A., J. successfully expressed by using expression system and then evaluated for use in serodiagnosis of alveolar echinococcosis. RecEm18 was recognized by 27 (87.1%) and 28 (90.3%) of 31 serum samples from clinically and/or pathologically confirmed alveolar echinococcosis patients by enzyme-linked immunosorbent assay and immunoblotting, respectively. Of 33 serum samples from cystic echinococcosis patients, 1 was recorded as having a weak positive reaction to RecEm18; however, none of the serum samples which were tested from neurocysticercosis patients (= 10) or healthy people (= 15) showed positive reactions. RecEm18 has the potential for Rabbit Polyclonal to MSK2 use in the differential serodiagnosis of alveolar echinococcosis. Alveolar echinococcosis (AE), caused by the larval stage of by accidental ingestion of eggs excreted with the feces of carnivores harboring the adult tapeworm of this species. The eggs hatch in the small intestine of the human host releasing the oncosphere which migrates via the portal system into various organs, mainly the liver, and differentiates into the metacestode stage. The metacestodes propagate asexually like a tumor, leading to organ dysfunction. Since clinical symptoms usually do not become evident until 10 or more years after initial parasite infection, early diagnosis and treatment are important for the reduction of morbidity and mortality (1, 9). At present, diagnosis of AE is primarily based on imaging techniques, including echography, computed tomography, and magnetic resonance imaging. These imaging techniques are sometimes Isochlorogenic acid A limited by the small size of visualized lesions and atypical images, which are difficult to distinguish from abscesses or neoplasms. Therefore, efforts have been directed toward identification and characterization of specific antigens of metacestodes for development of immunodiagnostic test that can detect specific antibodies (8, 9, 10, 16-18, 20-22, 25, 27, 33-35, 37). Using molecular and immunological techniques many researchers have attempted to identify protoscolex library by screening with a pool of sera from AE patients. The clone, designated II/3, comprised an incomplete copy of the associated mRNA, and the expressed protein was shown to have potential for use in the serodiagnosis of AE. Muller et al. (30) subcloned a fragment of this cDNA, referred to as II/3-10, which retained the diagnostic epitopes but was more suited to use in immunoassays. Neither publication included DNA or protein Isochlorogenic acid A sequence data. Subsequently, Frosch et al. (5) characterized a full-length mRNA from protoscoleces, including the DNA sequence, and showed that the expressed antigen, designated EM10, had potential for use in the diagnosis of AE. Contemporaneously, Hemmings and McManus (11) characterized a partial cDNA, designated EM4, encoding an antigen which they also found to be potentially useful for serodiagnosis of AE. The II/3 and EM4 proteins were subsequently confirmed as being fragments of EM10. This full-length recombinant antigen and its associated full-length native antigen are hereafter referred to as EM10, whereas the designations EmII/3 or EmII/3-10 are retained for the fragments of EM10 described by Vogel et al. (41) and Muller et al. (30), respectively. Recently, we reported another novel antigen, termed Em18 (18-kDa protein under reducing condition), partially purified by preparative isoelectric-focusing electrophoresis (IEFE) from protoscoleces and demonstrated its usefulness for highly sensitive and specific diagnosis of AE by either enzyme-linked immunosorbent assay (ELISA) or immunoblotting (16-18, 22). The sensitivity and specificity of Em18 for AE are very compatible to those of Isochlorogenic acid A recombinant EM10 or truncated fragments thereof (18, 21), raising the question as to whether EM10 and Em18 are antigenically or otherwise related. We here describe a partial amino acid sequence of the native Em18 antigen, confirming that Em18 is a fragment of the EM10 protein, and demonstrate that recombinant Em18 is highly effective in immunoassays for serodiagnosis of AE. MATERIALS AND METHODS Preparation of parasite material. (Furano isolate, Hokkaido, Japan) metacestode material was obtained from laboratory reared Mongolian gerbils infected by intraperitoneal passage. Crude antigen extract was prepared from fresh whole cyst tissue materials. The parasite organism was lysed with three times volume of neutral lysis buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS]) or acidic lysis buffer (0.1 M citrate buffer [pH 5.0], 150 mM NaCl, 1% Triton X-100, 0.1% SDS). After one freeze-thaw cycle Isochlorogenic acid A and centrifugation at 10,000 for 30 min at 4C, the supernatant was recovered and kept at ?80C until use. Patient serum samples. A total of 31 serum samples of patients with AE confirmed by image analysis and/or serology of immunoblot analysis with the native Em18 purified by IEFE from protoscoleces of were examined for this study. Seven and twenty-four serum samples were from patients in Alaska and Hokkaido, Japan, respectively. Serum samples from other.