Isolation of species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii for immunodiagnosis and immunoprophylaxis

Abstract

A simple procedure for the selective isolation of the protective species-specific protein antigens (SPAs) of Rickettsia typhi and Rickettsia prowazekii was developed to permit use of the SPAs in the immunodiagnosis and immunoprophylaxis of typhus infections. Although the SPAs were readily extracted from lysozyme- or detergent-treated rickettsiae, as measured by rocket immunoelectrophoresis, other polypeptides were also present, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast, both water and seven buffers, each at a 10 mM concentration and pH 7.6, were nearly equally effective in the selective release of the SPAs from whole cells by extraction for 30 min at 45 degrees C. High-ionic-strength buffers and MgCl2 abolished this SPA release, thus suggesting that divalent cations were important in the binding of the SPAs to the cell envelope. The efficacy of the dilute buffer extraction procedure for isolation of large amounts of SPAs was tested by further characterization of the supernatants obtained by centrifugation (200,000 x g) of two successive tris-(hydroxymethyl)aminomethane-hydrochloride buffer (Tris) extracts. With this procedure, between 10 and 15 mg of SPA was obtained from 100 mg of purified rickettsiae. Although low-molecular-weight ribonucleic acid fragments were released into the Tris extracts in significant amounts, only the SPAs were detected, in significant quantities, as measured by polyacrylamide gel electrophoresis and rocket immunoelectrophoresis. The Tris extracts contained the same major and minor SPA polypeptides as those observed previously in SPA preparations obtained by extensive diethylaminoethyl-cellulose column chromatography, but the Tris SPAs were more satisfactory antigens in an enzyme-linked immunosorbent assay.

Antigenic relationships among the rickettsiae of the spotted fever and typhus groups

“…Antibodies to this protein were mainly species-specific and groupspecific; weaker reactions occurred with 135-kDa antigens of the heterologous groups. The prominence in Coomassie blue-stained gels and the patterns of migration and immunoreactivity suggest that the protective species-specific antigens (SPA) of the typhus rickettsiae described by Dasch et al [6,15] belong to this family of antigens.…”

“…Besides distinctive group antigens, the rickettsiae also have cross-reacting antigens [4]. The rickettsial antigens shown to elicit immunity are proteins with molecular masses between 100 and 200 kDa [5,6]. Monoclonal antibodies against some of these antigens have been used to study the antigenic relationships among the rickettsiae [7,8].…”

The 120 kilodalton outer membrane protein (rOmp B) of Rickettsia rickettsii is encoded by an unusually long open reading frame: evidence for protein processing from a large precursor

“…The analogous protein in TG rickettsiae has been referred to as SPA. an acronym for which there are several documented meanings (Ching e^ ai, 1990;Carl et ai, 1990a,b;Dasch, 1981). To alleviate confusion in terminology, we have termed this rickettsial outer membrane protein, rOmp B.…”

Expression and Purification of the Crystalline Surface Layer Protein of Rickettsia typhi

“…Although its functional activity is poorly understood, some physical and immunological characteristics have been studied. It is known as a species-specific and protective antigen (1,3). It also elicits dominant antibody reaction demonstrated by polyclonal antisera which were employed in rocket immunoelectrophoresis (4).…”

“…About 10 mg of recombinant protein was obtained from a 1-liter culture of E. coli. Previously, it was reported that the SLP is a protective antigen and elicits dominant antibody response which could be exploited for clinical use (1,3). However, as R. typhi is an obligate intracellular parasite, it is very difficult to prepare the natural SLP.…”