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Characterization and Application of WDSSB5 MonoclonalAntibody for the Detection of Dengue Virus In C6/36 CellLine Using Immunocytochemical Method

Nurminha1,2, Sitti Rahmah Umniyati3, Wayan T. Artama4

1Postgraduate Study Program in Tropical Medicine, Faculty of Medicine, Universitas GadjahMada, Yogyakarta, Indonesia; 2Health Polytechnic Tanjungkarang, Lampung, Indonesia;3Departement of Parasitology, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta,Indonesia; 4Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.

Corresponding author: nurminha31@yahoo.com

ABSTRACTIntroducition: Dengue Fever (DF) and Dengue Hemorrhagic Fever (DHF) are caused by Dengue virus thatconsists of 4 serotypes of Dengue Virus (DENV) 1, 2, 3 and 4. Isolation of Dengue virus using C6/36 cell isconsidered as a gold standard for the diagnosis of Dengue virus infection. Dengue Team of Gadjah MadaUniversity successfully produced monoclonal antibodies of DENV 3 originating from hybrid cells of DSSC7,DSSE10 and WDSSB5. The detection of Dengue virus’s antigens of Ae. aegypti in human blood smear withStreptavidine Biotin Peroxide Complex (SBPC) immunocytochemistry method using DSSC7 primary antibodyis highly sensitive and specific, whereas using WDSSB5 monoclonal antibody yet to be characterized.Objective: The study aimed to identify characterization and application of WDSSB5 monoclonal antibody asprimary antibody for detection of Dengue virus originating from serum of patients with Dengue infectionwhich was inoculated in C6/36 cell line using SBPC immunocytochemistry method.Methods: The study was experimentally designed. Propagation of WDSSB5 hybridoma cell was performed invitro and in vivo. The characterization consisted of classification of WDSSB5 monoclonal antibody,examination of WDSSB5 ascites protein level, sensitivity and specificity of immunocytochemical SBPC methodusing WDSSB5 primary antibody and specificity of monoclonal antibody against Dengue antigen with DotBlot method. Dengue virus obtained from patients was inoculated in C6/36 cell. Detection of Dengue virusantigen was performed by SBPC immunocytochemistry method with WDSSB5 monoclonal antibody as primaryantibody. Positive control was made using C6/36 cell infected with DENV 1, 2, 3, 4 and inoculated in C6/36cell, whereas negative control uses cell C6/36 not infected with Dengue virus.Results: There was WDSSB5 monoclonal antibody detected in this research which was belonged to IgG classand IgG1 subclass. The least content of WDSSB5 monoclonal antibody that can detect Dengue antigen in C6/36 cell was 2.2 µg/µL. The WDSSB5 monoclonal antibody was sensitive to detect DENV 1, 2, 3, 4 antigens inC6/36 cell using SBPC immunocytochemistry method.Conclusion: There was WDSSB5 monoclonal antibody specific againts Dengue virus identified in this study.WDSSB5 monoclonal antibody belonged to class IgG and subclass IgG1. WDSSB5 Monoclonal antibody canbe applied to detect Dengue virus originating from serum of patients positively carrying Dengue virus inoculatedin C6/36 cell using SPBC immunocytochemistry method.

Keywords: Dengue Virus, immunocytochemical, monoclonal antibody, C6/36 cell

Nurminha et al., Characterization and Application of WDSSB5 Monoclonal Antibody for the Detection ofDengue Virus in C6/36 Cell Line Using Immunocytochemical Method

INTISARIPendahuluan: Demam Dengue (DD) dan Demam Berdarah Dengue (DBD) disebabkan oleh virus Dengue yangterdiri dari 4 serotype virus Dengue (DENV) 1, 2, 3 dan 4. Isolasi virus Dengue menggunakan kultur sel C6/36merupakan baku emas untuk menegakkan diagnosis infeksi virus Dengue. Team Dengue UGM telah berhasilmemproduksi antibodi monoklonal terhadap DENV 3 antara lain yang berasal dari sel hibrid DSSC7, DSSE10dan WDSSB5. Deteksi antigen virus Dengue pada organ nyamuk Ae.aegypti dan pada sediaan apus darahmanusia dengan metode imunositokimia Streptavidine Biotin Peroxide Complex (SBPC) menggunakan antibodiprimer DSSC7 mempunyai sensitivitas dan spesifisitas yang tinggi, sedangkan sel hibrid WDSSB5 belumdikarakterisasi.Tujuan: Tujuan penelitian ini adalah untuk melakukan karakterisasi dan mengaplikasi antibodi monoklonalWDSSB5 sebagai antibodi primer untuk mendeteksi virus Dengue dari serum pasien yang positif mengandungvirus Dengue yang diisolasi pada sel C6/36 dengan metode imunositokimia SBPC.Metode: Desain penelitian ini eksperimental. Pada penelitian ini propagasi sel hibridoma WDSSB5 dilakukansecara in vitro dan in vivo. Karakterisasi yang dilakukan meliputi uji klasifikasi antibodi monoklonal WDSSB5,pemeriksaan kadar protein asites WDSSB5, sensitivitas dan spesifisitas metode imunositokimia SBPCmenggunakan antibodi primer WDSSB5 serta spesifitas antibodi monoklonal terhadap antigen Dengue denganDot Blot. Virus Dengue yang berasal dari serum pasien yang positif mengandung virus Dengue diinokulasipada sel C6/36. Deteksi antigen virus Dengue dilakukan dengan menggunakan metode imunositokimia SBPCdengan antibodi monoklonal WDSSB5 sebagai antibodi primer. Kontrol positif digunakan sel C6/36 yang diinfeksivirus DENV 1, 2, 3, 4 dan diinokulasi pada sel C6/36, sedangkan kontrol negatif adalah sel C6/36 yang tidakdiinfeksi virus Dengue.Hasil: Hasil penelitian didapatkan antibodi monoklonal WDSSB5 yang termasuk kelas IgG dan sub subkelasIgG1. Kadar antibodi monoklonal WDSSB5 terkecil yang dapat mendeteksi antigen Dengue pada sel C6/36adalah 2,2 µg/µL. Antibodi monoklonal WDSSB5 sensitif dan spesifik mampu mendeteksi antigen DENV 1, 2,3, 4 pada sel C6/36 dengan metode imunositokimia SBPC.Simpulan: Didapatkan antibodi monoklonal WDSSB5 spesifik terhadap virus Dengue. Antibodi monoklonalWDSSB5 termasuk kelas IgG dan subkelas IgG1. Antibodi monoklonal WDSSB5 dapat diaplikasikan untukmendeteksi virus Dengue yang berasal dari serum pasien yang positif mengandung virus Dengue yang diisolasipada sel C6/36 dengan metode imunositokimia SPBC.

