Hemadsorption Immunosorbent Technique for the Detection of

JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1986, p. 17-174 95-1137/86/117-5$2./ Copyright 1986, American Society for Microbiology Vol. 23, No. 1 Hemadsorption Immunosorbent Technique for the Detection of Dengue Immunoglobulin M Antibody K. GUNASEGARAN,' T. W. LIM,2 A. AHMED,' J. G. AASKOV,3 S. K. LAM,4* AND T. PANG4 Department of Genetics and Cell Biology' and Department of Medical Microbiology,4 University of Malaya, and Institute for Medical Research,2 Kuala Lumpur, Malaysia, and Queensland Institute for Medical Research, Brisbane, Australia3 Received 3 June 1985/Accepted 7 October 1985 We developed a highly specific, sensitive, and economical hemadsorption immunosorbent technique for the detection of dengue-specific immunoglobulin M (IgM) antibody. The technique is based on the reaction of human sera with anti-human IgM immobilized onto a solid phase followed by the detection of dengue-specific IgM by the addition of a known quantity of dengue virus hemagglutinin and goose erythrocytes. Denguespecific IgM-positive sera showed hemadsorption. IgM antibody specific for dengue virus was detected in 22 of 39 (56%) convalescent-phase sera from primary dengue infections and 8 of 1 (8%) convalescent-phase sera from secondary dengue infections. Additionally, 32 of 76 single sera from patients were positive for dengue IgM; these sera were previously uninterpretable by the hemagglutination inhibition test, as only a single serum specimen was available. No false-positive results were obtained with sera that were negative by the hemagglutination inhibition test for dengue virus. Crude dengue virus hemagglutinin preparations could be used without purification. Dengue-specific IgG did not interfere with the results, nor was there any cross-reactivity between dengue hemagglutinins and IgM specific for other viruses. Some cross-reactivity of the dengue-specific IgM was observed with Japanese encephalitis virus hemagglutinins, but this did not present any problems in the interpretation of results. This test is specific, inexpensive, highly reproducible, and simple to perform. Dengue fever (DF) and dengue hemorrhagic fever (DHF) contribute significantly to morbidity and mortality in many parts of the tropical world. An important factor in the management of DF and DHF and in the prevention and control of outbreaks is the availability of a rapid, sensitive, specific, and economical laboratory diagnostic test to confirm dengue infection. However, currently available tests based on virus isolation and serology are still unsatisfactory in this regard. With regard to serology, most laboratories use conventional methods such as hemagglutination inhibition (HAI), the complement fixation test, and the neutralization test to diagnose dengue virus infections. These serological tests are of diagnostic importance if a seroconversion or fourfold or greater rise in antibody titers between acute- and convalescent-phase sera can be demonstrated. Very often the collection of paired sera is not well spaced to show a rise in titer of diagnostic value, or only a single serum specimen is collected. It would thus be of value to develop a specific and economical test which would be positive early in the infection and, preferably, require only a single specimen. The detection of dengue-specific immunoglobulin M (IgM) may be consistent with these considerations. Because IgM generally appears early in the onset of disease and is relatively transient, its detection would be an indication of an ongoing or recent infection. Conventional methods of detecting IgM antibody require the physical separation of IgM from IgG. These methods, besides being costly, are tedious to perform and time-consuming. A significant advance in the rapid diagnosis of viral infections was the development of solid-phase immunoassays for the detection of virus-specific IgM antibody (4, 6, 15, 16). The basis * Corresponding author. 17 for such assays is the "capture" of IgM by an anti-,. chain antibody bound to a solid phase. The presence of virusspecific IgM is demonstrated by the addition of viral antigen and an indicator system, e.g., erythrocytes. The development of such an approach obviates the need for physical separation of IgM and IgG molecules. Krech and Wilhelm (7) reported the development of the solid-phase immunosorbent technique for the detection of anti-rubella IgM antibody. In this immunoassay, sheep erythrocytes were used as the indicator system. The solid-phase immunosorbent technique was further modified by van der Logt et al. (13) to the more sensitive hemadsorption immunosorbent technique (HIT) for the determination of rubella and mumps IgM. IgM antibody capture immunoassays have also been developed for a number of other arboviruses (1, 2, 9), including dengue. We