Total saturation or poor development?

[Guest SoCal Elispot]

Hi Mabtech,

I ran an Elispot earlier this week using two hyrbidoma lines and the parent myeloma line.  We wanted to PoC the total IgG Elispot method before we start looking at B cell secretion with it.  I used MSIP plates and followed the Protocol II 1b for total IgG detection (24 hour incubation with cells). I started with 100k cells/well and titered down to 12.5k cells/well.  For the development I allowed only 15 minutes as the hybridoma wells had such an intense signal.  I have run Elispots in the past on stimulated PBMCs and was able to positively detect cells with low background.  Any help is appreciated.

 

Thanks,

SoCal Elispot

[Guest SoCal Elispot]

I forgot to add.

Pic 1 is myeloma cells, pic 2 hybridoma at 100k cell/well, pic 3 and 4 media and pbs, and pic 5 is 12k hyrbidoma/myeloma cells mixed at 5%/95% respectively.

Thanks,

[Christian@mabtech.com]

Dear Socal,

So you are looking at total IgG secretion in these elispot wells. Did you use Mabtech capture and detection antibodies? Also, did you coat overnight after doing etoh pre-treatment?

One thing comes to mind: hybridomas all secrete IgG. 12500 cells/well could be too much and you end up with totally confluent spots. In cases were all cells secrete the analyte of interest I would aim for adding in only 500-1000 cells/well. 

 

best,

Christian

[Guest SoCal Elispot]

Hi Christian,

I used the Mabtech mouse IgG basic kit and I did coat overnight at 4 C.  I am going to be coating plates again and will try 500-1000 cells/well.  Thanks for the help.

SoCal

[Christian@mabtech.com]

Very good! come back and tell us how it went! Images are always nice