Hi. Column A in the figure below is our experimental results, the plate is coated with mabtech antibody. The results were normal before, but recently, there were no spots for two times, and the plate wells showed picture results. Column B is the results of mabtech elispot kit as a control. What might be the reason?
Hi Sumu, and welcome to the forum!
The strange patchy pattern in column A might be due to substrate precipitation that has happened already in the flask. We recommend filtering the substrate through a 0.2um (syringe) filter before use. Did you filter the substrate prior to adding it to the wells?
//Jens
On 7/29/2024 at 5:48 PM, Jens@mabtech.com said:Hi Sumu, and welcome to the forum!
The strange patchy pattern in column A might be due to substrate precipitation that has happened already in the flask. We recommend filtering the substrate through a 0.2um (syringe) filter before use. Did you filter the substrate prior to adding it to the wells?
//Jens
Yes, the substrate solution is filtered before use, and the same substrate solution is used for column A and column B. The difference between column A and column B is that Column A is used the next day after being coated with antibodies, and column B is the pre-coated plate which is purchase from mabtech
Hi Sumu! Thanks again for your questions!
Your column B with the pre-coated plate kits looks good so we know the protocol is working which is great to see!
I want to confirm some details to get you the best help possible:
Get back to us with these details and we'll get to the bottom of this!
/Tyler
On 8/1/2024 at 4:42 PM, Tyler said:Hi Sumu! Thanks again for your questions!
Your column B with the pre-coated plate kits looks good so we know the protocol is working which is great to see!
I want to confirm some details to get you the best help possible:
- What pre-coated ELISpot kit are you using? Please list the product number or link to the product.
- What antibodies are you using for coating/detecting? Please list the product number or link to the product.
- What concentrations of the coating/detection antibodies are you using?
- Have these worked in previous experiments?
- Besides coating yourself, are the ELISpot protocols the same?
Get back to us with these details and we'll get to the bottom of this!
/Tyler
Hi. The number of pre-coated ELISpot kit we used was 3311-4APW-2 with the link ELISpot Plus: Mouse IL-4 (ALP) - Mabtech.
The number of antibody we using for coating was 3311-3-250 with the link Anti-mouse IL-4 mAb (11B11), unconjugated – Mabtech, and was used it at a concentration of 10 μg/ml.
The number of antibody we using for detecting was 3311-6-250 with the link Anti-mouse IL-4 mAb (BVD6-24G2), biotin – Mabtech, and was used it at a concentration of 1 μg/ml.
These worked in previous experiments, so I don't think it's a matter of raw materials.
Besides coating by myself,the rest of ELISpot protocols were same.