# A New Workflow to Generate Monoclonal Antibodies against Microorganisms
## 3 Results
### Successful Viral Protein (VP) Immunization Induces Immune Response against E. coli O157:H7  
According to the bioinformatic analyses, epitope-engineered VPs were generated and used for the immunization of Balb/c mice. The immune response against the VPs is shown in Figure 3 (second row). All immunization sera revealed a strong signal for the VP in nearly the same intensity with the exception of epitope 5, which showed a reduced immune response overall. In order to see if mice developed a specific immune response against the target microorganism _E. coli_ O157:H7, inactivated cells were stained with the immune serum and measured by flow cytometry. The specific staining plots are shown in Figure 3 (third row). For four out of five epitopes, significant positive stainings could be measured, whereas the preimmune sera showed no staining. Sera from mice immunized with epitope 5 showed no positive staining in flow cytometry, which correlates to the weaker ELISA signals for the corresponding VP. Therefore, the data let us conclude that we could induce an _E. coli_ O157:H7-specific immune response in four out of five samples without immunization with whole bacteria cell extracts. Establishment of a Cell Staining Panel for the Sorting of Antibody-Producing Hybridomas against E. coli O157:H7  
Due to the successful initiation of a specific immune response against _E. coli_ O157:H7, the fusion of splenocytes and the subsequent sorting of hybridoma cells was performed. For this, we chose our novel selection system based on transgenic myeloma cells [16]. The artificial cell surface marker used in this system allows the linking of an antibody capture matrix to catch the secreted antibody. If the antibody binds specifically to the target, a detection with fluorescently labeled secondary antibodies can be established. The positively stained hybridomas can be easily sorted by flow cytometry. We have established a cell staining panel with whole inactivated bacteria cells (Figure 4f) allowing us to verify the antibody's ability to bind the native structure on the bacterial cell surface directly during the sorting process. Figure 2: Schematic representation of the workflow for the generation of monoclonal antibodies specific for _E. coli_ O157:H7 using bioinformatic epitope prediction in combination with novel immunization and selection tools. Figure 3: Overview of the five selected epitopes and their induced immune response. Five epitopes with their amino acid sequences and their location on the target protein (red) were shown using PyMOL. The crystal structure of the target proteins is received from the PDB database. The protein surface of the outer membrane porin G (PDB: 2 iwv), as well as FimH together with FimC (orange) (PDB: 1 QUN), is shown in green. The serum of the immunized mice was tested for epitope-engineered VPs in ELISA. The signals were detected with an anti-mouse coupled HRP antibody and measured at 488 nm. Furthermore, inactivated _E. coli_ O157:H7 cells were stained with sera (1:100, green) and measured in flow cytometry. We used polyclonal PE-conjugated goat anti-mouse Ab for detection. A polyclonal rabbit anti-_E. coli_ antibody (dark gray) served as a positive control. As a negative control (light gray), the cells were stained with the detection antibody only. Splenocytes of mice that showed positive titers were used for fusion with transgenic myeloma cells to generate hybridoma cells producing _E. coli_ O157:H7 serotype-specific antibodies. We started to test the different conditions and controls to establish a bacteria cell-specific staining panel and to eliminate non-specific background signals. For the whole panel, there is a positive control using a PE-conjugated donkey anti-mouse secondary antibody to detect the successful binding of the antibody capture matrix (Figure 4a). This control setup was also used before fusion to ensure that the transgenic myeloma cells express the artificial cell surface marker to get a high sorting efficacy in the fusion pool.