Anti-dengue IgM antibodies were measured by the indirect ELISA and IgM-capture ELISA using type 2 and 3 dengue antigens on dengue patients\u27 sera. The assay results depended more strongly on the assay method than the serotype of dengue antigen used in the test. The results indicated that amounts of antibodies directed against virion and nonstructural viral antigen were not uniform in individual test serum

Balasubramaniam Vijayamalar

Igarashi Akira

Kheong Chew Tong

Sinniah Mangalam

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Trop. Med., 35 (2), 75-81, June, 1993 75Comparative Assay on Anti-dengue IgM Antibodiesby the Indirect ELISA and IgM-capture ELISAChew Tong KHEONG1,2, Vijayamalar BALASUBRAMANIAM2, Mangalam SINNIAH2and Akira IGARASHI1department of Virology, Institute of Tropical Medicine, Nagasaki University,12-4 Sakamoto- 1 -chome, Nagasaki 852 Japan2Division of Virology,Institute for Medical Research, Jalan Pahang, Kuala Lumpur,MalaysiaAbstract: Anti-dengue IgM antibodies were measured by the indirect ELISA and IgM-cap-ture ELISA using type 2 and 3 dengue antigens on dengue patients'sera. The assay resultsdepended more strongly on the assay method than the serotype of dengue antigen used inthe test. The results indicated that amounts of antibodies directed against virion andnonstructural viral antigen were not uniform in individual test serum.Key words: Dengue, IgM-ELISA, virion, nonstructural viral antigenINTRODUCTIONDengue fever (DF) and dengue hemorrhagic fever (DHF) have been major health pro-blems in many tropical countries especially in so叫east Asia (Halstead, 1966; 1980; 1992).Laboratory diagnosis on DF/DHF has been improved since the introduction of lgM-ELISA todetermine recently infecting viral agent(Bundo and lgarashi, 1985; Lam et al, 1987; Innis etal, 1989). It has generally been considered that the indirect ELISA, although its method issimple, could provide false negative results in the presence of high titered lgG class an-tibodies which compete with less amount of lgM antibodies to bind the assay antigen.However, as shown in the accompanying paper (Chew et al, 1993), pretreatment of test serawith lgG adsorbents did not improve the efficiency to detect anti-dengue lgM antibodies inthe indirect ELISA. On the contrary, such treatment apparently removed certain amount ofanti-dengue lgM together with lgG antibodies, resulting in the reduced number of positivespecimens. Theoretically, indirect ELISA using purified virion as assay antigen could detector measure such antibodies that were directed against virion structural proteins. In the caseof dengue and other鮎viviruses, the envelope glycoprotein (E) is the most dominant an-tigenic component. while in the conventional lgM-capture ELISA (MAC-ELISA), assay an-tigens were either virus-infected cell culture伽id or extracted by sucrose-acetone methodfrom infected mouse brain homogenate. when enzyme-conjugated detecting antibodiesReceived for publication August 2 1993.Contribution No. 2871 from the Institute of Troical Medicine, Nagasaki University76prepared from high titered DHF patients'sera (Innis et al, 1989) were used in the MAC-ELISA with mouse brain-derived antigen, such a system could detect or determine not onlythe antibodies against virion structural protein but also those against nonstructural protein aswell. It could be expected that the immune reaction in each individual patient may not beuniform and producing varying amount of anti-virion as well as anti-nonstruct:ural viral an-tibodies.In this communication, dengue patients'sera were assayed for their anti-dengue lgMantibodies by 2 different ELISA, indirect and MAC, u血g type 2 (D2) and type 3 (D3) an-tigens to see whether the assay result depends more on the assay method or serotype ofdengue antigen used in the test.MATERIALS AND METHODSserum specimens: One hundred serum specimens of dengue patients were selectedfrom the stock in the Division of Virology, Institute for Medical Research (IMR), Kuala