# A New Workflow to Generate Monoclonal Antibodies against Microorganisms
## 3 Results
### Successful Viral Protein (VP) Immunization Induces Immune Response against E. coli O157:H7
Figure 4b-e show various control setups of the staining panel that differ in the absence of several important components. Compared to the final setup, these controls prove the general possibility of the panel. Furthermore, non-specific background staining caused by dead cells was reduced using the live-dead marker 7-AAD. Nevertheless, the negative control shown in Figure 4b where the mouse serum was left out showed a low background signal. About 2% of the living cells are stained non-specifically, even after repeated washing steps. This might be because _E. coli_ cells can attach to cell surfaces or to the antibody capture matrix non-specifically if the binding sites are not blocked with serum antibodies or the later secreted antibodies. In comparison, the full panel (Figure 4f) where the immune serum from VP-EC3 was used showed a second emerging cell population that has a significantly higher PE signal. Despite the mouse serum, which consists of a multitude of polyclonal antibodies, about 6% of the cells could be stained in this way. These cells were able to bind a sufficient number of _E. coli_ O157:H7 cells, which made a second population visible in the flow cytometric analysis. Evaluation of the Hybridoma Sorting Approach with Inactivated E. coli O157:H7 Cells Serving as Antigen SUBSECTION  
For final sorting, we stained cells from the fusion pools generated by immunizations with VP-EC2, VP-EC3, and VP-EC4 and fused them with our transgenic myeloma cells. In general, we were able to stain cells in each fusion and sort around 7% of hybridomas  
Figure 4: Establishment of a staining protocol for the sorting of specific antibody-producing hybridomas for the antigen _E. coli_ O157:H7. Illustrated are the schematic sorting assembly and their flow cytometric measurements. The dot plot shows the live–dead marker 7 AAD against the PE signal. (**a**) Detection of the antibody capture matrix on transgenic myeloma cells. (**b**) The sorting approach without the secreted antibody (imitated by the mouse serum). (**c–e**) Sorting approach without the antigen _E. coli_ O157:H7, rabbit anti-_E. coli_ Ab, or the antibody capture matrix. (**f**) Overall structure of the final sorting approach. The cells have an antibody capture matrix. The secreted antibody of the hybridoma cell is imitated by the VP-EC3 mouse serum. Inactivated _E. coli_ O157:H7 cells represent the antigen. The antigen is bound by the polyclonal rabbit anti-_E. coli_ antibody, which in turn is detected by the polyclonal goat anti-rabbit antibody coupled to PE. specifically with a PE signal greater than 10\({}^{4}\) intensity. From fusion with VP-EC2, we gained 13,000 specific hybridoma cells out of 1 \(\times\) 10\({}^{6}\) cells in total (Figure 5; first column). The sorting gates were set very strictly around 10\({}^{4}\) to sort hybridomas with very high signals. For the VP-EC3 fusion, 21,000 cells out of 6 \(\times\) 10\({}^{5}\) cells could be sorted positively. From the fusion VP-EC4, we could sort around 75,000 cells from a pool of 1.35 \(\times\) 10\({}^{6}\) hybridoma cells. For further analysis, the cells were cultivated in a monoclonal and polyclonal manner in 96-well plates. Stable cell clones were tested regularly in flow cytometry with the same setup described above. We received the highest number of positive hybridomas from the VP-EC4 fusion, which already showed a significant signal in the serum assay. Overall, 98% of the seeded polyclones (154 out of 156) and about 80% of the seeded monoclonals (44 out of 55) were tested positive for _E. coli_ O157:H7. From the VP-EC2 fusion, 30% of the monoclonals and 20% of the polyconals were tested as specific, although the serum test showed weaker results compared to immunization with VP-EC3 and VP-EC4. The VP-EC3 fusion showed the lowest number of established monoclonals producing specific antibodies, but 35 polyconals showed a positive signal in this fusion.