Hostname: page-component-7b7567c8c-kwt9d Total loading time: 0 Render date: 2026-11-12T05:23:15.851Z Has data issue: false hasContentIssue false
  • English
  • Français

Diagnosis of alveolar echinococcosis using immunoblotting with plural low molecular weight antigens

Published online by Cambridge University Press:  01 March 2009

K. Yamano,
A. Goto,
M. Miyoshi,
K. Furuya,
Y. Sawada and
N. Sato
K. Yamano*
Affiliation:
Department of Biological Science, Hokkaido Institute of Public Health, North 19, West 12, Kitaku, Sapporo060-0819, Japan
A. Goto
Affiliation:
Department of Biological Science, Hokkaido Institute of Public Health, North 19, West 12, Kitaku, Sapporo060-0819, Japan
M. Miyoshi
Affiliation:
Department of Biological Science, Hokkaido Institute of Public Health, North 19, West 12, Kitaku, Sapporo060-0819, Japan
K. Furuya
Affiliation:
Department of Parasitology, National Institute of Infectious Diseases, Tokyo162-8640, Japan
Y. Sawada
Affiliation:
Tenshi College, Sapporo065-0013, Japan
N. Sato
Affiliation:
First Department of Surgery, Hokkaido University Hospital, Sapporo060-8648, Japan
*
**Fax: +81-11-736-9476yamano@iph.pref.hokkaido.jp
Rights & Permissions [Opens in a new window]

Abstract

Alveolar echinococcosis (AE) is endemic to Hokkaido, Japan. For the past 20 years, detection of AE among inhabitants has involved serological screening using an enzyme-linked immunosorbent assay (ELISA) followed by Western blotting (WB). Between the years 1987 and 2000, antigens targeted on 66, 55 and 30–35 kDa bands were routinely used in the WB step of AE diagnosis. However, since 2001 diagnosis has been dependent on three smaller molecular weight antigens (26–28, 18 and 7–8 kDa). Due to its higher sensitivity, this improved WB approach has been used as a confirmation step in the screening process and also for the testing of suspected AE cases in hospital outpatients. Using the improved WB technique, a total of 1745 serum samples were examined in 2001–2006 with 81 patients detected and registered with AE. Interestingly, sera from 76 of the 81 diagnosed AE patients (93.8%) demonstrated reactivity with all three antigens. However, sera from the remaining five patients (6.2%) demonstrated no reactivity with the 18 kDa antigen, even though they exhibited clearly detectable levels of reactivity with the 26–28 and 7–8 kDa bands. These results suggest that medical practitioners need to pay particular attention to the specific reactions to some different diagnostic antigens to minimize the risk of misdiagnosing AE patients. In turn, these results may also provide important diagnostic information for cystic echinococcosis (CE).

Information

Type
Research Papers
Information
Journal of Helminthology , Volume 83 , Issue 1 , March 2009, pp. 57 - 61
Copyright
Copyright © Cambridge University Press 2009

Introduction

Human alveolar echinococcosis (AE) is a severe parasitic zoonosis caused by infection with the metacestode Echinococcus multilocularis. In Japan, E. multilocularis infection is prevalent in Hokkaido where local red foxes serve as the definitive host and small rodents serve as the intermediate host. In order to detect AE in Hokkaido, mass screening of residents for this disease has been undertaken over the past 20 years as a part of administrative countermeasures (Kimura et al., Reference Kimura, Furuya, Kawase, Sato, Yamano, Takahashi, Uraguchi, Ito, Yagi and Sato1999).

The initial screening process utilized to detect AE is divided into two steps. The first step involves the use of an enzyme-linked immunosorbent assay (ELISA) (Sato et al., Reference Sato, Mitamura, Arai and Kumagai1983a, Reference Sato, Mitamura, Arai and Kumagaib), and the second step consists of both a Western blotting (WB) (Furuya et al., Reference Furuya, Sato and Kumagai1987, Reference Furuya, Sasaki, Honma, Kumagai, Sato, Takahashi and Uchino1989, Reference Furuya, Nishizuka, Honma, Kumagai, Sato, Takahashi and Uchino1990; Nagano et al., Reference Nagano, Sato and Furuya1995) and ultrasound-based tests. These tests are often used in provisional confirmation prior to surgical treatment. The final diagnosis for most AE patients is based on histopathological findings following surgery (Condon et al., Reference Condon, Rausch and Wilson1988; Furuya et al., Reference Furuya, Sato, Kawanaka, Takahashi, Saeada, Honma and Tamura2001). The Hokkaido Institute of Public Health (HIPH) has played an important role in the serological aspect of the mass screening process. Furthermore, the procedures utilized in the detection of AE have also brought about numerous improvements in the testing of serological samples at a more general level.