Lum-pur, Malaysia. An aliquot of each serum was brought by the first Author to the Departmentof Virology, Institute for Tropical medicine, Nagasaki University. The sera were diluted 1:100with PBS-T (phosphate buffered saline, pH 7.4, containing 0.05% Tween 20 and 0.02% NaN3)for lgM ELISA. A pool of high-titered DHF patients'sera was also brought by the firstAuthor and used to prepare anti-flavivirus lgG.preparation of purified dengue virion antigen and infected culture fluid:Dengue virions of type 2 (D2) New Guinea B strain and type 3 (D3) H87 strain wereprepared from infected C6/36 cell culture fluid as described in the accompanying paper. Apart of the infected culture fluid was kept aside to use as crude assay antigen in MACELISA.Indire¢ IgM ELISA: The procedure was already described in the accompanyingpaper, except that not only D2 but also D3 antigen was used.preparation of anti-flavivirus lgG and conjugation with HRPO: The hightitered DHF patients'serum pool was dialyzed against 100 volume of 5mM sodiumphosphate buffer, pH 7.0, at 4℃ overnight. The dialyzed serum was passed through No. 2filter paper before loading to 20 ml volume of DEAE Sephacel (Pharmacia) which had beenequilibrated with the same buffer. Fractions of 5 ml volume were collected by eluting the col-umn with the same buffer and OD28o was measured for each fraction. Fractions showing highoD28o were pooled and dialyzed against 0.01 M sodium carbonate buffer, pH 9.0, and con-jugated with HRPO by Wilson and Nakane's method (1978).Indirect ELISA: The procedure and interpretation of the results we】re described inthe accompanying paper.MAC ELISA: The flat-bottom 96 well ELISA plate (Nunc) was coated with anti-human lgM (mu chain-specific, Cappel) at 1:200 dilution in a coating buffer (0.05 M sodiumcarbonate buffer, pH 9.6, containing 0.02% NaN3) at 4℃ overnignt. The j'late was mac-tivated by incubation with Blockace (Yukijirushi) at 37℃ for 1 hr. The plate was emptied and77washed successively 4 times with PBS-T using Microplate Washer (Titertek, M96V, Flow),and reacted with diluted test serum along with the standard positive and negative sera at37℃ for 1 hr. The plate was emptied and washed as above, and assay antigen of infectedC6/36 cell culture加id was distributed to incubate at 37℃ for 1 hr. The plate was emptiedand washed as above and HRPO-conjugated anti血vivirus lgG was distributed and the platewas incubated at 37℃ for 1 hr. The optimal dilution of the HRPO-conjugated detecting an-tibody was predetermined by a checkerboard titration. The plate was emptied and washed asabove and substrate solution (0.5 mg/ml叩henylenediamine dihydrochloride and 0.02% H202in 0.05 M citrate-phosphate buffer, pH 5.0) was distributed. The plate was incubated at roomtemperature for 1 hr in the dark and the HRPO reaction was stopped by adding 1 N H2SO4.The OD474 of each well was recorded by an ELISA Analyzer (ETY-96, Tohyoh) using OD630as a reference wavelength. The cut-off value of the MAC ELISA is the same as the indirectELISA, that is double the average OD474 obtained for 8 wells of negative control. TheELISA OD of each test specimen was divided by the cut-off value to obtain P/N ratio. Anyspecimen with P/N ratio equal to or greater that 1.0 were considered as positive.Statistical me也od: The methods described by Snedecor (1951), were used.RESULTSP/N ratio obtained by indirect and MAC ELISA using D2 and D3 antigensAll the test sera were examined for their anti-dengue lgM antibodies by the indirectand MAC ELISA, and the results were summarized in Fig. 1 and Table 1. In Fig. 1, eachdot represents each test serum with their P/N ratio on the X-axis and Y-axis. It can be seenthat significant correlation was observed between the P/N ratios obtained by the same assaymethod (indirect in Panel C, or MAC in Panel F), even when the serotype of assay antigen isdifferent (D2 or D3). The correlation coefficient (R) was 0.455 for the indirect (Panel C), and0.360 for MAC ELISA (Panel F), respectively. On the other hand, almost no correlation wasobserved between the P/N ratio obtained by the same serotype of assay antigen when dif-ferent assay methods were used. R value between indirect and MAC ELISA was 0.009 forD2 (Panel A), and 0.039 for D3 (Panel D), respectively. The R value was further lower forTable 1. Average and standard deviation (SD) of P/N ratio of anti_dengue lgM antibodies obtained by indirect and MACELISAAssay Assay　　　　　　　/N ratioAntigen MethodAverage s DD2D2D3D3MAC　　　　　　　2. 