We are setting up a variety of virus stimulations as positive controls for adding to human PBMCs. My goal is to measure IgG induced by the virus so while total IgG is easy to measure via ELISA, what is the best way to measure viral specific IgG? I could coat plates with a viral antigen for an ELISA but will there be enough induced over a 5-7 day stimulation if viral specific B cells are not frequent, or will they expand enough? We will assume people have seen the viral antigen at least once before as not Ebola or something foreign. I have never run an ELISPOT but know that is another option but from my reading you have to prime the cells with other stimuli like CpG and then are looking at for example influenza IgG or tetanus IgG after coating the plate with the viral/bacterial antigen. Thanks in advance for any tips/advice as just worried there wouldn't be enough viral specific IgG induced for whether an ELISA would be sensitive enough as I am finding more papers on just looking ex vivo after vaccination.
Yes, ELISPOT is good option. I would suggest following
1) Competetive ELISA where you block specific antibodies (using specific antigen) and then measure Total antibody in Plate - the decrease in total response is specific antibody
2) If you are ready to do multiple ELISA then you can run each antigen coated on plate and detect-
3) About ELISPOT -- the reading will not be antibody concentration it will be number of cells producing antibody. For restimulation we normally use same antigen.. like for Influenza use Influenza (HA) antigen etc. But hear also you need to coat specific antigens.
I hope these helps ...
I would also suggest collecting the supernatant from stimulations and conducting ELISAs using plates coated with the disrupted virus.
For my master's project I've tried doing something very similar. I stimulated PBMCs for seven days with virus and collected the supernatants in hopes of looking for the production of virus specific IgG. I was able to detect total IgG by ELISA, but despite work with another post doc in the lab we were unable to develop an ELISA sensitive enough to detect virus specific IgG in the virus stimulated PBMC samples. However, in some individuals we could detect weak amounts of virus specific IgG in PBMCs stimulated with CpG, SAC and PWM (CpG cocktail). The post doc hasn't given up trying to develop this assay, but my time as a student is coming to an end. I've since moved to ELISPOTs and have been successful in detecting virus specific IgG after both CpG cocktail (as I too saw in the literature this was a method used) and virus stimulation of PBMCs (for seven days). I'm using PBMCs isolated from donors who haven't been re-exposed to the virus for over 6 years and some over 10 years. I hope this little bit of info helps.
Thanks very much for your responses. Andrea I was afraid of your type of response but will just collect supernatants and see I guess. Hoping to avoid ELISPOTS but if you were able to get ASCs without the huge stimulus of CpG cocktail it may work. I may also need to use vaccine proteins and not live virus so not sure if that will give a strong enough response but will hope for the best!
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