Development of IgM-capture Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Dengue using Beta-propiolactone-inactivated Dengue Viral Antigens Masaru Nawa* , Tomohiko Takasaki**, Ken-Ichiro Yamada**, 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, Shinjyuku-ku, Tokyo 162-8640, Japan #
by
Abstract Dengue viral antigen was treated with beta-propiolactone (BPL) and used in immunoglobulin M (IgM)capture enzyme-linked immunosorbent assay (IgM-capture ELISA) for the serodiagnosis of dengue. Dengue viruses lost infectivity after treatment with 0.2% BPL at 37oC for 10 minutes and longer. Fortyeight human serum samples, including both positives and negatives, were tested by IgM-capture ELISAs with untreated and BPL-treated dengue antigens. The results obtained by IgM-capture ELISA with BPL-treated dengue antigens were consistent with those obtained by ELISA with untreated dengue antigens. This IgM-capture ELISA system with BPL-inactivated dengue antigens is safe and more suitable for use in diagnostic laboratories. Keywords: Dengue, IgM-capture ELISA, Beta-propiolactone.
Introduction Dengue fever (DF)/dengue haemorrhagic fever (DHF) is a serious public health problem in many tropical and subtropical areas of the world(1). In recent years, epidemics of DF/DHF have dramatically increased in the Western Pacific, South-East Asia and South America because of rapid #
population growth, uncontrolled urbanization and increased frequency of air travel(2). DF/DHF will continue to be a major public health problem in the world unless effective control measures are taken. Dengue surveillance is essential to monitor epidemics, both at national and international levels. Laboratory diagnosis is necessary to confirm dengue virus infections. We have
For correspondence: mnawa@saitama-med.ac.jp
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previously reported about dengue IgMcapture ELISA with a high sensitivity(3). Four types of dengue viruses were combined and used as a tetravalent dengue antigen in the IgM-capture ELISA. Dengue viruses are a human pathogen, and the use of infectious viruses as antigens can be a potential problem in the assay. In the present study, we used beta-propiolactone (BPL)inactivated dengue viruses as antigens in IgM-capture ELISA to access its specificity and sensitivity vis-à-vis infectious viruses as antigens.
Erembodegen-Aalst, Bergium), and then conjugated with horseradish peroxidase (EZLink Plus Activated Peroxidase kit®, Perbio Science). Prototype dengue viruses were inactivated by incubation with BPL at a final concentration of 0.2% (v/v) for 30 min at 37oC. The infectivity of the virus was titrated by the focus-forming method in Vero cells(6).
Results and discussion Prototype dengue virus strains: type 1, Hawaii; type 2, New Guinea C; type 3, H87 and type 4, H241, were propagated in mosquito cell clone, C6/36, and then inactivated by BPL. The infectivity of the viruses obtained before and after BPL treatment is presented in the Table. Dengue viruses lost infectivity after treatment with BPL for 10, 30 and 60 minutes (Table). In order to prepare the assay antigen, BPL-treated antigen titration was carried out by an antibody sandwich ELISA according to the previously described method(3). (data not shown). The tetravalent antigen was prepared by mixing equal volumes of each monovalent antigen according to the method described previously(3,4). The reactivity to H-IgG and 4G2 IgG was compared between the untreated and BPL-treated dengue antigens (Figure 1). The reactivity of the BPL-treated virus to H-IgG was low, while that to 4G2 IgG was high enough to be used as antigen in IgM-capture ELISA.
