Hello

I performed a standard ELISA with the following elements

- Coating: IgG-hFc specific

- Primary antibody + VEGF-biotin complex (start 10nM, 12 points, 1/2 dilution factor)

- Secondary antibody: streptavidin-HRP

I then performed an in vitro transcytosis with MDCK cells to test the transcytosis of my antibody + VEGF-biotin complex from the basal to the apical side. I treated the cells with 100nM of the complex.

ELISA of the apical side was performed by adding original 50uL apical media and diluting the rest by 1/5 (8 points).

I want to know how I can quantify the amount of antibody+VEGF-biotin complex in the apical side.

I tried different methods such as forming y=ax+b or by interpolating the standard curve and using prism to calculate the concentration. Prism shows a low concentration such as 0.046nM and excel shows a minus result.

Thank you in advance