113　　　　　2. 068indirect 1. 226　　　　　0. 759MAC 1. 176　　　　　0. 774indirect　　　　　　2. 369　　　　1. 081784321y - 1.1510 +3.4928e-2x RA2 -0.009BE)0EbElH aBrffl H　　　　　　ロB 0ロ"HQ)005 ｢･･｢EDA10㌔　ロ　2.4082-1.8162e-2x R∧　0.001EQ4　品HH cjHS E正H慣昆｡a2惜冨666亀ロ｡oEb9　　E)Dl　鳴招巳鼻E00543210E]HQHn　　ロ13　BQQHB54ロ　y=2.6927-0.27469x RA2=0.039E) ㌔aド巳昏ロQDQH�"きiロ+BO取口㌔2≒…=BQElEI HHtヨHQ QHBH HGQ ElロBIH aロロ95　電｡02043210y = 1.1937+ 0.96023x RA2 = 0.455Q BElaaHO3ロ　匂日日　ロDEP日　田口亜軸o ｣i*lH5>"&a:回日田鼻El<ra aB o Q回　QLJ　　ロロO Q B♂甘　　口DEIB HEltヨn DCロBH HロEl回すQQQatヨ2　　　　　3　　　　　4QElH臣】y=1.2183+5.56206-3x R∧　o.oeoOBoElロB QE)B QQ=終日｡【ロ占官,BBロ　t)　_　】　ロ-　　　　　　　　　_　　!≡ rnEIl　鳴管□｡8　=　　　8OQ Eか期c~ = :落-=モ02　　　　　3　　　　　402054ロ3　　　　Ela21　010o　　　　1　　　　2　　　　　3　　　　　4　　oy ≡ o.70088 + 0.22461x R∧2 ≡ 0.360tdalヨE)HH Q 0E)回B�"育=育H中^別ijjntl飼い!3し乃J!覗El105520DロEDFFig. 1. Correlation between the P′N ratio of anti-dengue lgM antibodies obtained by indirect andMAC ELISA using D2 and D3 antigensEach dot represents each test serum, with its PIN ratio obtained by indirect o∬ MAC ELISAusing D2 or D3 antigen. Panel A: D2 MAC on X-axis and D2 indirect on Y-axis; Panel B:D2 MAC on X-axis and D3 indirect on Y-axis; Panel C: D2 indirect on X-axis -and D3 in･direct on Y-axis; Panel D: D3 MAC on X-axis and D3 indirect on Y-axis; Panel E: D3 MACon x-axis, D2 indirect on Y-axis; Panel F: D2 MAC on X-axis, D3 MAC on Y-axis, respec-tively. and MAC IgM ELISA.79the combinations of different assay method using different serotype of assay antigen 【0.001between D2 MAC and D3 indirect in Panel B, and 0.000 between D3 MAC and D2 indirectin Panel E, respectively]. The average and standard deviation (SD) of P/N ratio werecalculated for each assay method and assay antigen as shown in Table 1. The statistical test(t-test) showed that the difference between the average P/N ratio was significant betweenfollowing combinations: D2 MAC and D2 indirect (P<0.01); D2 MAC and D3 MAC(P<0.01); D2 indirect and D3 indirect (P<0.001).Serodiagnosis by MAC and indirect lgM ELISAThe number of test serum which showed positive or negative results by the MAC andindirect lgM ELISA using D2 and D3 antigens was summarized in Table 2. The agreementwas higher for the same assay method using different serotype of antigens 【0.673 for D2 in-direct and D3 indirect; 0.752 for D2 MAC and D3 MAC】 than the different assay method us-mg the same serotype of antigen 【0.475 for D2 indirect and D2 MAC; 0.366 for D3 indirectand D3 MAC】. Further reduction of the agreement was not observed for the combinations ofTable 2. Number of positive specimens obtained by the indirect and MAC ELISA usingD2 and D3 antigens and agreement of the test resultscompare<assayant♀1results,genand-ethod芸-aa田�"ree-enti｡D2 MAC (+)D2 MAC (-)D2 MAC (+)D2 MAC (-)D2 MAC (+)D3 MAC (-)D3 MAC (+)D3 MAC (-)D2 indirect (+)D2 indirect (-)D2 indirect (-)D2 indirect (+)D3 indirect (+)D3 indirect (-)D3 indirect (-)D3 indirect (+)D2 indirect (+); D3 indirect (+)D2 indirect (-); D3 indirect (-)D2 indirect (+); D3 indirect (-)D2 indirect (-); D3 indirect (+)D2D2D2D2MAC (+); D3 MAC (+.)MAC (-); D3 MAC (-)MAC (+); D3 MAC (-)MAC (-); D3 MAC (+)D2 indirect (+); D3 MAC (+)D2 indirect (-); D3 MAC (-)D2 indirect (+); D3 MAC (-)D2 indirect (-); D3 MAC (+)D3 MAC (+); D3 indirect (+)D3 MAC (-); D3 indirect (-)D3 MAC (+); D3 indirect (-)D3 MAC (-); D3 indirect (+)35133113544123154IE231423424121233324325ll5348　　　　　　　0. 47558687644370. 5740. 6730. 7520. 4360. 