Kata kunci: virus Dengue , imunositokimia, antibodi monoklonal, sel C6/36.

INTRODUCTIONDengue Disease (DD) and Dengue

Hemorrhagic Fever (DHF) are caused by Denguevirus, an Arbovirus group B which belongs tothe family of Flaviviridae, genus of Flavivirus andis composed of four serotypes (DENV 1 to 4).Dengue virus infections in humans result in aspectrum of clinical manifestations ranging fromthe mild undifferentiated febrile illness, dengue

fever, Dengue Hemorrhagic Fever (DHF) andDengue Shock Syndrome (DSS).

Dengue virus is spread all over the worldincreasingly mixed each other of Dengue 1,2,3,4following human mobility. All serotypes are stillendemic in Indonesia. The number of denguecases in Indonesia, according to WHO dataduring 2006 to 2008, were 106,425 cases ofdengue in 2006 with the mortality rate was 1,132

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(Case Fatality Rate (CFR) was 1.06%). In the year2007 there were 188,115 cases (CFR was 1.01%),whereas in 2008 there were 101,656 cases (CFRwas 0.73%).

Course of the DHF is very rapid so if notimmediately treated, it is often fatal. Until now,the diagnosis of Dengue fever in Indonesia isstill primarily based on clinical diagnosis withoutfollowed by virological diagnosis confirmation.Virological confirmation actually can be doneearly since viremia occurred 2 days before thefever and during fever. However, diagnostic toolfor this purpose has not been affordable for themost patients.

Laboratory tests that may be performed forconfirmation of dengue virus infection are virusisolation, detection of viral genome usingPolymerase Chain Reaction (PCR), viral antigendetection and serological tests. Virus isolationusing cell culture is the gold standard fordefinitive diagnosis of dengue virus infection.Cell cultures which widely used are Ae. pseudoscutellaris (AP/61) cells, Ae. albopictus (C6/36) andToxorrhynchites amboinensis (TRA-284). Cultureresults are usually identified using DirectImmunofluorescent Assay (DFA) or IndirectImmunofluorescent Assay (IFA) methods.Although it is the best way to ensure Denguevirus infection, this method requiressophisticated laboratory equipment andrequires a long time. The advantage of denguevirus isolation is it produced local dengue virusisolates that can be used for further research.

An immunocytochemistry method commer-cially available now is Streptavidin BiotinPeroxidase Complex (SBPC). This technique isusing biotin-labeled with secondary antibodythat can recognize the primary antibodies(monoclonal antibodies or polyclonal anti-bodies) and using enzyme-labeled streptavidinconjugated to horseradish peroxidase. A

substrate of chromogenic mixture is used todetect antigens on the cell or tissue with a veryhigh sensitivity so that the low levels of antigencan be detected. The main basis of SBPCimmunocytochemistry reaction is a strong bondbetween streptavidin and biotin. Immuno-cytochemistry method can be used to detectDengue virus in various organs of the mosquito,paraffin tissue or on buffycoat, whereas thosein C6/36 cell have not been reported.Examination using immunocytochemistry pre-parations can be done with a light microscopethat is widely available in the laboratory,whereas immunofluorescent preparationsshould be examined under an expensivefluorescent microscope and rarely available inthe laboratory. This method has advantagesbecause it does not require special equipmentsince it can be done with a light microscope thatis widely available in laboratories and does notrequire any particular skills. It is required specificmonoclonal antibodies against dengue virus asa diagnostic reagent. Dengue Team ofUniversitas Gadjah Mada has successfullyproducing a monoclonal antibody against theDENV 3 secreted by hybrid cells (clone) ofWDSSB5.

