Dual ELISpot

[Guest Giune Padilla]

Hello,

 

I am developing a Dual ELISpot assay. I have work assay (with match pair of antibody). But when I compare a single coated IFN gamma against Dual coated IFN Gamma and IL-2 antibodies there is a big difference in number of spot. What is the acceptable percentage difference between single and dual coated wells? Any advice?

[Christian@mabtech.com]

Welcome to the Mabtech Forum!

 

 

I think there may be at least a couple of reasons as to why you are not getting the same number of spots when analyzing both IFN-gamma and IL-2 in the same well in a dual ELISpot as compared to analyzing each cytokine separately in a regular ELISpot: 

 

 

Capture effects. As you know, antigen-specific T cell activation is dependent on several factors in order to work properly. This includes the presence of APCs capable of presenting antigen as well as providing costimulatory signals. Some of these costimulatory signals are in the form of cytokines and eliminating or reducing these may affect the level of response. In the dual ELISpot that you describe, both IL-2 and IFN-g will be quite effectively removed from the medium as they will be bound by the capture antibodies.  For the majority of cytokine combinations that we have tried, this removal of a cytokine does not present a problem but in the case of IL-2 it will. Thus, removal of IL-2 will influence not only the IFN-g response but also that of some other cytokines (e.g. IL-4 and IL-5). In the FluoroSpot assay, which we for several reasons prefer over dual ELISpot (see below), we have solved this by including a costimulatory anti-CD28 antibody which appears to effectively compensate for this capture effect (https://www.mabtech.com/products/3608-1-50_anti-human-cd28-mab-cd28-purified#tabs-min-1). Another approach would be to first preincubate the cells for a few hours in the presence of stimuli (e.g. antigen) before transferring them to the ELISpot plate. This seems to have a very similar effect but, of course, complicates the assay procedure.   

 

 

Weaknesses of the dual ELISpot. Another reason for obtaining fewer spots in the dual ELISpot may be linked to some inherent problems with this technique. Thus, contrary to the FluoroSpot assay, which allows simple and accurate detection of two or more cytokines, the dual ELISpot is simply more complicated to get to work properly. One of the problems is that the two substrate reactions need to be performed sequentially in two steps but, even if performed separately, they may interfere with each other and as a consequence lead to a reduced number of spots. Normally, if working with the enzymes ALP and HRP, it has proven beneficial to first perform the ALP substrate reaction before adding the HRP substrate but, even then, results may be suboptimal.  
 

Another problem with the dual ELISpot is that, to our knowledge, none of the existing ELISpot readers are particularly good at defining double secreting cells. Thus, while capable of properly evaluate and count cells which only secrete one of two analytes, (for instance the case if looking for IgG and IgM response in a B cell ELISpot), evaluation of T cells secreting several cytokines is more challenging and may result in incorrect results.  

 

Due to these problems with the dual ELISpot, we have instead focused our efforts on developing the FluoroSpot assay which does not suffer from these problems and, unlike the dual ELISpot, is also compatible with not only reading two but multiple analytes. Therefore, unless having specific reasons for using the dual ELISpot we would recommend considering to instead try the FluoroSpot.

 

Here is link to our human IFN-gamma/IL-2 FluoroSpot kit based on pre-coated plates:

 

https://www.mabtech.com/products/fsp-0102-2_human-ifn-γ-il-2-fluorospot-kit-pre-coated#tabs-min-1

 

A warm recommendation if you are going to evaluate the method for the first time!

 

 

Please comeback with questions in the future. The more questions and answers we accumulate in here, the better!

 

 

best regards,

Christian

 

 

 

[Christian@mabtech.com]

Hello again,

 

 

I just wanted to follow up on this forum-question again by posting a Powerpoint file from AID demonstrating the inherent limitations of trying to analyze a dual ELISpot well, as compared to the fluorescent based FluoroSpot technique.

 

This 2-slide Powerpoint is of good pedagogic value in explaining the main issue involved: Limitation of dual ELISpot explained by AID.pptx

 

The slides shows how two cells secreting the same 2 cytokines, but at different levels, will in the enzymatic assay lead to bias towards single stained spots. Dual spots will here simply be missed in situations where one the cytokines is secreted at a low level compared to the other (ie in the case of Cell 2 in the attached powerpoint). This does not happen not in FluoroSpot. 

 

best,

Christian

[Guest Caroline D]

Dear Christian,

I am interested in the Fluorospot IFN gamm/IL-17 plates. I need to test these two cytokines on samples containing a mixture of Tcells, B cells and APCs. 

I have a question related to what you said previously in the previous post. What would be the advantages (apart from samples and time saving) of the use of combined IFN gamma/Il-17 Fluorospot compared to use two single ELISPOT ? And what would be the disadvantage ? What about the consumption your mentioning in your post ? Does that have an impact in the case of Il-17 ?

