Figure 1.
Strategy applied to the selection of antibodies using both phage and yeast display.
Antibodies are first selected against Ag85 using two rounds of phage display, after which the whole selection output is cloned into a yeast display vector using homologous recombination. A further one or two rounds of sorting by flow cytometry allow the subsequent isolation and testing of single clones.
Figure 2.
Yeast cell analysis before and after sorting.
2A shows the yeast population after cloning the Ag85 selected phage display output. The upper right quadrant (Q2) representing those yeast that are displaying scFv and binding antigen contains 2.1% of all yeast cells, of which the top 1%, as indicated by the sort gate, were sorted. 2B shows the outcome of the first sort after yeast were collected and grown up. Q2 now contains 25.1% of all yeast cells, of which the top 1% were again sorted as shown. 2C shows the outcome of the second sort: Q2 now contains 35.5% of all yeast cells. 2D and 2E show the background streptavidin binding clones that represent, for both the first and the second sort, 3.2% of the total population.
Figure 3.
Specificity of selected monoclonals.
48 different monoclonals chosen from among the 111 identified were tested for specific binding to Ag85, as well as two irrelevant antigens (myoglobin and ubiquitin). All were positive for Ag85, showing signal to noise ratios exceeding 8.
Table 1.
CDR3 protein sequences of the different scFvs obtain after one or two round of yeast display sorting.
Figure 4.
Binding of selected monoclonals to different Ag85 subunits.
Each of the individual antibodies was tested for binding to the complete Ag85 complex, and the values obtained used to normalize the recognition of the 48 different scFvs for the three different Ag85 components. The arrowed clones were taken for further testing.
Table 2.
Affinities of 14 antibodies determined directly on the surface of yeast.
Figure 5.
Enzyme linked immunosorbant assay (ELISA) of selected antibodies.
14 of the scFvs were recloned into a yeast expression vector as scFv-Fc fusions [28], using a rabbit Fc domain, and tested for recognition of Ag85 directly adsorbed to plastic in an ELISA format using standard anti-rabbit antibodies as secondary reagents.
Figure 6.
Sandwich ELISA of selected antibodies.
Yeast displaying eight selected antibodies were used as Ag85 capture reagents, and tested with the same eight antibodies expressed as scFv-Fc fusions detected using fluorescent anti-rabbit secondary antibodies. Individual histograms show binding of the designated scFv-Fc fusion antibodies to individual yeast cells displaying the indicated antibodies in the presence of 100 nM Ag85. Fluorescence (i.e. the amount of scFv-Fc bound per individual yeast cell) is shown on the X axis, while the yeast count at each fluorescence level is shown on the Y axis. Yeast to the right of the vertical line in each plot are fluorescent as a result of a positive sandwich assay.
Figure 7.
Ag85 detection limits in a sandwich assay.
106 yeast displaying the indicated antibodies were induced, washed and resuspended in serial antigen dilutions. Binding was detected by flow cytometry following the addition of the indicated scFv-Fc fusion antibodies as induced culture supernatants, and fluorescent anti-rabbit secondary reagents. The indicated detection limits are defined as the minimal antigen concentration giving a signal three times greater than background. A: Detection in phosphate buffered saline. B: Detection in 1∶50 human serum diluted in PBS.
Figure 8.
Stability test of yeast expressed scFv-Fc fusions.
Unpurified scFv-Fc fusions were tested for binding to Ag85 by ELISA (Ag85 was adsorbed to plastic) at regular periods over 4 weeks. Results are expressed as percent signals obtained on the first day.