# A New Workflow to Generate Monoclonal Antibodies against Microorganisms
## 4 Discussion
coli_ in this pilot study, we postulate that by using structural prediction tools such as iTasser [25], it will be possible to select surface-related epitopes from less characterized target proteins as well. We previously started with the first experiments in this direction to see if our workflow is suitable for rare characterized targets as well. In the next part of the study, we developed a bacteria-specific staining and sorting protocol for antibody-producing hybridomas based on the selection principle that was published recently [16]. In this method, we used our novel transgenic myeloma cell lines, which express an artificial cell surface marker. This marker allows an antigen- or isotype-specific screening of hybridomas by linking the produced antibody to the corresponding producing mother cell. This selection principle can be performed after two weeks of HAT selection and displays an innovative new technology to identify and select specific mAb-producing hybridomas. In comparison to conventional screening methods via ELISA and the limited dilution of cells, combined with existing problems of coating ELISA plates with whole microorganisms or unspecific binding to plastic, this new approach can turn the selection process into a highly efficient task. The setup enabled a selection of nearly \(1\times 10^{5}\) positive hybridomas specific for _E. coli_ strain O157:H7 epitopes. For the fusion of EC4, we could select up to 75,000 specific hybridomas. From 55 monoclonals and 156 polyclonals plated on 96-well plates, 44 monoclonals and 154 polyclonals could be established as specific, which represents an optimal case. For the fusions EC2 and EC3, an output of 18% positive hybridoma cells could be achieved. With these results, we saw differences between the single epitopes and also the general instabilities after fusion and selection, but with this fast sorting, a high number of potential mAb candidates can be selected shortly and effectively. The obtained antibody candidates were further characterized for a possible cross-reactivity against other bacterial strains present in drinking water such as _Legionella pneumophila_, _ Bacillus_ ssp., and _E. coli_ K12 [data not shown]. Except for H4-2-F6, which shows a slight cross-reactivity to _L. pneumophila_ cells, all other antibody candidates were negative for tested bacterial species and solely positive for _E. coli_ O157:H7. It has to be noted that _E. coli_ K12 has coding genes for OmpG. However, previous studies have already shown that the expression is almost non-existent under laboratory conditions [26]. With the pilot approach shown in this study, the generation and selection of MO-specific antibodies seem to be a reliable and fast alternative without the necessity of using whole bacterial cell extracts or cell-based ELISA screenings. The generated antibodies could serve as potential candidates for POC systems in the field of food or drinking water analyses and are currently validated for upcoming developments. The following are available online at [[https://www.mdpi.com/article/10.3390/app11209359/s1](https://www.mdpi.com/article/10.3390/app11209359/s1)]([https://www.mdpi.com/article/10.3390/app11209359/s1](https://www.mdpi.com/article/10.3390/app11209359/s1)), Table S1: Primer for VPs generation, Table S2: Overview of identified surface proteins related to bacterial strain _E. coli_ O157:H7 EDL933. Conceptualization, M.G. and K.H. ; methodology, M.G., M.L., A.S. and K.M. ; software, M.G. ; validation, M.G. ; formal analysis, M.L. and M.G. ; investigation, M.G. ; resources, M.L. ; data curation, A.H.; writing--original draft preparation, M.G. and K.H. ; writing--review and editing, K.M. ; visualization, M.G. ; supervision, K.M. ; project administration, K.H. ; funding acquisition, K.H. All authors have read and agreed to the published version of the manuscript. **Funding:** This research was funded by the EU directive StaF for universities in the state of Brandenburg (85009560).