B-cell ELISpot IgG dark background staining

[Guest Michelle]

Hi there Mabtech,

we are trying to setup a B-cell IgG Elispot but have a hard time getting it right.

we use PVDF plates type MSIP from Milipore, and activate  the plate with 35% ethanol.

block the plate with 10% FBS for 30 min. wash with only PBS,

we use 25.000 PBMCs (fresh or frozen)

the at the file we diluted the detection anti-body ( MT78/145-biotine) from 1ug/ml till 0.125ug/ml the last concentration the spots look the best.

but what's really irritates my is the background. this was developed for 10 min. with filtered BCIP/NBT supstrate, I got not do it shorter because the spots where not strong enough. 

do you have any suggestions?

greetings,

Michelle

 

 

 

 

 

20190401_B_Cell_IgG_20190409101028.XLS

[Christian@mabtech.com]

Hello there Michelle! 

The background does look very high. A few questions/comments:

1. Did you pre-activate the B-cells for 3 days together with R848+IL2 in order to get the memory B-cells going? I will pressume yes but just checking. 

2. Prior to adding the cells to the ELISpot plate, did you wash them twice? There is a crazy amount of IgG in the supernatant of these precultures and it is important to wash away all IgG, otherwise they will give a general background to the membrane. Washing 3x can be smart. 

3. Did you use tween anywhere in the assay? If you used the general ELISA wash during the detection phase of the ELISpot, it is easy to forget that ELISA wash do in fact contain tween. Tween gives weird backgrounds if you etoh treat your plates. No benefit. 

4. Do the images generated in the reader match what you see with your own eyes? Maybe a strange question, but something is seriously weird in the images you are providing. There seems to be some optical effect in many of the wells. Like there is dust in the lens of the reader or something? Maybe the bad background only exist in the reader?