I was wondering if it would create any problem using a PBS, 1%BSA buffer as a wash buffer for a luminex based immunoassay. So far I have been using a wash buffer that comes as part of a Bio-Plex Reagent kit: (Bio-Rad, Cat. 171-304000JA), but the wash buffer is becoming a limiting factor as it gets finished soon leaving behind a large volume of each of the other reagents in the kit. Accordingly, I am planning to replace the kit wash buffer with an in-house prepared PBS,1%BSA and want to see your kind comments and suggestions on such a move. Thank you.
Dear Tareg,
In my opinion, PBS with 1% BSA as washing buffer contains too much BSA (I use such concentration for blotting techniques). In my experience with bead-based immunoassays PBS with 0.1% BSA was sufficient. I would recommend you to compare results of some commonly used test/experiment with commercial buffer then PBS with 1% BSA buffer and PBS with 0.1% BSA buffer. You will see if some differences occure. If it does not, 0.1% BSA in PBS is sufficient. If it does even in 1% BSA, some other supplements might be considered (e.g. tween 20).
Good luck
Zuzana
Dear Tareg,
we apply the bead-based Luminex technology for about two years. In the first time we varied the composition of the washing and dilution fluids:
1. As dilution fluid we use PBS, 1% BSA, 0,02% Tween 20, 0,1% NaN3 (for dilution of sample, anti Ig, Streptavidin-Phycoerythrin)
2. As washing fluid for the microtiter plates we also use PBS, 0,02% Tween 20, 0,1% NaN3
3. As washing fluid for the Luminex200 instrument we always use “Flow Cytometry, BD FACSFlow Sheath Fluid”
Pos. 1 and 2 were filtered by pore size of 0,2µm.
I hope, these details will help you in your research.
With best regards
Joachim Hoernig
Tareg,
we nominally buy buffer separately about 1L and add about 20-25% of DI water before each use.
Thank you all for the insigftul suggestions. Today I just tried the PBS, 1% BSA buffer and although the generated FI was not adversely affected, there was much bubble produced. Next time I will try to see what good use I may be able to make of your valuable suggestions. I would be happy if Nikunj Gevariya can take the time to let me know the details of the buffer and some recommended online vendor.
This is very old but I'll chime in since there doesn't seem to be the 'best' answer: PBS+1% BSA is going to be fine for some assays, but not all assays. Changing any buffer in an immunoassay can impact the assay performance and characteristics, though the wash buffer is probably the least important in most assays.
The most common wash buffer for xMAP assays is PBS + 0.05% Tween 20 + 0.05% (sodium azide or ProClin 300). As far as I know, this is what Bio-Rad's wash buffer is.
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