Dengue

Dengue Bulletin Volume 28 (2004)

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Detection of Dengue Virus Serotype-specific IgM
by IgM Capture ELISA in the Presence of
Sodium thiocyanate (NaSCN)

Masaru Nawa*^#, Tomohiko Takasaki**, Mikako Ito**, Ichiro Kurane**
and Toshitaka Akatsuka*

*Department of Microbiology, Saitama Medical School, 38, Moroyama, Saitama 350-0495, Japan
**Division of Vector-Borne Viruses, Department of Virology 1, National Institute of Infectious Diseases,
1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan

Introduction

Dengue is currently one of the most important arboviral disease in humans. Dengue viruses, belonging to the family Flaviviridae, are comprised of four antigenically cross-reactive serotypes and are responsible for epidemics in tropical and subtropical countries. Since the dengue outbreaks in Osaka, Kobe, Hiroshima and Nagasaki from 1942 to 1945, dengue has not occurred in an epidemic form in Japan[1]. However, imported dengue cases have been reported[2]. Approximately five million Japanese people annually visit countries in  tropical and subtropical areas and nearly two million people visit Japan from these areas. Therefore, dengue fever (DF) and dengue haemorrhagic fever (DHF) has become an infectious disease of significance and worthy of more attention from the medical community in Japan.

We have earlier reported the laboratory diagnosis of dengue by reverse transcription polymerase chain reaction (RT-PCR) and IgM-ELISA[3-5]. We demonstrated that IgM-ELISA was a reliable diagnostic method and that IgM responses were generally serotype cross-reactive but often highest against infecting virus serotype in most Japanese cases[4]. The serotype specificity of IgM responses in dengue patients has been controversial[6-8]. Burke had reported that serotype-specific IgM responses corresponding to the isolated virus type were detected in primary dengue virus infection[6]. Gubler had reported that in dengue infection, frequent monotypic IgM responses were not correlated with the virus serotype isolated from patients[7]. In 1984, Inouye et al.[9] demonstrated a new technique for the differentiation of antibody avidity after virus infection, i.e. rubella, rota and Japanese encephalitis viruses. They estimated the antibody avidity to viral antigen using a low concentration of a protein-denaturing agent, guanidine hydrochloride, in the diluent of antibody in the ELISA. They concluded that the “stringent immunosorption” technique was useful for investigating the antigenic relationship among closely-related viruses.

In the present study, we examined serotype-specific IgM responses under stringent conditions in the presence of a chaotropic agent, sodium thiocyanate (NaSCN), in the reaction mixture of dengue viral antigens and patients’ sera. The development of a simple method to distinguish serotype-specific reaction from cross-reaction will be useful not only for laboratory diagnosis but also for seroepidemiological studies.

Materials and methods

Twenty-eight serum specimens from 18 confirmed Japanese dengue cases were used in the study. Serum samples from 22 Japanese subjects with other illnesses, who had never been to areas where dengue was epidemic-prone or endemic, were used as the control. These sera were obtained for diagnostic purposes in clinics and hospitals in Japan from 2000 to 2002 and sent to the Department of Virology 1, National Institute of Infectious Diseases, Tokyo, Japan.

Prototype dengue viruses were propagated in the Aedes albopictus mosquito cell clone C6/36, and the infected cell culture supernatants were inactivated by incubation with beta-propiolactone at a final concentration of 0.2% for 30 minutes at 37 °C as previously reported[10]. Tetravalent and four monovalent dengue viral antigens were prepared by the method as previously reported[4,5].

The IgM capture ELISA was carried out according to the method described previously[3-5]. Anti-human IgM (µ chain specific) goat serum and peroxidase-conjugated anti-human IgM were purchased from Sigma-Aldrich, Inc, USA. The IgG fraction of the flavivirus-specific monoclonal antibody, D1-4G2, was prepared by the Protein G affinity chromatography kit (ImmunoPure G IgG Purification kit, PIERCE, USA), and then conjugated with horseradish peroxidase by a commercial kit (EZ-Link Plus Activated Peroxidase kit, PIERCE, USA).

In order to detect the serotype-specific reaction between dengue viral antigen and dengue virus-specific IgM antibody, patient serum was treated with the Protein G affinity chromatography kit described above. The IgG fraction in the serum specimen was removed by adding Protein G beads according to the manufacturer’s instruction. Unless otherwise stated, data were presented as the mean of independent two-to-three assays.

