Fixing and permeabilising the cells in ELISA is done for the same reasons that it’s done in IHC or FACS. It ensures free access of the antibody to its antigen by immobilising it. Permeabillisation will allow the antibody to detect internal antigens and/or internalised antigens.

But is the antigen not accesible without fixinf the cells? Can't one do ELISA with permealized cells/cell lysate without fixation?

Hi. I believe that the main reason for the fixation is to retain cell shape and morphology. Without a fixing step, cells on the plate will loose their structure or be solubilized by detergents in the buffers and washed away.

Yes, they are accesible as long as the cell does not loose its confirmation. Fixation would help immobilize the antigens by retaining cellular structure and thus enabling unhindered access of antibodies to cellular and subcellular compartments.

Hi Anette!Indeed, you should first fix the cells and then permeabilize them. The reason is that you need to maintain the cell substances "fixed" in their cellular locations before you permeabilize the cells. If you didn't fix the cells you can imagine that the cells components would " flow away" after cell permeabilization. There is an issue though, some proteins change morphology after fixation (You can again imagine them as becoming "too stiff" ) and therefore they might not be recognized by the same antibody that would recognize their native-unfixed- form.

Hi Annette, Antigona is right.. The cells need to fixed to the internal surface of the plate, in order not be "washed away" in the series of wash buffers applied during the process. Imagine that the primary and secondary antibodies bind to the antigens and all are "in solution". Upon applying the wash buffers, they would simply be decanted out of the plate and then while detecting, nothing would show.