I am running a test to detect antibodies to myeloperoxidase (MPO) in patient sera. When I use different lots of wash buffer on the same test kit, the reactivity changes significantly, sometimes over 50% less reactive.
The test is run on a magnetic bead with MPO attached to it. Patient serum is applied to the beads, decanted, and then washed 3 times with wash buffer. Isoluminol is used to detect antibodies that are left on the beads after the washes. I have already ruled out all of the other components in the test except the lot of wash buffer that is used. Generally, it seems like older lots of wash buffer lead to less reactivity, but the trend is not very strong.
The wash buffer consists of 10mM potassium phosphate buffer, 120mM potassium chloride, 0.05% Tween 20 (detergent), and 0.09% sodium azide.
Hey Lindsay,
First a question: Are the antibodies detected with a secondary HRP-labeled antibody or by the inhibition of the MPO? So which enzyme converts the Isoluminol? Maybe the MPO is interfering with the marker enzyme?
Are you doing it in a white Mikrotiterplate? Sometimes the wells can "cross talk" if you don't use white plates. Also you should be aware, that you should do all incubation steps in the dark. These white plates somehow (Don't ask... its magic... I did not want to believe it myself the first time either!) the surrounding light and release it when they are in the dark detection device.
Have you tried to use your own washing buffer? Maybe PBS +Tween 20? I am sure you know, that Azide Ions inhibit Peroxidases, as well as CN, CO, a too high concentration of H2O2 and you your case maybe Chlorid-ions?
How did you notice the loss in sensitivity? Do you have a standard antibody, that you can spike in buffer?
I am just guessing, but maybe that helps you?
Thanks Carl. The enitre test is run in a machine called QuantaFlash, so everything is done in the dark. The isoluminol is conjugated to an anti-human antibody and then the machine reads the intensity of the isoluminol, although I am not sure which enzyme is used to convert it. (I don't work with the machines often, but I was brought in from another research group to try and fix this problem.)
We have not tried another buffer so far, because we were trying to nail down the source of the problem first. The sensitivity discrepancies were noticed by our customers.
I do know that the azide inhibits peroxidases. I was wondering if the chloride ion might also be interfering. We use the same wash buffer for about 40 different tests and this is the only one we're having this problem with (and also the only peroxidase.)
Like this one?
Article Development and performance evaluation of novel chemilumines...
Ok this is read-out is quite new to me. But is it possible, that the MPO reacts with the isolumniol and catalysis the oxidation before the signal read-out? So it "flashes" before it is supposed to be luminescent?
But maybe its not the read-out itself, maybe the secondary antibody is a little bit old and is sensitive to change in conditions?
Those are great ideas. It could explain the fact that everything works fine with a brand new wash buffer. As the azide breaks down, it stops inhibiting the MPO. I'll have to look further into that. Thanks
Peter Carl Thanks for the help! We realized that the components in the wash buffer inhibit MPO, but if the wash buffer is exposed to UV light, something in the buffer is breaking down to the point that the MPO is no longer inhibited and the "flash" is happening before the detector is taking the reading. Your answer was very helpful.
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