 

Thanks for your help,

 

Best wishes

 

Caroline

[Christian@mabtech.com]

Hi there Carolione, 

One advantage of using the IFNg/IL-17A FluoroSpot is that you get insight to the number of dual secreting T-cells. This population is rather small but it does exist. With ELISpot you cannot detect the dual secreting cells naturally. 

Mabtech has recently launched our own FluoroSpot reader called IRIS. With this machine you have the benefit in fluorospot of being able to detect the spot volume, that is how much relative cytokine has been produced by each cell.

Before with previous readers one disadvantage of using FluoroSpot was that sensitivity was not as good as with ELISpot. You simply detected more spots in ELISpot compared to FluoroSpot, depending on stimuli. With some postive ctrls you had equal numbers but with antigen stimulation, you lost sensitivity. However, with the new Mabtech IRIS reader based on image RAW captures, this now changes. Sensitivity between the two methods are very comparable. 

 IL-17 does not have strong capture effects when combined with IFNg. But there can be small shifts compared to single coated ELISpot. I will look through our data a bit and try to find a nice comparison. Hold on. In the meantime, start looking Mabtech IRIS grants. It really is the game changer we have been waiting for:

 

[Caroline]

Dear Christian,

Thank you very much for your answer. Unfortunately we don't have the new mabtech reader available on site.

I started my first comparison experiment last week and it's very difficult for a beginner to decide which gain etc to put on the machine. 

I tried to compare my ELISPOT and fluorospot counts (IFNg Elispot and IFNg/IL-17 Fluorospot) and there is a much high number of spots with the Fluorospot. I tried to correct them manually but there still very high and it is honestly quite difficult when it is super bright to make a difference between 2,3,5 spots. I have a few questions : 

- Do you usually recommand not to correct the spots counts and to leave the machine countings (apart from the obvious artefacts ?)

- Do you know if there is a way to find tutorial for manual correction (I didn't find any) ?

- Can the coating of the plate have an impact on this ? 

- What about auto-fluorescence ?  I read my two cytokines plate on a machine set up for 3 CK and still had some spots on the third cytokine whereas there was no staining there ..

- Regarding the Il-17 control what would you consider as a good positive number of spots for this CK ? 

 

I have attached the general results of my ELISPOT but happy to discuss more detailed wells if you think it's relevant.

- The controls are 4 A-D and H, same for 8 and for 12.

- The first four colums are cells with 48 hours incubation before transfering to the plate.

- The 4-8 colums are after 24hours in incubation before transfering to the plate

- The 9-12 colums are samed day transfer to the plate

 

Thanks a lot

 

Best wishes

 

Caroline

IL 17 Cleaning deep636851713438585698.docx

[Christian@mabtech.com]

Dear Caroline! 

Quote

I started my first comparison experiment last week and it's very difficult for a beginner to decide which gain etc to put on the machine. 

Your frustration about reading is exactly the reason why we developed the Mabtech IRIS reader based on RAWSpot Technology. By reading in Image RAW there are no camera settings to consider except exposure, and because it is image RAW our dynamic range for exposure is crazy good: 0-16376 per each pixel, instead of 0-255 like in our competitor machines. 

On 2/11/2019 at 12:43 PM, Caroline said:

- Do you usually recommand not to correct the spots counts and to leave the machine countings (apart from the obvious artefacts ?)

I only remove obvious artifacts. No point in spending time manually correcting the machine. Unfortunately, the other machines are rather bad at detecting spot-centers correctly and accurately counting spots. Their raw material is simply to bad  

 

On 2/11/2019 at 12:43 PM, Caroline said:

- Do you know if there is a way to find tutorial for manual correction (I didn't find any) ?

No for CTL I have never seen any tutorials online. People tell me it is a rather complicated process. 

 

On 2/11/2019 at 12:43 PM, Caroline said:

- Can the coating of the plate have an impact on this ? 

No I think your results look technically perfect. Very nice. Coating seems to have been optimal. 

 

On 2/11/2019 at 12:43 PM, Caroline said:

- What about auto-fluorescence ?  I read my two cytokines plate on a machine set up for 3 CK and still had some spots on the third cytokine whereas there was no staining there ..

 We know from experience that our three color kits do not work on many CTL readers. They have some setup were our LED550 fluorophore bleeds over into LED640. This is the effect you are seeing. 

On 2/11/2019 at 12:43 PM, Caroline said:

- Regarding the Il-17 control what would you consider as a good positive number of spots for this CK ?

The frequency of IL-17A secreting T-cells is much lower compared to IFNg. I think your results look very reasonable. 

 

Overall, I think you should look into getting an IRIS reader from us instead. It is trully a game changing product. I attach a PPT we got from an IRIS customer that upgraded from the same reader you got I think. Look in the comparison. ImageRAW vs standard 8-bit analysis.

best,

Christian

 

 

IRIS vs Competitor.pptx