The vast majority of quantitative Luminex-assays that I have come across relies on a biotinylated detection antibody that is subsequently coupled to streptavidin-phycoerythrin requiring additional incubation and washing steps, as compared to using a detection antibody that is directly labelled with phycoerythrin.
There may be many possible reasons why the biotinylated versions are preferable e.g. PE-labels are large and may impede antigen binding, biotin gives a more flexible design that allows use of different PEs, etc., but I have not been able to find well-documented examples of biotinylated vs. directly labelled detection antibodies.
Of course this may vary from assay to assay, but if anyone has knowledge beyond the purely speculative as to why biotin should be preferred over direct labelling, I would be very interested in hearing about it.
Thanks in advance!
Dear Christian,
Going for the biotinylation amplifies your signal. When you also keep your background value or noise under control, this can significantly increase the analytical sensitivity of your assay. Due to the different possibilities of biotin over antibody ratio during the biotinylation procedure, you can conjugate multiple biotins on your antibody and hence, create multiple interaction sites for the streptavidin-PE in your assay. This gain in range of your quantitative assay mostly compensate the indeed required additional steps during the protocol.
Kind regards
Dear Ann.
Thank you for the answer. That makes sense, but I was under the impression that the ratios for biotin and PE would be more or less the same if conjugations are optimized correctly? At least some conjugation kit-vendors state the same range of ratios for both.
Could you perhaps point me to some good article(s) that supports this?
BR
Christian
Hi Christian,
I do not have any comparison studies at hand, but based on my own experience you can conjugate many biotins on your antibody. The small size of biotin (0.24 kDa) enables the implementation of high ratios. Since PE is quite larger (+/- 240kDa), those ratios are not possible (average kDa of IgG Abs is 150kDa). Optimizing a biotinylation seems more straightforward than a PE-conjugation.
Hope this helps.
Kind regards.
I fully agree: Chemical biotinylation -even though different chemistries are available - will never be a precise method. In the resulting antibody preparation, some will carry one, others carry two, others will carry three others more biotins. I think that the makers have optimized their protocol to have maximum biotinylation while keeping the antibody fully able to bind to the target. Sorry, no reference, only gut feeling.
Dear both.
Your answers make sense and you are probably right, but what I am looking for is a well-documented explanation.
I completely agree, that it must be possible to conjugate a greater number of biotins given its smaller size, but unless the resulting biotin-streptavidin complex provides some kind of distance-extending 'linker' enabling a higher total number of PE-molecules to attach to the detection ab then all the additional conjugated biotin molecules might be of limited use, and I guess that none of us can say for sure?
It also seems likely, that the sequential binding of the detection ab to its target, followed by biotin-mediated binding of the much larger SA-PE moiety could help avoid some of the steric hindrance that might otherwise impede binding of a heavily PE-labelled detection ab to its target, but again I have not been able to find any documentation.
It should be fairly straight forward to show at least some of this experimentally, so I think I will do some tests.
Do you have any preferred reliable methods for assessing biotin:antibody and PE:antibody ratios?
Best regards
why not try to titrate the binding of SA--PE to the biotinylated antibody using free biotin. well, this is probably most easily done in an ELISA setup. the biotin concentration needed to block binding, together with the protein concentration of the antibody, will give you a rough estimate of how many biotins are attached to the antibody.
The commonly used biotinylation reagents come with a linker that put biotin in some distance to the reactive group. Thus, steric hinderance of the binding of streptavidin is not an issue.
if you want to validate your reagent in an ELISA you can use the same reagent for ELISA and CBA. Doing ELISAs with PE is not the method of choice.
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