Teknik imunositokimia dengan antibodi monoklonal DSSC7 untuk kajian patogenesis infeksi dan penularan transovarial virus dengue serta surveilansi virologis vektor dengue

UMNIYATI, Sitti Rahmah, Promotor Prof. dr. Sugeng Juwono Mardihusodo, M.Sc

2009 | Disertasi |

Latar Belakang. Penyakit Demam Berdarah Dengue (DBD) merupakan masalah kesehatan yang penting di negara tropis dan sub tropis termauk Indonesia. Kondisi ini mungkin disebabkan oleh peran vektor DBD dalam menularkan virus Dengue secara transovarial selama dan pasca Kejadian Luar Biasa (KLB) DBD. Evaluasi pengendalian vektor dan surveilansi vektor di Indonesia selama ini ditujukan pada angka kepadatan stadium pra-imago saja yang berupa Angka Bebas Jentik (ABJ) yaitu persentase bangunan (rumah) yang bebas jentik (larva) Aedes tanpa diikuti konfirmasi uji virologis. Metode untuk deteksi virus pada nyamuk antara lain direct fluorescent –antibody (DFA) test pada jaringan nyamuk, biasanya otak, dan kelenjar saliva atau sediaan pencet kepala (head squash) dan reverse transcriptase polymerase chain reaction (RT-PCR), tetapi metode imunofluresensi memerlukan waktu yang lebih lama, dan memerlukan mikroskop fluoresens dan cryofreezer yang tidak selalu tersedia di laboratorium-laboatorium. Metode lain yang lebih sederhana yang menggunakan konjugat enzim seperti perokdidase dan fosfatase serta antibodi monoklonal atau antibodi poliklonal dan hanya memelukan mikroskop cahaya adalah metode imunositokimia. Antibodi monoklonal anti DEN-3 strain H-87 sebagai salah satu reagen diagnostik infeksi DEN telah diproduksi oleh Tim Dengue UGM melalui tiga kali fusi. Dari beberapa sel hibrid yang ditumbuhkan ternyata titer antibodi terhadap virus DEN-3 yang disekresikan sel hibrid DSSC7 meningkat secara signifikan pasca kloning. Nyamuk Aedes aegypti Linn. merupakan vektor penyakit DBD dan Chikungunya, sehingga untuk deteksi virus DEN pada Ae. aegypti perlu dipertimbangkan adanya reaksi silang terhadap virus Chikungunya. Berdasarkan penjelasan di atas penelitian laboratoris bertujuan untuk: (1) melakukan karakterisasi antibodi monoklonal DSSC7 yang akan digunakan sebagai antibodi primer untuk deteksi infeks virus Dengue berdasarkan teknik munositokimia (2) melakukan uji sensitivitas, spesifisitas, validitas, dan reliabilitas teknik imunositokimia SBPC (Streptavidin Biotin Peroxidase Complex) dengan menggunakan antibodi monoklonal DSSC7 sebagai antibodi primer untuk mendeteksi infeksi virus DEN pada nyamuk Ae. aegypti dan mengaplikasikan teknik tersebut untuk, (3) mengkaji patogenesis infeksi dan penularan transovarial virus Dengue pada nyamuk Ae. aegypti yang telah diinfeksi dengan virus DEN dan progeninya (4) penelitian di lapangan bertujuan untuk mengaplikasikan teknik imunositokimia tersebut untuk surveilansi virologis vektor DBD melalui deteksi infeksi transovarial virus DEN di daerah endemis DBD selama periode inter epidemi DBD. Metode: (1) Karakterisasi antibodi monoklonal dikerjakan melalui serangkaian kegiatan sebagai berikut: (a) preparasi antibodi monoklonal DSSC7 in vivo dalam cairan asites mencit Balb/c, (b) klasifikasi antibodi dengan metode ELISA (Enzym Linked Immunosorbent Assay), (c) uji spesifisitas antibodi berdasarkan metode Westernblotting dengan menggunakan antigen DEN-1, DEN-2, DEN-3, DEN-4 dan antigen Chikungunya, (2) Evaluasi teknik imunositokimia dilakukan dengan serangkaian kegiatan: (a) Titrasi antibodi monoklonal DSSC7 dilakukan pada pengenceran (1:10), (1:50) dan (1:100) terhadap sediaan irisan jaringan parafin beku nyamuk Ae. aegypti yang diinfeksi virus DEN-3 per oral dengan masa inkubasi 1 hari berdasarkan teknik imunositokimia SBPC, (b) uji sensitivitas, spesifisitas, (c) uji validitas, dan reliabilitas teknik imunositokimia SBPC dengan menggunakan antibodi monoklonal DSSC7 sebagai antibodi primer untuk mendeteksi infeksi virus DEN pada nyamuk Ae. aegypti. Sensitivitas dan spesifisitas prosedur imunositokimia divalidasi dengan menggunakan kontrol negatif dan kontrol