In the original WB method undertaken between 1987 and 2000, diagnosis of AE was based on the presence of 66, 55 and 30–35 kDa bands (Furuya et al., Reference Furuya, Sasaki, Honma, Kumagai, Sato, Takahashi and Uchino1989). Subsequent to this time and due to a higher sensitivity and specificity, the presence of 26–28, 18 and 7–8 kDa bands is now used for the provisional confirmation of the disease (Furuya et al., Reference Furuya, Kawanaka, Yamano, Sato and Honma2004; Yamano et al., Reference Yamano, Yagi, Furuya, Sawada, Honma and Sato2005).

On the basis of diagnosis using these low molecular weight antigens, numerous serum samples from people previously screened and from patients suspected to have AE in hospitals were examined between 2001 and 2006. As a result, we were able to identify 81 new AE cases in this 6-year period. However, 5 of the 81 cases demonstrated no reactivity with the 18 kDa antigen, although reactivity with one or both of the 26–28 and 7–8 kDa antigens was evident. In this paper, we report on the five registered AE patients with interesting serological characteristics and discuss the importance of making diagnoses based on the overall reactivity against plural antigens.

Materials and methods

Serum samples

A total of 278,696 serum samples were collected for the primary screening (ELISA) process undertaken between 2001 and 2006. From these samples, 848 individuals were recalled for a secondary examination involving WB, ultrasound, computed tomography and/or magnetic resonance imaging. In addition, 897 sera were also tested as part of a routine examination of hospital-suspected cases.

Antigen production

ELISA and WB tests were undertaken employing a crude antigen extracted from E. multilocularis cysts. To prepare the antigen, protoscoleces were collected from cysts in cotton rat liver which had been experimentally inoculated with metacestodes of E. multilocularis. The protoscoleces were repeatedly washed with phosphate-buffered saline (PBS, pH 7.4) and treated with 0.2% Triton X-100 in PBS. This solution was then centrifuged for 15 min at 500 rpm and the supernatant dialysed against PBS for 24 h at 4°C to remove detergent. The total protein concentration of the antigen solution was set to 12 μg ml− 1.

ELISA protocol

ELISAs were performed as described previously by Sato et al. (Reference Sato, Mitamura, Arai and Kumagai1983a, Reference Sato, Mitamura, Arai and Kumagaib) with some minor modifications. Flat-bottomed microplates (EIA/RIA 8-well strip, high-bind type, Corning, New York, USA) were coated with crude antigen (100 μl/well) for 4 h at 37°C. Serum samples diluted at 1:250 in 0.05% Tween–PBS (100 μl/well) were then added to the wells of the microplates and incubated overnight at 4°C. After washing, 100 μl of anti-human IgG alkaline phosphatase conjugated antibody (Sigma, St Louis, Missouri, USA; 1:2500 in 0.05% Tween–PBS) was added and incubated for 4 h at 37°C. After further washing, bound antibodies were detected by the presence of a reaction for alkaline phosphatase on p-nitrophenyl-phosphate (0.1 mg/well) in 10% diethanolamine buffer (pH 9.8) following an incubation period of 8 min at 37°C (100 μl/well). The reaction was stopped with the addition of 50 μl of 3N NaOH and the optical density (OD) values read at 405 nm on a microplate reader (Model 680, BIORAD, Hercules, California, USA). Between each incubation step, plates were washed three times with 0.05% Tween-PBS (300 μl/well).

