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Pyruvate dehydrogenase complex-enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies

GND
1225388163
Affiliation/Institute
Abteilung Biotechnologie, Institut für Biochemie, Biotechnologie und Bioinformatik, Technische Universität Braunschweig
Moreira, Gustavo Marçal Schmidt Garcia;
GND
1225396190
Affiliation/Institute
Abteilung Biotechnologie, Institut für Biochemie, Biotechnologie und Bioinformatik, Technische Universität Braunschweig
Köllner, Sarah Mara Stella;
GND
1175043907
Affiliation/Institute
Abteilung Biotechnologie, Institut für Biochemie, Biotechnologie und Bioinformatik, Technische Universität Braunschweig
Helmsing, Saskia; Jänsch, Lothar; Meier, Anja; Gronow, Sabine; Boedeker, Christian;
GND
111685311
Affiliation/Institute
Abteilung Biotechnologie, Institut für Biochemie, Biotechnologie und Bioinformatik, Technische Universität Braunschweig
Dübel, Stefan; Mendonça, Marcelo; Moreira, Ângela Nunes; Conceição, Fabricio Rochedo;
GND
124252605
Affiliation/Institute
Abteilung Biotechnologie, Institut für Biochemie, Biotechnologie und Bioinformatik, Technische Universität Braunschweig
Hust, Michael

The genus Listeria comprises ubiquitous bacteria, commonly present in foods and food production facilities. In this study, three different phage display technologies were employed to discover targets, and to generate and characterize novel antibodies against Listeria: antibody display for biomarker discovery and antibody generation; ORFeome display for target identification; and single-gene display for epitope characterization. With this approach, pyruvate dehydrogenase complex-enzyme 2 (PDC-E2) was defined as a new detection target for Listeria, as confirmed by immunomagnetic separation-mass spectrometry (IMS-MS). Immunoblot and fluorescence microscopy showed that this protein is accessible on the bacterial cell surface of living cells. Recombinant PDC-E2 was produced in E. coli and used to generate 16 additional antibodies. The resulting set of 20 monoclonal scFv-Fc was tested in indirect ELISA against 17 Listeria and 16 non-Listeria species. Two of them provided 100% sensitivity (CI 82.35-100.0%) and specificity (CI 78.20-100.0%), confirming PDC-E2 as a suitable target for the detection of Listeria. The binding region of 18 of these antibodies was analyzed, revealing that ≈ 90% (16/18) bind to the lipoyl domains (LD) of the target. The novel target PDC-E2 and highly specific antibodies against it offer new opportunities to improve the detection of Listeria.

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