IFN-g ELISpot using TMB

[Guest Hayan]

Hello.

I have an inquiry about the recovery protocol you recommend when some wells are blank. 

You recommend SA-ALP antibody in the protocol, but I used ELIspot PLUS(HRP) precoated kit.

I wonder if I could rescue the wells using HRP antibody instead of SA-ALP.

Also, I wonder whether the recovery protocol works even after completely drying the plates because the picture beside the protocol described counting spots. 

 recovery protocol below:

1. Wash the blank wells 5x with 200ul PBS.
2. Add detection antibody at 1ug/ml in PBS containing 0.5% FCS and incubate for 2h.
3. Wash the blank wells 5x with 200ul PBS.
4. Add SA-ALP in PBS containing 0.5% FCS and incubate for 1h.
5. Wash the blank wells 5x with 200ul PBS.
6. Add substrate and incubate for 10min.

 

[Christian@mabtech.com]

Welcome to the forum!

Yes, using SA-HRP should in principle work, however I have not tested it for recovery. It is therefore possible it does not work as well as it does for SA-ALP. HRP tends to develop spots quicker compared to ALP and in a recovery situation that is maybe not advantageous. A stronger/quicker development might lead to a decrease in membrane and spot contrast, but that is a total speculation. 

The recovery protocol can indeed work even if the plate is completely dry. However, the older the experiment, the less likely the recovery protocol to work great. You typically get "something" back on 3 monts old plates but certainly not as much as on 3 weeks old plates. 

One thing: time in substrate in the recovery protocol can vary alot depending on the condition of the bound cytokine. Sometimes I have recovered for 30-40 minutes, sometimes only 10min. I tend to monitor the development process. You donts want to go too long because eventually the membrane will become so dark that any recovered spots will be difficult to decipher.