenocytes.

    Another aspect, you stimulate the cells in the ELISpot plate with R848+IL2. However, if the antigen specific B-cells need to be pre-activated for 3 days to get going with the secretion, I strongly recommend doing this in 15ml tubes or culture plate. Here you can increase the cell density to 3million cells/ml and the B-cells get optimal cell to cell contact. Then after 3 days of activation with R848+IL2, wash extensively and seed the OVA coated ELISpot plate. Titrate the cells. Incubate overnight. No need to include r848+Il2 in the ELISpot plate itself.