conceptual advantage of the use of split GFP to label scFvs is that only the complemented GFP1-10 is fluorescent. Since the only source of GFP11 is that fused to the scFv, this should improve signal to noise, by eliminating extraneous fluorescence. Functional analysis of the scFv-D1.3-GFP11 construct was first performed using purified protein, to correlate the fluorescent signal with a precise amount of scFv. In this first experiment two sets of beads were used, one coated with the specific antigen lysozyme and one with myoglobin as a negative control. The fluorescence values obtained showed excellent correlation between the concentration of the fluorescently labeled scFv and the signal (Figure 7a), even down to relatively low concentrations (5 nM). Furthermore, titration of the GFP complemented scFv allowed us to calculate the approximate affinity by nonlinear regression (using PRISM), as 2964 nM, similar to the values obtained above by ELISA and FLISA. In general, the most time-consuming aspect of affinity determination is purification of the scFv and the determination of concentration. Given that we were able to obtain the concentration of scFv-D1.3-GFP11 in the bacterial lysate using the standard curve in figure 4 (5.6 mg/ml) we also attempted to determine the affinity of the D1.3 scFv using crude samples, rather than the purified samples described above. Complementation was carried out with a molar excess of GFP1-10 added to serial dilutions of POP culture (EMD) extract. The results clearly demonstrate the ability to detect antibody bound to specific antigen (lysozyme) and not to the negative controls (myoglobin and IgER) (Figure 7b), without the need for purification. This allowed us to approximate the affinity of D1.3 scFv in the crude extract to 3867 nM, similar to the value of 29 nM previously obtained using purified scFv (Figure 7a). This indicates the value of the split GFP system to calculate approximate scFv affinities that can be used in screening, particularly in high throughput multiplex processes in which protein purification can be avoided. Discussion Fluorescently labeled antibodies can be extremely useful research and diagnostic tools. Full-length antibodies have been chemically linked to different fluorophores, including FITC, TRITC CY-3 and CY-5. This sometimes leads to reduced binding to target antigen, especially when conjugation occurs within the antigen binding site [42]. It is also difficult to control the number of labels incorporated, with considerable variability from antibody to antibody. Direct conjugation of scFv molecules to fluorescent dyes (e.g. CY-5) has also been carried out [43], although this requires prior scFv purification. Many reports describing direct fusion of GFP to scFvs have been published [15,16,18,44–46]. However conventional scFv are prone to aggregation and the fusion between scFv and GFP augments these scFv aggregation tendencies. Furthermore, careful reading of these reports reveals that the yields for scFv-GFP fusions are extremely low, due to the incompatible folding requirements of the two moieties: antibody fragments prefer oxidizing environments, while fluorescent proteins prefer reducing environments. One exception to this general trend has been the fusion of GFP to scFvs that can fold and are functional in the cytoplasm [46]. More recently recognition molecules based on GFP with antibody-like binding proprieties have been described [47], representing an interesting new approach to the creation of fluorescent affinity reagents. The split GFP system is able to overcome problems of expression and folding while providing fluorescent labeling characteristics to the scFv. As the GFP11 tag was evolved to be Split GFP Labeling of scFvs Figure 7. Affinity determination. A. Affinity determination by multiplexed assay using purified protein. After 2 hours of complementation with the GFP1-10 fragment, the scFv-D1.3-GFP11 shows specific binding to its antigen, chicken lysozyme, and no binding activity to the negative control, myoglobin. Fluorescence values are plotted versus the different concentrations of purified scFv-D1.3-GFP11 giving a Kd value of 2964 nM. Data are fit with a nonlinear least squares regression and are visualized with the concentration values on a log scale. B. Affinity determination by multiplexed assay using serial dilution of bacterial lysate (POP culture extract) obtained from induced cells. Unpurified scFv-D1.3-GFP11 obtained from induced bacteria shows specific binding to lysozyme, but not to myoglobin or IgE receptor. Fluorescence values are plotted versus the different concentrations of scFv-D1.3-GFP11 present in crude bacteria extract calculated by reference to the GFP complementation standard (figure 4), resulting in a Kd value of 3867 nM. Data are fit with a nonlinear least squares regression and are plotted with the concentra- tion values on a log scale. doi:10.1371/journal.pone.0025727.g007 minimally intrusive [19], its fusion to the C terminus of proteins, including scFvs, has little effect on expression, solubility or folding. This property should expand the choices of cellular compartments PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e25727