I am working on capturing E.coli in immunoassy with streptavidin nanoparticle. Capture antibody linked beads and detection antibody linked nanoparticle. Through gas generation to read out, it shows like PBS has a high value than E.coli sample. To avoid unbinding beads, I used different concentration(0.5,1,3%) of BSA and increase blocking times(1,6,12hrs). Neither works. How could I get stable value in PBS and E.coli? when people uses E.coli antibody, how does it work?
What I used is like this ;
antibody beads conjugated
add e.coli (reaction time 30min)
wash in PBS
add detection antibody conjugated biotin-streptavidin nanoparticle (10min reaction time)
wash in PBS
gas generation
Hey! Could you explain a little bit more? Your antibodies are in vivo biotinilated? Do you remove the free biotin from your sample?
when antibody -biotin conjugation follows the process, I also use centrifugation with 3KD filtering tube to remove unbounded biotin.
biotinilated process :
BNHS in DMF has the reaction time with e.coli antibody in Na2co3.
Centrifuged with a filtering tube
Remaining samples in the tube conjugates with Streaptavidin nanoparticle
Hi Eunyeong, most capture assays detect the bound bacteria by various methods, but I have never heard of detection of captured E. coli by gas generation.
Could you elaborate?
it is similar with other detection method like detection antibody conjugated with gold- or platinum nanoparticle. However, when I tried the conjugation of detection antibody with nanoparticle, it didn't work. so I uses biotinilated antibody. Finally, on the flow strip, it will capture e.coli (not aptamer) captured with capture antibody immobilized on the strip. To read out the data, I am supposed to use gas generation reaction.
Not very clear. On one hand you are saying Capture antibody linked beads and detection antibody linked nanoparticle are being used but on the other hand this has been mixed with working on capturing E.coli in immunoassy with streptavidin nanoparticle. If you can properly and scientifically elaborate on this may help to resolve the problem faced by you.
With best wishes.
the research plan is using a flow strip which is capture antibody immobilized. so sample will be shown in the test line on the strip and while detection antibody linked biotin streptavidin nanoparticle flows on the trip, it will capture the target. As nanoparticle has the color, it will work out as reading the results but I use gas generation principal with nanoparticle reaction. Before immobilized capture antibody on the strip, I work Beads-antibody form to confirm whether it works or not. what I am working on now is beads-antibody as capture antibody. However, on this step, I couldn't get stable results especially on PBS sample as PBS has high pressure than E.coli Sample.
Still it is not clear. Immunocomponents those you you are using does not seems to be compatible. You say detection antibody linked biotin streptavidin nanoparticle flows on the trip. It is either biotin - nanoparticle or streptavidin-nanoparticle may be used. In your assay protocol which component is linked simply with either biotin or streptavidin that needs clarification and against that you are using nanoparticle coupled biotin or streptavidin against it.
it is very simple. antibody linked with biotin, streptavidin linked with nanoparticle. antibody-biotin linked with streptavidin-nanoparticle. so biotin makes link with antibody and streptavidin nanoparticle.
Are you adding step wise detection antibody conjugated biotin (10min reaction time) wash in PBS followed by streptavidin conjugated nanoparticle. If yes than your protocol looks alright. If you are adding together than have you titrated the combination properly and how that need elaboration.
How you are translating this reaction on lateral flow that remains to be understood in proper sequence.
With best wishes.
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