Flow Cytometry
A
Figure 6
Hybridoma screening using a combination of fluorescent bar-coding and high
throughput flow cytometry. A: Supernatants are combined with detecting antibodies and
fluorescently bar coded cells in assay plate wells (from
www.intellicyt.com). B: Four separate populations of cells are identified using different concentrations of calcein AM. C: Histograms showing the
levels of test Mab binding to each of the four populations of cells
B
C
more complex heterogeneous populations, such as those found in PBMCs.
One example where we have deployed the HyperCyt is for the routine analysis of GRO
48
stimulated CD11b upregulation in neutrophil pop- ulations in whole human blood (Figure 5). CD11b is expressed on the surface of many leucocytes including granulocytes. Functionally it regulates leukocyte adhesion and migration to mediate the inflammatory response13 and its upregulation in granulocytes can be used as a surrogate marker for chemotaxis, similar to actin polymerisation. The assay only requires 10ul of whole blood per well in a 96-well plate, reducing blood usage and avoiding costly neutrophil purification procedures. Plates are stacked on the Biomek deck and automatically transferred on to the HyperCyt. Typical plate read time is less than 10 minutes using a HyperCyt, whereas at least 60 minutes is required using the FC500 system. The multiparametric nature of flow cytometry allows the detection of CD11b directly on neutrophils using a combination of light scat- tering and CD16 surface maker labelling. Other invaluable information is also captured, such as cell number and morphology giving an indication of potential compound toxicity. We have also been exploring the applications of HTFC for antibody screening. Traditionally, anti- body screening for cell surface target antigens often consists of several sequential steps. Each step involves different tests for binding and specificity followed by cell-based assays. Screening hybrido- ma supernatants for specific antibodies that bind cell-based antigen is a critical component of mon- oclonal antibody generation and often a bottleneck in the process. Binding of monoclonal antibodies to whole cells expressing target protein is preferred over solid phase methods such as standard ELISA- based technologies. It is highly advantageous if cell-based assays can be performed at the primary screening stage in the monoclonal antibody selec- tion process. This is because in cell-based assays ligands remain in their natural confirmation. The FMAT (Fluorometric Microvolume Assay Technology) is recognised as a gold standard plat- form for primary therapeutic antibody screening using whole cells expressing the target protein of interest14. Since thousands of clones are routinely screened, flow cytometry was not viewed as a prac- tical option for primary high throughput hybrido- ma screening. However, flow cytometry offers sev- eral advantages over the FMAT such as sensitivity and its ability to measure several parameters simul- taneously. The simple use of flow cytometry light scattering allows the discrimination of dead cells from test populations and thus reduces the num- bers of false positives, a constant issue we identify with the FMAT. Previously throughput has limited flow cytometry use to secondary confirmation
Drug Discovery World Spring 2013
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80