ELISA calls for help
Deitiker, Philip R
(by pdeitik from bcm.edu)
Wed Apr 13 12:08:19 EST 2011
If you really need linearity across a broad range, ELISA is not the way to go. The development of ELISA like any similar chromatic development method is non-linear, and linearity exists over a short range and lacks accurate reproducibility for large samples. For ELISA this means you need to titer one of factors being tested at close intervals to join (by computation) results that will appear over a broad spectrum. For ELISA do not assume linearity over more than a 10 fold range, however near linearity may exist over 100 fold range. In addition because the development process in a kinetic process, there are edge effects on assay plates that cause non-linearity, unless the material is 'plated' in humidity/temperature controlled environment.
If you really need linearity one can use 125-I labeled detection methods, with these methods linearity can be achieved in excess of 1000 fold provided (in my experience backgrounds in the 300 range and signals as high as 150,000 CPM) one compensates for various forms of non-specific binding. The reason for the differences is that ELISA reactions tend not to reach end point, you are stopping the reaction under a reaction profile in which temp, temperature, humidity, lighting are variable factors in the development. With 125-I detection methods, the label, generally at much higher molar amounts than the target, reaches near saturation. In the assays time I have run, 125-I protein A, saturation is generally reached within minutes even though typical incubation times are for 90 minutes, therefore time and temperature are largely factored out.
One other thing, with assays and replication, variable replicates in samples is almost the surest sign of pipetting errors. Repeatingly using pipette tips in forward mode tends to show increase in counts in the replicate, pre-equilibrating the tip or using reverse mode can reduce this problem. The relative amount of protein (antigen, antibody or developing agent) bound to the tip changes with tip and volume pipetted, and for protein solutions with high protein concentrations, the volume pipetted will differ from the set volume in the forward mode during the second replication. The problem in linearity is often not the procedure but the handling issue. You can test your technique by using color standards on ELISA plates to verify that you are pipetting correct volumes and adding protein to the color standards may also reveal shifts in tip behavior during replication. In some cases you may even detect colorant binding to the tip wall itself. With 125-I methods these errors are simply found (apparently fluid-free tips that have 20% of the signal) and easily compenstated for.
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of WS
Sent: Wednesday, April 13, 2011 1:36 AM
To: methods from magpie.bio.indiana.edu
Subject: Re: ELISA calls for help
actually, there is no need to use chemiluminesce. Probably much too
sensitive in this case, and very tricky to record, as the signal is
not linear with time, when you pipet the substrate manually. Do you
have the possibility to use TMB? Do you also get high background then?
What are the values you expect for positive signals - do you include a
positive control? If in doubt, just coat a serial dilution of mouse
IgG (or serum).
Can you be a bit more specific and describe your experiment, please? I
suppose that you immunized a mouse an want to check if it was
successful. before starting monoclonal production.
Is your antibody against a protein or some sort of small molecule?
Please also post some details about buffers, blocking procedure,
washing steps etc.
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