Evaluation of an Enzyme-Linked Immunosorbent Assay (ELISA) with Affinity-Purified Em18 and an ELISA with Recombinant Em18 for Differential Diagnosis of Alveolar Echinococcosis: Results of a Blind Test

Abstract

Alveolar echinococcosis (AE) is the most potentially lethal parasitic zoonosis of the nontropical areas in the northern hemisphere, where cystic echinococcosis (CE) is also endemic. Both AE and CE are highly endemic in China, and both serologic detection of echinococcosis, either AE or CE, and differentiation of AE from CE are crucial problems. Evaluation of Western blot analysis (WB) and enzyme-linked immunosorbent assay (ELISA) for the Em18 antigen, using affinity-purified and recombinant Em18, was carried out "blindly" using 60 human sera from patients diagnosed in France. The results were compared with those obtained using a commercially available Echinococcus WB immunoglobulin G (IgG) kit developed in France. The Em18 WB and Echinococcus WB IgG showed very similar results for detection of AE. Both affinity-purified Em18 or a recombinant Em18 WB and Echinococcus WB IgG seem useful for identification of AE, and the latter seems appropriate for both AE and CE, whereas affinity-purified Em18 ELISA and the newly developed recombinant Em18 ELISA appear to be suitable for detection of AE, especially for epidemiological surveys.Alveolar echinococcosis (AE), caused by the metacestode of the fox tapeworm, Echinococcus multilocularis, is the most potentially lethal parasitic zoonosis of the nontropical areas in the northern hemisphere, where cystic echinococcosis (CE), caused by the metacestodes of the dog tapeworm, E. granulosus, is also endemic. For example, both AE and CE are highly endemic in China (8,19) and serological detection of echinococcosis, either AE or CE, and differentiation of AE from CE are crucial problems, since the pathogenicity of these two types of echinococcosis and the treatment of patients with these diseases are critically different (14,16).In Japan and France, immunoblotting (Western blotting [WB]) assay systems have been developed for differentiation of AE from other diseases (4-7). The Asahikawa Medical College (AMC) group in Japan has focused on the detection of antibody response to the Em18 antigen (approximately 18 kDa) extracted from protoscoleces of E. multilocularis (4, 9, 15) and has tried to purify Em18, which shows a single band in WB, and to make it available for enzyme-linked immunosorbent assay (ELISA), using preparative isoelectric focusing (PIEF). Since purification of Em18 by PIEF takes longer and the yield is not as great, we have shifted to purification of Em18 by affinity chromatography (AffEm18) and production of a recombinant Em18 (RecEm18) for WB and ELISA (7, 9, 10, 13, 18). Echinococcus WB immunoglobulin G (IgG) (EchWB IgG; LDBIO Diagnostics, Lyon, France), which has a high sensitivity for the detection of both AE and CE, is basically very similar to the AMC system since it also focuses on differentiation of AE and CE based on different banding patterns including antigen B (most predominant at 8 and 26 to 28 kDa), Em16, and Em18 in crude antigens. The merit of the latter system is that it detects both AE and CE on a single strip based on the difference in the banding...

Development of an Immunochromatographic Test To Detect Antibodies against Recombinant Em18 for Diagnosis of Alveolar Echinococcosis

“…This is not an incomprehensible result, because it is known that a few CE patient sera react to rEm18 even though rEm18 is highly specific antigen for AE (11,12,16,18). These results indicated that the ICT is a sensitive and specific method for the diagnosis of E. multilocularis infection.…”

“…Therefore, immunological tests have been considered important methods to confirm clinical findings, to give diagnostic help by providing information on the parasite in case of unclear images, or to survey in areas of endemicity where imaging techniques are not readily available (4,9,11). Previously, we have reported an enzyme-linked immunosorbent assay (ELISA) and an immunoblot analysis (IB) by using recombinant E. multilocularis 18-kDa antigen (Em18), the breakdown product of ezrin-radixin-moesin-like protein (2) that is also known as EM10 (8), EM II/3 (7), or EM4 (10) by the cysteine peptidase, and demonstrated that these two tests have a high potential for differentially diagnosing AE (1,12,16,18). However, these two methods are time-consuming and require special materials and equipments, which make them not suitable for clinical applications.…”

Serological diagnosis of echinococcosis: the diagnostic potential of native antigens

“…These antigens have diagnostic sensitivities ranging between 81 and 100% and discriminate between AE and CE with a reliability ranging from 80 to C95% [15,26,27,[43][44][45][46]. The native and partially purified antigens tested in our study did not discriminate between AE and CE: they all recognised all AE and at least 81.3% of CE cases.…”

“…Antigen B (AgB) was prepared as described elsewhere [24,25], the Em2(G11) antigen was prepared as described by Deplazes et al [15], and the recombinant Em18 antigen was provided by the Institute of Parasitology in Bern, Switzerland [26,27].…”

Molecular Cloning, Expression, and Serological Evaluation of an 8-Kilodalton Subunit of Antigen B from Echinococcus multilocularis

“…Serum samples were collected from the following groups of patients with AE and CE after appropriate ethical approval was obtained and informed consent was obtained from the patients: 30 serum samples from patients with CE in France (14) and 30 serum samples from patients with CE in China confirmed by surgery; 20 serum samples from patients with CE in Australia confirmed by serology with Ag5 with or without surgery (17,18), followed by WB and ELISA with AgB of E. granulosus hydatid cyst fluid at Asahikawa Medical College (AMC) (20) (Table 1); 45 serum samples from patients with AE diagnosed by surgery plus Em18 serology at Hokkaido University Hospital and AMC; and 19 serum samples from patients with AE in France diagnosed by surgery plus serology with a commercially available kit (14,16). The major target antigens were Em18, Em16, and AgB in a whole larval extract of E. multilocularis (10,12,14,16). These serum samples were obtained from patients who had been confirmed to have CE or AE and do not necessarily represent samples taken from patients prior to their commencement of treatment surgically and/or chemotherapeutically.…”