I am biotinylating my monoclonal antibodies at the moment.
However, I have biotinylated two batches of them and the reactivity & background(non-specific binding) seems to be inconsistent.
Checking for reactivity, I used direct ELISA (coat plate with antigen, followed by biotin-labeled antibody, and finally streptavidin-HRP).
On the other hand, to check for the background or non-specific binding, I also used direct ELISA like the one above but changed the coated antigen to 3%BSA.
According to the manual (EZ-Link® Sulfo-NHS-LC-Biotin, Pierce Biotechnology), the steps of biotinylation seems to be quite easy. It says to mix the biotin solution with your desired antibody at a certain ratio, leave it on ice for 2hrs, and that's it.
Afterwards, I dialysed the labeled antibodies in PBS for 5 times, 1 hr each.
PS.It is understandable that antibodies from different batches would give different results, but the strange thing is, I biotinylated 2 batches of a monoclonal antibody from the same tube and they gave slightly different results (both reactivity and background), especially the reactivity. I think that the problem is from the biotinylation step as mentioned above, but I have no idea how to fix the problem right now.
For antibodies from different batches, I intend to check for antibody purity via SDS-PAGE prior to biotinylation just to make sure that there aren't other contaminants that might interfere with the biotinylation step.
Thanks in advance. I really appreciate all the advice.
Pongpawan,
What degree of variation are you encountering? Is it 10-20% or >2 fold?
Most probably the removal of the activated biotin in incomplete.
You can increase dialysis time, as well as number of changes.
Alternatively, you can use a desalting column.
There is some possibility that your biotin coupling reaction is incomplete and differences in two batches are due to the degree of incompleteness. If so, test a batch with longer reaction time (try1.5X - 2x increase) and thorough clean up post coupling as mentioned above.
Finally, some batch to batch variations always occur in chemical modifications. If you have large enough amount modified in one batch to finish all the experiments that need to be compared within the group, you will not be introducing an unpredictable variable in your analysis.
Thank you Tausif,
Btw, I have tried longer dialysis time to remove the excess biotin but this doesn't help much (non-specificity interaction doesn't decrease). Is there any way to get rid of the excess biotin other than dialysis?
Thank you vr much.
Extensive dialysis should work well, just as a desalting column, as I mentioned above.
The other reason for perceived high background as well as variability may have to do with your familiarity and proficiency with ELISA protocol itself.
When you are amplifying the signal, even the smallest details do matter. There are some protocols in my lab where I insist that plates are washed at least 8-10 times before and after adding the enzyme-conjugated antibody.
You may want to look into that side of the problem too...
Try to use EZ-Link NHS-PEO Solid Phase Biotinylation Kit, from Thermo Scientific. I had excellent and reproducible results when I used this kit. It label the antibodies immobilized into a column and the biotin excess is removed during the process without any additional step.
Oscar,
thank you vr much for your advice. I'll absolutely go and have a look. Thanks again!
Our group has been using this method which is, essentially, labeling + purification + desalting in a single column, and which doesn't dilute your sample too much. Give it a try! "Amicon® Pro Purification System enables fast, efficient antibody biotinylation"
Ohhhh wow, thank you very much. I'll have a close look at it. ^___^
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