Results and discussion

One of the authors has previously reported that the addition of NaSCN to the reaction mixture of ELISA highlights serotype-specific reaction between crude dengue viral antigen and anti-dengue hyperimmunized mouse sera[11]. The antigen-antibody reaction was affected not only by the concentration of NaSCN but also by the procedures of the NaSCN treatment. A concentration higher than 0.7 M inhibited the reaction non-specifically, while a concentration lower than 0.3 M had no effect on the discrimination of serotype-specific reaction from the cross-reactive one. According to these results, we decided to use NaSCN in IgM-capture ELISA at the following conditions: (i) NaSCN was included at a final concentration of 0.5 M in 10% normal calf serum-PBS; (ii) peroxidase-conjugated flavivirus- specific monoclonal antibody D1-4G2 (D1-4G2) was diluted in 0.5 M NaSCN; and (iii) viral antigens captured by patients’ IgM were detected by the detection antibody in 0.5 M NaSCN.

Figure 1. The effects of NaSCN treatment on antigen-antibody reaction

(a) Human serum was serially diluted from 1:50 to 1:400 with 10% CS-PBS containing 0.5 M NaSCN, and IgM was captured on anti-human IgM goat serum-coated wells. IgM was detected with peroxidase-conjugated anti-human IgM goat serum. (b) Serially diluted tetravalent dengue viral antigen was captured on the solid phase sensitized with D1-4G2 IgG, and then detected with peroxidase-conjugated D1-4G2 IgG in the presence of 0.5 M NaSCN.

Figure 1 shows the reactions between anti-human IgM and patients’ IgM (a), and dengue viral antigen and the detection antibody (b), in the presence of 0.5 M NaSCN in the ELISA. These two reactions were not affected by 0.5 M NaSCN, suggesting that IgM capture and detection of dengue viral antigens were not affected in IgM-capture ELISA.

Figure 2 shows anti-dengue IgM titration curves in the presence or absence of 0.5 M NaSCN in the ELISA. The IgG fraction in the serum specimen was removed by adsorption with the Protein G beads (UltraLink Immobilized Protein G Plus, capacity: ~25 mg IgG/ml of the gel, PIERCE). The levels of cross-reaction between dengue viral antigens and patients’ IgM antibody (a) were decreased after the treatment with 0.5 M NaSCN in the ELISA (b): The level of reaction with homologous dengue-1 antigen was less affected. The results suggested that the addition of 0.5 M NaSCN to the reaction mixture of ELISA may highlight a serotype-specific reaction between crude dengue viral antigen and anti-dengue IgM antibody.

Figure 2. Titration curves of dengue virus specific IgM antibody in the presence

or absence of 0.5 M NaSCN in the ELISA

We examined the IgM levels by ELISA with the antigen of 4 dengue virus serotypes. The table below shows the results of 28 serum samples from 18 Japanese dengue patients. The infecting dengue virus serotypes were determined by RT-PCR. The data were presented as the index value according to the method described previously^[4]. We defined index values of 2.28 or greater and 26.60 or greater as positive, respectively, in the absence and presence of 0.5 M NaSCN in the ELISA. In all the tested cases, the serotype-specific IgM levels were the highest against the infecting dengue virus serotype than against three other serotypes in the presence of NaSCN. The serotype-specific IgM responses were more highlighted in the presence of NaSCN than in its absence. These data suggest that IgM responses to the infected dengue virus serotype determined by RT-PCR in primary dengue infection were the highest. These results agreed with the report by Burke^[6].

Table. Serodiagnosis of dengue by IgM-ELISA with and without 0.5M NaSCN

D1 to D4, dengue virus type 1 to 4, respectively. ND, not determined.
* index values were calculated by the formula A₄₉₂ with the viral antigen/A₄₉₂ with uninfected control antigen.

The chaotropic agent, NaSCN, is known to denature protein structure and inhibit the formation of immune complexes as was exploited for immunoaffinity chromatography^[12,13]. There are multiple factors which affect NaSCN’s ability to highlight serotype-specific reaction in comparison with cross-reaction in the ELISA. One of the factors is a characteristic of dengue viral antigens, which are prepared from the infected mosquito cell culture. It was reported that a rapidly sedimenting haemagglutinin (RHA) was cross-reactive and labile, but the soluble complement-fixing antigen (SCF) was serotype-specific and relatively stable against the NaSCN treatment in the ELISA^[11,14]. The predominant polypeptides in RHA and SCF fractions represent the envelope glycoprotein and NS1, respectively^[11]. Trent et al.^[15,16] observed three antigenic determinants on the envelope glycoprotein: (i) flavivirus group- reactive; (ii) complex-specific; and (iii) serotype-specific. Henchal et al.^[17] characterized four antigenic determinants on the envelope glycoprotein: (i) flavivirus group-reactive; (ii) dengue complex-specific; (iii) dengue subcomplex-specific; and (iv) dengue serotype-specific.

