Directly labeled antigen detection

[Guest Josh]

Hi Mabtech,

 

I will be running a fluorospot assay where we will be looking at detecting secreted antibody from plated plasma cells using two isomers of an antigen.  In the past we have seen issues with labeling these antigens and I would like to know if we were to directly label with fluorophores what kind of signal loss would we lose in comparison to performing a two step detection?  We have tested the antigens via flow cytometry and know of two fluors that work well with them that have excitation/emission in the range necessary for reading.  Thanks for your time.

 

Sincerely,

 

Josh

[Jens@mabtech.com]

Hi Josh,

Welcome to the forum! Sounds like an exciting project!

I can't say for sure that it would be impossible, but I think the signal loss might be substantial if you were to utilize direcly fluorophore-conjugated antigens. We have done similar experiments in-house, some of which have been published in Jahnmatz et al 2016: https://www.ncbi.nlm.nih.gov/pubmed/26930550. In this paper, we applied a two-step detection system using peptide-tagged antigens and subsequent anti-tag detection with fluorophore-conjugated mAbs (see attached image).

The peptide tags - whose amino acid sequences are published in the paper - were recombinantly expressed with the antigen and we developed the fluorophore-labeled anti-tag mAbs, now QC:ed and readily available. 

We did not even consider testing directly conjugated antigens for three reasons:

1. There is a risk that the fluorophores hide the relevant epitopes of the antigens, essentially rendering them useless. Perhaps that is the issue you have been seeing in the past? A peptide tag can be positioned specifically, e.g. N-terminally and thus won't hinder antibody binding. 

2. It can be hard to get consistent fluorophore conjugations on different batches of antigens. With our setup, we can work with large batches of QC:ed fluorophore-labeled antibodies instead.

3. With a peptide-tagged antigen, there is no need to purify the antigen; you can just use the antigen-supernatant. 

To my knowledge, there are only a few other papers utilizing similar setups, among them Hadjilaou et al 2015: https://www.ncbi.nlm.nih.gov/pubmed/26320246. The authors of this paper used fluorophore-labeled antigen-specific mAbs, and thus also they used a two-step detection setup. Two reasons for taking the Jahnmatz 2016-approach instead is that (i) tagged antigens circumvent that very need for antigen-specific detection mAbs, and (ii) that anti-tag detection mAbs won't compete for binding the same epitopes that the plasma cell secreted antibodies might recognize. 

The anti-tag mAbs are not yet officially launched, but send me an email (jens@mabtech.com) if you want to continue the discussion and/or would like to try those anti-tag mAbs. 

Kind regards,

Jens

 

 

[Jens@mabtech.com]

Hi again Josh,

There is actually a paper, Luque et al 2018, in which the authors utilize a one-step detection system for FluoroSpot: https://www.ncbi.nlm.nih.gov/pubmed/30075182

However, they don't detect fluorophore-labeled antigen, but fluorophore-labeled HLA mulitmeres (thus looking for anti-HLA IgG in a transplantation setting).

The HLA multimeres might have a higher binding capacity and bind more fluorophore molecules than a single antigen would, potentially increasing the signal, but at least it's some data showing the feasibility of a one-step detection system.

Kind regards,

Jens