The purpose of this study was tocharacterize and apply WDSSB5 monoclonalantibody as the primary antibody for detectionof dengue virus from serum of patients positivefor dengue virus that was isolated in cell C6/36with SBPC immunocytochemistry method.

MATERIALS AND METHODSThis study was an experimental study. In this

study, the propagation of hybridoma WDSSB5was performed in vitro and in vivo. In in vitropropagation, hybridoma clones cultured in RPMIPBS-enriched culture medium, penicillin-streptomycin and antifungal amphotericin B on

24 wells microplate and incubated at 37 °C with5% of CO2. Cell growth was observed every dayunder an inverted microscope until reachinglogarithmic phase. Then, recloning was done toproduce hybridoma cell line. Classification ofmonoclonal antibody was determined using anantigen-mediated ELISA method. Specificity ofWDSSB5 monoclonal antibodies was analyzedusing Dot Blot method.

Isolation of viruses from patient’s serumperformed on mosquitoes and cell C6/36. Serumwas diluted with PBSBA (1: 5). Samples wereintrathoracically inoculated into each ofToxorrhyinchites sp aged 2-4 days. Afterinoculation, mosquitoes were incubated at 32°Cwith 75-85% humidity for 9-10 days and fed with10% sugar solution. The mosquitoe’s heads werecut and other body parts are crushed and madea suspension with buffer containing proteinstabilizers and antibiotics so it becomes a 10%suspension, then centrifuged and filtered.

The confluent’s C6/36 cell taken from themedium and spared little and the suspensionwas inoculated on C6/36 cell, then adsorbed for60 minutes in laminary flow hood and shakenevery 10 minutes. Five mL of maintenancemedium were added then the cells wereincubated at 29-30°C for 4-5 days without CO2.Every day the infected cells were observed underan inverted microscope and compared withcontrol. After the cells on each bottle showedsymptoms of infection include the presence ofgiant cell or cell lysis has begun, cells werescraped and cell then fit with the medium into15 mL centrifuge tube and centrifuged at 450 gat temperature of 4°C for 10 minutes.Supernatant was stored at temperature of -84°C.The pellets were made into preparation for

infectivity examination with immunocyto-chemistry techniques. Uninfected cells or controlis also made into preparation on poly-L-lysineglass objects. Once dried, the preparation werefixed with cold methanol and dried up at roomtemperature. The WDSSB5 monoclonal anti-bodies of 2.2 ug/mL was used as primaryantibodies and then the results were observedunder a light microscope. Positive controls wereprepared using C6/36 cells infected with DENV1,2,3,4 and the negative controls were C6/C36cells that were kept uninfected. After inoculation,the cells were incubated for 1-4 days. The C6/36cell preparations were infected by dengue virusis not given the primary antibody the primaryantibody at the time of SBPC immuno-cytochemistry test. The results are called positivefor dengue antigen if the cell cytoplasm is brown,while the results are called negative if the cellcytoplasm is blue.

RESULTS AND DISCUSSIONRecloning of WDSSB5 hybridoma cell clones

The propagation of WDSSB5 hybridomaclones results in obtaining monoclonalantibodies in WDSSB ascites fluid in volume of19.5 mL.

Figure 1. Producer of hybridoma cell line under aninverted microscope

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Characterization of WDSSB5 monoclonalantibodies

Classification Test Classification test ofmonoclonal antibody was conducted using anantigen-mediated ELISA method. The resultshows that the WDSSB5 monoclonal antibodybelong to the IgG class and IgG1 subclasses.

Figure 2. Result of classification test of WDSSB5monoclonal antibody (B5, well number 5and 6 arise brown color)

Examination of WDSSB5 ascites proteinlevels

Examination results obtained WDSSB5ascites protein levels was 11 mg/mL.

Optimization results obtained WDSSB5 ascitesprotein levels was 2.2 mg/mL. This level isoptimal for detecting dengue virus in C6/36 cellpreparations based on the method ofimmunocytochemistry SBPC with a positive rateof 100% (Table 1). The results of microscopicexamination of SBPC immunocytochemistryusing WDSSB5 as a primary antibody successfullydetect dengue antigens in of C6/36 cellspreparations and showed a positive reactionindicated by the brown color of the cytoplasmof the cell.

Negative reaction is indicated by theappearance of blue color in the negative controland in C6/36 cell were infected by dengue virusbut not given the primary antibody when SBPCimmunocytochemistry test was performed. Theresults of microscopic examination of the SBPCimmunocytochemistry of optimization WDSSB5ascites protein levels as the primary antibodyon C6 / 36 cell preparations are presented inTable 1.

Table 1. SBPC immunocytochemistry examination for optimization of WDSSB5 primaryantibody levels in C6/36 cell preparations which were infected by DENV 3 andincubated for 4 days

Photomicrographs of SBPC immunocyto-chemistry of C6/36 that were infected by DENV3 and incubated for 4 days for optimization

WDSSB5 monoclonal antibody levels arepresented in Figure 3.