Preliminary experiments undertaken in 1983 gave a mean OD value of 0.94 ± 0.21 for 46 confirmed AE patients. Therefore, the diagnostic criteria for the current study were defined based on the mean OD and mean OD minus 2 standard deviations as follows: OD 0–0.50 = negative; 0.51–0.99 = quasi-positive; over 1.00 = positive (Sato et al., Reference Sato, Mitamura, Arai and Kumagai1983a). A negative mean OD value (n = 471) was defined as 0.29 under these conditions.

Western blot protocol

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) was undertaken as described by Laemmli (Reference Laemmli1970), utilizing a crude E. multilocularis antigen preparation. Antigens were separated electrophoretically on SDS–15% polyacrylamide gels and the separated antigens were electroblotted on to a nitrocellulose membrane. The membrane was subsequently blocked in 5% skimmed milk in PBS for 1 h at room temperature (RT) and then incubated with patient sera at 1:100 in PBS containing 0.05% Tween 20 and 5% skimmed milk for 1.5 h at RT. After washing, the membrane was treated with an anti-human IgG alkaline phosphatase conjugated antibody (Sigma), diluted at 1:2500 in 0.05% Tween–PBS, for 1 h at RT. After further washing, the membrane was exposed to BCIP/NBT substrate solution (PerkinElmer Life Sciences, Gaithersburg, Maryland, USA) for colour development. We adopted multiple markers including 26–28, 18 (the main marker utilized) and 7–8 kDa bands in our WB testing. The criteria for judgement are presented in table 1.

Table 1

Results of Western blot (WB) testing and a summary of echinococcosis detection between 2001 and 2006.

CE, cystic echinococcosis.

a Type A and B are positive and Type C is quasi-positive.

* The first step of the screening process by ELISA.

In addition, the commercially available immunoblot assay kit, ECHINOCOCCUS Western Blot IgG (Ldbio Diagnostics, Lyon, France) was also utilized in this study. The operation of this kit and the interpretation of subsequent results were undertaken in accordance with the manufacturer's recommendations.

Results

An outline of examination results and echinococcosis detection

Table 1 presents an outline of the examination results and echinococcosis detection. Confirmative WB tests were carried out on a total number of 1745 sera between 2001 and 2006, including both secondary examinations undertaken as part of the Hokkaido serological mass screening system and the regular examination of suspected outpatients in hospitals. Under the ‘law concerning prevention of infectious diseases and medical care for patients of infections’, 103 patients from Hokkaido were newly reported to the Infectious Disease Surveillance Center as having echinococcosis in these 6 years. From our review study, it was identified that HIPH directly participated in the detection of at least 82 cases (79.6%), including one cystic echinococcosis (CE) patient. Among these 81 AE patients, 79 cases were found to be positive and two quasi-positive in our WB testing procedure. In addition, 21 of the newly registered 103 patients included cases that were diagnosed by other systems undertaken by some facilities, and were detected only via primary screening using ELISA, or were identified during surgery associated with other diseases.

As outlined in table 1, 81 AE patients detected by our WB testing were classified into three groups. Type A comprised 76 positive cases that demonstrated reactivity against the 18, 26–28 and 7–8 kDa antigens, Type B consisted of three positive cases showing reactivity against the 26–28 and 7–8 kDa antigens, and Type C included two quasi-positive cases that reacted only to the 26–28 kDa antigen. The Type A cases, that demonstrated reactivity to the 18 kDa antigen, are thought to represent typical positive AE patterns. In contrast, Types B and C (n = 5) represented patients with active AE and with characteristic sero-negativity for antibody directed to the 18 kDa antigen.

Five atypical cases

Five atypical patients of Types B (n = 3) and C (n = 2) were comprehensively diagnosed based on serological examination, diagnostic imaging and histopathological analysis. These patients characteristically lacked the presence of an 18 kDa band on their immunoblots (fig. 1), and similar results were also obtained using a commercially available immunoblot kit.

Fig. 1

Immunoblot analysis of patients showing sero-negativity against the 18 kDa antigen. Lanes: M, the molecular size marker; 1, a positive control (Type A); 2 and 3 are sera from Type B patients (Cases 2 and 3); 4 and 5 are sera from Type C patients (Cases 4 and 5). The diagnostic bands are indicated by arrows.

