Monospecific and bispecific antibodies against E. coli O157 for diagnostics.

Escherichia coli O157:H7 is a serious human pathogen that causes hemorrhagic colitis, and occasionally hemolytic uremic syndrome. Identification of the O157 antigen is an essential part of the detection and management of E. coli O157:H7. A quadroma P126 secreting a bispecific hybrid MAb (bsMAb), which recognizes both E. coli O157 and horseradish peroxidase in one molecule was produced by somatic hybridization of hybridomas specific for E. coli O157 and HRPO molecule. A bridge ELISA was used to select the quadromas obtained for bispecific monoclonal antibody purification and characterization. Benzhydroxamic-acid agarose (BHA) affinity co-chromatography was used as a convenient one-step method for purifying the HRPO-bsMAb complex for ultrasensitive diagnostic applications. Sandwich ELISA for detecting E. coli O157:H7 with HRPO-bsMAb allows quick one step detection of spiked E. coli O157:H7. The detection sensitivities were 100 CFU, 750 CFU and 500 CFU per 1 ml of tap water, lake water and apple juice respectively by microtiter assay. E. coli O157:H7 detection with immunofilter ELISA and immunomagnetic ELISA formats was approximately 1 CFU/ml and 10 CFU/ml respectively. BsMAbs avoid enzyme conjugation, has highest specific activity and molecular uniformity without aggregates and contribute to good signal to noise ratios. This new bispecific antibody can be generated and purified from quadroma cultures by affinity co-chromatography in one step and can be used to develop a new generation of assays for public health applications in water, food and human sample testing.

Production and characterization of monoclonal antibodies against shope fibroma virus superoxide dismutase and glutathione-s-transferase.

PURPOSE: The superoxide dismutase (SOD) like proteins encoded by Leporipoxviruses play a role in regulating the redox status of infected cells. The biological function of these proteins is unclear. Why poxviruses encode these proteins are still unknown. Exploiting standard hybridoma techniques, we developed a monoclonal antibody (MAb) against shope fibroma virus superoxide dismutase (sfvSOD) to be used in diagnostics and as tools to understand the role of SOD-like proteins in pathogenesis. METHODS: Hybridoma cell fusion technology was used for production of MAbs. Balb/c mice were immunized with sfvSOD-GST fusion protein. Hybridoma clones were screened using indirect enzyme linked immunosorbent assay (ELISA). Specificity and reactivity of the MAbs were determined by Western blot analysis (WBA) and indirect ELISA. Protein G affinity chromatography was used for the purification of MAbs. RESULTS: Two stable hybridoma clones producing MAbs against the two domains of the fusion protein were obtained. The anti-GST (glutathione-s-transferase) and anti-sfvSOD MAbs were found to react specifically with GST and sfvSOD proteins respectively, in addition to the sfvSOD-GST fusion protein. Isotypes of these MAbs were identified as IgG2b heavy chain and k light chain. CONCLUSION: The anti-sfvSOD MAb (P115.SOD MAb) has been successfully used in studying the enzymatic and biochemical properties of a SOD homolog encoded by sfv. We also developed a strong anti-GST MAb which was also cloned and characterized P115.GST MAb. The anti-GST MAb might be useful in analyzing GST fusion proteins and in immunoaffinity chromatography purification of GST fusion proteins.

Molecular zipper assays: a simple homosandwich with the sensitivity of PCR.

PURPOSE: The purpose of this study is to develop a simple and inexpensive method for detection of viral load or antigens present in the body fluids as for diagnosis or monitoring of infectious diseases. For example, in case of viral infection, nucleic acid based quantitative PCR/RTPCR are sensitive in measuring viral load to follow the course of therapy or infection. The key limitations of such assays include the need for sample extraction, susceptibility to inhibitors, and high cost. METHODS: A molecular zipper assay based on the simple homosandwich concept for repeated epitopes was developed where the analyte or virus is sandwiched between the same antibodies for detection. A comparative study of the lower limit of detection of M13 model virus was performed with various substrates. RESULT: Homosandwich molecular zipper assay captured the model virus with high avidity resisting multiple rounds of washing. Detection of the virus by enzyme labeled MAb in combination with chemiluminescent substrates provided practical assay sensitivities of 7-15 phages and a theoretical detection sensitivity of one virus particle. CONCLUSION: The significance of our results on the molecular zipper assay relates to the development of ultrasensitive pathogen assays at low cost. Such assays could be developed for pathogenic bacteria and viruses, especially HIV & HCV viruses, which are ravaging impoverished continents of Africa, Asia and Latin America.