Generation and characterization of murine monoclonal antibodies to recombinant YopM, YopB and LcrV proteins of Yersinia pestis Rekha Khushiramani, Urmil Tuteja, Jyoti Shukla, Anupama Panikkar and H.V. Batra *,  Division of Microbiology, Defence R & D Establishment, Jhansi road, Gwalior 474002, India * Author for correspondence: Tel.: +91-0751-2233491, Fax:+91-0751-241148, E-mail: h_v_batra@rediffmail.com   Present address: Microbial Containment Complex, Sus Road, Pashan, Pune 21 Received 8 October 2004; accepted 30 November 2004 Keywords: Monoclonal antibody, Y. pestis, Yops Summary YopM, an effector, YopB, a translator, and LcrV, a regulator, are proteins forming important componants of type III secretion system of Yersinia pestis. Recombinant truncated YopM of 32 kDa, YopB of 28 kDa and LcrV of 31 kDa sizes were utilized for priming BALB/c mice for the generation of monoclonal antibodies following standard poly-ethylene glycol (PEG) fusion protocol. Nine, 10 and 6 stabilized hybridoma cell lines could be generated against YopM, YopB and LcrV proteins, respectively. All these monoclonal antibodies were found reactive to Y. pestis strain A1122 and did not show any cross-reactivity to Y. enterocolitica, Y. pseudotuberculosis, Y. kristensenii, Y. frederiksenii, Y. intermedia, Klebsiella pneumoniae, Escherichia coli, Salmonella typhi, Salmonella abortus-equi and Staphylococcus aureus tested by ELISA and Western blotting. Monoclonal antibodies also exhibited reactivity to their corressponding native protein antigens in Y. pestis i.e. 42 kDa for YopM, 41 kDa for YopB and 37 kDa for LcrV in immunoblotting. Reactivity of monoclonal antibodies was further assessed on 26 Y. pestis isolates including 18 from 1994 plague outbreak regions (11 from pneumonic patients, 7 from rodents) and 8 from rodents of Deccan plateau of Southern India by Western blotting as well as by sandwich ELISA. The monoclonal antibodies could specifically locate the expression of yopM, yopB and lcrV genes among these Indian Y. pestis strains as well. Results obtained with sandwich ELISA and Western blot were identical to those observed by PCR. Monoclonal antibodies to Yops, therefore, can be employed for an early and reliable identification of virulent Y. pestis strains. Introduction Yersinia pestis, the causative organism of bubonic and pneumonic plague uses a type III secretion system for the translocation of effector molecules into the target host cells. The type III secretion system of Y. pestis secretes the majority of virulence factors including the 12 Yersinia outer proteins (Yops), Yop secretion proteins (Ysc), a few specific Yop chaperons (Syc) that are contributed by the 70 kb middle plasmid (Cornelis 2000). A uniform nomenclature has been introduced for yopB, yopD, yopE, yopH, yopJ, yopM, yopO, yopT and lcrV genes and their respective proteins. The LcrV protein, known since the mid- 1950s is a protective antigen against plague. YopE and YopT are responsible for cytotoxicity. YopH was found to inhibit phagocytosis of bacteria by macro- phages (Perry & Fetherston 1997). YopJ induces apoptosis, YopO inhibits signal transduction. The YopM protein is among the major virulence effectors (HOPEMT) responsible for anti-phagocytic response during the time of infection. YopB, YopD and YopK are the translocators that help in translocation of effector Yops. Likewise, the LcrV protein acts as a regulator of low calcium response and is also immu- nogenic (Cornelis 2000). Among the various Yops, murine monoclonal antibodies have been reported for only LcrV protein using conventionally purified V antigen for priming of mice. These monoclonal antibodies to V antigen have been attempted for use in therapy, immunogenicity and epitope mapping studies but not for detection of Y. pestis and its strains (Sato et al. 1991; Hill et al. 2003). Generation of murine monoclonal antibodies to three important Yops (YopM, YopB and LcrV) in the present study was undertaken for developing simple immunoassays for identification and strain typing of Y. pestis, employing truncated recombinant proteins as antigen for the sensitization of mice and for screening of hybridoma. The monoclonal antibodies thus generated could recognize their corresponding epitopes in the Y. pestis strains and could also be utilized in simple immunoassays (ELISA and Western blot) for detect- ing the expression of these proteins. World Journal of Microbiology & Biotechnology (2005) 21: 955–960 Ó Springer 2005 DOI 10.1007/s11274-004-6984-5