Case 1 was a 70-year-old Japanese male of Type B, the details of whom have already been reported (Yamano et al., Reference Yamano, Yagi, Furuya, Sawada, Honma and Sato2005).

Case 2 was a 49-year-old Japanese male of Type B, who was detected via the Hokkaido screening system in 2003. His ELISA result was quasi-positive (OD 0.658) in the primary screening process, and a WB test undertaken as part of a secondary confirmation step was judged to be positive for the 26–28 and 7–8 kDa bands. When he consulted a physician for a thorough examination, several cysts were identified in S2/3, S5 and S7 of the liver using ultrasonography and computed tomography. He subsequently underwent surgery in September 2003, and histopathological confirmation was carried out.

Case 3 was a 43-year-old Japanese male of Type B. In this patient, several cysts with calcification were identified in S6/7 and S8 of the liver using ultrasonography and computed tomography following a medical examination. A serum sample for serological testing was collected in September 2004. The subsequent ELISA test demonstrated an OD value within the negative range (OD 0.417). Further WB tests identified the presence of 26–28 and 7–8 kDa protein bands and were judged to be positive. The cysts were surgically removed in December 2004 and histopathological confirmation was carried out.

Case 4 was a 71-year-old Japanese female of Type C. Upon consultation with a physician for ureteral calculus, a phyma of 5 cm in diameter was identified in S8 of the liver using computed tomography. A subsequent serological test was undertaken on a serum sample collected in February 2005. Both ELISA and WB analysis gave a quasi-positive result, with an OD value of 0.731 and the presence of a 26–28 kDa band, respectively. This patient received a resection of phyma in March 2005, and a diagnosis of AE was confirmed following histopathogical analysis.

Case 5 was a 19-year-old Japanese male of Type C. Following a medical examination for stomach ache, a phyma with calcification was identified in the left lobe of the liver using ultrasonography, computed tomography and magnetic resonance imaging. A serological test was undertaken on a serum sample collected in February 2005. An ELISA test demonstrated an OD value within the positive range (OD 1.51); however, a WB test identified the presence of only the 26–28 kDa band and was thus judged to be quasi-positive. The patient underwent surgery in May 2006 and a diagnosis of AE was confirmed following histopathological analysis.

Discussion

AE is prevalent in the forest and farming areas of the northern hemisphere and is endemic to the Hokkaido region of Japan. CE, which is caused by infection with E. granulosus, occurs most often around areas of the world involved in the raising of livestock and is generally regarded to be of minor importance in imported cases in Japan (Furuya et al., Reference Furuya, Kawanaka, Sato, Honma and Tamura2000). Therefore, the accurate diagnosis of AE is a major priority in Japan, especially in Hokkaido.

A serological screening system using ELISA (Sato et al., Reference Sato, Mitamura, Arai and Kumagai1983a, Reference Sato, Mitamura, Arai and Kumagaib) and WB (Furuya et al., Reference Furuya, Sato and Kumagai1987, Reference Furuya, Sasaki, Honma, Kumagai, Sato, Takahashi and Uchino1989, Reference Furuya, Nishizuka, Honma, Kumagai, Sato, Takahashi and Uchino1990; Nagano et al., Reference Nagano, Sato and Furuya1995) tests has been established previously to detect patients with AE residing in Hokkaido. These methods employ a crude antigen extracted from E. multilocularis cysts which detects numerous antibodies directed specifically against E. multilocularis antigens.

