Cross-reactivity/ staining of membrane by secondary antibody

[Guest Amalie]

Hi, 

I've been optimizing a Mouse B cell ELISPOT assay for looking at antigen specific cells vs total IgG cells. I am coating MSIP (Millipore plates) with anti-IgK Universal Binding, then plating whole spleen single cell solutions and am getting good results in both 'Total Ig' and my antigen-coated wells. 

However, when I now change the secondary antibody to look for antibodies with a FLAG- or Strep-tag I get dark wells with no spots in the anti-Ig-coated wells. I'm using 1ug/mL of antibody, which works fine for antigen-coated wells

I've tried a number of different anti-FLAG and Strep antibodies (All HRP conjugated) and have found some FLAG ones that can work (see attached image). But I can't find any Strep antibody that works. 

Because of the suspected cross-reactivity with using my anti-IgK for coating I also tried Mabtech anti-mouse IgG K/L (two types of monoclonals)(Used in the attached image). However, using this anti-IgG to coat only created cross-reactivity in my "IgG secondary" wells too. 

I am also using ELISPOT HRP substrate from Mabtech.

I would prefer to keep my 'Total Ig' control wells in the experiment, also to control for detection differences between my two types of secondary antibody. 

 

Do you have any input on what could be causing the supposed membrane staining/cross-reactivity with coating antibody or how to get rid of it?  

 

Best wishes, 

Amalie

 

[Christian@mabtech.com]

Hey welcome!

I have some difficulty understanding your scientific question and set up. Am I correct in this statement:

- You have mice with B-cells secreting antibodies that are from the get-go labeled with FLAG or STREP? Have you achieved this with some sort genetic technique? Fascinating if yes

 

I am sceptical because when you coat with a capture antibody that captures all IgG it becomes very strange to then in the detection step add an antibody. This will inherently short-circuit the set-up, or have I misunderstood?

In general, many antibodies that work in ELISA does not work in ELISpot. It is common that have 5 decent working ELISAs (ie 10 antibodies) and when you assess all those in ELISpot only 1 pair gives a good result. Many combos will result in rubbish. 

In your uploaded image what is different in A, B, C, D, E, F, G, H? Different donors? 

[Guest Amalie]

Hi Christian, 

Thank you for your quick response. I know the setup might be a bit unusual but yes, I have mouse B cells that secrete antibodies that are genetically altered to carry either a FLAG or a Strep tag. 
I can efficiently detect these antibodies in ELISA assays. 

I have based my protocol on this published protocol to detect total Ig and RBD (in my case DNA)
https://www.sciencedirect.com/science/article/pii/S2666166723000886
- In this protocol they coat the well with anti-Ig and also use a detection antibody against Ig. I did find this strange but seems to work and I need to coat my plate with something. Do you know of alternative strategies for detecting total Ig secreting cells?

In my case of detecting FLAG- and Strep-tagged antibodies, I am coating with anti-IgK or DNA, plating total splenic cells (in all wells but less in Ig-coated wells than in DNA-coated wells), and detecting with anti-FLAG-HRP or anti-Strep-HRP (Detecting with anti-IgG/anti-IgA/anti-PanIg has worked fine in this setup so far)

I guess, that if an antibody doesn't work well in ELISA I would expect it to not work in both the Ig- and the DNA-coated wells. In this case it seems to work OK for the antigen-specific coated wells. That's why I'm suspecting some sort of "short-circuiting" as you called it

All wells in the image I attached have the same cells plated (more in DNA than in Ig). The difference between them is the antibodies used for detection (ie. the schematic on the left)

 

Thank you again, 

 

Amalie

[Christian@mabtech.com]

The paper you link have evaluated total IgG secretion, but also in parallell used the "reverse B-cell ELISpot" detection protocol to assess RBD. 

For RBD you coat with anti-IgG in the bottom and capture all secreted antibodies. Then in the detection step you add RBD labeled with a small peptide tag called WASP. The advantage is that sensitivity often goes up compared to when coating with the antigen since epitopes are better conserved with labeled antigens used in the detection phase only. 

My understanding of your setup is that you want to emulate this but you have the issue of using an antibody for detecting your FLAG labeled IgGs. If these antibodies are (ie anti-FLAG 1/2/3/4 and anti-STREP 1/2/3/4) are mouse antibodies it will fundamentally not work (ie short-circuit the assay) unless you do some very extravagant blocking steps. By contrast a rat antibody against flag or strep will not cross react with the coating mab and should in principle work. Are they mouse or are they rat mabs?

When coating the PVDF membrane with an antigen you change the B-cell assay. Here you don't have the problem described above. However, DNA is a very small molecule and difficult to attach to the PVDF membrane unless you have it coupled to a carrier protein of some sort. What has been your strategy?