positif sebagai indikator bahwa antibodi berikatan dengan struktur yang cocok. Sediaan imunositokimia head squash nyamuk Ae. aegypti yang telah diinfeksi dengan virus DEN-3 intra torakal maupun per oral dibuat pada masa inkubasi 11 hari dengan antibodi monoklonal spesifik protein E DEN-3 atau antibodi monoklonal spesifik DEN komersial dari Laboratorium Chemicon sebagai kontrol positif. Sediaan head squash tanpa antibodi primer dan sediaan head squah nyamuk Ae. aegypti normal koloni laboratorium serta sediaan head squash nyamuk bukan vektor DEN (Anopheles) dengan antibodi monoklonal DSSC7 digunakan sebagai kontrol negatif. Kesepakatan hasil pendeteksian antigen DEN di bawah mikoskop cahaya pada perbesaran 400x dan 1000x, begitu pula hasiil pendeteksian antigen DEN antara pemeriksa I dan II diuji berdasarkan Formula Landis dan Koch; (3) Patogenesis infeksi dan penularan transovarial virus DEN pada nyamuk Ae. aegypti diobservasi dengan mendeteksi antigen DEN pada berbagai organ nyamuk Ae. aegypti yang telah diinfeksi dengan virus DEN-3 pe oral pada irisan jaringan parafin beku, abdomen squash dan head squash. Penularan transovarial-transstadial virus DEN dilakukan dengan pendeteksian sediaan head squash Ae. aegypti yang telah diberi pakan darah pendeitita DBD akut dan progeninya pada stadium telur, larva, serta pendeteksian antigen DEN pada sediaan head squash Ae. aegypti yang telah diinfeksi dengan virus DEN-2 per oral dan progeninya pada stadium imago berdasarkan teknik imunositokimia dengan ntibodi monoklonal DSSC7 sebagai antibodi primer; (4) surveilansi virologis vektor DEN dilakukan dengan observasi infeksi transovarial virus DEN pada Ae. aegypti berdasarkan metode imunositokimia SBPC pasca KLB DBD 2004 di Yogyakarta dan menganalisis secara statistik hubungan antara prevalensi infeksi transovarial virus DEN dan angka infeksi virus DEN pada nyamuk Ae. aegypti yang tertangkap pada stadium imago pada bulan November 2006-Januari 2007 di Kabupaten Bantul, Sleman, dan Kota Yogyakata dengan metode chi square. Hasil: (1) Antibodi monoklonal DSSC7 berhasil diproduksi in vivo dalam cairan asites 2 ekor mencit Balb/c sebanyak 50 ml. Hasil analisis isotipe antibodi monoklonal menunjukkan bahwa antibdi monoklonal DSSC7 termasuk Klas IgG 1sotipe IgG1. Hasil uji spesifitas antibodi monoklonal DSSC7 menunjukkan bahwa antibodi monoklonal DSSC7 dari sediaan asites (1:100) memperlihatkan reaksi imunologis yang kuat dengan antigen DEN-1, DEN2, DEN3, dan DEN 4 pada berat molekul kira-kira 48.000 Dalton (48 kDa) dan tidak bereaksi silang dengan antigen Chikungunya berdasarkan metode Western blotting. (2) Evaluasi. Kontrol positif. Antigen DEN terdeteksi sebagai deposit granular berwarna kecoklatan yang tersebar di antara jaringan otak nyamuk Ae. aegypti yang diinfeksi dengan virus DEN-3 per oral dengan masa inkubasi 11 hari dan nyamuk Ae. aegypti yang diinfeksi dengan virus DEN-2 per oral dengan masa inkubasi 13 hari menggunakan antibodi monoklonal spesifik DEN komersial dari Laboratorium Chemicon. Antigen DEN juga terdeteksi sebagai deposit kecoklatan pada sitoplasma sel otak yang erinfeksi dengan antibodi monoklonal spesifik protein E DEN-3 2D3B10 (1:50). Kontrol negatif. Hasil negatif terdeteksi sebagai warna kebiruan pada jaringan otak nyamuk Ae. aegypti infeksiosa dan nyamuk normal tanpa antibodi primer. Hasil negatif ditunjukkan pula pada otak nyamuk Ae. aegypti koloni laboratorium yang tidak diinfeksi virus DEN dengan antibodi monoklonal DSSC7 untuk membuktikan bahwa antibodi monoklonal DSSC7 tidak mengenal otak nyamuk. Nyamuk Ae. aegypti yang diinfeksi dengan virus DEN-2 per oral dengan masa inkubasi 13 hari menunjukkan hasil positif pada bagian otak sebagai deposit granular kecoklatan yang tersebar di antara jaringan otak berdasarkan teknik imunositokimia SBPC dengan antibodi monoklonal DSSC7 sebagai antibodi primer dengan indeks infeksi sebesar 93,33%. Deteksi antigen DEN pada sediaan head squash berdasarkan metode imunositokimia SBPC pada pebesaran 