Dark "clumps" in peptide-stimulated samples

[Guest Tetiana]

Dear Mabtech Customer Service Team!

We're performing your protocol for Human IFN-y ELISpot PLUS kit (HRP) on human PBMC with two pools of peptides. Our peptides are SARS-Cov-2 SNMO pool and SARS-Cov-2 S1 pool, we use CD3 for positive control and CD28 as co-stimulatory antibody.

We use media RPMI 1640 with 10% FBS for our cells incubation, don't use Tween in our washing buffer, and add all peptides and antibodies directly before incubation of cells in ELISpot wells.

Our problem is that in some wells with peptides we saw strange dark clumps, but in control wells and in wells with cells of other people everything was okay. I thought the problem could be in damaged membrane or specific reaction on peptides from some people's cells. So what can be our problem? Maybe you can give us some kind of advice?

First two uploaded photos are samples with clumps, which were stimulated by peptides, and next two are negative and positive control wells of this sample.

20 S1.tif 20 SNMO.tif 20 neg.tif 20 pos.tif

[Christian@mabtech.com]

Dear Tetiana,

We know from experience that some patient-samples can sometimes generate strange backgrounds in ELISpot that look like the ones seen in your images. One common theme is that the patients analyzed has an ongoing inflammation in their body from a disease state such as cancer, sepsis or autoimmune disease. I have some speculation as to what could be happening:

1. During widespread inflammation cells in the blood become sticky and can form clumps. After Ficoll separation this is often seen when taking out the PBMC band. The inherent stickiness of the cells leads to more debri being left on the ELISpot membrane during incubation.

2. Red blood cells often contaminate the PBMC band after Ficoll separation. A lot of red blood cells can lead to very strange backgrounds, but not always. It needs to be red blood cells of certain kind (like often seen in inflammatory patients). Since you have many contaminating red blood cells in the ficoll separation it is high liklihood you have plenty of platelets to. These are said to act as spunges in the blood picking up cytokines and releaseing them on the ELISpot membrane during incubation. Platelets bahavior changes in patients compared to healthy donors. 

3. HRP is a great senstive enzyme for detection in immune assays. However, I do prefer ALP in ELISpot. One reason why is because cells can release endogenous peroxidases during the incubation. These enzymes react with the TMB substrate in ELISpot and can form small tiny spots on the membrane. In healthy donors this is most often no problem but in patients the cells are more prone to acting strange like described above. One way to counteract endgenous peroxidase activity is to treat the membrane with a low concentration of hydrogen peroxidase before adding the detection antibody. 

4. Neutrophils increase in numbers during inflammation and become a bit like baloons during inflammation. As a result, they easily contaminate the PBMC band during Ficoll separation. Neutrophils inhibit T-cell responses and releases peroxidases. They are also sticky cells after activation. Furthermore, if they get counted as PBMC the proportion of T-cells analyzed in each well goes down. 

 

How do you isolate your PBMC? There are ways to get rid of contaminating granulocytes for example.