1. Which kind of ELISpot plate do you recommend?

Answer: We recommend the ELISpot plate with PVDF membrane and pretreated with ethanol. It could combine enough capture antibodies effectively.

2. Why the ELISpot plate needs to be pretreated with ethanol before coating?

Answer: Because the ELISpot plate membrane is hydrophobic, and treated with ethanol turns it into hydrophilic. Only a hydrophilic membrane could combine enough capture antibodies effectively, and enough quantity of capture antibodies is the precondition to get high-quality spots.

3. Is the color of the ELISpot plate important? What color should I choose, white or transparent?

Answer: Both white and transparent ELISpot plate use PVDF membrane. The two colors have just a little difference on the outer tube, which makes no influence on the result.

4. How to pretreat the ELISpot plate with ethanol?

Answer: Usually, we pretreat the ELISpot plate with ethanol shortly. This step is very important. ELISpot plate with different PVDF membrane requires different pretreated methods.

5. Why the coating antibodies concentration which ELISpot recommend is much higher than ELISA?

Answer: ELISpot plate contains PVDF membrane, and the porous structure of the PVDF membrane could combine many capture antibodies, which helps monitor the secretory proteins when local high concentration appears. When capture antibodies is less, weak and dispersive large spots would appear, which is bad for the outcome assessment. Different systems coat different quantities of antibodies, but usually the amount of coated antibodies each well is 1-1.5µg. The quantity of coated antibodies on the ELISA plate is one-tenth of this amount approximately.

6. What kind of cells fit for the ELISpot analysis?

Answer: ELISPot and FluoroSpot are applicable to all kinds of cells.

7. Could the frozen cells be analyzed by ELISpot?

Answer: Frozen storage cells could be analyzed by ELISpot, but the results depend on the quality of cells.

8. How many cells should be added in each well when doing ELISpot analysis?

Answer: It depends on the type of cells and the frequency of cells secretion expected. When the antigen-specific T cells are detected, the amount pf peripheral mononuclear cells shouldn’t less than 200000 each well. Other types of cell should not higher than 500000 each well, or the layers of cells may generated, which would cut down the spot quality.

9. Could I use the human serum to do the ELISpot analysis?

Answer: We do not recommend doing ELISpot analysis with human serum. For the following reasons: 1. The cytokine we want to detected may contains in the serum as well, such as IFN-γ, and it would combine with the capture antibodies and detect antibodies. 2. Human serum may contains heterophil antibodies, and it could combine with immunoglobulin of any other species.

10. What’s the difference between ALP and HRP conjugates?

Answer: Both ALP and HRP are common enzymes in immunoassay. They catalyze different reactions and require different substrates. In ELISpot, the sensitivities of two enzymes are the same, but the speed of reaction catalyzed by HRP is faster.

11. What kind of substrate should I use in ELISpot?

Answer: ELISpot needs precipitated substrates. ELISA substrates couldn’t used in ELISpot because it would not produce spots. We recommend using BCIP/NBT-plus substrate with ALP and TMB substrate with HRP.

12. Why the TMB spots turned yellow or almost vanished after one day’s test?

Answer: When you wash the plate with tap water, the normal blue spots may turn into almost invisible yellow, and that is caused by the ionic compound in the water. We recommend using deionized water or distilled water. And the similar problem would not occur in the BCIP/NBT-plus substrate with ALP.

13. Why the non-specific spots could be observed in the control well without cells added?

Answer: The non-specific spots appear rarely. One reason is the precipitation would produced after the storage of biotinylated antibodies. The other is that there is dust exists in buffer or contamination.

14. Do you have any images of ELISpot expected for reference?

Answer: You could see the images expected via the website below.

https://www.mabtech.com/knowledge-center/assay-principles/elispot-assay-principle/elispot-images

15. Could I use the ELISpot plate to do FluoroSpot?

Answer: To avoid the autofluorescence, you could use hypofluorescence PVDF membrane plate from Millipore.

16. How long is best to read the FluoroSpot plate after the experiment finished?

Answer: The fluorescence of FluoroSpot plate is very steady in a dry and dark environment and it could store for a few months. But we recommend reading the plate in a week after the experiment finished.