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How to determin suitable tumor antigen concentration and incubate time

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#1 Guest_Yestin Yang_*

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Posted 02 July 2014 - 10:03 AM

Hi Mabtech,




This time we want to compare the different response to tumor antigen between two effect T cells by your IFN-g ELISPOT kit.




We boiled the tumor tissue and extracted supernatant for tumor antigen.




Would you please share your experience on the tumor antigen concentration and incubate time setting?




Thank you very much!

#2 Staffan@mabtech.com



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Posted 16 July 2014 - 06:37 PM

Hi Yestin,
While no expert in the field, I will just try to provide some general reflections regarding the use of tumor cell lysates when studying tumor-directed T cell responses. Hopefully, someone with more direct experience of working with tumor antigens can give further advice and insight.  
Although most studies of tumor-directed T cells have employed purified protein or peptides, some people have also used more crude preparations of antigen such as tumor cell lysates of the type you describe. These have been used both for vaccination and for monitoring the resulting immune response often in the context of preloaded autologous dendritic cells.
A problem with using cell lysates rather than more characterized proteins or peptides is that T cells require a certain concentration of antigen to be efficiently triggered.  However, the concentration of individual antigens in a cell lysate is rarely known and may also differ between preparations. Due to this, titration of the tumor cell lysate with one or more positive donors may be recommended in order to define a suitable concentration.  Typically, concentrations of a few hundred µg/ml have been used but in some studies the amount of added lysate has instead been related to the number of cells from which the lysate was prepared using, for instance, a 1 to 1 mixture of PBMC and tumor cells.
With regard to the incubation time in the IFN-g ELISpot assay, it might be recommended to extend this from the overnight incubation often used with peptides to two nights in order to accommodate for the more complex antigen processing and presentation. The use of separately generated autologous dendritic cells may also help to optimize conditions.
Best regards / Staffan 

#3 Guest_Yestin Yang_*

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Posted 23 July 2014 - 11:00 AM

Dear Staffan,

Thank you very much for this comprehensive answer.

It's so complicated in the antigen recognition. The peptide is a good choise in the tumor vaccine study in vitro.But the situation will become involute in vivo.

I agree with your propsal that we should titrate the tumor cell lysate. Is it mean of the protein concentration detection? And how can we konw which is the function department? 


Thank you!



#4 Christian@mabtech.com


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Posted 21 August 2014 - 03:44 PM

Dear Yestin,



You are absolutely right, the immunology of antigen recognition is certainly complicated.



When it comes to the tumor cell lysate, a normal setup would involve extracting the lysate and measuring the total protein concentration in micrograms/ml. The lysate would then be titrated in cell culture medium and added to the ELISpot plate together with the PBMC from a donor that you are interested in evaluating.


Alternatively, the tumour cell lysate could simply be based on cell numbers instead of protein concentration. For example, the lysate could be prepared from 1 million cultured tumour cells. This lysate would in turn be diluted into a volume of 1 ml (in cell culture medium). In this case, your stock concentration would be 1 million tumour cells per milliliter of stock lysate.


In either of these cases a major problem surrounding this setup is knowing weather the potential ELISpot response is originating from the tumor antigen or some other protein found within the lysate. 



I hope this clears up some of your questions Yestin.  



best regards,


Christian Smedman
Senior scientist, R&D
My aim is to accurately describe our official policies and recommendations when it comes to ELISA, ELISpot and FluoroSpot. Nonetheless, my viewss are in many cases my own and may not necessarily reflect those of Mabtech.


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