A Serodiagnostic Assay by Microenzyme-Linked Immunosorbent Assay for Human Anisakiasis Using a Monoclonal Antibody Specific for Anisakis Larvae Antigen Atsuhito Yagihashi, Noriyuki Sato, Shuji Takahashi, Hajime Ishikura, and Kokichi Kikuchi

From the Department of Pathology. Sapporo Medical College. Sapporo. and Ishikura Hospital, Iwanai, Hokkaido, Japan

Anisakiasis is caused by infection with the Anisakis larvae (AL) and may be acquired by eating raw fish (sushi or sashimi) [I]. The clinical manifestations of this disease are thought to be caused by penetration of AL into the mucosal and muscular layer of the stomach and intestine [2, 3]. Endoscopic examinations frequently can identify AL in patients, usually those with gastric anisakiasis. However, the clinical diagnosis is often difficult, especially if the organism is in the small intestine, which is inaccessible to diagnostic endoscopy [4]. Many physicians have noted the need for a serodiagnostic test. However, such assays have lacked specificity, possibly due to lack of an antibody reacting specifically against AL. Hence, there is an increasing demand for a more specific serodiagnosis assay system using patient serum. We have previously raised monoclonal antibodies (MAbs) that could specifically react with AL antigen [5, 6]. Here, we describe the successful establishment of a specific serodiagnostic assay system using MAbs in a microELISA. Debate continues about the development of anisakiasis in patients after ingestion of AL [7]. Tanaka and Torisu [8] suggested that acute anisakiasis is mainly due to a hypersensitivity reaction to specific Anisakis antigens [8]; however, the antigens responsible for the development of anisakiasis remain unknown. Thus, our present work may contribute to the clarification of the immunobiologic significanceof antigens for anisakiasis.

Received 9 June 1989; revised 14 November 1989 Reprints and correspondence: Dr. N. Sato, Department of Pathology, Sapporo Medical College, S. 1, W. 17, Chuo-ku, 060 Sapporo, Japan. The Journal of Infectious Diseases 1990;161:995-998 © 1990 by The University of Chicago. All rights reserved. 0022-1899/90/6105-0029$01.00

Patients, Materials, and Methods Monoclonal antibodies. We have already established MAbs, namely Anl-An7, that can react with the antigens expressed in AL [5]. Among these MAbs, AN2 with IgGl isotypecan react specifically with AL but not with other helminths such as Ascaris suum, Pseudoterranova decipiens larvae, Toxocara cati, Trichinella spiralis, Echinococcus multilocularis, and Dirofilaria immitis. It also does not cross-react with human tissues. The An2-defined antigen has been thought to be composed of 40- and 42-kDa heterodimers with a disulfide bond. An immunofluorescence study showedthat the An2defined antigen was distributed in muscle, renette cells, and intestines of AL. Further, this antigen has been suggested as one of the excretory-secretory antigens of AL. In our study, we used the An2 MAb for the serodiagnostic assay of human anisakiasis. Patient and control sera. Anisakiasis was confirmed by endoscopic and histologic examinations. Ninety-three serum samples were obtained from patients with anisakiasis at various clinical stages at our institution and others (see Acknowledgment). Patients were 26-68 years old. Most complained of severe abdominal pain on the first day of illness. Serum samples were obtained at various dates after the onset of clinical anisakiasis, from day 1 through week 12. Control sera were collected from 45 healthy persons, aged 19-52 years. Thirty-four ofthe controls had often eaten raw fish, although they lacked clinical symptoms that suggested acute or chronic anisakiasis. MicroEUSA. About 100 ",Iof PBScontaining 100 mglml ofMAb An2 was adsorbed at 37°C for I h in wells of microELISA plates (MS-3596F/H; Sumitomo Beiklight, Tokyo). The MAb was first purified by a method previously described [9, 10] using protein A Affi-Gel beads (Bio-Rad Laboratories, Hercules, CA). The plates were washed with PBS, and 50 ",I ofthe antigen containing 100 ",g protein/ml that was obtained by centrifuging the homogenized AL in PBS at 3000 rpm was applied to each well for 1 h at 37°C. Plates were then washed several times with PBS. Diluted patient or control sera (100 ",I; lOx dilution) was then applied at 37°C for 1 h before washing. Finally, peroxidase-conjugated goat anti-human IgG, IgA, and IgM

