Detection and Isolation of Yersinia pestis Without Fraction 1 Antigen by Monoclonal Antibody in Foods and Water
ABSTRACT Most available immunoassays for Yersinia pestis are based on the detection of fraction 1 antigen (F1) when yersiniae are grown at 37°C. A monoclonal antibody (MAb) was developed based on the detection of surface antigens that are not F1. F1-deficient Y. pestis cells were induced and used to immunize BALB/c mice from which MAb (immunoglobulin G1), which specifically recognizes Y. pestis, with or without F1, was obtained. This MAb (6B5) did not cross-react with enteric bacteria, including Yersinia enterocolitica. Enzyme-linked immunosorbent assay results revealed that MAb 6B5 is specific for Y. pestis, with the exception of a minor cross-reaction with Yersinia pseudotuberculosis. Western immunoblot analysis revealed that MAb 6B5 recognizes a Y. pestis outer membrane protein of ca. 30 kDa. Magnetic beads that were coated with MAb 6B5 were compared with beads coated with polyclonal antibody (PAb; rabbit) against Y. pestis for the isolation of Y. pestis in food and water samples by using a PATHATRIX cell concentration apparatus. Enrichment cultures of Y. pestis in different foods by using two different times (6 and 24 h) in brain heart infusion broth at 37°C were evaluated. Results revealed MAb 6B5-coated magnetic beads were equivalent to magnetic beads coated with PAb against Y. pestis A1122 whole cells in concentrating Y. pestis for isolation, especially when samples were enriched for 6 h. However, the selectivity for Y. pestis of the magnetic beads coated with MAb 6B5 was greater than that coated with PAb.
- SourceAvailable from: Dario De Medici[Show abstract] [Hide abstract]
ABSTRACT: Abstract Microbiological analysis is an integral part of food quality control, as well as of the management of food chain safety. Microbiological testing of foodstuffs complements the preventive approach to food safety activities based mainly on implementation and application of the concept of Hazard Analysis and Critical Control Points (HACCP). Traditional microbiological methods are powerful but lengthy and cumbersome and therefore not fully compatible with current requirements. Even more, pathogens exist that are fastidious to cultivate or uncultivable at all. Besides immunological tests, molecular methods, specifically those based on polymerase chain reaction (PCR), are available options to meet industry and enforcement needs. The clear advantage of PCR over all other rapid methods is the striking analytical principle that is based on amplification of DNA, a molecule being present in every cell prone to multiply. Just by changing primers and probes, different genomes such as bacteria, viruses or parasites can be detected. A second advantage is the ability to both detect and quantify a biotic contaminant. Some previously identified obstacles of implementation of molecular methods have already been overcome. Technical measures became available that improved robustness of molecular methods, and equipment and biochemicals became much more affordable. Unfortunately, molecular methods suffer from certain drawbacks that hamper their full integration to food safety control. Those encompass a suitable sample pre-treatment especially for a quantitative extraction of bacteria and viruses from solid foods, limited availability of appropriate controls to evaluate the effectiveness of the analytical procedure, the current inability of molecular methods to distinguish DNA from viable cells and DNA from dead or non-cultivable cells, and the slow progress of international harmonisation and standardisation, which limit full acceptance of PCR-based methods in food control. The aim of this review is to describe the context and the prospects of PCR-based methods, as well as trends in research and development aimed at solving the next decade challenges in order to achieve full integration of molecular methods in food safety control.Food Analytical Methods 07/2014; DOI:10.1007/s12161-014-9915-6 · 1.80 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Yersinia pestis which is the causative agent of pneumonic plague and distributed in all continents has led to many deaths during the history. Because of its high mortality rate, it must be diagnosed and treated at the earliest time post infection and therefore, rapid diagnostic tests are required. In the present study, we cloned the coding sequence of F1 capsular antigen of the bacteria in the pBAD/gIII plasmid for later expression and purification of the protein to produce poly and monoclonal antibodies against this antigen, and subsequently to develop rapid and efficient diagnostics tools for Y. pestis infections.Research in pharmaceutical sciences 11/2014; 10(1):75-80.