IFN-g/IL-2 FluoroSpot - BSA or FCS and antibody filtering questions!

[Guest Lucia]

Hi,

We are now doing FluoroSpot with your IFN-g/IL-2 kit, when we read the protocol, we have such questions ,could you please help to give some comments:

 

The step of dilution of detection antibody, it was written with PBS-0.1%BSA. Can we use PBS- serum instead?  What is the suggested concentration of serum?
And for the step of filtering the antibodies, must the filter be sterile one? Is non-sterile one ok? Why we need to filter the antibodies? I want to know what is filtered by this step? 

Thanks!

[Christian@mabtech.com]

Hello Lucia, and welcome to the Mabtech Forum!

 

Yes, you can use "PBS-serum" instead of the recommended "PBS-0.1% BSA". However, it is always advised to follow the instructions found in the protocol.  

 

But if BSA is missing from your lab I would use a concentration of 0.5% fetal bovine serum (FBS). That is, a mixture 250µl FBS in 50ml of PBS. It is important to point out that you want to avoid the use of human AB serum as this may contain heterophilic antibodies that can interfere in the detection process. In addition, human serum can also, in theory, contain the analyte of interest (Either IFN-gamma or IL-2). Since both of these analytes will bind to the capture and detection antibodies in the FluoroSpot assay, they can in this scenario cause a fluorescent background of the membrane.

 

 

Fetal bovine serum, also referred to as Fetal calf serum (FCS), is the preferred choice. Always make sure that you buy your FBS/FCS from a high quality source. 

 

 

The 0.2µm filters (low protein binding) used for the filtering of the detection antibody solutions in the FluoroSpot assay do not need to be sterile. However, pretty much any filter for this purpose that you buy these days will come in a sterile package. You want to avoid reusing a filter that has been used for filtering some other antibody solution. In addition, make sure that it does not contribute in adding dust or particles into the FluoroSpot wells.

 

Dust, hairs and particles often autofluorescence and will therefore lead to unnecessary backgrounds as artefacts on the membrane when reading the plate in a FluoroSpot reader.

 

 

best regards,

Christian