400x memberikan reaksi sahih dan terandalkan, karena menunjukkan nilai kesepakatan Kappa yang baik (0,64) dengan hasil pemeriksaan pada perbesaran 1000x. Hasil uji kesahihan dan keterndalan antara pemeriksa I dan II sangat baik karena menurut Landis dan Koch (1977) kesepakatan dipandang sangat baik bila nilai kappa 0,81-1,00. Hasil perhitungan sensitivitas dan spesifisitas menurut formula Hermannn (1995) berturut-turut sebesar 96% dan 97%. Hasil positif terdeteksi sebagai deposit granular kecoklatan pada jaringan otak berdasarkan hasil pemeriksaan sediaan imunositokimia SBPC head squash di bawah mikroskop cahaya. Hasil negatif terlihat sebagai warna kebiruan pada jaringan otak; (3) Antibodi monoklonal DSSC7 dalam asites mencit Balb/c titer (1:50) optimum untuk mendeteksi antigen DEN pada nyamuk Aedes sp yang telah diinfeksi virus DEN-3 peroral, antara lain di bagian sitoplasma sel usus tengah pada masa inkubasi 1-3 hari, hemosit pada masa inkubasi 1-2 hari, lemak (1-2) hari, ovarium (1-2) hari, kelenjar saliva (2 hari) dan otak (1-2) hari berdasakan metode munositokimia SBPC pada irisan jaringan parafin beku nyamuk; (3) hasil penelitian menunjukkan bahwa infeksi transovarial virus DEN terdeteksi pada sediaan abdomen squash di oosit pada masa inkubasi 4 hari dn 5 hari. Hasil penelitian juga menunjukkan bahwa infection rate abdomen squash dan head squash pada masa inkubasi 5 hari berturut-turut sebesar 75% and 33,33%. Di samping itu diketahui pula bahwa antigen DEN terdeteksi pada jaringan otak nyamuk Ae. aegypti yang telah menghisap darah pasien DBD pada masa inkubasi 8 hari dan progeninya pada stadium telur, larva, dan pupa. Indeks infeksi nyamuk Ae. aegypti yang telah diinfeksi dengan virus DEN-2 per oral dengan masa inkubasi 13 hari sebesar 93,33%, sedangkan indeks infeksi progeninya pada stadium imago umur sminggu tanpa diberi pakan darah sebesar 82,22%. Hasil tersebut didapat berdasarkan metode imunositokimia SBPC dengan menggunakan antibodi monoklonal DSSC7 sebagai antibodi primer; (4) hasil investigasi di lapangan pasca KLB DBD 2004 mengindikasikan bahwa infeksi transovarial secara alamiah terjadi di daerah endemis DBD Strata I di kota Yogyakarta tepatnya di kelurahan Terban dan Klitren dengan TIR (Transovarial Infection Rate) sebesar 26,67%. Hasil investigasi di lapangan pada akhir tahun 2006-awal tahun 2007 menunjukkan bahwa TIR Ae. aegypti terhadap virus DEN di Kabupaten Bantul, Sleman, dan Kota Yogyakarta berturut-turut sebesar 10,87%, 29,31%, dan 16,16%. Hasil analisis statistik menunjukkan ada perbedaan yang sangat bermakna TIR Ae. aegypti menurut lokasi (P=0,003<0,05)). Data investigasi juga menunjukkan tidak adanya perbedaan yang bermakna antara Indeks Infeksi Transovrial (TIR) dan Indeks Infeksi Ae. aegypti terhadap virus DEN pada nyamuk yang tetangkap pada stadium imago di Kabupaten Bantul (P=0,6>0,05), Kabupaten Sleman (P>0,05) dan di Kota Yogyakarta (P>0,05). Simpulan: (1) Antibodi monoklonal DSSC7 termasuk Klas IgG sub-kas IgG1 dan menunjukkan reaksi spesifik terhadap protein viral DEN NS-1 serta tidak bereaksi silang dengan virus CHIK. (2) Teknik imunositokimia SBPC dengan menggunakan antibodi monoklonal DSSC7 sebagai antibodi primer memberikan reaksi spesifik, peka, sahih, dan terandalkan untuk mendeteksi infeksi virus DEN pada nyamuk Ae. aegypti di bawah mikroskop cahaya. Teknik tersebut menunjukkan kesepakatan yang sangat baik (Kappa = 0,92) antara pemeriksa I dan II, dengan sensitivitas dan spesifisitas berturut-turut sebesar 96% dan 97%. (3) Teknik imunositokimia SBPC dengan menggunakan antibodi monoklonal DSSC7 berhasil diaplikasikan untuk mendapatkan gambaran patogenesis infeksi dan penularan transovarial virus DEN pada nyamuk Ae. aegypti di bawah mikroskop cahaya. (4) Teknik imunositokimia SBPC dengan menggunakan antibodi monoklonal DSSC7 sebagai antibodi primer berhasil diaplikasikan untuk surveilansi virologis vektor DBD melalui deteksi infeksi transovarial alamiah virus DEN selama inter epidemi DBD.