In our original WB testing procedures, protein bands of 55–66 kDa and a broad smear band including polysaccharides ranging from 30–35 kDa were identified as markers for AE when separated by SDS–PAGE (8% polyacrylamide gel). More recently, Em18, which is located in the 18 kDa region of immunoblots, has been reported to be a good marker for AE detection (Ito et al., Reference Ito, Nakao, Kutsumi, Lightowlers, Itoh and Sato1993, Reference Ito, Schantz and Wilson1995, Reference Ito, Sako, Yamasaki, Mamuti, Nakaya, Nakao and Ishikawa2003). It was subsequently demonstrated that the Em18 antigen is the breakdown product of EM10 (55–66 kDa antigen) following proteolysis by cysteine proteinases (Sako et al., Reference Sako, Nakao, Nakaya, Yamasaki, Gottstein, Lightowlers, Schantz and Ito2002). These results confirmed that Em18 and 55–66 kDa antigens were basically equal in the diagnosis of AE with WB testing. In addition, a commercially available immunoblot kit that detects bands ranging from 7 to 28 kDa has been developed and manufactured in France (Liance et al., Reference Liance, Janin, Bresson-Hadni, Vuitton, Houin and Piarroux2000) for the detection of echinococcosis. Thus, we began to investigate the use of low molecular weight antigens in the identification of AE (Yamano et al., Reference Yamano, Yagi, Furuya, Sawada, Honma and Sato2005). Our improved WB method monitors the presence of three marker bands ranging from 7 to 28 kDa. This concept is similar to that utilized by the above-mentioned commercially available kit. However, in the utilization of crude antigen, the 7–8 kDa band is usually accompanied by an approximately 12 kDa band which appears to be related to antigen B (AgB) (Leggatt et al., Reference Leggatt, Yang and McManus1992). Moreover, some CE cases may be detected due to cross-reaction with the 26–28 and/or 7–8 kDa antigens, a result that may also occur with the commercially available kits (see table 1).

In the previously utilized WB testing procedures, we identified patients demonstrating sero-negativity for the 55/66 kDa antigens. Similarly, we detected five patients showing sero-negativity for the 18 kDa antigen over a period of 6 years, using the application of low molecular weight markers. The prognosis for these five patients is good as a result of surgical treatment, suggesting that precise serodiagnosis aids in the improvement of curative effects.

It has recently become popular to use low molecular weight antigens as a diagnostic marker for AE. However, 6.2% (5 of 81) of patients showed sero-negativity against the 18 kDa antigen, a main marker for AE. Unfortunately this frequency is quite significant. We have also reported previously that AE patients demonstrating sero-negativity against the 18 kDa antigen were detected at a frequency of 21.7%, due to the presence of the 26–28 kDa and/or 7–8 kDa bands when using the commercially available immunoblot kits (Furuya et al., Reference Furuya, Kawanaka, Yamano, Sato and Honma2004). Similar findings regarding sero-negativity against the 18 kDa antigen were also reported for Chinese and French AE patients (Nirmalan & Craig, Reference Nirmalan and Craig1997; Liance et al., Reference Liance, Janin, Bresson-Hadni, Vuitton, Houin and Piarroux2000). These findings may indicate that the positive rate against the 18 kDa antigen in sera from AE patients is not sufficiently high and that the detection of multiple bands on an immunoblot test may be required for the correct diagnosis of AE.

The recent development of recombinant antigens has also contributed to an improvement in serodiagnostic examinations. These antigens include EmII/3 (Vogel et al., Reference Vogel, Gottstein, Muller and Seeback1988; Felleisen & Gottstein, Reference Felleisen and Gottstein1994; Kouguchi et al., Reference Kouguchi, Suzuki, Yamano, Honma and Sawada2005), EM10 (Frosch et al., Reference Frosch, Frosch, Pfister, Schaad and Bitter-Suermann1991; Helbig et al., Reference Helbig, Frosch, Kern and Frosch1993; Hubert et al., Reference Hubert, Cordero, Frosch and Solomon1999) and Em18 (Ito et al., Reference Ito, Xiao, Liance, Sato, Sako, Mamuti, Ishikawa, Nakao, Yamasaki, Nakaya, Bardonnet, Bresson-Hadni and Vuitton2002). Unfortunately, no single antigen identified to date has proven successful in detecting all AE patients. Therefore, it is concluded that we need to pay particular attention to the specific reactions to plural different diagnostic antigens such as the 26–28 and 7–8 kDa proteins to minimize the risk of inaccurate diagnosis of AE.

