A New Workflow to Generate Monoclonal Antibodies against Microorganisms

Abstract

Monoclonal antibodies are used worldwide as highly potent and efficient detection reagents for research and diagnostic applications. Nevertheless, the specific targeting of complex antigens such as whole microorganisms remains a challenge. To provide a comprehensive workflow, we combined bioinformatic analyses with novel immunization and selection tools to design monoclonal antibodies for the detection of whole microorganisms. In our initial study, we used the human pathogenic strain E. coli O157:H7 as a model target and identified 53 potential protein candidates by using reverse vaccinology methodology. Five different peptide epitopes were selected for immunization using epitope-engineered viral proteins. The identification of antibody-producing hybridomas was performed by using a novel screening technology based on transgenic fusion cell lines. Using an artificial cell surface receptor expressed by all hybridomas, the desired antigen-specific cells can be sorted fast and efficiently out of the fusion cell pool. Selected antibody candidates were characterized and showed strong binding to the target strain E. coli O157:H7 with minor or no cross-reactivity to other relevant microorganisms such as Legionella pneumophila and Bacillus ssp. This approach could be useful as a highly efficient workflow for the generation of antibodies against microorganisms.

Automated determination of 8-OHdG in cells and tissue via immunofluorescence using a specially created antibody

“…Fourteen days after fusion, antigen-specific screening was performed with hybridoma cells as previously described [56] , [57] , [58] . Before sorting, the presence of biotinylated artificial surface receptors on hybridoma cells was checked by flow cytometry using phycoerythrin-conjugated streptavidin (SAV, Merck , Darmstadt, Germany).…”

Response to Comments on “Monoclonal Antibody Discovery Based on Precise Selection of Single Transgenic Hybridomas with an On-Cell-Surface and Antigen-Specific Anchor”

“…We agree that the transgenic fusion cell line first constructed by Listek’s group is great work for hybridoma cell selection based on the flow cytometry sorting strategy . The authors stated that the hapten of FITC with a single epitope and the protein of OVA and pathogenic strain of E. coli O157:H7 with multiple epitopes as model molecules were used to select the antigen-specific hybridoma cells in Listek’s work in 2020 and 2021 . However, they just verified the cell supernatant of a transgenic hybridoma by enzyme linked immunosorbent assay (ELISA), while the antibody produced by ascites induction or in vitro expression were not evaluated, and they did not purify the antibody and analyze the performances of the antibody.…”

Comment on “Monoclonal Antibody Discovery Based on Precise Selection of Single Transgenic Hybridomas with an On-Cell-Surface and Antigen-Specific Anchor”

“…The selection of hybridomas producing E. coli -specific antibodies was published previously in ref . Briefly, 1 × 10 6 cells were incubated with a mouse-specific antibody capture matrix (goat-anti mouse IgG coupled to avidin) for 20 min at 4 °C.…”

“…To get rid of cold capturing events during staining, we preferred to use a self-build antibody-capture matrix to catch released antibodies and detect the cells with fluorescently labeled antigen and/or Tag-specific antibodies as published in refs and . This setup is shown in Figures – in different variants.…”

“…The selection system, which was named selma, is meanwhile broadly accessible. , Since 2020, we developed the system further for different antigen classes such as proteins, haptens, and microorganisms. One recent publication from our group by Göthel et al in 2021 describes the use of the transgenic Sp2/0 cells to identify potent mAbs against E. coli (Figure , published in ref ). By using an in silico epitope prediction combined with selma as a selection tool, a comprehensive and fast workflow was established to generate species-specific antibodies with minor cross-reactivity to other related microorganisms.…”