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Heat inactivation of fbs necessary for ELISA? - (Dec/08/2009 )

in order to prevent high background is heat inactivated fbs better? I am using j774.2 macrophages. Any other suggestions for preventing background are welcome.


Thanks

-prof. moriarty-

heat inactivation is usually used for inactivation of complement. Many elisas test for analytes in serum or plasma...which contains complement.

If you can detail exactly what "background" you are observing it may help us answer your question.

-sgt4boston-

sgt4boston on Dec 9 2009, 09:59 AM said:

heat inactivation is usually used for inactivation of complement. Many elisas test for analytes in serum or plasma...which contains complement.

If you can detail exactly what "background" you are observing it may help us answer your question.


I am testing for the cytokine TNF-a - I haven't started the ELISA so I haven't observed background, I am just trying to take a preventive step.

-prof. moriarty-

Background can be due to

non-specific sticking of conjugte to wells (incomplete blocking)
conjugate sticking to capture ab (washing not optimized or too high conc of conj)
conjugate bound by capture ab (cross reactivity of capture ab)
conjugate binding to capture ab (cross reactivity of conjugate ab)
sample sticking to wells
sample sticking to capture ab/ blocking protein

etc.

these are just a few of the many conditions that can create assay "background"

-sgt4boston-