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A serodiagnostic assay system to detect human anisakiasis is described. This assay uses a microenzyme-linked immunosorbent assay with monoclonal antibody An2 that recognizes the antigen specifically found in Anisakis simplex larvae. The data showed that sera from infected patients reacted strongly with the A. simplex larvae antigen that was immobilized with monoclonal antibody An2 on microplates. Patients' IgG, 19A, IgM, and 19E reacted strongly with An2-defined antigen at 4 or 5 weeks and at 1 or 7 days, respectively, after the onset of clinical symptoms of anisakiasis. However, sera from healthy persons did not react, even though they often ate raw fish. This indicates a high diagnostic specificity of this assay system for clinically manifested anisakiasis.

Yagihashi et al.


JID 1990;161 (May)

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Figure 1. Reactivity by microELISA of serum IgG, 19A, and IgM to the antigenimmobilized by monoclonal antibody An2. Data shown are various phases in patients with anisakiasis and controls.

Figure 2. Reactivity of serum IgE to the antigen immobilized by monoclonal antibody AnZ at various phases as shown by microELISA. Data are shown in patients with anisakiasis and controls.

or anti-human IgE serum was reacted at 37°C for I h in each well and then washed. Next, I00 ~I of solution containing 400 ~g of OPD (O-phenylenediamine dihydrochloride; Nakarai Chemical, Kyoto, Japan) in I ml of substrate buffer (0.05 M citric acid, pH 4.0) was added and reacted in the presence of om % H20 2 at 37°C for 30 min. Each well was counted for optical density (OD) at 490 nm.

a high titer, although the number of samples that were assessed was not large. Criteria for the serodiagnosis ofanisakiasis. To establish the diagnostic criteria in this microELISA system on the basis of the data demonstrated in figures I and 2 and the data from sera from II, 8, and 2 patients obtained, respectively, at 2,3, and 5 weeks after onset of disease, we set a value for IgG, IgA, and IgM and ofIgE by calculating the OD of each anisakiasis patient's serum and dividing by the OD of sera from 45 healthy controls. As indicated in table I, for IgG, IgA, and IgM, when a value of 1.86was given for the serodiagnosis in microELISA, 100% diagnostic efficiency was obtained at 4 and 5 weeks after onset of the disease. In contrast, all control sera were reasonably included in the negative cases. Table 2 shows a value for the diagnostic criteria as IgE of sera detected by this microELISA. With a value of 1.72, all control sera were clearly categorized into the negative cases, and the diagnostic efficiency was 82 % and 93 % at 1 day and 1 week, respectively, after the clinical onset of anisakiasis. If a cutoff value of 1.60 were to be used, the sensitivity of the IgG, IgA, and IgM test would improve to 46% (18/39). For the IgE test, a cutoff of 1.45 would yield a sensitivity of90% (35/39). The specificity at these levels would be 98 % for the IgG, 19A, and IgM test (44/45) and 100% for the IgE test.

Results Reactivity ofpatient and control sera. Our previous study [5,6] showed that MAb An2 could react specifically with the antigen of AL but not with those of other helminths including A. suum, P. decipiens larvae, Tox. cati, Tr. spiralis, E. multilocularis, and D. immitis. This evidence suggests that sera from patients with anisakiasis could react strongly with An2defined antigen and that a microELISA using MAb An2 could provide relatively specific serodiagnostic assays for anisakiasis. We obtained 93 serum samples from patients with anisakiasis. Of these, 39, 15, 8, 3, and 7 were taken at day I and at 1,4, 8, and 12 weeks, respectively, after onset of disease. Figure I shows the reactivity of serum IgG, IgA, and IgM from patients and 45 control samples. The data clearly indicated that most patient IgG, IgA, and IgM reacted strongly with the An2 antigen at I and 4 weeks after onset of anisakiasis. Even at I day, many samples clearly reacted with the antigen. At 12 weeks after onset of disease, 6 of 7 serum samples showed nearly the same range of reactivity as the control sera. Figure 2 shows the reactivity 01 IgE from patients with anisakiasis and healthy controls. The results showed that many patients reacted strongly with the An2 antigen as early as 1 day or 1 week after disease onset. At 8 weeks after onset, the IgE of patients with anisakiasis did not clearly react with

Discussion The consumption of raw fish (sushi, sashimi) has become more common in many countries. As a result, the incidence of diseases caused by parasites transmitted from fish is increasing [1-4, II, 12].

