Molar ratios of IgM and IgG antibodies in in vitro assays (View forum version)



dgreen

Posted 28 November 2010 - 06:42 PM

Hi: I'm interested in performing in vitro assays to compare the activity of IgM versus IgG antibody molecules that bind to the same target. Obviously IgM is able to bind to 5 targets compared only one target with an IgG. Can anyone offer advice on how to deal with this issue when setting up in vitro assays for example bioassays etc. I think I need to correct for molar ratios but I'm not sure.

BioMiha

Posted 28 November 2010 - 11:39 PM

Hi,

IgM does not bind five targets, because even though the secreted form is indeed pentameric it has a single specificity. If you really want to go into the very details, you should know that IgM binds 10 identical epitopes (cross-reactiviy aside) and IgG binds 2. But it is not the molar ratio you should consider, rather it is the avidity effect (synergistic effect of multiple binding sites), which I think is in reality impossible to correct for, because not all binding sites are actually occupied.
The real problem in immunoassays with IgM is that because it can compensate for lack of affinity with high avidity there is a lot of non-specific binding. If you take an ELISA plate and coat it with any protein and test the human IgM response, you will see a lot of antibodies, even in negative control sera. I frequently observe titers of 1/5000 and more with negative control sera, which completely masks the true antibody response.
I would advise you to first optimize your immunoassay and then calculate the IgM concentration based on an IgM standard and do the same for IgG. That way you will not have to correct for anything.

dgreen

Posted 29 November 2010 - 04:27 PM

Hi: Thankyou for your response. It is a difficult question. I understand that I should optimize assay with IgM standard and then with an IgG standard. So in essence the concentrations could be different, but it is still feasible to compare the activity between an IgM and IgG binding to the same target.

Cheers,


Hi,

IgM does not bind five targets, because even though the secreted form is indeed pentameric it has a single specificity. If you really want to go into the very details, you should know that IgM binds 10 identical epitopes (cross-reactiviy aside) and IgG binds 2. But it is not the molar ratio you should consider, rather it is the avidity effect (synergistic effect of multiple binding sites), which I think is in reality impossible to correct for, because not all binding sites are actually occupied.
The real problem in immunoassays with IgM is that because it can compensate for lack of affinity with high avidity there is a lot of non-specific binding. If you take an ELISA plate and coat it with any protein and test the human IgM response, you will see a lot of antibodies, even in negative control sera. I frequently observe titers of 1/5000 and more with negative control sera, which completely masks the true antibody response.
I would advise you to first optimize your immunoassay and then calculate the IgM concentration based on an IgM standard and do the same for IgG. That way you will not have to correct for anything.


BioMiha

Posted 30 November 2010 - 07:09 AM

If you can prepare the Fab fragment of each of your Abs, that will give you the affinity of a single binding site.