report here our attempts to develop the HIT for determining specific IgM antibody in dengue virus infections. MATERIALS AND METHODS Sera. Three different groups of sera were used in screening for IgM antibody to dengue virus. Group 1 consisted of 6 pairs of sera from dengue patients, 4 single high-titered dengue convalescent-phase sera (high HAI antibody titers), and 76 single sera from dengue patients. The 6 pairs of sera from dengue patients consisted of acute- and convalescent-phase sera from primary (39 pairs) and secondary (1 pairs) dengue infections as defined by the World Health Organization guide (14) and 11 pairs of sera negative by the HAI test. All acute-phase sera were collected between days 1 and 5, and convalescent-phase sera were collected between days 7 and 15 after the onset of symptoms. As all these sera were collected during an epi-

VOL. 23, 1986 HIT FOR THE DETECTION OF DENGUE-SPECIFIC IgM ANTIBODY 171 a- >, 128- Fm 64- z 4 32- x cm 16- w 8- Rfi 4 FI FE <4 ' a) Antibody Titres Against DEN 1 ;:. 128' m 64- F z 32-2 ) 16 w cr 8- F: E 4 - I- IC <4- b) Antibody Titres Against DEN 2 1-% ` ' 128 co 64' F: z < 32 16' I- Fe 4' I <4' 'I 2 4 8 16 32 64 128 c) Antibody Titres Against DEN 3 2 4 8 16 3264 128 _ D 128 o 64 z 32 2 16 R 8 w,- 4- i-. <4- I 2 4 8 16 3264128 256 HAI TITRE (TOTAL AN11 BODY) d) Antibody Titres Against DEN 4 2 4 8 16 3264128 256 FIG. 1. Comparison of dengue IgM and total antibody titers in 39 convalescent-phase sera from primary dengue infections. Acute-phase sera were collected between days 1 and 5, and convalescent-phase sera were collected between days 7 and 15 after the onset of symptoms. All cases had a clinical diagnosis of DF and DHF and a clinical history consistent with this diagnosis. DEN 1, dengue type 1 virus. demic period, a clinical diagnosis of DF or DHF (14) was made by the clinicians responsible for blood collection. All patients had a clinical history consistent with DF or DHF (14). All sera were tested by HAI, and when positive, they showed reaction with all four dengue serotypes. The 76 single sera were used to screen for dengue IgM to determine whether a conclusive diagnosis could be made and also to test the cross-reactivity of dengue IgM with Japanese encephalitis virus (JEV) hemagglutinins. These single sera were previously uninterpretable by the HAI test, as only a single serum specimen was available. Group 2 consisted of 61 individual sera from non-dengue patients known to contain antibodies against the following antigens: Salmonella typhi, Proteuts OXK (scrub typhus), Leptospira spp. rubella, microsomal antigen, and thyroglobulin. These sera were confirmed positive by standard serological tests. Group 3 consisted of a panel of IgM cross-reacting sera kindly provided by U. Krech of the Institut fur Hygiene und Medizinische Mikrobiologie, University of Bern, Bern, Switzerland. It included one serum specimen from an Epstein-Barr virus infection, two specimens from cytomegalovirus infections, and three specimens from enterovirus infections. All six sera had IgM antibodies which cross-reacted broadly with one or more viruses and were used to assess the specificity of the HIT for dengue virus. s. Crude dengue types 1 to 4 virus and JEV hemagglutinin antigens were prepared from suckling mouse brain by the method of Clarke and Casals (3). Purified dengue virus antigens were prepared by concentrating supernatants of dengue virus-infected C6/36 cell cultures with polyethylene glycol (PEG 6). HIT for dengue. The HIT used was similar to that for rubella (9) with some modifications. Briefly, 125 RI of rabbit anti-human IgM (p. chain specific; Dako Immunochemicals, Copenhagen, Denmark) at a 1:4 dilution in borate-saline buffer (ph 9.) was added to each well of polystyrene U-bottomed microtitration plates (Immulon 2; Dynatech Laboratories, Inc., Alexandria, Va.). After an overnight incubation at 4 C, the coated plates were washed three times with distilled water containing.5% Tween 2 and shaken dry. Sera were treated by extraction with acetone to remove hemagglutinin inhibitors and incubated with goose erythrocytes to remove heteroagglutinins (3). The extracted sera were serially diluted in anti-human IgM-coated plates in.4% bovine albumin-borate-saline buffer (ph 9.) with an initial dilution of 1:4. The final volume of the diluted serum was 1,u1 per well. The plates were incubated for 2 h at 37 C. They were then washed three times with distilled water and tapped dry, and 5 of dengue hemagglutinating antigen at 1 hemagglutinating unit was added to each well except the controls and incubated at 4 C overnight. A 5-,ul sample of a.6% suspension of goose erythrocytes in phosphate buffer, at a ph optimal for the hemagglutinating activity of the respective dengue virus antigens, was added and incubated for 1 h at 37 C. The plates were then centrifuged at 65 x g for 1 min to facilitate reading. A positive

