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High background in Ni chelate ELISA

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#1 bachai



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Posted 14 April 2009 - 05:12 PM

I'm using Ni chelate plates for ELISA detection of Abs to an Ag that I expressed with 6His tag. Following the manufacturer's protocol the plates were rinsed 3 times with PBS/Tween20 (PBST), then the Ag diluted in 0.01M KCl was allowed to immobilized on wells overnight at 4oC. Primary Abs were human sera (1/250-1/500) and secondary Ab was an anti-human IgG AP (1/5000); the diluent was PBST, which was used also for extensive wash (5 times) between incubations. The 'experts' that the company introduced me advised that if I strictly adhere to their protocol there should be minimal background, however by this protocol I had a hugh background binding of IgG from human sera in the absence of antigen (OD>0.6 in only 20min!).
When I was getting back to my old habits of using BSA for blocking the plates after having the Ag sitting on the plates, and adding some BSA to the Ab diluent, the background was much reduced (OD0.1-0.2 in 1h). For some individual sera I still had higher background (about double) compared to the majority of tested sera, but this was OK provided high binding to wells with antigen.
I'm writing this to share my experience but still curious if someone had similar problems?

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