Affinity determinations for crude antigens from conditioned media.

<p>(A) Dual-curve analysis in the fixed antibody assay orientation of recombinant rhesus DKK4 in conditioned media of 293F cells. (B) Overlay plot of the curves obtained in the fixed antigen assay orientation using recombinant human DKK2 (black squares) and cyno DKK2 (red circles) from conditioned media of 293F cells, each diluted threefold with running buffer. The blue curve shows the cyno DKK2 assay performed at a fourfold higher flow rate and the green curve represents a control titration of purified mouse DKK1 spiked into a threefold dilution of media from untransfected cells. (C) Dual-curve analysis in the fixed antigen assay orientation of native human DKK1 in the media of a Hepatocarcinoma cell line, diluted 1000-fold (red) and 20-fold (black) with running buffer. The apparent K_D values were (A) 2.1 pM – rhesus DKK4, (B) 110 nM – human DKK2; 120 nM (red) and 170 nM (blue) – cyno DKK2, and (C) 0.16 pM – human DKK1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036261#pone-0036261-t001" target="_blank">Table 1</a>).