Hi all,
I am currently optimizing an ELISA to detect myeloperoxidase in human serum. I currently use 1% BSA/PBS to block and as a diluent for the standards/samples and antibodies. My wash buffer is PBS+0.1% Tween. I currently coat the plate overnight and, following this, incubate standard/samples overnight. This protocol has worked well for detecting MPO in cell culture media of PMA-stimulated granulocytes.
However, I get poor linearity-of-dilution for my serum samples: the matrix effect is overcome by a 1:8 dilution, but the absorbance values for this level of dilution falls outside of the linear range of this assay. Does anyone have any suggestions as to how I can overcome the matrix effect in serum without diluting to 1:8?
Does anyone recommend using 10-50% animal serum instead of BSA as a standard/sample diluent?
Does anyone recommend doing a simultaneous incubation of the target antigen with the enzyme-conjugated detection antibody?
Thank you in advance for your help!
I don't know about for MPO but for other enzymes a concentration by ultracentrifugation step can be added prior to analysis so then you can discard supernatant and dilute in PBS avoiding serial dilutions of the serum.
On the other hand, you might have already considered this but why not measuring MPO enzymatic activity instead or besides MPO quantity?
Thank you for your responses, much appreciated! Thilini Madurangika Jayasinghe, we are looking into standard addition at the moment alongside figuring out what other options might be available, given the labour-intensive nature of standard addition. Luis Antonio Ochoa-Ramírez could you expand on the process of concentration by ultracentrifugation please? I should also clarify, we are interested in looking at MPO-DNA complexes alongside other DNA-protein complexes and individual proteins. We are generally having the same problem (matrix effect) with all our ELISAs.
I haven't done the procedure myself but here is where I got the tip:
https://www.caymanchem.com/pdfs/707002.pdf
Check the troubleshooting part (page 21). They recommend Amicon concentrators but I found this website with some protocols that might be useful for you considering you are not looking for just MPO:
https://www.sigmaaldrich.com/life-science/protein-sample-preparation/protein-concentration/amicon-ultra-centrifugal-filters/protein-sample-ultrafiltration/ultrafiltration-protocols.html
Sorry for not being able to give an expert response, I hope it is of use to you though.
Thank you very much, Luis, I will look into the Amicon filters, they look like a great option!
I had problems with high background with patients serum in my ELISA. To reduce the background I diluted the serum by 1:2000 (instead of standard 1:100) and added ELAST from PerkinElmer to enhance the signaling. It worked great. Background was down and specific signal was still up. Maybe this can work for you as well.
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