Figure 3. Photomicrographs showing brown color (positive) for dengue virus antigen in the cytoplasm ofC6/36 cells (magnification of 100 x 10 times). B (2.2 µg/µL), C (1.1 µg/µL), D (0.22 µg/µL), E (0.11µg/µL). A and F showing C6/36 cell cytoplasm with negative reaction (blue).

Sensitivity and Specifity Tests of WDSSB5monoclonal antibody by SBPC immunocyto-chemistry method

The results of examination SBPC immuno-cytochemistry showed that 2.2 ug/mL of

WDSSB5 primary antibody was able to detectinfection of DENV 1, 2, 3, 4 in C6/36 cell from 1-day of incubation period with a positive rate of100%. The results are depicted in Table 2.

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Table 2. The results of SBPC immunocytochemistry on C6/36 cell which were infected by DENV 1, 2, 3, and4 with the incubation period of 3-4 days

Table 2 shown that 2.2 ug/mL of WDSSB5primary antibody was able to detect infectionof DENV 1, 2, 3, 4 in C6/36 cell from 1-day ofincubation period with a positive rate of 100%.This suggests that the WDSSB5 primary antibodyis sensitive and able to detect infection of DENV1, 2, 3, 4 from the onset of infection.

Photomicrographs of SBPC immunocyto-chemistry of C6/36 cell that were infected byDENV 1,2,34 and incubated for 1 to 4 days forare presented in Figure 4.

Figure 4. Photomicrographs of SBPC immunocytochemistry preparations of C6/36 cells that wereinfected by DENV 1, 2, 3, 4 and incubated with 2.2 µg/µL of WDSSB5 primary antibody(incubation days 1, 2 3, and 4) showing brown color (positive) for dengue virus antigenin the cytoplasm of C6/36 cells (magnification of 100 x 10 times). B (2.2 µg/µL), C (1.1µg/µL), D (0.22 µg/µL), E (0.11 µg/µL). A, B, C, D = incubation days 1,2,3,4 of denguevirus type 1; E, F G H = incubation days 1,2,3,4 of DENV 2; I, J K, L = incubation days1,2,3,4 of DENV 2; M N, O, P = incubation days 1,2,3,4 of DENV 4.

Figure 4 shows that those WDSSB5monoclonal antibodies as primary antibodiesare capable of recognizing all serotypes of denguevirus. The C6/36 were infected by DENV 1, 2, 3, 4and incubated for 1-4 days showed a positivereaction (brown cytoplasm). The negativecontrol and C6/36 cells were infected by denguevirus but not given the primary antibodyshowed blue cytoplasm. At the microscopicobservation brown color intensity in C6/36 cell

which were incubated 1 to 2 days showed browncytoplasmic and more cell number, whereas cellswhich were incubated 3-4 days showed positivedark brown cytoplasmic. In C6/36 cell which wereincubated 4 days showed less cell number dueto lysis of cells and there are many brown spotsaround the cells and even the nuclei also appearbrown. This is due to a longer incubation timeso that the titer of virus was getting higher.

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Specificity test of monoclonal antibody usingDot Blot method

The results showed that the WDSSB5monoclonal antibody showed a positive reactionwith the formation of black color against denguetype 1 2, 3, 4 antigens. This test used denguetype 1 2, 3, 4 antigens as a positive control,dengue type 1, 3, 4 are from local isolates,whereas the negative control using PBS. Resultof specificity test of the monoclonal antibodytest with Dot blot method were presented inFigure 5.

Figure 5. Positive reaction to DENV type 1 and 2antigens showing black dot color.

Isolation of Dengue Virus from Toxorrhyn-chites sp mosquitoes and C6/36 Cells

Photomicrographs of SBPC immunocyto-chemistry of mosquito’s head squash showinga positive reaction. The results showed thatgranule brown colored like sand on thecytoplasm of mosquitoes’s brain cells whichwere injected with Dengue virus (Figure 6).

Figure 6. Photomicrographs of SBPC immunocyto-chemistry of head squash of Toxorrhyn-chites sp mosquitoes which wereintrathoracally injected with the serum ofpatients positive for Dengue virus. A & Bshowing positive results (brown colorcytoplasm). Negative control (C) shows ablue cytoplasm.

Results of virus inoculation in C6/36 cellshowed a positive reaction to an infection, thepresence of giant cells and cells began to lyse.Photomicrographs of C6/36 cells were presentedin Figure 7.

Figure 7. Photomicrographs under an inverted microscope (magnification of 100 x 10times) show sign of infection. A: the confluent of C6/36 cell which wereuninfected by dengue virus; B: the presence of cytophatic epec in C6/36 cellwhich were inoculated with the serum of patients positive for dengue virus;C: C6/36 cell which were inoculated with dengue virus type 4.

Application of WDSSB5 monoclonal an-tibodies to detect dengue virus from serumof patients positive for dengue virus whichwere inoculated in C6/36 cell using SBPCimmunocytochemistry method

The results of microscopic examination ofSBPC immunocytochemistry of C6/36 cellspreparations which were derived from the serumof patients are presented in Table 3.

