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Francesca2016-11-22T15:33:38Z

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Dear all

I would like to perform an Elispot to measure the memory B cells in patients who received (1 year ago) the 13 valent pneumococcal vaccine (prevenar). I’ll use the kit from mabtech with code MBT-3850-2A (ALP) and I’ll coat the 96-plate (Millipore MSIPS4510 treated with EtOH) with the antigen , consisting in each serotype included in prevenar (pneumococcal polysaccharide serotype 1, 3, 4, 5, 6A , 6B , 7F, 14, 18, 19A, 19F, 23F). I’ll coat a plate with single 13 serotypes (in triplicate 13 X3 =39 wells), control negative and IgG (as a positive control).

-I wonder if I need to conjugate each pneumococcal polysaccharide with human methylated albumin during the coating.

-do I need to assay a negative control for each serotype? Can I use in the plate just one well with negative control  coated with one serotype?

Thanks a lot

Francesca

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Christian@mabtech.com2016-11-23T15:29:47Z

Admin

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Dear Francesca, 

 

Welcome to our forum, I will do my best to give you some support on this situation:

 

1. Fantastic that you are using EtOH pre-treatment for your B-cell ELISpots - essential in obtaining optimal results!

 

2. I am uncertain if you need to conjugate the polysaccharides to albumin in order to achieve optimal coating. This could be the case, but I have no experience with polysaccharides. Maybe it does not matter and you can get good coating with the free antigens. You should test.  However, one thing to consider if you go the route of conjugating the antigens is to use albumin without contaminating IgG. Standard Albumin contain IgG so if you are going to use this you need to make sure you buy specially purified. Otherwise you will have membrane background when doing the elispot. An option could be to use BSA instead. BSA contains IgG as well but the origin is not human. 

 

3. I do not feel you need to use negative control for each serotype. Since you will be looking for memory B-cell responses, the PBMC isolated from the patients should be incubated with R848+recombinant IL-2 for 3 days. Wash cells and add them into the B-cell ELISpot plates. I would want to include 2 negative controls. One were you have coated with some irrelevant protein, like BSA for example. Another well were you have coated an irrelevant polysaccharide. Now, I do know if such a polysaccharide exists but it would be a very relevant negative control. Of course, you need a polysaccharide that you know humans do not have IgGs against. Do that exist? If not, I would be ok with only using the BSA negative control.