# A New Workflow to Generate Monoclonal Antibodies against Microorganisms
## 4 Discussion  
The generation of antibodies for the detection of MOs is associated with several issues. Identifying suitable hybridomas whose antibodies bind solely to strain-specific targets is extremely challenging. In particular, for MOs, it is difficult to identify species-specific epitopes due to a high overlap between highly conserved regions and the lack of appropriate cross-reactivity screens [19]. Screenings for suitable binders generally use ELISA/ELISPOT techniques and Western or dot blotting methods. However, these methods also possess certain disadvantages related to the purity of used components, unspecific binding to plastic, or the high levels of cross-reactivity between the strains. A flow cytometry approach is a useful and sensitive alternative to screen for potential binders due to its ability to gate on whole bacteria and identify mAbs that bind on cell surface-related targets [20]. In addition, flow cytometry staining of bacteria is a simple and reliable process. In this study, we focused on the combination of three aspects to establish a comprehensive workflow for MO-specific mAbs--first, a bioinformatic one for the identification of unique and strain-specific epitopes, a second one for an epitope-specific immunization with engineered VPs, and a third one with a flow cytometry-based selection system for the identification of specific antibody-producing hybridomas. For the bioinformatics analysis, we compared the genomes of eight indicator MOs that are commonly associated with contaminated drinking water [21], as well as the pan-genome of _E. coli_ with the reference strain _E. coli_ O157:H7. The panel of MOs used for this proof-of-concept study provides a valid database to demonstrate our workflow. However, for future applications, we would like to point out that the panel can still be adapted and extended. To guarantee the accessibility of these epitopes, we combined several prediction and localization tools such as IEDB, PSORTb, and BioCyc. The combination of these different tools has already been proven in several studies to identify new vaccines against MOs [22; 23]. The selected epitopes were cloned into our VP system, which allows a surface-related presentation of the epitope sequence on a virus-derived carrier protein. Another advantage  
Figure 7: Specific epitope binding of the mAbs to the epitope-engineered VPs. The mAb binding to epitope-engineered VPs with integrated epitopes was measured in immunoassay. Per well, 50 \(\upmu\)L of a 5 \(\upmu\)g/mL VP solution was coated overnight. The wells were washed, blocked with PBS/NKS (5%), and incubated for 1 h with 5 \(\upmu\)g/mL Ab solution. Again, the wells were washed and incubated with HRP-conjugated goat anti-mouse Ab for 30 min. The signals were measured at 450 nm 15 min after adding TMB substrate. The P157 (anti-VP) mAb was used as a positive control. n = 3. * \(p\) < 0.05, *** \(p\) < 0.001 compared to the control group by one-way-ANOVA. of using such a system for immunization is a very rapid and specific immune response to the inserted epitopes, as shown in Figures 3 and 7. Four out of five selected epitopes were able to induce a very specific titer within 4 weeks without the need of immunizing whole bacterial cell extracts and adjuvant. The use of inactivated bacteria or bacterial cell extracts for immunization is accompanied by certain pitfalls, as it was described earlier in [PERSON] et al. 2011 [24]. Many bacterial species show phenotypic alterations dependent of their growth phase, culture media, or other environmental factors, which is influencing the presentation of specific epitope sequences. Using the bioinformatic workflow described in this study in combination with the VP system for immunization, a targeted and reliable induction of strain-specific mAbs can be achieved. Although we choose characterized and well-defined protein targets from _E.