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Table 3. Microscopic examination of SBPC immunocytochemistry of C6/36 cells preparations which werederived from the serum of patients

Table 3 shows that with SBPC immunocyto-chemistry method, the primary antibodyWDSSB5 successfully detect dengue antigens onthe preparations C6/36 cell which were infectedwith Dengue virus from serum of patientspositive for DENV 1, 3, and 4. Positive levels vary

depending on the concentration of dengue virusand time of incubation of C6/36 cell. Of the 29patients whom serum were inoculated on C6/36 cell, 96.5% showed positive results (28 positiveand 1 negative results).

Figure 8. Photomicrographs of SBPC immunocytochemistry of C6/36 cell which wereinoculated with serum of patients positive for dengue virus showing a positivereaction (brown cytoplasm). A (positive for DENV 1), B (positive for DENV 1 & 3), C(positive for DENV 3), D (C6/36 cell which were infected by dengue virus showingcell cytoplasm with negative result (blue). E (negative control) showing a bluecytoplasm, F (C6/36 cell which were infected by dengue virus without primaryantibody) also showing a blue cytoplasm.

Photomicrographs of SBPC immunocyto-chemistry of C6/36 cell which were inoculatedwith serum of patients positive for dengue viruswere presented in Figure 8. The results showeda positive reaction indicated by the brown colorin the cytoplasm of positive control and C6/36cell which were infected with dengue virusderived from patient serum. Negative reactionis indicated by the blue color of the negativecontrol, as well as in the cytoplasm of C6/36 cellswhich were infected by dengue virus and not

given the primary antibody when SBPCimmunocytochemistry test was performed. Inthis study, immunocytochemistry examinationfound C6/36 cells that showed false positiveresults, namely the cells C6/36 preparation whichwere not infected by the dengue virus butshowed positive results. Nonetheless,preparations which show false positive resultscan still be distinguished from positivepreparations which containing antigens (Figure9).

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Figure 9. Photomicrographs of SBPC immunocytochemistry of C6/36 cell. A (false positive),B (C6/36 cell which were infected by dengue virus showing cell cytoplasm withpositive result (brown), C (negative control of C6/36 cell which were not infectedby dengue virus) showed a blue cytoplasm.

The propagation of WDSSB5 hybridomaclones

For in vitro propagation, WDSSB5 hybri-doma clones were cultured in RPMI mediumcontaining 20% of FBS. After the cells are inoptimal growth, culture was continued usingRPMI medium with 10% of FBS. After the growth,recloning process was conducted to yield theproducers of hybridoma cells where there is onlyone cell in each well of a 96-microplate and fromone cell derived hybridoma producer clones lineswhich producing monoclonal antibodies. Afterthe growth of producer of hybridoma cells in aculture medium, subsequent propagationperformed in vivo in Balb/c mice which havebeen sensitized with 0.5 mL of pristan intra-peritoneally for 4 times at intervals of 2 weeks.The pristan is an immunosuppressive agent andaccelerate the formation of ascites fluid. Thenthe mice were injected with suspension contains106– 107 of producer clones cells per animal.Once abdomen was enlarged, ascites fluid wastaken until the it becomes tumor. In this study,the volume of ascites fluid produced was 19.5mL with twice-tap. This supports previous

research conducted by Umniyati et al.10, whohave succeeded in producing monoclonalantibodies derived from DSSC7 hybrid cell in vivoin Balb/c mice.

According to Goding17, productions ofmonoclonal antibodies in vitro and in vivo haveboth advantages and disadvantages. Concentrat-ion of monoclonal antibody in vitro culture wereabout 1-10 µg/mL, whereas the concentrationof in vivo culture was 1000-fold (mg/mL) thanthat of in vitro culture. Levels of immunoglobulinproduced in vitro is more advantageous in termsof purity, as only slightly contaminated withanother protein derived from the medium,whereas in vivo may be contaminated with otherproteins derived from mice. So for the furtherdevelopment of monoclonal antibodies such asmonoclonal antibodies labeling, purification isnecessary to obtain a pure one.

Characterization of WDSSB5 monoclonalantibodies

Specific character of monoclonal antibodyis a major requirement for a variety of immuno-logical reaction techniques. The main properties

of monoclonal antibodies is determined by thestructure of the antibody molecule, which isdetermined by the amino acid sequence,including the presence of class and subclass ofan antibody18.

In this study, the results of classification testsshowed that WDSSB5 monoclonal antibody isincluded in IgG class and IgGI subclass. This resultsupports research by Sutaryo et al12,19,20, whichhas successfully produced monoclonalantibodies against DENV 3 derived from DSSC7hybrid cells. The antibodies included in the IgG1class and can recognize dengue antigens withthe molecular weight of 48 kDa based onepitopes analysis by Western Blotting method.

In this study, we conducted a specificity testof monoclonal antibodies by Western Blottingmethod, but the results could not be concludedbecause the epitope was not detected. This islikely due to conformational difference ofepitopes. In addition, another possibility is theabsence of denaturation prior to SDS transfer,so there is no fragmentation of proteins andafter immunoblotting, the antibodies do notrecognize epitopes. In this study, to determinethe specificity of monoclonal antibodies againstdengue antigens, a Dot Blot method was carriedout. The drawback is Dot blot method can onlyknow the type of antigen but cannot determinethe molecular weight of the protein. Thismethod is good enough to use as a screening alarge sample16.

