I am trying to bind lipid extractions from mycobacteria to Nunc PolySorp 96 well plates to perform ELISA's.
Lipids are insoluble in water, so when I try and dilute down my lipid fractions (in 2:1 chloroform:methanol) into carbonate buffer I am seeing them precipitate out.
Based on some papers I have read I have tried air drying the lipids at RT after adding to the plates in 2:1 chloroform: methanol, but this seems to be starting to perish the plates.
Does anyone have any suggestions of how to bind lipids to ELISA plates?
Many Thanks,
Rachel
I think I gave up on that idea. While at one time I thought that using 9:1 hexane:ethanol probably worked... and it's what we use to coat plates for cell culture. But I never got the ELISA to work... It might be becuase I never had any antibodies... or the lipids got washed off during the subsquent steps... I never invested enough time to figure that out.
Hi Rachel,
I think that it will be better for you to use covalent (crosslinking) immunoassay binding plates. They are similar to the traditional ELISA plates but have some functional gruops that can be activated to convalently linked your antigen to the plate. I have used CovaLink™plates (NUNC) to immobilized a small peptide and tested in a conventional indirect ELISA and it worked fine. The protocol for the activation/linking was very simple (incubate ON the diluted Ag in 10μL of disuccinimidyl suberate (2 mg/mL) in dimethyl sulfoxide) and I guess you can use organic solvents to dilute the lipids thus, you will should have any problems.
I leave you a page from ThermoFisher where you can find more details on the functional groups and different CovaLink plates but information can be found in other places as well. https://www.thermofisher.com/fr/fr/home/life-science/protein-biology/protein-assays-analysis/elisa/elisa-microplates-plasticware/covalent-crosslinking-immunoassay-plates.html
Hope this help.
Good luck!
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