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lID 1990;161 (May)

Serodiagnosis of Anisakiasis

Table 1. Assessment of criteria for the serodiagnosis of anisakiasis as detected in patient and control IgG, IgA, and IgM.


Table 2. Assessment of criteria for the serodiagnosis of anisakiasis as detected in patient and controllgE.

Values and ranges for criteria * Sera









21/39 5/15 0/11 1/8 0/8 0/2 0/3 5/7

10/39 2/15 2/11 2/8 0/8 0/2 0/3 1/7

2/39 2/15 3/11 3/8 1/8 112 2/3 1/7 +

6/39 6/15 6/11 2/8 7/8 112 1/3 0/7 ++


Sera Healthy controls Anisakiasis patients I day I week 2 weeks 3 weeks 4 weeks 5 weeks 8 weeks 12 weeks Reactivity









4/39 0/15 1111 2/8 118 0/2 0/3 2/7

3/39 1/15 0/11 2/8 2/8 0/2 0/3 3/7

24/39 9/15 8/11 3/8 4/8 112 3/3 2/7 +

8/39 5/15 2111 1/8 1/8 1/2 0/3 0/7 ++


NOTE. -, negative, ±, equivocal, +, positive, + +, strongly positive. * Values for diagnostic criteria for the IgG, IgA, and IgM detection assay were calculated as follows: OD of patient's serum divided by the mean OD of sera from patients without anisakiasis.

NOTE. -. negative, ±, equivocal. +, positive, + +, strongly positive. * Values for diagnostic criteria for IgE detection assay were calculated as follows: OD of patient's serum divided by the mean OD of sera from patients without anisakiasis.

Anisakiasis, a parasitic disease, is caused by the larvae of Anisakis species. If ingested with raw fish, they enter the stomach and intestine. Patients with acute anisakiasis frequently complain of severe abdominal pain. Clinically, however, the diagnosis may be difficult. Endoscopic examinations seldom detect helminths directly. Also, in the chronic phase of the disease, granulomatous lesions are observed in some patients, which necessitate differentiation from neoplastic lesions. Thus, a more precise differential diagnostic system has been in demand, especially one that uses patient serum. Several attempts have been made to develop a diagnostic system by using a polyclonal rabbit anti-Anisakis serum or using extracted Anisakisantigens [13, 14]. However, the results had several drawbacks in terms of the specificity of reaction. We previously developed a microELISA system to detect patients' antibodies to crude Anisakis antigens that were immobilized on ELISA plates. This system, however, indicated a relatively high background for patients without anisakiasis and for sera from healthy persons (data not shown). In the present work, we attempted to establish a more specific serodiagnosis system in microELISA using MAbs already obtained by our laboratory [5, 6]. When MAb An2 was used in this serodiagnostic microELISA system, the data indicated that the reaction was highly specific for anisakiasis patients and the serodiagnostic efficiency was extremely high. When we set an adequate number for the diagnostic criteria from the data obtained from micro ELISA, the efficiency was 100% for IgG, IgA, and IgM detection, particularly at 4 and 5 weeks after the onset of anisakiasis. This was also mostly true for the IgE detection system, especially within 1 week of onset. These data indicate that our present microELISA technique using patients' sera could be very effective for the specific diagnosis of anisakiasis at various clinical stages after onset of the disease. However, the test is not without weakness. Anisakiasis often presents as an acute abdomen; therefore,

a test is needed that is accurate at the onset of symptoms, not 4-5 weeks later. The IgG, IgA, and IgM test had a sensitivity of only 20.5 % at day 1 (8/39), severely limiting its usefulness. The IgE test was better; it had a sensitivity of 82 % (32/39). Further, there was no clear correlation between the reactivity of patients' sera to An2 antigen and the serum level of concentration of IgG, IgA, and IgM and IgE in these patients. Our previous study suggested that the An2-defined antigen, with a heterodimer of 40- and 42-kDa molecules, was one of the excretory-secretory antigens. Our present study indicated that sera from anisakiasis patients could also recognize the An2-defined antigen. However, we lack data on the immunobiologic and immunopathologic significance of the An2defined antigen. Several reports [8, 12] have analyzed the functions of excretory-secretory antigen molecules. These indicate that a major function of these molecules is a strong proteinase-like activity, suggesting a relationship between excretory-secretory antigens and the capability of tissue destruction by AL. It will be of interest to determine whether the An2 molecule is responsible for the pathologic development of this disease. There is also controversy about the severity of clinical manifestations and infection by this specific organism [1, 8]. It has been observed that primed hosts may demonstrate acute anisakiasis with more severe clinical manifestations. However, it is not certain which antigens, such as the granuloma formation factor, are responsible for development of pathologic lesions in anisakiasis. Our establishment of the specific serodiagnosis may give insight into these problems in human anisakiasis. Acknowledgment For sera and data from anisakiasis patients, we thank T. Ooiwa, Ooiwa Gastrointestinal Hospital; H. Ohtaki, Ohtaki Gastrointesti-

