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Biotinylating Ag for B Cell ELISpot

  • s
    sebbeols2017-01-31T14:56:16Z

    Hi Mabtech!

     

    I am working on optimising a B cell ELISpot for a vaccination study. I have previously worked with coating Ag directly on plates or utilising biotinylated protein Ag. For the current study there is unfortunately no protein Ag currently identified as an Ab/B cell target that we could use for the pathogens vaccinated for (different live attenuated vaccines). Thus, we only have the vaccine itself or formalin-fixed virions to work with. My attempts at coating directly with the vaccine have been fruitless and it may be because of the low concentration of virions in the vaccine (1.5-2.4 ug/0.5mL). 

     

    In your brochure for the B cell ELISpot, you have a figure about the Pandemrix vaccine (coat vs biotin, Fig 4). Is the biotinylated Ag used there an HA protein or is it the actual vaccine that you have managed to biotinylate? Do you guys have any experience in biotinylating vaccines or virions for use as probes in ELISpot? Or do you believe that coating directly with the formalin-fixed virions is a better way to go (have not yet tried this)?

     

    Best regards,

    Sebastian

  • C
    Christian@mabtech.com2017-01-31T18:00:06Z
    Admin

    Hey Sebastian, welcome back!

     

     

    In Fig 4, It was in fact the actual vaccine Pandemrix that we managed to biotinylate using a standard protocol.

     

    In your particular situation there are no real "words of wisdom", except for: try the biotinylation and see what that gives you.

     

    I am big believer in the reversed protocol in general. Is it possible to also biotinylate the formalin-fixed virions? 

  • C
    Christian@mabtech.com2017-02-02T06:49:51Z
    Admin

    Hey again, I got a hold of Gun in our R&D. She did the biotinylation of the Pandemrix used in Fig 4. Here is her info:

     

    Hi Sebastian.

    I am the one at Mabtech who did the biotinylation of Pandemrix :-)

    Here is my protocol;

     

    Biotinylation:

    Pandemrix: A/California/7/2009 (H1N1)v-like virus (GlaxoSmithKline, lot 0071052)

     

    Biotin used: Sulfo-NHS-LC/ 21355 lot CAS 127062-22-0 :

    1 ul (5.57 mg/ml H20) to 150 ul Pandemrix

     

    Incubated 30 min in RT

    Dialyzed on. against PBS/azide (0.02%) in +4oC using Slide-A-lyzer from Pierce: MWCO 10000

    I got 75% recovery

     

    Good Luck!

    Gun Kesa

    PhD

    Senior Scientist R&D

  • s
    sebbeols2017-02-02T09:00:31Z

    Thanks for your replies Christian and Gun!

     

    I agree with you Christian that the reversed protocol is great. I do try to conduct as many of my future ELISpots this way, if I can get my hands on the reagents. 

     

    I'll make an attempt to biotinylate the vaccine or the formalin-fixed virions once I can get a hold of either of these from our collaborators. I'll get back to you with the results!

     

    Gun: What molar ratio did you use of biotin to Pandemrix? I don't know what concentration the virus comes in the Pandemrix vaccine.

     

     

    Thanks again!

    /Sebastian

  • C
    Christian@mabtech.com2017-02-02T15:24:20Z
    Admin

    Hey again Sebastian,

     

     

    So I talked to Gun again and she says she did not know the Molar ratio of virus in Pandemrix. This was not disclosed by the manufacturer. However, she made sure she used an excess of biotin, since any left overs will removed anyway by dialysis. 

     

    I have also attached a PDF file with some detailed descriptions on the biotinylation process we use. I hope it will be of help to you and to others reading this forum. Feel free to share.

     

    Biotinylation Guideline.pdf

     

    best,

    Christian