Pathogen-specific antigen target for production of antibodies produced by comparative genomics

Abstract

Listeria monocytogenes continues to be a major public health risk and there is a need for improved rapid detection methods. New and highly specific L. monocytogenes antibodies are needed to advance current detection and meet the needs of industry. This research compared the L. monocytogenes genome with that of L. innocua (a nonpathogenic species of Listeria) and identified nine surface proteins specific to L. monocytogenes. Protein sequences were collected from the database and properties such as hydropathy profile and transmembrane topology were analyzed using TMpred software. Nine peptide sequences were chosen, and synthetic peptides were made and administered to rabbits for antibody production. All nine antibodies were screened against a panel of L. monocytogenes, nonpathogenic Listeria, and nonListeria bacteria. Two of the nine antibodies, ie, the Lm404 and LmC639 polyclonal antibodies, showed a specific reaction to L. monocytogenes internalin B and actin polymerization protein, respectively, and were characterized further by enzyme-linked immunosorbent assay, Western blot, and transmission electron microscopy. In Western blot, both antibodies reacted with the targeted protein and did not cross-react with other Listeria spp. The Lm404 polyclonal antibody showed a high reaction with the panel of 41 L. monocytogenes strains while the LmC639 polyclonal antibody showed a weak reaction. Both the Lm404 and LmC639 polyclonal antibodies showed potential for use in immunoassays for specific detection of L. monocytogenes. This study further indicates that comparative genomics could be used to select pathogen-specific antigen for antibody production.

Protocol to reveal the binding partner of secreted housekeeping enzymes in Listeria monocytogenes via in silico prediction to in vivo validation

“… 9 N/A Antibodies Rabbit polyclonal α-InlB (Lm404) (1:1,000 dilution) Our lab, Lathrop et al. 10 , 11 N/A Mouse monoclonal α-LAP (EM-H7) (1:2,000 dilution) Our lab, Pandiripally et al. 12 N/A Goat anti-rabbit HRP conjugated IgG Cell Signaling Technology Cat # 7074, RRID: AB_2099233 Horse anti-mouse HRP conjugated IgG Cell Signaling Technology Cat # 7076, RRID: AB_330924 Critical commercial assays and chemicals Pierce BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23225 Halt protease inhibitor cocktail (100X) Thermo Fisher Scientific Cat# 78429 Phusion high-fidelity DNA polymerase NEB Cat# M0530 Platinum Hot Start PCR master mix (2X) Thermo Fisher Scientific Cat# 130000 T4 DNA ligase NEB Cat# M0202S BamHI NEB Cat# R0136 SphI NEB Cat# R0182 DNA clean & concentrator-5 Zymo Cat# D4013 ZymoPURE plasmid miniprep Zymo Cat# D4208T NEB-5-alpha competent E. coli NEB Cat# C2987H Softwares and algorithms SnapGene GSL Biotech Version 6.0.2; https://www.snapgene.com/support/downloads ClusPro Boston University and Stony Brook University Version 2.0; htt...…”