A lot publications are using secondary antibody. But I was wondering, if it will compromised the sensitivity of the assay if I am using labelled primary antibody? I wanted to make the method simpler as the whole process is too long.
The sensitivity may be reduced in your proposed DAFIA for immunogen quantification within your alhydrogel formulation however you need to run the proposed assay to determine performance limits. Titrate your labeled primary antibody against your immunogen hydrogel to select optimal concentration for discriminating signal to noise and make your decision based on the data. A good labeled primary antibody should be sensitive in a direct assay down to 10 ng immunogen without difficulty. I suspect that most of the DAFIA use a two step assay secondary to the lack of availability of labeled primary antibody. A good method for DAFIA is published in :
J Immunol Methods. 2009 May 15; 344(1): 73–78. doi:10.1016/j.jim.2009.03.005.
There are many variations for this assay. Good luck
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