Results of the protein levels examinationby the Biorad micro assay method showed thatlevels of WDSSB5 ascites protein was 11 mg/mLusing Bovin Serum Albumin (BSA) as a standardsolution. This proves that the in vivo productionof monoclonal antibodies yields a high

concentration which is 1000 times higher thanthat of in vitro17. The weakness in this study wasthat protein purification is not performed so thatthe levels of proteins obtained are still crudeproteins which influenced by other proteinsderived from mice.

In this study, the optimization of proteinascites dilution rate is conducted to establishthe smallest levels (highest dilution) of a WDSSB5monoclonal antibody that detects Dengueantigens optimally based on SBPCimmunocytochemistry method. Optimizationwas performed using C6/36 cells infected byDENV 3 since WDSSB5 hybrid cells is the resultof a fusion between splenocytes of immune miceagainst DENV 3. The results of microscopicexamination of SBPC immunocytochemistry(Table 1) showed that 20 mL of monoclonalantibody as the primary antibody withconcentration of 2.2 mg/mL successfully detectDENV 3 antigens which were inoculated in C6/36 cells for 4-days incubation with the positiverate of 100%.

Previous studies conducted by Umniyati etal10 using a DSSC7 monoclonal antibodies with1:10 concentration; 1:50 concentration; 1: 100concentration as the primary antibody withSBPC immunocytochemical methods to detectdengue virus infection in the abdomen and headsquash of Ae. aegypti after 5-day incubationyielded positive results of 33.35% and 75%. Thisstudy showed better results than those ofprevious study because of a shorter incubationtime.

In this study, the sensitivity and specificitytests of WDSSB5 monoclonal antibodies wereconducted by SBPC immunocytochemistry usingC6/36 cells which were infected by dengue virus

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type 1, 2, 3, 4 with an incubation time of 1-4days. The results of microscopic examination ofSBPC immunocytochemistry (Table 2) showedthat the WDSSB5 monoclonal antibody as theprimary antibody able to detect dengue type 1,2, 3, 4 antigens in C6/36 cells and sensitive since1 day of incubation. This suggests that theWDSSB5 monoclonal antibody as the primaryantibody with a concentration of 2.2 ug/mL withSBPC immunocytochemistry method can detectdengue antigens early before the virus life cycletakes place perfectly. Thus, WDSSB5 monoclonalantibodies recognize common epitopes ofDengue antigen. The weakness of this test is thatspecificity test using C6/36 cells infected byChikungunya virus was not performed, so thespecificity of the Chikungunya virus is not known.However, the immunocytochemistry examinat-ion always includes a positive and negativecontrols.

Dengue virus isolation in the Toxorrhyn-chites sp mosquitos and C6/36 cell lines

Inoculation in the Toxorrhynchites spmosquito aims to increase virus titers in serum,because the virus titers in serum are very low.In addition, to avoid contamination from othermicroorganisms due to the filtering process ofscours mosquitoes, a 0.22 ì filter is used so thatall other micro-organisms can be filtered unlessthe virus. It can prevent contamination at thetime of inoculation of C6/36 cells.

In a previous study by Samuel and Tyagi(2006), inoculation performed on Toxorrhyn-chites sp and Aedes sp mosquitoes for denguevirus isolation, especially for increasing the viraltiter. The density of dengue virus isolation inC6/36 cells was 36%, while the inoculation ofmosquitoes were 80%.

In this study, result of inoculation of the virusin C6/36 cells was 96.5% positive. Of the 29positive serum samples (by RT-PCR), afterinoculation of C6/36 cells and microscopicexamination of SBPC immunocytochemistry, theresults showed that 28 samples were positiveand 1 sample was negative. Inoculation of C6/36 cells needs a different incubation time rangedfrom 5-12 days, it depends on the onset of signsof infection such as the presence of giant cell orcell lysis. In samples that showed a positivereaction, cytophatic are evident, but not for thesamples showed a negative reaction.

The C6/36 cells is a clone of cells line derivedfrom Aedes albopictus mosquitoes that are mostwidely used for growing Arboviruses in cellculture media. C6/36 cells proved to have highsensitivity for dengue virus infection, easy tohandle, has high stability and has the easyoptimum temperature for growth thanmammalian cells such as BHK-21, LLC-MK2 andVero cells21,22,23.

Previous research by Paula24 and Osman25

showed that Dengue virus isolation derived frompatient serum in C6/36 cells and the detectionof virus were compared using IFAT and RT-PCRmethods. Virus detection by RT-PCR takes 1 day,whereas the IFAT requires 4 days after the viruswas isolated.

Application of WDSSB5 monoclonalantibodies to detect dengue virus from theserum of patients positively carrying denguevirus which were inoculated in C6/36 cell linesusing SBPC immunocytochemistry method.