172 GUNASEGARAN ET AL. result was shown by a hemadsorption pattern, whereas a button indicated the absence of IgM antibody. All four serotypes of dengue hemagglutinating antigens were used for each assay. HAI test for dengue. The dengue HAI test was performed by the method of Clarke and Casals (3). Removal of IgM. A 1-,ul sample of dengue convalescentphase serum (with IgM antibody) was preincubated with an equal volume of anti-human IgM-coated immunobeads (Bio- Rad Laboratories, Richmond, Calif.), at a concentration of 1 mglml in borate-saline buffer, at 4 C for 1 h. The serum was then pretreated by acetone extraction and adsorbed with goose erythrocytes. The HIT was performed with these sera on plates coated with anti-human IgM (IgM capture) to show the effect of the removal of IgM on the IgM titers obtained in the HIT. RESULTS HIT for dengue. In all of the acute-phase sera from the 6 pairs in group 1, no anti-dengue IgM was detected by the HIT. 1gM antibody to all four serotypes of dengue virus was detected in 22 of 39 (56%) convalescent-phase sera from primary dengue infections, and all of these had HAI titers of at least 1:64 (Fig. 1). Moreover, 8 of the 1 (8%) convalescent-phase sera from patients with secondary dengue infections had IgM antibody titers to dengue ranging from 1:8 to 1:1,28 or more (Table 1). None of the 11 convalescent-phase sera from the HAI-negative cases had detectable levels of IgM antibody to dengue. Also, 32 of 76 single sera TABLE 1. Comparison of dengue IgM (HIT) and total antibody (HAI) titers in 1 convalescent-phase sera from secondary dengue infections Specimen Antibody titers' Assay no. DEN1 DEN 2 DEN 3 DEN 4 1 HAI 64 2,56 64 64 HIT 1,28 64 1,28 1,28 2 HAI 32 1,24 1,28 1,24 3 HAI 1,28 1,28 64 2,56 HIT 1,28 32 16 32 4 HAI 32 2,56 64 2,56 5 HAI 64 5,12 5,12 5,12 6 HAI 32 2,56 5,12 5,12 7 HAI 64 5,12 5,12 1,24 8 HAI 64 2,56 64 2,56 HIT 2.1,28 1,28 2.1,28 1,28 9 HAI 32 2,56 64 2,56 HIT.1,28.1,28.1,28.1,28 1 HAI 64 2,56 1,28 5,12 "DEN 1, Dengue type 1 virus. TABLE 2. IgM titers detected by the HIT to dengue type 2 virus and JEV in 32 single sera inconclusive by the HAI" Specimen IgM HIT titers Specimen IgM HIT titers no. DEN 2 JEV no. DEN 2 JEV 1 1,28 <2 17 1,24 16 2 32 <2 18-82,72 5,12 3 2,56 <2 19 1,24 <2 4 16 <2 2 1,24 <2 5 1,28 <2 21 1,24 <2 6 1,24 <2 22 2,56 <2 7 32 8 23 82,72 2,56 8 8 2 24 41,36 1,24 9 5,12 64 25 1,28 16 1.82,72 1,24 26 1,24 5,12 11 1,24 1,28 27 1,28 32 12 1,24 1,28 28 1,28 1,28 13 2,48 5,12 29 1,24 1,24 14 1,24 2,56 3 5,12 5,12 15 5,12 8 31 64 64 16 1,24 2,56 32 2,56 2,56 "Only the 32 positive sera of the 76 single sera from group 1 are presented here. DEN 2, Dengue type 2 virus. (42%) were positive for dengue 1gM with titers ranging from 1:8 to more than 1:8, (Table 2). Specificity of the HIT. The cross-reactivity of dengue IgM with JEV antigens was examined by testing 32 single sera from dengue patients previously shown to possess dengue IgM (Table 2). Of these 32 sera, 1 (31%) showed no cross-reactivity, and 22 (69%) showed cross-reactivity with JEV antigens (Table 2). Of the latter 22, 17 (77%) showed higher titers to dengue type 2, and 5 (23%) had similar titers (Table 2). None of the sera had higher titers to JEV. To further test the specificity of this assay, we screened a batch of sera from other febrile human diseases. All sera in group 2 were tested against the four dengue antigens in an attempt to see whether there was any cross-reactivity, i.e., whether these sera would cross-react with the dengue antigens to give false-positive results. None of these sera reacted with dengue antigens in the HIT. The six sera from group 3 were characterized by crossreacting IgM antibody to a variety of viruses (e.g., herpesvirus, enterovirus, cytomegalovirus, measles virus, etc.). None of the six sera tested showed any cross-reaction in the HIT against the dengue antigens. Effect of removal of IgM on the HIT. It was also of interest to determine the effect of the removal of IgM on the HIT. The four dengue convalescent-phase sera from group 1 (with high HAI titers), previously shown to possess IgM antibody titers, were used in this experiment. The removal of IgM resulted in a drop in IgM HIT titers (Table 3). Use of crude and purified virus antigens in the HIT. In another experiment, with 1 randomly chosen individual sera from dengue patients, it was shown that crude dengue virus antigen preparations could be used instead of purified antigen in the HIT, and no difference in the results were apparent (Table 4). DISCUSSION J. CLIN. MICROBIOL. A rapid and inexpensive diagnostic method is highly desirable in some arboviral diseases such as DHF, as these diseases are life threatening and can be managed more rationally once the nature of the infecting agent is known.

