Characterization of surface proteins of Cronobacter muytjensii using monoclonal antibodies and MALDI-TOF Mass spectrometry

Abstract

BackgroundCronobacter spp. is a newly emerging pathogen that causes meningitis in infants and other diseases in elderly and immunocompromised individuals. This study was undertaken to investigate surface antigenic determinants in Cronobacter spp. using monoclonal antibodies (MAbs) and MALDI-TOF Mass spectrometry.ResultsSpleenocytes from mice that were immunized with heat-killed (20 min, 80°C) Cronobacter cells were fused with SP2 myeloma cells. Five desirable MAbs (A1, B5, 2C2, C5 and A4) were selected. MAbs A1, B5, 2C2 and C5 were of IgG2a isotype while A4 was an IgM. Specificity of the MAbs was determined by using immunoblotting with outer membrane protein preparations (OMPs) extracted from 12 Cronobacter and 6 non-Cronobacter bacteria. All MAbs recognized proteins with molecular weight ranging between 36 and 49 kDa except for one isolate (44) in which no OMPs were detected. In addition, MAbs recognized two bands (38-41 kDa) in four of the non-Cronobacter bacteria. Most of the proteins recognized by the MAbs were identified by MALDI-TOF peptide sequencing and appeared to be heterogeneous with the identities of some of them are still unknown. All MAbs recognized the same epitope as determined by an additive Index ELISA with their epitopes appeared to be conformational rather than sequential. Further, none of the MAbs recognized purified LPS from Cronobacter spp. Specificity of the MAbs toward OMPs was further confirmed by transmission electron microscopy.ConclusionsResults obtained in this study highlight the immunological cross-reactivity among Cronobacter OMPs and their Enterobacteriaceae counterparts. Nevertheless, the identity of the identified proteins appeared to be different as inferred from the MALDI-TOF sequencing and identification.

Detection of Cronobacter Genus in Powdered Infant Formula by Enzyme-linked Immunosorbent Assay Using Anti-Cronobacter Antibody

“…As a result, IgG developed by LPS immunogen showed no titer for any Cronobacter spp. These results agree with previous findings of Luk and Lindberg (1991) and Jaradat et al (2011) , who were unable to obtain stable antibodies against LPS from Cronobacter and Salmonella due to the structure and composition of LPS obtained from bacterial cells of Cronobacter and Salmonella .…”

Strategies for the Identification and Tracking of Cronobacter Species: An Opportunistic Pathogen of Concern to Neonatal Health

“…Hochel and Škvor ( 14 ) developed an indirect competitive enzyme immunoassay for the detection of Cronobacter species using polyclonal antibodies. The surface antigenic determinants in Cronobacter species using monoclonal antibodies (MAbs) and MALDI-TOF Mass spectrometry were also investigated ( 15 ). A sandwich ELISA was undertaken to detect C. muytjensii in PIF ( 16 ).…”

Diversity of O Antigens within the Genus Cronobacter: from Disorder to Order

“…LPS was prepared following the method described by Jaradat et al (37) with minor modifications. Briefly, cells (250 ml) were harvested in the exponential phase of growth by centrifugation (5,000 ϫ g, 10 min), washed 3 times, and resuspended in 5 ml of 50 mM sodium phosphate buffer (pH 7.0).…”