36680different assay method using different serotype of antigen [0.574 for D2 MAC and D3 in-direct; 0.436 for D2 indirect and D3 MAC].DISCUSSIONour results showed that the result of anti-dengue lgM ELISA was influenced moresignificantly by the assay method (MAC or indirect) than by the serotype of assay antigen(D2 or D3). Such a result indicates that in some patients'sera lgM antibodies were preferen-tially produced against virion antigen, probably E protein, while, in other patients antibodiesdirected against nonstructural protein may predominate. This reasoning waits verification byfu仙er experiments using other methods, such as western blotting on several test sera which血owed significantly different P/N ratios by MAC and indirect ELISA. In the conventionalwestern blotting, however, it should be kept in mind that the assay antigen has to bedenatured before loading to the SDS-PAGE, and the procedure may miss to detect such an-tibodies directed to the conformational-dependent epitopes. Nevertheless, it may be in-teresting to see whether the different reactivity of the patient's sera to different viral an-tigenic components may be related with the clinical manifestation of DF/DHF, since im-munopathological processes have been postulated to play important roles in the pathogenesisof severe DHF or dengue shock syndrome (Russell and Brandt, 1973; Halstead and 0 Rouke,1976; Halstead, 1988).AcKNOWLEDGMENTSThe first author was supported by the Japan International Cooperation Agency (JICA)for his travel and stay in Japan under the IMR-JICA Project for the Research on SelectedTropical Diseases in Malaysia, in the fiscal year 1992.REFERENCE S1 ) Bundo, K., and lgarashi, A. (1985): Antibody-capture ELISA for detection of immunoglobulin M an-tibodies in sera from Japanese encephalitis and dengue hemorrhagic fever patients. J. Virol.Methods, ll: 15-22.2 ) Chew T. -K., Balasubramaniam, V., Sinniah, M., & Igarashi, A. (1993): Pretreatment of test serawith three kinds of lgG-adsorbents did not improve sensitivity in the indirect ELISA to detect anti-dengue lgM antibodies. Trop. Med. 35: 65-73.3 ) Halstead, S. B. (1966)‥ Mosquito-borne haemorrhagic fevers of south and southeast Asia. Bull.WHOリ35: 3-15.4 ) Halstead, S. B. (1980): Dengue haemorrhagic fever - a public health problem and a field forresearch. Bull. WHO., 58: 1-21.5 ) Halstead, S. B. (1988): Pathogenensis of dengue: challenge to molecular biology. Science, 239:476-481.816 ) Halstead, S. B. (1992): The XXth century dengue pandemic: need for surveillance and research.Wld. Hlth. Statist. Quart., 45: 292-298.7 ) Halstead, S. B., & O'Rouke, E. J. (1976): Antibody-enhanced dengue virus infection in primateleukocytes. Nature (London), 265: 739-741.8 ) Innis, B. L., Nisalak, A., Nimmannitya, SリKusalerdchariya, S., Chongswasdi, VリSuntayakorn, S.,Puttisri, P., & Hoke, C. H. (1989): An enzyme-linked immunosorbent assay to characterize dengueinfections where dengue and Japanese encephalitis co-circulate. Am. J. Trop. Med. Hyg., 40:418-427.9 ) Lam, S. K., Devi, S., & Pang, T. (1987): Detection of specific lgM in dengue infection. SoutheastAsian J. Trop. Med. Publ. Hlth., 18: 532-53声.10) Russell, P. K., and Brandt, W. E. (1973): Immunopathological processes and viral antigensJ;associated with sequential dengue virus infection. pp263-277. In M. Pollard (ed.). Persistent Virallnfections. Perspect. Virol., vol. 8.1 1) Snedecor, G. W. (1951): Statistical Methods Applied to Experiments in Agriculture and Biology. Thelowa State College Press, Iowa.12) Wilson, M. B., & Nakane, P. K. (1978): Recent developments in the periodate method of con-jugating horse radish peroxidase (HRPO) to antibodies. In W. Knapp, K. Holubar, & G. Wick (ed.).Immuno加orescence and Related Staining Techniques. Elsevier / North-Holland Biochemical Press,Amsterdam.

Comparative Assay on Anti-dengue IgM Antibodies by the Indirect ELISA and IgM-capture ELISA

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Comparative Assay on Anti-dengue IgM Antibodies by the Indirect ELISA and IgM-capture ELISA

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