References

Condon, J., Rausch, R.L. & Wilson, J.F. (1988) Application of the avidin–biotin immunohistochemical method for the diagnosis of alveolar hydatid disease from tissue sections. Transactions of the Royal Society of Tropical Medicine and Hygiene 82, 731–735.CrossRefGoogle ScholarPubMed
Felleisen, R. & Gottstein, B. (1994) Comparative analysis of full-length antigen II/3 from Echinococcus multilocularis and E. granulosus. Parasitology 109, 223–232.CrossRefGoogle ScholarPubMed
Frosch, P.M., Frosch, M., Pfister, T., Schaad, V. & Bitter-Suermann, D. (1991) Cloning and characterization of an immunodominant major surface antigen of Echinococcus multilocularis. Molecular and Biochemical Parasitology 48, 121–130.CrossRefGoogle ScholarPubMed
Furuya, K., Sato, H. & Kumagai, M. (1987) Application of Western blotting method for immunoserologic diagnosis of hydatidosis. Journal of Clinical and Experimental Medicine (IGAKU NO AYUMI) 141, 41–42(in Japanese).Google Scholar
Furuya, K., Sasaki, S., Honma, H., Kumagai, M., Sato, N., Takahashi, M. & Uchino, J. (1989) Serologic investigations of human alveolar hydatid disease by Western blotting and indirect histo-immunoperoxidase techniques. Japanese Journal of Parasitology 38, 184–193.Google Scholar
Furuya, K., Nishizuka, M., Honma, H., Kumagai, M., Sato, N., Takahashi, M. & Uchino, J. (1990) Prevalence of human alveolar echinococcosis in Hokkaido as evaluated by Western blotting. Japanese Journal of Medical Science and Biology 43, 43–49.CrossRefGoogle ScholarPubMed
Furuya, K., Kawanaka, M., Sato, N., Honma, H. & Tamura, M. (2000) Has Echinococcus granulosus settled in Hokkaido? The Journal of the Japanese Association for Infectious Diseases 53, 176–177.Google ScholarPubMed
Furuya, K., Sato, N., Kawanaka, M., Takahashi, K., Saeada, Y., Honma, H. & Tamura, M. (2001) Immunohistochemical examinations of echinococcal liver lesions from patients with alveolar hydatid disease in Hokkaido. Report of the Hokkaido Institute of Public Health 51, 1–6(in Japanese).Google Scholar
Furuya, K., Kawanaka, M., Yamano, K., Sato, N. & Honma, H. (2004) Laboratory evaluation of commercial immunoblot assay kit for serodiagnosis of Echinococcus infections using sera from patients with alveolar hydatidosis in Hokkaido. The Journal of the Japanese Association for Infectious Diseases 78, 320–326(in Japanese).Google ScholarPubMed
Helbig, M., Frosch, P., Kern, P. & Frosch, M. (1993) Serological differentiation between cystic and alveolar echinococcosis by use of recombinant larval antigens. Journal of Clinical Microbiology 31, 3211–3215.CrossRefGoogle ScholarPubMed
Hubert, K., Cordero, E., Frosch, M. & Solomon, F. (1999) Activities of the EM10 protein from Echinococcus multilocularis in cultured mammalian cells demonstrate functional relationships to ERM family members. Cell Motility and the Cytoskeleton 42, 178–188.3.0.CO;2-3>CrossRefGoogle ScholarPubMed
Ito, A., Nakao, M., Kutsumi, H., Lightowlers, M.W., Itoh, M. & Sato, S. (1993) Serodiagnosis of alveolar hydatid disease by Western blotting. Transactions of the Royal Society of Tropical Medicine and Hygiene 87, 170–172.CrossRefGoogle ScholarPubMed
Ito, A., Schantz, P.M. & Wilson, J.F. (1995) EM18, a new serodiagnostic marker for differentiation of active and inactive cases of alveolar hydatid disease. The American Journal of Tropical Medicine and Hygiene 52, 41–44.CrossRefGoogle ScholarPubMed
Ito, A., Xiao, N., Liance, M., Sato, M.O., Sako, Y., Mamuti, W., Ishikawa, Y., Nakao, M., Yamasaki, H., Nakaya, K., Bardonnet, K., Bresson-Hadni, S. & Vuitton, D.A. (2002) 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. Journal of Clinical Microbiology 40, 4161–4165.CrossRefGoogle Scholar