Detection of DENV 1, 2, 3, 4 isolated fromC6/36 cells was first performed by Igarashi21 byusing a fluorescent antibody staining technique.Positively Dengue antigen results as a green

fluorescent. In this study, the detection of DENV1, 2, 3, 4 antigens was conducted using SBPCimmunocytochemistry. Positive results werecharacterized by the presence of brown color inthe cytoplasm of infected cells15.

Dengue antigen detection by SBPCimmunocytochemistry method using C6/36 cellswhich were infected by dengue virus type 1, 2,3, 4. The basic principle is the presence of 40and 45 kDa glycoprotein on the cell surface whichis receptor for dengue virus26. Dengue virusreplication process takes place in the cytoplasmof cells2. Dengue virus antigen localized to thecytoplasm of cells will bind to the WDSSB5monoclonal antibody that will be recognized bybiotin-labeled secondary antibody. Furthermore,with the addition of conjugate enzyme-labeledstreptavidin radish peroxidase and chromogensubstrate solution, the antigen can be detectedby the presence of brownish color in thecytoplasm of infected cells15.

In this study, a WDSSB5 monoclonalantibody as the primary antibody can detectdengue antigens on the C6/36 cells which wereinfected by dengue virus derived from serumpositive for dengue virus in the acute phase ofthe disease include fever day 1 to day 7. Feverday or day of sampling related to the presenceof dengue virus antigen that can be detected bySBPC immunocytochemistry test. AccordingWHO27, after the onset of the disease denguevirus can be detected in serum, plasma, bloodcells and other tissues for 4-5 days. During thisphase, virus isolation, nucleic acid detection anddetection of dengue antigens can be used fordiagnosis of dengue virus infection.

The results of microscopic examination ofSBPC immunocytochemistry of C6/36 cells (Table

3) showed that there were 13 positive sampleswhich had an average rate of 100%.Observations results showed a strong positivereaction with the brown color is very clear inthe cytoplasm of cells, many giant cells, irregularcell shape, cell nuclei shrink and brown cellnucleus, cell lysis, and there are patches antigenaround cell. The positive samples with averagerate of 35.29% showed weakly positive reactionwith the brown color not so clearly seen. Thismay be due to the less long incubation time sothat less infection occured. At negative sample(T24) with incubation time of ten days, at thetime of harvesting the cell culture, there are noobvious symptoms of infection. This is likely dueto a less concentration of virus compared tonumber of Toxorrhynchites sp mosquito thatsuccessfully injected only one who lives despiterepetitions were performed 3 times. The sampleswith positive results showed more number ofmosquitoes that live more than 3-5 mosquitoindicating the higher virus titers.

This study found the false-positives resultof SBPC immunocytochemistry due to severalfactors such as the presence of non-specific colorreaction, which is caused by the presence ofendogenous peroxidase produced by tissuesand cells normal28.Another factor that can causea false positive is a non-specific binding occursbetween streptavidin with biotin endogenouscomponents contained in cell28,29. In this study,we make an effort to minimize the reaction byadding peroxidase blocking solution to eliminateendogenous peroxidase activity, but have notbeen able to remove endogenous peroxidase.In immunocytochemistry test, the less cleanwashing factors also affect the occurence of falsepositive results. However, false positive results

TMJ Vol. 02 No. 2 171 - 189

still can be distinguished by the positive resultsof dengue virus infection.

During this time, SBPC immunocyto-chemistry test has been developed to detectdengue virus in various organs such as the headsquash mosquitoes, abdominal squash, paraffintissue or on bufficoat also in human bloodsmears. On the other hand, detection of denguevirus in C6/36 cells is one of the important partsin the development of a monoclonal antibodyas a diagnostic tool.

SBPC immunocytochemistry examinationcan be used as one of the methods of detectionof Dengue virus in cell culture. This methodproved to have high sensitivity and specificityfor detecting antigen of DENV 1, 2, 3, 4 in C6/36cells. Immunocytochemistry method has theadvantage since it uses light microscope anddoes not require special skills, relatively simple,inexpensive and can be done in a simplelaboratory.

CONCLUSIONThere was WDSSB5 monoclonal antibody

specific of Dengue virus identified. WDSSB5monoclonal antibody belonged to class IgG andsubclass IgG1 and can be applied to detectDengue virus originating from serum of patientspositively carrying Dengue virus inoculated inC6/36 cells using SPBC immunocytochemistrymethod.

REFERENCES1. Depkes. RI. Pedoman Tatalaksana Demam

Berdarah di Indonesia, Jakarta, 1999.2. Soegijanto, S. Patogenesis dan Perubahan

Patofisiologi pada Infeksi Virus Dengue.Dalam: S. Soegijanto, Demam Berdarah

Dengue, Edisi 2. Airlangga University Press,Surabaya, 2006;61-79.

3. Listiyaningsih, Prediksi Evolusi Genetik VirusDengue di Indonesia. Dalam Seminar KajianKLB dari Biologi Molekuler sampai Pem-berantasannya. Pusat Kedokteran Tropis,Fakultas Kedokteran UGM, Yogyakarta, 2005.

