PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST THE RECOMBINANT P60 ANTIGEN OF LISTERIA MONOCYTOGENES
Keywords:
listeriosis, Listeria monocytogenes, p60, recombinant antigen, monoclonal antibodies, hybridoma, enzyme-linked immunosorbent assayAbstract
Listeriosis is an infectious disease affecting humans and animals caused by Listeria monocytogenes. One promising approach to improving immunological methods for pathogen detection is the production of specific monoclonal antibodies against immunogenic bacterial proteins. The aim of this study was to generate and characterize monoclonal antibodies against the recombinant p60 antigen of L. monocytogenes. Purified recombinant p60 protein was used for immunization of BALB/c mice. Immune splenocytes were fused with X63-Ag 8.6.5.3 myeloma cells using PEG-1500. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA). Following five independent hybridizations, 121 (10.1%) of 1,198 hybridomas showed primary specific reactivity to recombinant p60. Multi-stage screening resulted in the selection of seven stable hybridoma clones producing specific monoclonal antibodies. Antibodies produced by clones 1H8 and 2A3 exhibited the highest activity, with titers of 1:256 in culture supernatants and 1:204,800 in ascitic fluid. No cross-reactivity with the tested heterologous recombinant antigens was detected. Western blot analysis confirmed the specific recognition of recombinant p60 by monoclonal antibodies produced by clone 1H8. The obtained monoclonal antibodies represent promising candidates for the further development of immunodiagnostic reagents for the detection of L. monocytogenes.
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