CHARACTERIZATION AND APPLICATION OF WDSSB5 MONOCLONAL ANTIBODY FOR THE DETECTION OF DENGUE VIRUS IN C6/36 CELL LINE USING IMMUNOCYTOCHEMICAL METHOD

ABSTRACTIntroducition: Dengue Fever (DF) and Dengue Hemorrhagic Fever (DHF) are caused by Dengue virus that consists of 4 serotypes of Dengue Virus (DENV) 1, 2, 3 and 4. Isolation of Dengue virus using C6/36 cell is considered as a gold standard for the diagnosis of Dengue virus infection. Dengue Team of Gadjah Mada University 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 with Streptavidine Biotin Peroxide Complex (SBPC) immunocytochemistry method using DSSC7 primary antibody is 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 as primary antibody for detection of Dengue virus originating from serum of patients with Dengue infection which was inoculated in C6/36 cell line using SBPC immunocytochemistry method.Methods: The study was experimentally designed. Propagation of WDSSB5 hybridoma cell was performed in vitro and in vivo. The characterization consisted of classification of WDSSB5 monoclonal antibody, examination of WDSSB5 ascites protein level, sensitivity and specificity of  immunocytochemical SBPC method using WDSSB5 primary antibody and specificity of monoclonal antibody against Dengue antigen with Dot Blot method. Dengue virus obtained from patients was inoculated in C6/36 cell. Detection of Dengue virus antigen was performed by SBPC immunocytochemistry method with WDSSB5 monoclonal antibody as primary antibody. Positive control was made using C6/36 cell infected with DENV 1, 2, 3, 4 and inoculated in C6/36 cell, 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 class and 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 in C6/36 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 can be applied to detect Dengue virus originating from serum of patients positively carrying Dengue virus inoculated in C6/36 cell using SPBC immunocytochemistry method. Keywords: Dengue Virus, immunocytochemical, monoclonal antibody, C6/36 cel

SENSITIVITY AND SPECIFICITY OF MONOCLONAL ANTIBODY DSSE10 IN HEAD SQUASH TOXORHYNCHITES SPLENDENS USING IMMUNOHISTOCHEMICAL PEROXIDASE TECHNIQUE

Dengue virus are transmitted from human to human by the bites of infective female Aedesmosquitoes from subgenus Stegomyia. One of the way to detect Dengue virus antigen is by usingimmunohistochemical technique. This method was reported to detect dengue vims antigen in lowlevels. The aims of this study is to measure sensitivity and specificity of monoclonal antibodyDSSE10 using SBPC to detect antigen Dengue virus in head squash Toxorhynchites splendenswere infected with dengue patient serum and RT-PCR as gold standart. Artificially-infected Tx.splendens mosquitoes with serum positif dengue virus were used as infectious samples and noninfectedTx. splendens mosquitoes were used as control negative. The immunohistochemichalSBPC assay using monoclonal antibody DSSE10 then applied in mosquitoes head squash todetect Dengue vims antigen. RT-PCR as a gold standart was applied in each mosquito thorax.The result were analyzed by descriptive stasistic test and 2x2 diagnostic test table. Monoclonalantibody DSSE10 using immunohistochemical SBPC assay in head squash Tx. splendens wasgave sensitivity 87,09% and specificity 92,5%. Conclussion of this study is DSSE10 Monoclonalantibodies can be used as primary antibodies for the detection of dengue vims antigen inmosquito head squashKeywords: Dengue viruses, SBPC, antibodies DSSE10, head squash, Toxorhynchitessplendens' Virus Dengue ditularkan dari orang ke orang melalui gigitan nyamuk Aedes dari subgenusStegomyia. Salah satu cara untuk mendeteksi antigen vims Dengue adalah dengan menggunakanteknik imunohistokimia. Metode imunohistokimia dilaporkan dapat mendeteksi antigen vimsDengue dalam kadar yang rendah. Tujuan penelitian ini adalah melakukan evaluasi sensitivitasdan spesifitas antibodi monoklonal DSSE10 dengan metode imunohistokimia Streptavidin BiotinPeroxidase Complex (SBPC) untuk mendeteksi antigen Dengue melalui scdiaan head squashnyamuk Toxorhynchites splendens yang diinfeksi dengan scrum penderita positif vims Denguedengan baku emas RT-PCR. Sampel penelitian adalah nyamuk Tx. splendens yang diinfeksisecara intrathorax dengan scrum positif vims Dengue dan serum negative vims Dengue, sedangkontrol positif adalah nyamuk yang diinfeksi virus Dengue serta nyamuk tanpa perlakuansebagai kontrol negatif. Sediaan head squash nyamuk Tx. Splendens yang dibuat, diperiksadengan uji imunohistokimia SBPC untuk mendeteksi antigen vims Dengue menggunakanantibodi monoklonal DSSE10. Pemeriksaan RT-PCR pada thorax nyamuk digunakan sebagai baku emas untuk uji ini. Hasil penelitian dianalisis dengan statistik deskriptif dan tabel ujidiagnostik 2x2. Hasil penelitian menunjukkan antibodi monoklonal DSSE10 dengan metodeimunohistokimia SBPC pada sediaan head squash nyamuk 73c. Splendens untuk mendeteksiantigen vims Dengue mempunyai sensitivitas 87,09% dan spesifisitas 80,44%. Kesimpulannyaadalah ntibodi monoklonal DSSE10 dapat digunakan sebagai antibodi primer untuk mendeteksiantigen virus Dengue pada head squash nyamuk.Kata Kunci: virus Dengue, SBPC, antibodi DSSE10, head squash, Toxorhynchitessplenden