4. Sutaryo. Dengue, Penerbit Medika. FakultasKedokteran Universitas Gadjah Mada,Yogyakarta, 2004:17-96.

5. World Health Organization. Dengue statusin South East Asia Region: An epidemio-logical perspective, 2008.

6. Available from url: http://www.searo.who.int/LinkFiles/Dengue_dengue-SEAR- 2008.pdf

7. Umniyati SR, Soeyoko, Mulyaningsih B.Pengembangan antibodi monoklonal antiDengue-3 produksi local Universitas GadjahMada untuk Deteksi infeksius Virus Denguepada nyamuk Aedes spp. Laporan penelitianHibah Bersaing X/l, Universitas GadjahMada, Yogyakarta, 2003.

8. Kao CL, King CC, Chao DY, Wu HL, and ChangGJJ. Laboratory diagnosis of dengue virusinfection : current and future perspectivesin clinical diagnosis and public health. J.Microbiol. Immunol. Infect, 2005:38;5-16.

9. Aryati. Aspek laboratorium DBD. Dalam:S.Soegijanto, Demam Berdarah Dengue,Edisi 2, p. 117-30. Airlangga University Press,Surabaya, 2006.

10. Anonim, 2005. Histology and Immunocyto-chemistry.Available from url: www.hmds.org.uk/histology.html

11. Umniyati SR, Sutaryo, Wahono D, ArtamaWT, Mardihusodo SJ, Soeyoko, MulyaningsihB, and Utoro T. Application of monoclonalantibody DSSC7 for detecting dengue

infection in Aedes aegypti based onimmunocytochemical streptavidin biotinperoxidase complex assay (ISBPC). DengueBulletin, 2008;32: 83-98.

12. Umniyati SR. Teknik imunositokimia denganantibody monoclonal DSSC7 untuk kajianpathogenesis infeksi dan penularantransovarial virus dengue serta surveilansivirologist vector dengue. [Disertasi],Universitas Gadjah Mada, Yogyakarta, 2009.

13. Sutaryo, Umniyati SR, dan Wahyono D.Produksi antibodi monoklonal terhadapvirus dengue-3 untuk Deteksi PenderitaDemam Berdarah Dengue dan vektornya.Laporan Penelitian RUT-3 Tahun I. FK UGM,Yogyakarta, 1996.

14. Wahyono D, Pichazyk M, Mourton C, MarieBastide M, Bernard PAU. Novel anti-digoxinmonoclonal antibodies with differen bindingspecificities for digoxin metabolites andother glycocides. Hibridoma, 1990: 9;619.

15. Kuno G, Gomez I, Gubler DJ. An ELISAProcedur for The Diagnosis of DengueInfection. J. Virol. Meth, 1991:33; 101-3.

16. Umniyati, SR, Sutaryo, Wahyono D, andArtama WT. Application of monoclonalantibody DSSC7 for early detection ofdengue infection in blood smear pre-paration based on immunocytochemicalstreptavidin botin peroxidase complexassay. Int Joint Symp Frontier Sciences fromgene to application. Faculty of Medicine.Universitas Gadjah Mada, 2008b.

17. Rantam AF. Metode Imunologi, Edisi I.Penerbit Airlangga University Press,Surabaya, 2003.

18. Goding JW. Monoclonal Antibodies Principleand Practice, Academic press, Inc, London,1983.

19. Artama WT. Pedoman Kuliah AntibodiMonoklonal, Teori, Produksi, Karakterisasidan Penerapan. PAU-Bioteknologi Univer-sitas Gadjah Mada, Yogyakarta, 1992.

20. Sutaryo, Umniyati SR, Wahyono D. Produksiantibodi monoklonal terhadap virusdengue-3 untuk Deteksi penderita DBD danvektornya. Laporan Penelitian RUT-3 TahunII. FK UGM, Yogyakarta,1997.

21. Sutaryo, Umniyati SR, Wahyono D. Produksiantibodi monoklonal terhadap virusdengue-3 untuk Deteksi penderita DBD danvektornya. Laporan Penelitian RUT-3 TahunIII. FK UGM, Yogyakarta, 1998.

22. Igarashi A. Isolation of a Singh,s Aedesalbopictus Cell Clone Sensitive to Dengueand Chikungunya Viruses. J. Gen. Virol,1978:40; 531-44.

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25. Paula D, Lima MD, Clotteau M. ImprovedDetection of Dengue -1 Virus from IgM-Positive Serum Samples Using C6/36 CellCultures in Association with RT-PCR.Intervirology, 2003:46;227-31.

26. Osman O, Fong YM, Devi S. PreliminaryStudy of Dengue Infection in Brunei. Facultyof Medicine University of Malaya. Jpn. J.Infect, 2007:Dis 60;205-08.

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28. World Health Organization. Dengue guide-lines for diagnosis, treatment, preventionand control, new edition. WHO, Geneva,Switzerland, 2009.

29. Miller RT. True positive vs. false positivestaining. The Focus Immunohistochemistry,2001;1-2.

30. Taylor CR, and Shi SR. Practical issues:fixation, processing and antigen retrieval. In:C.R. Taylor and Richard J. C. (ed.), Immuno-microscopy A Diagnostic Tool for the SurgicalPathologist, 3rd edition, p. 71-74. Elsevier Inc,Philadelphia, USA, 2006.

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