Subrat

Can you elaborate more on the specific assay your performing.  Are you performing an Anti-IgG (B cell) or Anti-IFN-gamma (T cell) ELISPOT?  How long is your culture period?  Concentration of detection antibody etc.

Greg Kirchenbaum

Hello Greg thanks for responding,

I am performing Anti-IgG (B cell) Elispot, culture period is 24hrs and and using 200ng/well detection antibody.

Kindly check if there is a mention on the substrate that you have to filter it before use. Try next time to filter with a 0.2 micron filter the substrate. some substrates like BCP/NBT need to be filtered everytime before you use . check sigma recommendation for this. Good luck

Dear Sambhuprasad,

                   Sorry I have not mentioned, but usually I filtered the substrate and used.

Subrat

I saw that your coating with 1µg/mL of protein/well, is it possible to increase your antigen concentration.  In the past, I have used 2-5µg/mL of protein for coating wells.  If the cells are secreting a low-affinity antibody, increasing the avidity of the Ab-Ag complex may result in greater antibody retention and larger spots.  Best of luck

Greg

I have tried with different concentration of coating antigen like 1ug/ml, 5ug/ml and 10ug/ml.

Subrat

Can you elaborate on the source of cells used for the assay?  How were they treated prior to culture in the ELISPOT plate? 

Greg

Greg

I am using mouse spleen cells.  First of all I am injecting 5ug purified protein (antigen) to the mouse, then after  15 days giving a booster dose and then harvest the spleen. Then I am making single cell suspension, counting and addition to the Elispot plate. 

Subrat

Prior to the boost, have you assess the quality/quantity of the antibody response by ELISA?  Is it possible that the mice are making a poor response to the antigen, and therefore you have few ASCs following the boost?  Have you consider performing total IgG+ ELISPOTs in addition to the Ag-specific ELISPOT assay?  

Greg

Greg

Yes I have checked the antibody response by ELISA and it is giving good response. I have also taken total IgG, but didn't get spots. 

Hi Subrat,

do you wash your plate thoroughly with water between ethanol prewetting and coating, do you wash off excess antigen after coating and do you block your plate with a standard procedure (e.g. growth medium containing FBS)? I assume there are no substances toxic to the cells (e.g. detergents or azide) in any of the solutions used from prewetting to the end of the cell incubation. Azide will also inhibit HRP.

Perhaps your anti-IgG detection antibody is not appropriate for ELISPOT? Your absence of total IgG spots may suggest this. From my experience I know that antibodies that work well in e.g. ELISA not necessarily work well in ELISPOT. You may want to check the literature and test a few of those mentioned there (if any). Alternatively, it may also be possible to buy antibodies (or expensive ready made kits) validated for ELISPOT (e.g. from Mabtech).

Best regards,

Geir

Hello Geir,

Yes wash the plate thoroughly. I have used antibodies  from Mabtech. But I don,t understand why spots are not appearing in total IgG coated wells. If you have suggestions  then please suggest.

You may consider to try TMB for membrane applications/IHC instead of AEC. TMB gives brighter spots (e.g. TMB-H from Moss). Have you tested your HRP conjugate?

Hi Geir,

I have been using this HRP conjugates for IFN-g also and it is giving good results, so I don't there is any problem with HRP-conjugate. Yes, I have never used TMB substrate for ELISpot though used in ELISA. So I shall try TMB. 

Hi with the Mabtech kit, do you filter the substrate every time ?  Its recommended to filter it before every use. If you are not doing this may be these spots are due to the particles in substrate. All the best !