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Healthy controls Anisakiasis patients I day I week 2 weeks 3 weeks 4 weeks 5 weeks 8 weeks 12 weeks Reactivity

Values and ranges for criteria *


Yagihashi et al.

nal Hospital; Y. Yamamoto, First Department ofInternal Medicine, Kochi Medical School; T. Tsukioka, Tsukioka Internal Clinic; G. Toyonaga,Second Department of Internal Medicine, Kurume University School of Medicine; and K. Kurokawa, Institute of Gastroenterology, Tokyo Woman's Medical College.





II. 12. 13. 14.

eds. Gastric anisakiasis in Japan: epidemiology, diagnosis, treatment. Tokyo: Springer-Verlag, 1989:103-111 Maizels RM, Phillip M, Ogilvie 8M. Molecules on the surface of parasitic nematodes as probes of the immune response in infection. Immunol Rev 1982;61:109-136 Tanaka J, Torisu M. Anisakis and eosiniphil. I. Detection of a soluble factor selectively chemotactic for eosinophils in the extract from Anisakis larvae. J Immunol 1978;120:745-749 Yagihashi A, Sato N, Torigoe T, Okubo M, Konno A, Takahashi N, Yamashita T,.Fujinaga K, Kuzumaki N, Kikuchi K. Identification of the transformation-associated cell surface antigen expressed on the rat fetus-derived fibroblast. Cancer Res 1988;48:2798-2804 Sato N, Sato T, Takahashi S, Okubo M, Yagihashi A, Koshiba H, Kikuchi K. Identification of transformation-related antigen by monoclonal antibody on Swiss 3T3 cells induced by transfection with murine cultured colon 36 tumor DNA. J Nat! Cancer Inst 1987;78:307-313 DeardorffTL, Fukumura T, Raybourne RB. Invasive anisakiasis: a case report from Hawaii. Gastroenterology 1986;90:1047-1050 Matthews FB. The source, release and specificity of Anisakis simplex larvae. J Helminthol 1984;58:175-185 Kobayashi A, Tsuji M, Wilbur DL. Probable pulmonary anisakiasis accompanying pleural effusion. Am J Trop Med Hyg 1985;34:310-313 Desowitz RS. Human and experimental anisakiasis in the United States. Hokkaido Igaku Zasshi 1986;61:358-371

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I. Asaishi K, Nishino C, Hayasaka H. Geographical distribution and epidemiology. In: Ishikura H, Hamiki M, eds. Gastric anisakiasis in Japan: epidemiology, diagnosis, treatment. Tokyo: Springer-Verlag, 1989:31-36 2. Van Theil PH, Kuipers FC, Raskam RH. A nematode parasitic to herring, causing acute abdominal syndromes in man. Trop Geogr Med 1960;2:97-113 3. Kliks MM. Anisakiasis in the United States: four new case reports from California. Am J Trop Med Hyg 1983;32:526-532 4. Pinkus GS, Coolidge C. Intestinal anisakiasis. First case report from North America. Am J Med 1975;59:114-120 5. Takahashi S, Sato N, Ishikura H. Establishment of monoclonal antibodies that discriminate the antigen distribution specifically found in Anisakis larvae type I. J Parasitol 1986;72:960-962 6. Takahashi S, Ishikura H, Hayasaka H. Monoclonal antibody, intradermal reaction, and Sarles' phenomenon. In: Ishikura H, Namiki M,


JID 1990;161 (May)

A serodiagnostic assay by microenzyme-linked immunosorbent assay for human anisakiasis using a monoclonal antibody specific for Anisakis larvae antigen.

A serodiagnostic assay system to detect human anisakiasis is described. This assay uses a microenzyme-linked immunosorbent assay with monoclonal antib...
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