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Questions from customers2017-04-24T09:20:45Z

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Dear Mabtech,  

 

Usually, we coat our plates ourselves but recently I had a chance to try the pre-coated sample kit and it made such a difference! But before moving forward I wanted to get your expert opinion on the data and whether I am correctly interpreting what I am seeing.

 

In the past, when I ran the assay on our plates I got a lot of background, and I had trouble getting the correct focus on the reader. For my comparison, I ran the assays at the same time, using exactly the same samples oriented exactly the same way on the plate.

 

After development, the pre-coated plates were much clearer. The spot resolution was much higher and there was much less background in the high responding wells, thereby increasing the contrast. I read the plates with the IFN count settings and did not adjust anything except the AOI. What I saw was much higher spot counts on the pre-coated plate. Visually, it is obvious that the level of specificity of the samples did not change, but the background is significantly decreased on the pre-coated plate. I’m guessing this then allows the camera to identify the spots more accurately.

 

 

My questions:

 

  1.  Am I correct in assuming that the decrease in background is resulting in more accurate spot counts?

 

  2.  I did not have to adjust the reader setting much to get most of the spots counted on the pre-coated plate. Is this

how the reader should perform, i.e. with minimal manipulation to settings?

 

  3.  Do you think the flexible underdrain on the manually coated plate could be contributing to poor spot       resolution?

 

  4.  The protocol we currently follow uses antibodies from Mabtech, but ALP and color development reagents from other companies. In addition to variability from coating the plate ourselves, do you think there is added variability when mixing the different reagents from different manufacturers? Also, do you think the conjugated ALP might be helpful not only to save time with the assay but also to minimize non-specific color development?

 

Thank you so much for your valued input!

 

Best,

R

S

Susi2017-04-24T09:32:00Z

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Dear R,

 

I am happy to hear that your results improved by using our pre-coated plates! You are absolutely right, by having a kit that generates lower background, spot-definition is improved and the reader identifies spots easier. However, in many cases, it looks like there were more spots in the pre-coated plate compared to the manual plate. Could you send me a PowerPoint with images of pre-coated and manual coated wells taken from the original saved folder? I have given some instruction in the attached PPT. By doing it this way, I can look at the high definition version of the wells and can better give feedback.

 

Your questions:

1. Yes, a decrease in background will make it easier for the software to count the spots accurately. However, to me it seems like you actually have an overall more sensitive system using the pre-coated plate. Fundamentally more spots are detected using the pre-coated plate. However, I would have to see the high-resolution images to really say that.

 

2. You should get a feeling about changing the spot count settings on the AID reader. There is no right or wrong! The reader should be regarded as your unbiased partner in plate reading.  If the reader is not counting 100% of the visual spots, only 70%, that is ok as long as you consistently use the same setting throughout the wells. You could for example exclude the weakest spots. In general, I think your setting looks good, although I would need to look at your high-resolution images to be absolutely sure.

 

3. Hard to say really. The flexible underdrain can lead to problems with leakage, which can dramatically affect the result of individual wells or section of the plate. However, it does not look like this is your issue as your suboptimal result for the manually coated plate seems consistent throughout all wells.

 

4. This is probably the main reason why you don’t see the same result in the manually coated plate. Mixing manufacturers is not recommended. Additionally, we make sure our streptavidin and substrate perform optimally for ELISpot.

 

The directly conjugated detection antibody 7-B6-ALP is a very reliable and good choice.

 

Best regards,

Susi

Save high resolution images.pptx

Qf

Questions from customers2017-04-24T09:49:43Z

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Thank you very much for the feedback! I am attaching the slides with the high-resolution images. I think you’re right about the pre-coated plate giving a higher number of spots in some cases. You mentioned that this is expected with the pre-coated plate. Is this because the plate improves the sensitivity of the assay? Meaning- can I rely on the higher spot counts as being correct? Or is it giving spots based on non-specific activity?

 

I have also attached slides with wells A 10- 12. Even though there is a lot of background on the edges of the manual plate, it seems like there is a general increase in the number of spots on the pre-coated plate.

 

Wells C 10-12 are also attached. This is an example of where I think the specificity is the same on each plate, but there is so much smearing of the spots and background that the spots can’t be counted properly.

 

precoat_v_manual.pdf

S

Susi2017-04-24T10:08:00Z

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Hello R,

 

the  pre-coated plates are done under optimal coating conditions. This can definitely make the assay more sensitive if you are running a suboptimal coating protocol. Perhaps more importantly in this case, with the pre-coated plate you use our 7b6-ALP and our substrate, this can lead to major improvement in sensitivity! Not all enzymes and substrates are the same, especially in the world of ELISpot. I think you are seeing a marked improvement in the actual number of spots as evident from how much the results improved in the positive control wells A10-A12. You go from seeing a “smear” to many distinct spots. One should not be able to remain skeptical in this comparison.

 

Now in some instances and stimuli, the activated T-cells secrete so much IFNg on a single cell level that the decrease in sensitivity really does not matter. Looking at B1-B3 we do not have a super difference but the Mabtech plate definitely has an edge over the manually coated one. If you were to adjust focus of your own plate and then play a bit with count-settings, the difference would be smaller, maybe a 10-20% increase in actual spots. I would always go for the more sensitive setup.

 

Kind regards,

Susi

Qf

Questions from customers2017-04-24T11:37:40Z

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Thank you very much!

 

The results have been so great with the pre-coated plates, I just wanted to be sure that it wasn’t too good to be true, especially before I made the decision to change everything completely.

 

Again I really appreciate you taking the time to answer my questions and being so accessible. These days there aren’t many companies that still give the kind of support Mabtech’s customer and technical team have given. That being said, I hope you won’t mind if I reach out to you in the future as a resource.

 

Best regards,

R

S

Susi2017-04-24T11:46:17Z

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You are more than welcome to always contact us for support!

 

Best,

Susi