Materials and methods IgM-capture ELISA was performed as previously reported(3,4). Forty-eight serum specimens from 33 Japanese dengueconfirmed cases were used in the study. The sera were obtained in clinics and hospitals in Japan from 1998 to 2000 and sent to the Department of Virology 1, National Institute of Infectious Diseases, Tokyo, Japan. The sera were stored at –80oC before use. The serum samples (0.1 ml) diluted at 1:101 were subjected to ELISA. The result was obtained with a positive to negative (P/N) ratio: P/N ratio = A492, reading with the viral antigen/A492, reading with uninfected control antigen. IgG purified from pooled dengue patients sera (H-IgG) and the flavivirusspecific monoclonal antibody, D1-4G2-4-15 (4G2) (5), were used as the detection antibodies. IgG was purified by protein G affinity column chromatography (ImmunoPure IgG Purification kit®, Perbio Science, 96
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Development of IgM-capture ELISA for Serodiagnosis of Dengue using BPL
Table. Effect of BPL on infectivity of dengue viruses Dengue virus serotypes Original virus titres FFU/0.025 ml 6.9 x 10 5
Incubation time (min) 10 30 60 10 30 60 10 30 60 10 30 60
No BPL treatment FFU/0.025 ml 6.9 x 105 6.2 x 105 5.9 x 105 1.2 x 105 1.1 x 105 1.0 x 105 1.3 x 104 1.3 x 104 1.3 x 104 4.6 x 104 4.6 x 104 4.6 x 104
0.2 % BPL FFU/0.025 ml < 1.0 x 102 < 1.0 x 102 < 1.0 x 102 < 1.0 x 102 < 1.0 x 102 < 1.0 x 102 < 1.0 x 102 < 1.0 x 102 < 1.0 x 102 < 1.0 x 102 < 1.0 x 102 < 1.0 x 102
DEN-1
DEN-2
1.2 x 10
5
DEN-3
1.3 x 10
4
DEN-4 FFU: focus formin unit
4.8 x 10
4
Figure 1. Reactivity of the tetravalent BPL-treated dengue viral antigen 3.0 2.5 2.0 ELISA O.D. 1.5 1.0 0.5 0.0 1x 2x 4x 8x 16 x 32 x 64 x Antigen dilution [Dengue antigen captured on the solid phase was detected by horseradish peroxidase-conjugated H-IgG (2.5 µg/ml, square) and 4G2 IgG (2.5 µg/ml, circle). Solid lines indicate mock-treated antigen, and broken lines indicate BPL-treated antigen.]
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Forty-eight human samples including positives and negatives were tested by IgMcapture ELISA with untreated and BPLtreated antigens (Figure 2). Positive(P)/ negative(N) ratios obtained by the ELISA with untreated dengue antigens were somewhat
higher than those obtained by the ELISA with BPL-treated antigens. However, the P/N ratios of these IgM-positive and negative serum samples were correlated (r2=0.824) between these two ELISA systems.
Figure 2. A comparison of results obtained by IgM-capture ELISA with BPL-treated and mock-treated antigens 20 y = 1.1824x - 0.5145 r2 = 0.824
Mock-treated Ag
15
10
5
0 0 5 10 BPL-treated Ag [Forty-eight dengue IgM-positive and negative serum specimens were subjected to IgM-capture ELISA. Results were presented as the P/N ratio.]
15
20
BPL induces alkylation of the proteins and inactivates viruses(7). The reaction is completed by incubating the viruses with BPL at 37oC, and degrades per se depends on hydration during the incubation period. In the present study, BPL inactivated dengue viruses, but did not change the reactivity to 98
the monoclonal antibody, 4G2. The results obtained by the IgM-capture ELISA with BPL-treated antigen were similar to those obtained by the IgM-capture ELISA with untreated antigens. Thus, the newlydeveloped ELISA system maintains high specificity and sensitivity. The new system Dengue Bulletin – Vol 27, 2003
Development of IgM-capture ELISA for Serodiagnosis of Dengue using BPL
also has the advantage in the prevention of possible laboratory infections. The IgMcapture ELISA with BPL-inactivated dengue antigen is, therefore, suitable for use in diagnostic laboratories in developing countries.
Acknowledgement This work was supported by grants from the Research on Emerging and Re-emerging Infectious Diseases, Ministry of Health, Labour and Welfare, Japan.
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