Monoclonal antibodies binding to the cell surface of Listeria monocytogenes serotype 4b.

Serotype 4b strains of the food-borne pathogen Listeria monocytogenes are responsible for a large portion of sporadic listeric infections and all major food-borne listeriosis outbreaks in humans. Hybridomas were produced from three fusions with lymphocytes of ND4 mice immunized either with the insoluble antigens of L. monocytogenes serotype 4b or with formalin-killed bacterial cells and screened for monoclonal antibodies (mAbs) reactive to L. monocytogenes serotype 4b. A set of 35 mAbs was identified by ELISA as having reactivity with both the insoluble antigen fraction and the whole-cell antigens. Thirteen of these mAbs belonged to immunoglobulin subclass G1 (IgG1), fifteen were IgG2a and seven mAbs were IgM. Only 20 out of the 35 mAbs were capable of detecting protein bands of various sizes ranging from 20 to 88 kDa in Western blots. Two of these mAbs, M2365 and M2367, were capable of binding to cell-surface antigens of live L. monocytogenes serotype 4b, as demonstrated by immunofluorescence microscopy and immunogold transmission electron microscopy. Immunofluorescence microscopy showed that M2365 and M2367 failed to bind to the cell surfaces of Escherichia coli O157 : H7, Salmonella enterica (serotype Typhimurium DT104) or Campylobacter jejuni. Evaluation of the cross-reactions of all 35 mAbs with whole-cell antigens of E. coli O157 : H7, S. Typhimurium, C. jejuni and Listeria innocua by ELISA indicated that the majority of the mAbs, including M2365 and M2367, did not cross-react with E. coli O157 : H7, S. Typhimurium or C. jejuni and showed no or a very weak reaction with L. innocua. Furthermore, M2365 and M2367 showed no reaction with whole-cell antigens derived from L. monocytogenes serotypes 1/2a, 1/2b and 3a, and from Listeria grayi, Listeria ivanovii and Listeria seeligeri, in an ELISA. Collectively, these data suggest that M2365 and M2367 have potential use in the development of immunological methods of laboratory diagnosis for L. monocytogenes serotype 4b in clinical or food samples.

Enzyme-linked immunosorbent assay based on a chimeric antigen bearing antigenic regions of structural proteins Erns and E2 for serodiagnosis of classical swine fever virus infection.

The antigenic region (residues 109 to 160) of classical swine fever virus (CSFV) protein E(rns) and the N-terminal antigenic region (residues 1 to 136) of protein E2 were constructed in the form of a fused, chimeric protein, C21E(rns)E2, for use as an enzyme-linked immunosorbent assay (ELISA) antigen for the serodiagnosis of CSFV infection. Tested with 238 negative-field (CSFV-free) sera from Canadian sources, the specificity of the ELISA was determined to be 93.7%. All 20 sera from experimentally infected pigs representing a variety of animals, virus strains, and days postinfection (dpi; range, 7 to 210) were detected as positive (100%). In contrast, an ELISA based on an E(rns) fragment (E(rns)(aa 109-160)) or an E2 fragment (E2(aa 1-221)) identified only 18 (90%) of 20 sera from infected pigs as positive, missing two targets collected at 7 dpi. These data suggest that use of the chimeric antigen C21E(rns)E2 would improve serodiagnostic sensitivity and allow for the detection of CSFV infection as early as 7 dpi.

An indirect enzyme linked immunosorbent assay for the detection of bovine antibodies to multiple Leptospira serovars.

An indirect enzyme linked immunosorbent assay was developed for the detection of bovine antibodies to multiple pathogenic Leptospira serovars, including canicola, copenhageni (represents icterohaemorrhagiae), grippotyphosa, hardjobovis, pomona, and sejroe. The antigen utilized in this assay was a sonicated mixture of equal parts of killed whole cells of each of the 6 serovars named above. A mouse monoclonal antibody against bovine immunoglobulin (Ig)G1 that was conjugated with horseradish peroxidase was used for detection of bound antibodies. This assay was evaluated with sera (n = 3107) that were microscopic agglutination test (MAT)-negative (at a 1:100 dilution) for each of the 6 serovars listed above and sera (n = 601) that were MAT-positive (at a 1:100 dilution) for 1, or any combination of the 6 listed serovars. In addition, sera from serial weekly bleedings of cows, which were individually experimentally infected with serovars hardjobovis, copenhageni, grippotyphosa, or canicola, were also tested in this assay. At an optimal cut-off point determined by receiver operating characteristic (ROC) curve analysis, the relative sensitivity and specificity of the assay were 93.5% (95% confidence interval = 91.2% to 95.3%) and 94.7% (95% confidence interval = 93.9% to 95.5%), respectively. This assay was able to detect antibody in the sera of animals experimentally infected with serovar hardjobovis as early as 1 week postinoculation.