ORIGINAL PAPER MarõÂa J. Pe rez-Alvarez á Ruth Larreta á Carlos Alonso Jose M. Requena Characterisation of a monoclonal antibody recognising speci®cally the HSP70 from Leishmania Received: 26 April 2001 / Accepted: 11 May 2001 / Published online: 7 July 2001 Ó Springer-Verlag 2001 Abstract Heat-shock protein 70 HSP70) is ubiquitously distributed along the evolutionary scale and has such an amino acid sequence conservation that it is considered the most evolutionarily conserved protein. In order to obtain immunological tools speci®c against Leishmania infantum HSP70, hybridomas were established that secreted monoclonal antibodies mAbs) against recom- binant L. infantum HSP70. One of them, named mAb 2B8D2, speci®cally reacted with the Leishmania protein and did not recognise HSP70 from the related kineto- plastid Trypanosoma cruzi. The use of synthetic peptides allowed us to determine the B-cell epitope recognised by this mAb, an epitope located in the divergent C-terminal domain of the protein. Remarkably, the mAb possesses the capacity to immunoprecipate HSP70 from promas- tigote extracts of L. infantum. The fact that human HSP70 is not recognised by this mAb assures the use- fulness of this antibody for diagnostic purposes and studies involving Leishmania infection of macrophages. Introduction Parasitic protozoa of the genus Leishmania, causative agents of leishmaniasis, exist as extracellular ¯agellated promastigotes within the gut of their sand¯y vectors and as intracellular a¯agellated amastigotes within the phagolysosomes of the mammalian host's macrophages. Transmission from the poikilothermic insect vector into a mammalian host includes a temperature upshift, which is a key trigger for the promastigote-to-amastigote dif- ferentiation reviewed by Zilberstein and Shapira 1994). Consequently, heat-shock proteins HSPs), whose ex- pression is induced by the temperature increase, are believed to play a role in the Leishmania morphological transition. Among HSPs, HSP70 constitutes the most conserved protein known to date that is present in all organisms Gupta and Golding 1993). HSP70s are involved in relevant physiological functions within cells, such as protein-folding and maturation Rassow et al. 1997). Furthermore, HSP70s have been described as dominant antigens of infectious agents, suggesting that they may play an important role in the host±parasite relationship. In particular, it has been demonstrated that the Leishmania HSP70 is a major target of the humoral response during human leishmaniasis MacFarlane et al. 1990; Quijada et al. 1998). In addition, a recent report has pointed out that L. infantum HSP70 is endowed with a remarkable capacity to stimulate the immune system of mice Rico et al. 1998). To build tools that may help us to understand the role of HSP70 in both Leishmania dierentiation and parasitic infection, we set forth the production of speci®c monoclonal antibodies mAbs) against this protein. Al- though commercially available antibodies exists that recognise Leishmania HSP70, they also bind to the host homologue. Here, we describe the production and characterisation of a mAb directed speci®cally against Leishmania HSP70 that does not recognise human HSP70 or even the HSP70 from the related parasite Trypanosoma cruzi. Materials and methods Recombinant proteins The construction of plasmids expressing the Leishmania infantum HSP70 LiHSP70) and the maltose-binding protein MBP)-Nt70 and MBP-Ct70 recombinant proteins have been described else- where Rico et al. 1998, 1999). MBP-Nt70 and MBP-Ct70 contain the L. infantum HSP70 amino-terminal and carboxyl-terminal do- mains, respectively, fused to the carboxyl-end of the Escherichia coli MBP. In order to express the human HSP70 HuHSP70) as a recombinant protein, the corresponding gene was cloned into the Parasitol Res 2001) 87: 907±910 DOI 10.1007/s004360100456 M.J. Pe rez-Alvarez á R. Larreta á C. Alonso á J.M. Requena &) Centro de BiologõÂa Molecular ``Severo Ochoa'', Universidad AutoÂnoma de Madrid, 28049 Madrid, Spain E-mail: jmrequena@cbm.uam.es Fax: +34-91-3974799