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ABSTRACT: Immunosensors based on the microgravimetric quartz crystal microbalance (QCM) technique have been developed for the detection of Salmonella species from serogroups A, B and D. Salmonella serogroup-specific murine monoclonal antibodies, respectively, raised against these serogroups were immobilized onto the silver electrodes of piezoelectric (PZ) crystals by cross-linkage via glutaraldehyde (GA) to the electrode surfaces pre-coated with thin polyethyleneimine (PEI) layer. The specific immunosensors developed gave responses in linear ranges from 10(5) to 5x10(8) cells per ml with no significant interference from other strains of Salmonella and Escherichia coli up to 10(8) cells per ml. They showed good repeatability and excellent linear range, achieving detection limits down to 10(4) cells per ml with ability to distinguish different strains of Salmonella. These biosensors exhibited an exquisite specificity evidenced by their ability to discriminate antigens, the structures of which differ only by the isomeric form of di-deoxyhexose. The antibody-modified crystals showed no loss in activity over 4 days under storage at 4 degrees C.
Biosensors and Bioelectronics 09/2002; 17(8):676-84. · 5.60 Impact Factor
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ABSTRACT: Serum samples were taken from 57 patients with sporadic non-A, -B, and -C (Non A, B, C) acute hepatitis at different times after onset of the disease and tested for the presence of the hepatitis E virus (HEV) RNA, IgM, and low avidity IgG antibodies. The viral antibodies were detected using two ELISA. One assay (GL) was produced using a mixture of recombinant peptides specified by ORF2 and ORF3 of the viral genome. The other was produced with an ORF2 specified peptide, pE2. The latter occurs naturally as homodimer, it is recognized strongly in its dimeric form by human sera and, in the primate model, it confers protection against experimental HEV infection. Nineteen samples were positive for one or more of these acute markers of HEV infection, 14 of which were acute sera with elevated ALT levels and 5 were convalescent sera with normal ALT level. The results showed that icteric phase of sporadic hepatitis lasts for about 17 days and it coincides with a period when viremia is subsiding as HEV antibodies are developing. Viremia was intermittent and all but one of the 5 instances were confined to the icteric phase with elevated ALT levels. On two of these occasions, viremia preceded detection of HEV antibody, on another 2 occasions it was concurrent with the detection of pE2 specific IgM and/or low avidity IgG and only in one case of protracted viremia was the viral genome detected concurrently with avid pE2 IgG antibody. Ten (71%) of the 14 acute sera were reactive for pE2 IgM, eight (57%) were reactive for low avidity pE2 IgG, and six (43%) for the GL IgM. The sensitivity for the diagnosis of acute hepatitis E may be increased to 87% by combining pE2 IgM and viremia. GL IgM was detected later, but persisted for a longer period of time than the pE2 antibodies, and it was the only acute antibody detected in the convalescent sera.
Journal of Medical Virology 02/2002; 66(1):40-8. · 2.82 Impact Factor
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ABSTRACT: Serum samples were taken from 57 patients with sporadic non-A, -B, and -C (Non A, B, C) acute hepatitis at different times after onset of the disease and tested for the presence of the hepatitis E virus (HEV) RNA, IgM, and low avidity IgG antibodies. The viral antibodies were detected using two ELISA. One assay (GL) was produced using a mixture of recombinant peptides specified by ORF2 and ORF3 of the viral genome. The other was produced with an ORF2 specified peptide, pE2. The latter occurs naturally as homodimer, it is recognized strongly in its dimeric form by human sera and, in the primate model, it confers protection against experimental HEV infection. Nineteen samples were positive for one or more of these acute markers of HEV infection, 14 of which were acute sera with elevated ALT levels and 5 were convalescent sera with normal ALT level. The results showed that icteric phase of sporadic hepatitis lasts for about 17 days and it coincides with a period when viremia is subsiding as HEV antibodies are developing. Viremia was intermittent and all but one of the 5 instances were confined to the icteric phase with elevated ALT levels. On two of these occasions, viremia preceded detection of HEV antibody, on another 2 occasions it was concurrent with the detection of pE2 specific IgM and/or low avidity IgG and only in one case of protracted viremia was the viral genome detected concurrently with avid pE2 IgG antibody. Ten (71%) of the 14 acute sera were reactive for pE2 IgM, eight (57%) were reactive for low avidity pE2 IgG, and six (43%) for the GL IgM. The sensitivity for the diagnosis of acute hepatitis E may be increased to 87% by combining pE2 IgM and viremia. GL IgM was detected later, but persisted for a longer period of time than the pE2 antibodies, and it was the only acute antibody detected in the convalescent sera. J. Med. Virol. 66:40–48, 2002. © 2002 Wiley-Liss, Inc.
