Hello,
I'm using your interferon gamma ELISPOT, and for practical reasons I will not be able to do the overnight capture antibody coating at +4°C, would you have an alternative coating protocol to recommend? (like 2h room temperature or something like that?)
Many thanks for your answer.
Best regards
Luc de Chaisemartin
INSERM U996, Paris, France.
Hello and welcome to the Mabtech Forum!
I have myself utilized a "quick" coating procedure of only 4 hours incubation at 37C (i.e inside of an incubator). ELISpot plates had in this case been pre-activated with EtOH and the capture antibody was added at a concentration of 1,5 micrograms/well. After 4h hours, the plates were blocked and cells were added.
When the experiment was done and the plates were developed, IFN-g spots came out looking ok (cells stimulated with a-CD3 and PHA). So it most definitely works doing it like this. However, in the same experiment, I also analyzed IL-2. These spots on the other hand suffered both in quality and numbers compared to the recommended protocol of leaving the capture antibody to coat overnight.
Consequently, changing the coating protocol can lead to a lowered ELISpot sensitivity and will affect different antibody systems to various degrees. Although I did not observe a degradation in the results for IFN-g, this might not have been the case had I looked at a low frequency, antigen-specific response, where both the number and amount of IFN-g secreted theoretically can be much lower from each responding cell in comparison to what is seen for PHA and a-CD3.
In essence, changing the time for coating your plates alters your protocol and may or may not affect the outcome of your ELISpot data. Only you can decide if it is worth it or not.
best,
Christian
Hello Luc,
We have done tests to see how the incubation time affects the coating efficiency. The tests were made at room temp and the amount of mAb bound to the membrane was determined by measuring the free mAb in the PBS (the buffer used for coating) at different times. In the graph shown we added 1.5 ug of mAb in 100 ul and after 8 h close to 100% of the added mAb was bound. Shortening the coating time to less than 8 h yielded less efficient coating and a 4 h incubation resulted in approximately 85% bound mAb. Please note that this was done with EtOH treated PVDF plates; the use of non-treated PVDF will result in much less efficient coating even at longer coating times.
To compensate for a shorter incubation time one can add more mAb to each well. Decreasing the volume to e.g. 50 ul/well (without decreasing the amount of mAb) is likely to also improve the coating at shorter incubation times.
The data in the graph were obtained using mAb 1-D1K against human IFN-g but I think one can assume that most mAbs will behave in a similar manner. But as Christian said, any deviation from the recommended protocol can impact the assay and should be properly validated.
Another way of getting around the issue of shortening the coating time is of course the use of pre-coated plates.
Best regards,
Niklas Ahlborg
Head of R&D at Mabtech
