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Human IgG Elisa variability problems - (Nov/09/2012 )

Hello everybody,
I registered to bioforum because we are really struggling with a human IgG elisa that was perfectly working a few weeks ago.
So we coat our own engineered peptide in CCB O/N at 10µg/ml, block with 1X PBS 0.1% BSA for 30min, add our diluted human sera for 30 min (usual dilution is 1/100 in wash buffer 0.1% BSA, usually works). During this step we incubate the biotinylated goat anti human IgG (1/10 000) with the streptavidin-HRP (1/4000) in 1ml diluent. Then we add the 1ml to the desired volume of diluent, incubate the mix on the plate for 30 min, add TMB for 10 min and stop the reaction with 2M H2SO4.
As I was saying this protocol was perfectly fine a few weeks ago but now OD values for the same control samples (we have tested several of our positive controls) are extremely low and not reproducible from one experiment to another and from one person to another.
We have changed everything: making fresh CCB, fresh coating peptide, changing secondary antibody, HRP antibody, TMB, water to prepare the buffers, we even have checked the plate reader. We have also checked other dilutions of secondary and HRP, diluting less the secondary and more the HRP gave higher OD values but again we were not able to reproduce it. Trying to decrease or increase the concentration of the coated peptide also did not change anything. The HRP/TMB seems to work because adding TMB to 1µL HRP in diluent gives a strong blue color. Nothing that we tried works.
Two weeks ago, the same type of elisa with almost the same type of coating peptide also started to go off, and now it is not working any more. On another hand, the same type of elisa with the same peptide on mouse serum seems to work as usual (although I should repeat to be sure). The only difference between the human and the mouse elisa is the serum and the secondary antibody. Given that the secondary antibody has been changed it could come from the serum, but even if we had lost the reactivity of our sera (which would be surprising on all of them), why can't we go back to stable values even if they are low?
I hope somebody has an idea of what I could try now, thank you in advance!


What is it that you are quantifying with your ELISA? Is it a therapeutic human IgG that binds to a target and your coating peptide is a portion of that target? Just trying to understand your ELISA.

The combination of your biotinylated antibody and streptavidin-HRP in a pre-incubation/complexing step may be unrobust and will need titering for each lot used or perhaps you should consider avoiding the complex thing completely. If each lot of biotinylated antibody has a varaible free biotin component (and the free component may increase with inappropriate storage), it will variably reduce the capacity of the streptavidin to bind the antibody. Also, the proportion of streptavidin in your streptavidin-HRP prep and number of available biotin binding sites on each streptavidin molecule (it's usually 4, but some will be blocked by the HRP), and the extent of biotinylation of your anti-human IgG (one or multiple biotins on each molecule), all will contribute the the size of your anti-human IgG-biotin-streptavidin-HRP complex and will ultimately affect the number of HRP molecules attached (indirectly) to your human IgG. If you manage to create a complex of 20 HRPs for example, then you will get a signal 20 x higher than if you don't generate a complex at all.

Someone probably once titered the streptavidin-HRP and anti-human IgG-bioin and came to the conclusion that the pre-incubation complex route gave greater sensitivity for THOSE LOTS OF REAGENTS. It may be beneficial to make things simple, and add the anti-human IgG biotin and streptavidin-HRP sequentially with a wash step between. In this way, there is no possibilty of free biotin left in the biotinylated antibody blocking the streptavidin, and no unreproducible complexes of biotinylated anti-human IgG/streptavidin-HRP to deal with.

So the critical question is: has there been a lot change in either the streptavidin-HRP or the anti-human IgG? Has the biotinylated antibody been stored incorrectly and released some of it's biotin?

More details of your assay may allow us to give additional suggestions,

good luck!

-Ben Lomond-

I really hate when my ELISA's stop working, many times I've found that it has something to do with the biotinylated secondary. I have problems with a batch of perfectly working secondary just going bad even in the -20C and over time have to dramatically increase the concentration.

As Ben says above, if you've changed lots or batches of any of the components, especially the coating or biotinylated antibodies, they need to be tested. Rarely in my lab, do different lots behave the same as previously used lots.