VOL. 23, 1986 HIT FOR THE DETECTION OF DENGUE-SPECIFIC IgM ANTIBODY 173 TABLE 3. Dengue convalescent-phase sera: removal of IgM with immunobeads Dengue IgM Anti-lgM for Specimen Specimen rntivgm ofo antibody titers f(hit) for DEN no.~ ~ ~ emvl ~~~~~IgMa _4 1 + <4 4 2 + <4 16 3 + <4 64 4 + <4 8 a Symbols: +, sera treated for the removal of IgM; -, sera not treated for the removal of IgM. b DEN 1-4, Dengue types 1 to 4 virus. Virus isolation attempts are often unsuccessful due to the improper collection and transport of specimens, the insensitivity of the host systems, or the presence of neutralizing antibodies, although some good isolation techniques have been reported (1, 12). Another possible approach to rapid diagnosis is the demonstration of dengue virus-specific IgM antibody, as this suggests a recent or ongoing infection (8) and requires only a single specimen. Specific IgM antibody is transiently produced during acute dengue virus infections and should appear in the serum between days 4 and 6 after the onset of illness (11). Although previous reports indicated that IgM is produced in very low quantities (11), especially during secondary dengue infections, it seems worthwhile to reexamine its presence by newer, more sensitive techniques. Recent reports have indicated the usefulness of this approach in a number of arbovirus infections (1, 2, 9). Methods used to date to detect virus-specific IgM antibody have been beset by a number of problems. Falsenegative results are possible due to the high concentration of viral IgG antibody which may block IgM antibody through competition with antigen. Conversely, the presence of rheumatoid factor may result in false-positive results. Thus, physical separation of the IgG from IgM antibody in the serum would be necessary for the assay of IgM antibody. Moreover, as was mentioned above, the conventional methods used are tedious and time consuming. The HIT is an improvement based on the antibody capture method and avoids some of the above-mentioned problems. Besides, the HIT uses erythrocytes and not enzyme-linked reagents as the indicator system. This makes the assay significantly cheaper. The results presented here indicated that no IgM was detected in any of the acute-phase sera which had no or very low titers of HAI antibodies (primary dengue infections) or which had significant titers of HAI antibodies (secondary dengue infections) (Table 1). Most of these acute-phase sera had been collected between days 1 and 5 after the onset of disease. Russell (11) reported that dengue IgM antibody appears in the serum between days 4 and 6 after the onset of illness in primary dengue infections. Thus, it is likely that the collection of the acute-phase sera had been done too early to demonstrate the presence of IgM antibody. Alternatively, the IgM level may have been below the sensitivity limit of the HIT. In contrast, 22 of 39 (56%) convalescent-phase sera from primary dengue infections and 8 of 1 (8%) convalescent-phase sera from secondary dengue infections possessed dengue-specific IgM as detected by the HIT (Table 1). Most of these sera were collected approximately 7 to 15 days after the onset of disease symptoms. It thus appears that in dengue virus infections, IgM appears in the serum only after about 7 days. Alternatively, as was mentioned above, the assay may be unable to detect lower concentrations of IgM present in the serum before day 7. The results obtained also have some direct relevance to the previous findings of Russell (11) who found that IgM responses in secondary infections appeared to be markedly suppressed, often to undetectable levels. He used the HAI test to detect the IgM antibody, and it is possible that with a more sensitive assay system, such as the HIT, even low levels of IgM can be detected. This was found to be the case in the present study in which seven convalescent-phase dengue sera had high levels of IgM antibody and one had IgM antibody to one dengue serotype (Table 1). Our results thus indicate