immunoassay Query - linearity (Aug/01/2007 )

Hi

Recently in one of our discussion one of my colleague mentioned that, immunoassay are inherently Non-linear, I would like to know is this true?
If so what is the reason for such a non-linearity??

I would also like to know in what all cases an assay could show up non-linearity??

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what kind of immunoassay? EIA, RIA, ELISA, dotblot, immunoblot, Ouchterlony, others?

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the knid of immunoassay in question will be ELISA and immunoblot...
but also i would like to know does this rule applies to other assay also???

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I do not know if this is really a rule for all immunoassays as they work different;


immunoblot: it is a question of development; BCIP/NBT or other colorimetric precipiation methods have really a poor linearity; better is autoluminography but films are also narrow-ranged in linearity; best is as I think for IB development an imager but not all labs have one as they are very expensive;

ELISA has a better linearity than film-dependent immunoblotting; here, fluorescence signaling is more sensitive than colormetric measurement which improves linearity

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hi,
your colleague was right. i hope you will be convinced with my reason.
all immuno assays are inherantly non linear
because antigen-antibody reactions are very specific and limited, for example if we recall the theory of antibody-antigen intereactions you have 3 stages
1-zone of antigen excess
2- zone of equivalance
3- zone of antibody excess


since in all immuno assays either antigen or antibody is limited, you will not have linearity after zone of quivalance stage. in addition to this you need to consider the limitations of HRP enzyme and its available substrate for color developement.

i hope i could convince you.

gud luk
welcome for your comments
sravan

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the model you suggest is right but presumes that both antigen and antibody are freely diffusing; ELISA or blotting techniques, however, at least antigens or antibodies are immobilized

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hi,
you are right, in ELISA and blotting techniques you have immobilized (limited) Antigen or antibody. that is the exact point we should be focusing!! because of limitation in one of the protein, the second system (either antibody or antigen and detection or capture) will get only limited interactions. which gives non linear curve.

welcome for comments
rgds
sravan

the model you suggest is right but presumes that both antigen and antibody are freely diffusing; ELISA or blotting techniques, however, at least antigens or antibodies are immobilized
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so there are at least two limitations of linearity (non-linearity): first, limited abundance of antigen or antibody per assay, and second, limitations by the detection method (colorimetric/fluorescent/luminescent/radioactive methods of detection); in any case, you have to find the range of linearity (calibration) to get reliable results

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