Hello,
I am currently optimizing an absorbance-based ELISA assay. The ELISA is a sandwich-type, with the final component being a secondary antibody fused to HRP. I then use Turbo-TMB to obtain absorbance values. As I am hoping to use the minimum amount of reagents in the ELISA, I have run a standard curve using a dilution series of one component of the assay which I hope to competitively inhibit. From this standard curve, I have determined the amount of the component suitable for the assay, however it is higher than I had hoped it to be. Will utilizing Ultra-TMB instead of Turbo-TMB have a dramatic change on the sensitivity of my assay and allow me to use less reagents in the sandwich?
Thank you!
Article A fluorescent peroxidase probe increases the sensitivity of ...
Article A fluorescent peroxidase probe increases the sensitivity of ...
Hi
The 100 ul of TMB substrate will form the expected fluorescence to each well, if your expecting low substrate use to my advise you have to run with your negative control as well with the standards provided by the kit, so that you can fix the TMB volume and further proceed with your ELISA. Hope this might help you
I have not experienced with the Ultra, I use 100uL of TMB-turbo and works fine, but you could perform an assay, as suggested by Gunapati.
In general, it is challenging to optimize an assay with constraints on reagents. One popular option is a smaller volume, e.g., 384-well plates versus 96-well plates.
A fluorescence readout will increase assay response. But higher readout levels often are associated with a similar increase in background noise. Note that sensitivity of an ELISA is usually limited by relatively high background rather than relatively low response. You may need to add more wash steps and/or use a more stringent wash buffer to keep background low.
I think that the substrate TMB should be fresh and clean, which reduces the effect on sensitivity
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