tion of the affinity for D1.3-GFP11 was calculated by nonlinear regression using the PRISM software package (version 5.0 Graph Pad) as 41.4 nM. This value is similar to that obtained when affinity was estimated by ELISA: Kd 25.2 nM. This falls within the range (3-500 nM) of most estimates of D1.3 affinity calculated using a number of different methods [34,37–41], indicating the further utility of GFP complementation in affinity determination. Although the absence of fluorescence in the non-complemented GFP1-10, ensures that only the scFv is fluorescent, this does not avoid a small degree of non-specific binding of the complemented scFv to the negative control antigen (myoglobin), and will depend upon the specific binding properties of each individual scFv. Yeast display Yeast display [3] is a technique in which antibodies are displayed on the surface of yeast particles and selected on the basis of their ability to bind to fluorescently labeled antigens. Clones showing increased binding can then be sorted from those showing less binding. In order to ensure that selected clones have higher affinity and not merely increased display levels, binding is usually normalized using an antibody recognizing a peptide tag at the C terminus of the antibody. Given the results we obtained above, it became clear that we may be able to use GFP complementation to carry out normalization of display rather than using anti-tag Split GFP Labeling of scFvs antibodies. We created yeast display constructs in which scFv-D1.3 was displayed on the surface, containing both the GFP11 peptide as well as the peptide epitope recognized by SV5 (Figure 1d). Biotinylated lysozyme was detected using Alexa 633 labeled streptavidin, and display levels were assessed using either GFP1-10 or phycoerythrin labeled SV5. The results in figure 6 show that labeling by GFP complementation was essentially as effective in normalizing display levels as the use of PE labeled SV5. The only major difference between the two labeling methods was that labeling was more intense with Phycoerythrin labeled SV5, reflecting the fact that each Phycoerythrin contains 34 fluoro- chromes while each GFP contains a single fluor [35,36]. This demonstrates the potential utility of using split GFP to label antibodies in this application as well. Multiplex In a recent paper we demonstrated the advantages of multiplex flow cytometry in the screening of scFvs following selection [24]. In that paper, scFvs were labeled using a fluorescent K coil that bound to an E coil appended to the C terminus of the scFv. Although we were able to obtain high signal to noise using this labeling system, one problem is that the fluorescently labeled K coil is fluorescent even when not bound to scFv, and under some circumstances demonstrated some binding activity of its own. One Figure 6. Yeast display of scFv-D1.3-GFP11. The scFv-D1.3-GFP11 containing both the GFP11 peptide as well as the peptide epitope recognized by SV5 was displayed on the surface of yeast cells. Biotinylated lysozyme was detected using Alexa 633 labeled streptavidin, and display levels were assessed using either GFP1-10 or phycoerythrin labeled SV5. Labeling by GFP complementation was essentially as effective in normalizing display levels as the use of PE labeled SV5. doi:10.1371/journal.pone.0025727.g006 PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e25727