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Primary antibody quantitation


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#1 chrisg

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Posted 09 November 2009 - 09:01 AM

I am in the process of developing an ELISA method, and wish to quantitate the concentration of primary antibody that has successfully bound to the well of the microplate during the initial plate coating. I want to do this to confirm that I have actually managed to bind antibody to the plate, and to determine the optimal concentration of antibody to coat with. Does anyone have a protocol or a link to such a method? Can the Bradford or Lowry assay be modified to assay solid-phase protein?

Thanks for your help in advance!

#2 Gerard

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Posted 09 November 2009 - 10:06 AM

I don't know any method to measure the bound antibody because the concentrations are to low, to determine the optimal concentration of your antibody you have to use the checkerboard methode.
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#3 wirly

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Posted 09 November 2009 - 06:53 PM

I am in the process of developing an ELISA method, and wish to quantitate the concentration of primary antibody that has successfully bound to the well of the microplate during the initial plate coating. I want to do this to confirm that I have actually managed to bind antibody to the plate, and to determine the optimal concentration of antibody to coat with. Does anyone have a protocol or a link to such a method? Can the Bradford or Lowry assay be modified to assay solid-phase protein?

Thanks for your help in advance!


Not sure about the Bradford or Lowry for this application, but would be interested to hear if it works. Do you really need to know the actual concentration of the antibody, or do you just want maximal binding capacity? If the latter is the case, then just a coating titration from very high 10+ ug/mL to very low should give you the range. Block the plate and do an anti-IgG ELISA holding the detecting Ab concentration constant and look for the plateau of signal.




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