Genus-specific epitope on the 60-kilodalton Legionella heat shock protein recognized by a monoclonal antibody

Abstract

A monoclonal antibody (MAb) immunoglobulin G2a (2125) was produced against a 60-kDa Legionella heat shock protein (HSP), recognizing a unique epitope common to all species of the genus Legionella. The antibody reacted in the immunoblot with 59 Legionella species and serogroups that were tested and showed no cross-reactivity with other bacteria, including Acinetobacter spp., Bordetella spp., Pseudomonas spp., Mycobacterium spp., and Escherichia coli. Two other MAbs (2122 and 2130) reacted with the 60-kDa Legionella protein as well but showed different cross-reactivities with other gram-negative bacteria in the same molecular mass range. The genus-specific MAb 2125 as well as the cross-reacting MAbs 2122 and 2130 were shown to be reactive with the expressed protein of the cloned gene of the 60-kDa HSP of Legionella micdadei and Legionella pneumophila. These antibodies demonstrate that Legionella-specific and nonspecific epitopes are present on this protein. A sandwich enzyme-linked immunosorbent assay (ELISA) in which the genus-specific MAb is used both as a capture antibody and as a biotinylated second antibody has been established. With this test it is possible to detect Legionella whole cells, sonicated cells, and cell fractions containing the 60-kDa HSP. The main part of the 60-kDa HSP is found in the cytoplasmic fraction. The sandwich ELISA can be used to demonstrate the increased expression of the 60-kDa protein in Legionella cells following heat shock as well as marked differences in the detection of the 60-kDa HSP on whole cells of different Legionella strains. The high specificity and sensitivity of the sandwich ELISA for sonicated cells might be very useful to screen on a genus level for Legionella cells or the 60-kDa antigen in environmental isolates or body fluids of patients.

Cloning and sequencing of the genes coding for the 10- and 60-kDa heat shock proteins from Pseudomonas aeruginosa and mapping of a species-specific epitope

“…Although the most striking features of hsp60 molecules are their immunudominance and broad cross-reactivity, it has been shown that these proteins also contain genus-and species-specific epitopes (31). The monoclonal antibody, MAb 2528, used in this study was shown in extensive tests to recognize a species-specific epitope on the hsp60 of P. aeruginosa (2).…”

Antigenic and genetic characterization of Leaionella Proteins: Contribution to taxonomy, diagnosis and pathogenesis

“…This is similar to the organization of the groE operon in E. coli, whereas the analogous proteins in M tuberculosis are expressed from different operons (Shinnick 1987, Shinnick et al 1989. We could, however, not demonstrate any convincing heat-shock induced increase in expression of the 60 kL3 L. micdadei GroEL in the recombinant by Western blotting with a Legionella CA-specific monoclonal antibody (Steinmetz et al 1991). Radioactive incorporation of 35Smethionin during heat shock did result in a stronger expression of a 60 kD protein, which, however, could represent both the E. coli and the L. micdadei GroEL.…”

“…The same investigators raised three monoclonal antibodies against the L. pneumophila CA (Sampson et al 1991), but none of these were 100 YO specific to the Legionellaceae, and their use in a diagnostic test has not yet been presented. German researchers produced a monoclonal antibody (Steinmetz et al 1991) reactive with a genus-specific epitope on the L. pneumophila CA. This antibody (MAb 2125) did not perform adequately in an immunofluorescence test, but was found to be usell in a membrane immunoassay for identifLing Legionella colonies (Steinmetz et a1.…”

Rapid identification of legionellae by a colony blot assay based on a genus-specific monoclonal antibody

“…L. pneumophila strains 040741 and 038695b.. legionellae without cultivation (6). In this study, we developed a straightforward and rapid colony blot assay based on this genus-specific MAb which makes it possible to screen hundreds of colonies in one test and to identify legionellae growing on agar medium in less than 2 h. Table 1 lists the strains used in this study.…”

“…The production and characterization of the genus-specific MAb 2125 has been described previously (6). MAb 2125 recognizes a genus-specific epitope on the 60-kDa heat shock protein of all Legionella species.…”