This study introduced a new strategy for selecting scFv specific to an unlabeled antigen. Antibody-mediated labeling enables selections with antigens that are difficult to biotinylate. It may also enable the display of antigens in more native conformations. Moreover, detection MAbs or scFv can be used to mask an epitope, thereby increasing the likelihood of isolating clones to different epitopes. The limitations of this method are that pre-existing antibodies (IgG, Fab, or scFv) are needed, and antibodies of low affinity may not bind to the antigen long enough to be detected.
A non-immune human scFv library was used for initial selections with the BoNT/A Hc
, and scFv clones that bound Hc
were detected by using labeled anti-BoNT/A IgG MAbs identified previously (Razai, et al., 2005
, Amersdorfer, et al., 2002
, Levy, et al., 2007
). Throughout selections, we incubated the yeast with excess antigen (100 nM Hc) for sufficient time (45 minutes) to allow antigen binding to proceed to saturation. This strategy assures the isolation of high-affinity as well as low-affinity clones.
In general, the scFv clones isolated with biotinylated MAbs tended to be more Hc specific, and fewer bound secondary reagents, compared to clones isolated with the A633-labeled detection MAbs. This may have been due to the intensity of the phycoerythrin portion of the SA-PE detection reagent. We also found that clones that were sorted from an apparently “higher affinity” gate, that is those clones with the highest mean fluorescence intensity for Hc binding, did not exhibit better affinity than clones isolated from a “lower affinity” gate and typically bound secondary reagents only. Additionally, selections performed at lower antigen concentrations also did not produce higher affinity clones, and similarly exhibited mostly secondary reagent binders.
Previous selections on BoNT/A immune phage libraries by Amersdorfer et al.
resulted in isolation of 28 scFv antibodies (Amersdorfer, et al., 1997
). Clones analyzed for affinity to Hc
using a flow cytometric assay ranged from 1 nM to greater than 100 nM. Two clones, S25 and C25 had affinities of 73 nM and 1 nM, respectively, and a third scFv isolated from a human immune library, 3D12, had an affinity of 37 nM (Amersdorfer, et al., 2002
). Each of these scFv bound different epitopes of the BoNT/A holotoxin (Mullaney, et al., 2001
Our selections using the non-immune human yeast-display scFv library identified 20 unique clones with affinities for the BoNT/A (Hc) that were similar to those seen with the immune library, ranging from 5 nM to 48 nM. Epitope binning revealed at least two non-overlapping epitopes on the Hc were bound by the three scFv, although they have not been mapped to the exact epitope. These observations demonstrate the utility of the non-immune library for isolating affinity reagents to novel epitopes.
Clones were isolated that represented three of the six VH
families and five of the eleven VL
families described by Feldhaus et al.
(Feldhaus, et al., 2003
). Moreover, the percent usage of each family in clones that we identified was consistent with the usage described for the original library, suggesting that antibody-mediated selections do not introduce a selection bias with respect to VH
Five of twenty scFv tested bound to the BoNT/A holotoxin [scFv F2B2, scFv F8B6, scFv 4–2 (G8C6), scFv 14–2 (G10C8), and scFv 19–3 (H3D1)]. Two of these (scFv F2B2 and F8B6) bound very poorly and had no functional light chain as determined by sequence analysis. All the other clones bound only Hc (data not shown). Of the three that bound holotoxin, scFv 4–2 (G8C6) is one of two scFv isolated from the negative sorts, and was only isolated with the 3D12-A633 detection MAb. By contrast, clone scFv 14–2 (G10C8) was isolated 14 times, had the lowest affinity by flow cytometry and Biacore, and was isolated at least once with all three detection MAbs. All three scFv that bound holotoxin were active following secretion from E. coli.
The utility of negative sorting was illustrated by an experiment in which we sorted clones from the R2 output following incubation with 100 nM Hc and MAb 3D12-A633, but did not perform a negative sort. We then screened 24 sorted clones despite the strong presence of nonspecific binders present in the antigen binding region of the bivariate plots. Our hypothesis was that within this pool of nonspecific binders existed clones that bound specifically to Hc. However, analysis of 24 clones from this sort identified 22 secondary reagent binders, and none bound Hc. In contrast, negative sorting yielded two Hc-binding clones, both of which were not isolated in any other selections or with the other two detection MAbs.
The three MAbs used in the selections are known to bind different epitopes on BoNT/A-Hc
(Mullaney, et al., 2001
, Razai, et al., 2005
, Levy, et al., 2007
). If a MAb is blocked from binding to Hc
following its capture by the yeast-displayed scFv, it could be inferred that the MAb and scFv share a common or overlapping epitope. ScFv 19–3 was observed to inhibit MAb B4 binding, but did not inhibit binding of MAbs 3D12 or AR1. Therefore, scFv 19–3 binds an epitope that overlaps the B4 epitope on BoNT/A. In contrast, scFv 4–2 and scFv 14–2 did not inhibit the binding of any of the three detection MAbs. Therefore, it can be predicted that scFv 19–3 could be used in an ELISA assay when paired with scFv 4–2 or scFv 14–2.
The fact that MAbs B4 and 3D12 were used in the first two rounds of the selection indicates that scFv 19–3 must have been isolated with MAb 3D12. This clone was eventually identified in the AR1-sorted round 3c output. This observation highlights the advantage of using multiple detection MAbs during early rounds of selections to optimize the likelihood of isolating clones that bind to many different epitopes. Taken together, the data indicate that there are at least five non-overlapping epitopes on the BoNT/A Hc including: the AR1 epitope, B4 epitope, 3D12 epitope, scFv 19–3 epitope, and scFv 4–2/14–2 epitopes.
The Endopep-MS experiment demonstrated the function of soluble scFv following production and purification. This assay has been used many times to detect BoNT toxins in biological samples (Barr, et al., 2005
, Kalb, et al., 2010
, Kalb, et al., 2005
, Kalb, et al., 2009
, Kalb, et al., 2006
). In our assay, the mass spectrometry signals observed with the polyclonal BoNT/A antiserum were consistently less intense than the signals observed when the three scFv were captured simultaneously. This may be due to the avidity of the three scFv working synergistically. However, the polyclonal BoNT/A antiserum has been shown to neutralize some activity of the toxin resulting in lowered peptide cleavage activity in the Endopep-MS assay (Kalb, et al., 2006
). We cannot rule out that the larger signals observed with the three scFv may not be due to increase toxin capture but rather may be due to failure to neutralize the toxin. However, it is difficult to conceive of a mechanism by which three different antibodies neutralize the toxin individually, but not in combination. Given that the three scFv bound at least two different epitopes on the antigen, synergistic binding effects are more likely to account for our observations.
In summary, this report described a novel antibody-mediated labeling technique for screening yeast display libraries for scFv that bind multiple epitopes on an unlabeled antigen. It also demonstrated synergistic binding of scFv antibodies to an enzymatically active antigen. Synergistic antigen capture could be a broadly applicable strategy for improving the performance and utility of monovalent antibody-like molecules such as scFv.