Ito, A., Sako, Y., Yamasaki, H., Mamuti, W., Nakaya, K., Nakao, M. & Ishikawa, Y. (2003) Development of Em18-immunoblot and Em18-ELISA for specific diagnosis of alveolar echinococcosis. Acta Tropica 85, 173–182.CrossRefGoogle ScholarPubMed
Kimura, H., Furuya, K., Kawase, S., Sato, C., Yamano, K., Takahashi, K., Uraguchi, K., Ito, T., Yagi, K. & Sato, N. (1999) Recent epidemiologic trends in alveolar echinococcosis prevalence in humans and animals in Hokkaido. The Journal of the Japanese Association for Infectious Diseases 52, 117–120.Google ScholarPubMed
Kouguchi, H., Suzuki, T., Yamano, K., Honma, H. & Sawada, Y. (2005) Characterization of various recombinant antigens from Echinococcus multilocularis for use in immunodiagnosis. The Protein Journal 24, 57–64.CrossRefGoogle ScholarPubMed
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.CrossRefGoogle ScholarPubMed
Leggatt, G.R., Yang, W. & McManus, D.P. (1992) Serological evaluation of the 12 kDa subunit of antigen B in Echinococcus granulosus cyst fluid by immunoblot analysis. Transactions of the Royal Society of Tropical Medicine and Hygiene 86, 1–4.CrossRefGoogle ScholarPubMed
Liance, M., Janin, V., Bresson-Hadni, S., Vuitton, D.A., Houin, R. & Piarroux, R. (2000) Immunodiagnosis of Echinococcus infections: confirmatory testing and species differentiation by new commercial Western blot. Journal of Clinical Microbiology 38, 3718–3721.CrossRefGoogle ScholarPubMed
Nagano, M., Sato, C. & Furuya, K. (1995) Human alveolar echinococcosis seroprevalence assessed by Western blotting in Hokkaido. Japanese Journal of Medical Science and Biology 48, 157–161.CrossRefGoogle ScholarPubMed
Nirmalan, N. & Craig, P.S. (1997) Immunoblot evaluation of the species specificity of Em18 and Em16 antigens for serodiagnosis of human alveolar echinococcosis. Transactions of the Royal Society of Tropical Medicine and Hygiene 91, 484–486.CrossRefGoogle ScholarPubMed
Sako, Y., Nakao, M., Nakaya, K., Yamasaki, H., Gottstein, B., Lightowlers, M.W., Schantz, P.M. & Ito, A. (2002) Alveolar echinococcosis: characterization of diagnostic antigen Em18 and serological evaluation of recombinant Em18. Journal of Clinical Microbiology 40, 2760–2765.CrossRefGoogle ScholarPubMed
Sato, H., Mitamura, H., Arai, J. & Kumagai, M. (1983a) Serological diagnosis of human hydatid diseases by enzyme-linked immunosorbent assay (part 1). Enzyme-linked immunosorbent assay by multilocular Echinococcus antigen. Report of the Hokkaido Institute of the Public Health 33, 8–15(in Japanese).Google Scholar
Sato, H., Mitamura, H., Arai, J. & Kumagai, M. (1983b) Serological diagnosis of human hydatid diseases by enzyme-linked immunosorbent assay (Part 2). Application and evaluation on the health examination. Report of the Hokkaido Institute of Public Health 33, 16–20(in Japanese).Google Scholar
Vogel, M., Gottstein, B., Muller, N. & Seeback, T. (1988) Production of a recombinant antigen of Echinococcus multilocularis with high immunodiagnostic sensitivity and specificity. Molecular and Biochemical Parasitology 31, 117–125.CrossRefGoogle ScholarPubMed
Yamano, K., Yagi, K., Furuya, K., Sawada, Y., Honma, H. & Sato, N. (2005) Active alveolar hydatidosis with sero-negativity for antibody to the 18 kDa antigen. The Journal of the Japanese Association for Infectious Diseases 58, 122–124.Google Scholar
Figure 0

Table 1Results of Western blot (WB) testing and a summary of echinococcosis detection between 2001 and 2006.

Figure 1

Fig. 1Immunoblot analysis of patients showing sero-negativity against the 18 kDa antigen. Lanes: M, the molecular size marker; 1, a positive control (Type A); 2 and 3 are sera from Type B patients (Cases 2 and 3); 4 and 5 are sera from Type C patients (Cases 4 and 5). The diagnostic bands are indicated by arrows.