How to decrease the background of a IgG ELIS

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How to decrease the background of a IgG ELIS

Postby lily0411 » May 20 2015 4:17 am

I'm using indirect ELISA to detect IgG antibodies against a specific pathogen. Antigen is also coated by us. The conjugate is an goat anti-human IgG (gamma) antibody. In the assay, healthy controls (derived from negative exploitation) give high background absorbance (1.0 in some cases) than positive patients sera.
The pathogen coated in those plates is not pure but derived from cultures. Can anyone tell me why?

Does anyone have suggestions on how to decrease background ?

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Re: How to decrease the background of a IgG ELIS

Postby mdfenko » Jun 10 2015 7:10 am

you don't give any specific information about your procedure so here are some thoughts which may or may not have already been taken into consideration.

the blocking agent and the solutions in which it is used may have a significant effect on the background.

i usually block with normal serum from the species from which the secondary antibody obtained. this helps rule out non-specific binding of the secondary.

if your primary is for detection of a phosphoprotein or if your detection system is biotin-(strept)avidin based then milk is not a good choice for blocking. bsa or normal serum or a combination would be a better block.

besides blocking the wells, the blocking agent should be included in the antibody solutions to preabsorb any antibodies which might bind to the blocking agent either specifically or non-specifically.

increased salt (up to 0.5M) may help reduce non-specific binding.

one other thing, the addition of up to 0.1% tween-20 also helps reduce non-specific binding.
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