Journal of Medical Virology 12/2001; 66(1):40 - 48. · 2.82 Impact Factor
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ABSTRACT: A monoclonal antibody (MAb), MO15, was raised against the lipopolysaccharide antigen of an epsilon15-lysogenized serogroup E(1) Salmonella strain. The O factor 15-specific MAb MO15, together with another serogroup E-specific MAb, can differentiate among phage lysogenization variants in serogroup E salmonellae. Their epitope specificities in relation to conventional O-antigenic structures are discussed.
Applied and Environmental Microbiology 01/2000; 66(1):419-21. · 3.83 Impact Factor
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ABSTRACT: We previously described an antigen capture enzyme-linked immunosorbent assay which makes use of monoclonal antibody T6, which recognizes an epitope on the outer core polysaccharide of Salmonella lipopolysaccharide molecules that is common to almost all Salmonella serovars. In this paper, we show that this assay can detect between 10(5) and 10(7) Salmonella cells per ml even in the presence of excess Escherichia coli. A total of 153 of 154 (99%) serogroup A to E strains and 51 of 78 (71%) serogroup F to 67 strains were reactive as determined by this assay. This corresponds to a detection rate of approximately 98% of all salmonellae known to affect humans. None of the 65 strains of non-Salmonella bacteria tested positive. Taking advantage of the O-factor polysaccharides also present on the antigen captured by the immobilized T6 antibody, we showed that 136 of 154 Salmonella serogroup A to E strains (88%) were correctly differentiated according to their serogroups by use of enzyme conjugates of a panel of O-factor-specific monoclonal antibodies. We evaluated this assay for the detection and serogroup differentiation of salmonellae directly from enrichment cultures of simulated food, eggs, pork, and infant formula milk. All 26 samples which had been contaminated with Salmonella spp. were detected by T6 (100% sensitivity), with only one false-positive result from 101 samples not contaminated by Salmonella spp. (99% specificity). The detection time was substantially reduced to between 17 and 29 h, depending on the enrichment methods used. Since there were no false-negative results, we concluded that this enrichment-immunoassay method can afford rapid screening for Salmonella spp. in food samples.
Applied and Environmental Microbiology 08/1996; 62(7):2294-302. · 3.83 Impact Factor
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ABSTRACT: Immunosensors based on the microgravimetric quartz crystal microbalance (QCM) technique have been developed for the detection of Salmonella species from serogroups A, B and D. Salmonella serogroup-specific murine monoclonal antibodies, respectively, raised against these serogroups were immobilized onto the silver electrodes of piezoelectric (PZ) crystals by cross-linkage via glutaraldehyde (GA) to the electrode surfaces pre-coated with thin polyethyleneimine (PEI) layer. The specific immunosensors developed gave responses in linear ranges from 105 to 5×108 cells per ml with no significant interference from other strains of Salmonella and Escherichia coli up to 108 cells per ml. They showed good repeatability and excellent linear range, achieving detection limits down to 104 cells per ml with ability to distinguish different strains of Salmonella. These biosensors exhibited an exquisite specificity evidenced by their ability to discriminate antigens, the structures of which differ only by the isomeric form of di-deoxyhexose. The antibody-modified crystals showed no loss in activity over 4 days under storage at 4 °C.
Biosensors and Bioelectronics.
