of known protein concentration fig4 shows a corresponding standard curve of a tbet elisa according to the exemplary embodiment for the performance of the tbet elisa the steps relate to the following in detail insert number of required wells into a frame of the 96 well plate addition of 50 u03bcl well assay buffer addition of 100 u03bcl well standard control sample incubation for 60 minutes on the shaker wash all wells 4 u00d7 with 400 u03bcl well of wash buffer addition of 100 u03bcl well anti tbet antibodies incubation for 60 minutes on the shaker wash all wells 4 u00d7 with 400 u03bcl well of wash buffer addition of 100 u03bcl well peroxidase marked anti tbet specific antibodies incubation for 30 minutes on the shaker wash all wells 4 u00d7 with 400 u03bcl well of wash buffer addition of 100 u03bcl well substrate incubation for 30 minutes stop reaction by addition of 100 u03bcl stop solution measurement of optical density at 450 nm with a microwell plate reader in order to consider differences in the sample preparation a standardization of the concentrations of gata 3 and tbet can be performed differences in the sample preparation can arise e g due to the following differing cell numbers to be lyzed differing lysis efficiencies of the individual samples or differing content in the different cell types within the cell preparations standardization to the total protein content of the cell lysate see under u201c measurement of the concentrations of gata 3 and tbet u201d standardization to.