Brandt et al.^[14] reported that the DEN-2 SCF antigen extracted from infected mouse brains was resistant to the treatment with protein denaturing agents. Falconar and Young^[18] reported serotype-specific epitopes on NS1. These results suggest that heterologous antigenic determinants on dengue viral antigens contribute to the cross-reactivity among the four dengue serotypes, and that the presence of a chaotropic agent in the reaction mixture induces changes of viral antigens and may decrease the cross-reactive antigenicity.

In conclusion, we demonstrated that the addition of NaSCN to IgM-capture ELISA highlighted the detection of serotype-specific IgM in comparison with serotype cross-reactive IgM. This procedure may be useful for determining infecting dengue virus serotypes, especially in primary dengue virus infection.

Acknowledgement

This work was supported by a grant for research on emerging and re-emerging infectious diseases from the Ministry of Health, Labour and Welfare, Japan.

References

1.      Hotta S. Twenty years of laboratory experience with dengue virus. In Saunders M and Lennnet EH (Eds.), 1965, Medical and Applied Virology. Green, St Louis: 228-256.

2.      Yamada K, Takasaki T, Nawa M, Nakayama M, Arai TY, Yabe S and Kurane I. The features of imported dengue fever cases from 1996 to 1999. Jpn J Infect Dis, 1999, 52: 257-259.

3.      Yamada K, Nawa M, Takasaki T, Yabe S and Kurane I. Laboratory diagnosis of dengue virus infection by reverse transcriptase polymerase chain reaction (RT-PCR) and IgM-cpature enzyme-linked immunosorbent assay (ELISA). Jpn J Infect Dis, 1999, 52: 150-155.

4.      Nawa M, Yamada K, Takasaki T, Akatsuka T and Kurane I. Serotype-cross-reactive immunoglobulin M responses in dengue virus infections determined by enzyme-linked immunosorbent assay. Clin Diag Labo Immunol, 2000, 7: 774-777.

5.      Nawa M, Takasaki T, Yamada K, Akatsuka T and Kurane I. Development of dengue IgM-cpature enzyme-linked immunosorbent assay with higher sensitivity using monoclonal detection antibody. J Virol Methods, 2001, 92: 65-70.

6.      Burke DS. 1983. Rapid methods in the laboratory diagnosis of dengue virus infections. In T Pang and R Pathmananathan (Eds.), Proceedings of the International Conference on Dengue/Dengue Hemorrhagic Fever. University of Malaya, Kuala Lumpur, Malaysia, pp. 72-84.

7.      Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev, 1998, 11: 480-496.

8.      Innis BL. Antibody responses to dengue virus infection. In DJ Gubler and G Kuno (Eds.), Dengue and dengue hemorrhagic fever, Wallingford: CAB International, 1997: 221-243.

9.      Inouye S, Hasegawa A, Matsuno S and Katow S. Changes in antibody avidity after virus infections: detection by an immunosorbent assay in which a mild protein-denaturing agent is employed. J Clin Microbiol, 1984, 20: 525-529.

10.   Nawa M, Takasaki T, Yamada K, Kurane I and Akatsuka T. Development of IgM-cpature enzyme-linked immunosorbent assay for serodiagnosis of dengue using beta-propiolactone-inactivated dengue viral antigens. Dengue Bulletin, 2003, 27: 95-99.

11.   Nawa M. An enzyme-linked immunosorbent assay using a chaotropic agent (sodium thiocyanate) for serotype specific reaction between cude dengue viral antigen and anti-dengue mouse antibody. Microbiol Immunol, 1992, 36: 721-730.

12.   Dandlinker WB and de Saussure VA. Antibody purification at neutral pH utilizing immunospecific adsorbents. Immunochemistry, 1968, 5: 357-365.

13.   Avrameas S and Ternyck T. Use of iodide salts in the isolation of antibodies and the dissolution of specific immune precipitates. Biochem J, 1976, 102: 37c-39c.

14.   Brandt WE, Cardiff RD and Russell PK. Dengue virions and antigens in brain and serum of infected mice. J Virol, 1970, 6: 500-506.

15.   Trent DW, Harvey CL, Qureshi A and Lestourgeon D. Solid-phase radioimmunoassay for antibodies to flavivirus structural and nonstructural proteins. Infect Immunol, 1976, 13: 1325-1333.

16.   Trent DW. Antigenic characterization of flavivirus structural proteins separated by isoelectoric focusing. J Virol, 1977, 22: 608-618.

17.   Henchal EA, Gentry MK, McCown JM and Brandt WE. Dengue virus-specific and flavivirus group determinants identified with monoclonal antibodies by indirect immunofluorescence. Am J Trop Med Hyg, 1982, 31: 830-836.

18.   Falconar AKI and Young PR. Production of dimer-specific and dengue virus group cross-reactive mouse monoclonal antibodies to the dengue 2 virus non-structural glycoprotein NS1. J Gen Virol, 1991, 72: 961-965.