I am working on developing a luminex sandwich immunoassay and discovered that the literature (luminex xMAPCookBook) recommends carrying out a coupling efficiency test. I used the same antibody pair I am using for the assay to carry out the coupling efficiency test but the resulting FI signal got exgteremly low (20-40). The capture antibody is mouce antihuman mAb and the detection antibody is rabbit antihuman polAb. I am writing to seek some suggestion on what the reason behind the very low FI signal might have been. Mainly I would like to know if it is appropriate to use a detection antibody different from that used in the assay to determine coupling efficieny of the capture antibody to the beads. Regards.
Does your detection antibody recognize your mouse capture mAb, or does it recognize the antigen that your mAb is meant to capture? To test for coupling, your detection antibody should recognize the capture mAb. This means your detection Ab would be different than the one you would use to detect the captured antigen (i.e. use rabbit anti-mouse IgG, not rabbit anti-human IL-6,Il-10,X,Y,etc). Furthermore, the your anti mouse detection Ab should be conjugated to PE, or biotin (which would require streptavidin:PE). Hope that helps.
I agree completely with Evan's response above, good advice. Using anti-IgG-PE allows you to read the coupling confirmation assay with the Luminex instrument, and is most recommended. I have used anti-IgG-HRP, for a far lower resolution comparison of coupling efficiencies between batches of conjugated beads. This was only done while waiting for anti-IgG-PE products to be delivered, and I wouldn't recommend it over using anti-IgG-PE as your coupling efficiency assay detection antibody.
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