that significant levels of IgM antibody are indeed produced during secondary dengue infections but its detection depends on collecting blood at an appropriate time. The usefulness of the HIT was also very clearly demonstrated when 32 of 76 (42%) single sera, ranging in HAI antibody titers from 1:1 to 1:64, were found to contain dengue-specific IgM and thus could subsequently be diagnosed as dengue infections (Table 2). A definitive diagnosis based on these single sera would not have been possible with the HAI test. It is also of importance to assess the cross-reactivity of TABLE 4. Use of purified and crude dengue virus antigens in the HIT Specimen a HIT titersb no. ' DEN 1 DEN 2 DEN 3 DEN 4 1 C <4 <4 <4 <4 2 C 64 64 64 64 P 1,28 64 64 1,28 3 C <4 <4 <4 <4 4 C.1,28.1,28.1,28.1,28 P.1,28-1,28.1,28.1,28 5 C <4 <4 <4 <4 6 C.1,28 1,28 1,28 1,28 7 C.1,28.1,28.1,28 1,28 P.1,28.1,28.1,28.1,28 8 C 64 64 64 64 P 64 64 64 64 9 C 1,28.1,28 1,28 1,28 1 C 1,28 1,28 1,28 64 a Symbols: C, crude antigen (suckling mouse brain); P. purified antigen (cell culture grown). b DEN 1, Dengue type 1 virus.

174 GUNASEGARAN ET AL. dengue IgM (as detected by the HIT) with JEV hemagglutinins, as these viruses are members of the flavivirus group which exhibit antigenic cross-reactivity and are endemic in the region. A total of 22 sera from the 32 single sera (69%) from group 1, which had IgM to dengue, cross-reacted in the HIT with JEV hemagglutinins (Table 2). However, 17 (77%) of these sera had higher dengue-specific IgM titers, and only 5 (23%) had similar IgM titers to dengue and JEV (Table 2); none had higher titers to JEV. There was no cross-reactivity in 1 of the 32 (31%) positive single sera (Table 2). Such cross-reactivity is to be expected due to the sharing by both viruses of flavivirus group reactive antigenic determinants and has been reported previously by other workers (2, 5). The specificity of the HIT for dengue was supported by the use of immune sera from a number of other febrile diseases, such as typhoid, typhus, leptospirosis, and rubella. All these sera showed no cross-reaction in the HIT against dengue antigens. The panel of IgM cross-reacting sera to other viral infections also illustrated that the HIT for dengue was specific. The six sera from Epstein-Barr virus, cytomegalovirus, and enterovirus infections had crossreacting IgM antibody to other infections, such as polio 1, mumps, measles, Legionella, Epstein-Barr virus, chlamydia, varicella-zoster virus, toxoplasmosis, and others (U. Krech, personal communication). None of these sera crossreacted with dengue antigens as determined by the HIT. An advantage of the HIT for dengue IgM antibody is that crude virus antigen preparations could be used. Moreover, high levels of IgG in the sera do not seem to interfere with the IgM capture method. Also, unlike the enzyme immunoassay (1) and radioimmunoassay (2) techniques of detection, the HIT does not require expensive reagents or special equipment. It is cheap, simple to perform, rapid, sensitive, and highly specific, and it requires only a single specimen. It can be routinely performed in any arbovirus laboratory, and we believe it can be a valuable tool in the diagnosis of dengue and other arbovirus infections. ACKNOWLEDGMENTS We appreciate and gratefully acknowledge U. Krech for his suggestions and for providing a panel of IgM cross-reacting sera and Victor Choo and B. G. Ooi of the Institute for Medical Research, Kuala Lumpur, Malaysia, for providing us with some of the dengue immune sera used in this study. This project was in part funded by the World Health Organization Regional Office for the Western Pacific, Manila, the Philippines, and the University of Malaya, Kuala Lumpur, Malaysia (vote F42/84). J. CLIN. MICROBIOL. LITERATURE CITED 1. Bundo, K., and A. Igarashi. 1985. Antibody capture ELISA for detection of immunoglobulin M antibodies in sera from Japanese encephalitis and dengue haemorrhagic fever patients. J. 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