Colors of IFNg spot

[Guest Tan Trieu Nguyen]

Hello,

I recently used mabtech Mouse IFNg ELISPOTplus kit (HRP) (Cat # 3321-4HST-2) and I have some questions regarding the color of the IFNg spots developed. From what I saw from the forum the spots are supposed to turn purple but mine have dark green color, purple only in some part of the membrane (bottom right portion in some wells). I wash with PBS between steps and deionised water to stop substrate development. I dry the wells for at least 2 days in a biosafety cabinet.

Additionally, how long can I keep these plates if I store them in a dry, dark place?

Thank you!

Best,

Trieu

 

[Christian@mabtech.com]

Hi there,

The kit you have evaluated uses HRP in combination with a TMB substrate from Mabtech. The spots come out looking greenish in color, just like in your image. It is a very typical result. The spots can be affected by the liquid used for turning off the substrate reaction. Use water and the spots tend to be greenish, but use PBS and the spots get a slight "bluish" tint. No impact on the performance of the kit. 

The purple spots I have referred to in earlier threads of this forum relate to ELISpot kits detected with ALP enzyme and BCIP/NBT Plus substrate from Mabtech. The get the characteristic purple color and is less affected by using either water or PBS for turning off the substrate reaction. 

HRP spots are more sensitive compared to ALP. HRP spots should be protected from dark and ought to be read within the first couple of days post development. After 2-4 weeks you can see clear degradation with HPR ELISpot but it will depend on the character of the spots.

By contrast, ALP spots will last for a very long time as long as the plates are protected from light. I have seen plates that are over 1 year old and the spotnumbers are the same as day1. 

As a result, I in general recommend ALP kits over HRP kits.