Background. Dengue Haemorrhagic Fever (DHF) is an important health problem in tropical and sub-tropical countries including Indonesia. This condition may be caused by the role of transovarial transmission of DEN virus in DEN vector during inter epidemic of DHF. Evaluation of vector control and vector surveillance is only based on population density of immature stage such as Angka Bebas Jentik (ABJ) in Indonesia without confirmed by virologic test. This entomologic indice is the precentage of house (premise) showing free from Aedes larvae. Aedes aegypti is the important vector of DHF and chikungunya fever, therefore for virus detection in the mosquito, an effort to minimize of the possibility of cross reactivity toward Chikungunya virus must be considered. There are several methods for virus detection in the mosquito such as the direct fluorescent –antibody (DFA) test on mosquito tissues, usually brain or salivary glands or head squashes, and reverse transcriptase polymerase chain reaction (RT-PCR). However the DFA method has the disadvantages of being labor-intensive and requiring fluorescent microscope and cryofreezer. Newer methods involving enzyme conjugates such as peroxidase and phosphatase in conjunction with either polyclonal or monoclonal antibodies are greatly improved. Monoclonal antibody against DEN-3 strain H-87 has been produced by Dengue Team of Gadjah Mada University through three time fusions. Among of the hybridoma cell lines generated from the third fusion, DSSC7 hybrid cells were still growing very well in the tissue culture, after storing it in the Liquid Nitrogen for several years. The antibody against DEN-3 secreted by the DSSC7 hybrid cells was significantly increasing after cloning. Regarding the above explanation the objectives of laboratory studies are aiming at (1) characterizing the monoclonal antibody DSSC7 for detection dengue infection based on immunocytochemical assay, (2) performing sensitivity, specificity, validity, and reliability tests of the immunocytochemical technique to detect DEN infection in Ae. aegypti mosquitoes using monoclonal antibody DSSC7 as primary antibody and applying the technique for studying, (3) pathogenesis of infection and transovarial transmission of DEN virus in orally infected Ae. aegypti. Field studies are aiming at applying the mmunocytochemical technique for virological surveillance through detection of transovarial infection in DEN vector during inter epidemic of DHF. Methods: (1) Characterization of monoclonal antibody DSSC7 was carried out through several activities as follows: (a) preparation of monoclonal antibody DSSC7 in Balb/c mice ascites, (b) determination of the antibody Class and subclass based on antigen mediated ELISA, (c) the specificity test of the monoclonal antibody DSSC7 based on Westernblotting method, using DEN-1, DEN-2, DEN-3, DEN-4, and Chikungunya antigen. (2) Laboratory studies for performing sensitivity, specificity, validity, and reliability tests were carried out through several activities as follows. The specificity of the immunocytochemical procedure is validated by negative controls and by positive controls that show that the antibody is binding to an appropriate structure. The presence of dengue antigen on head squash of intrathoracally infected male Ae. aegypti were detected based on ISBPC assay using mAb 2D3B10 and commercially mAb as positive controls. The mAb 2D3B10 is specific for DEN-3 and it reacts to viral E protein at the molecular weight of 57.9 kDa, whereas the commercially mAb reacts to DEN-1, DEN-2, DEN-3, and DEN-4. Negative control tissue specimens without primary antibody were used as negative controls. Positive result was detected as discrete brownish granular deposits throughout most fields having brain tissue. Negative result were detected as blue color throughout most field having brain tissue and no brownish color other than the chitinous mosquito tissues and non specific background distinctly different from specific positive result. The validity and reliability test was performed under light microscope at magnification of 400x and 1000x between two observers based on Landis and Koch’s Formula; (3) pathogenesis of infection and transovarial transmission of Ae. aegypti against DEN have been observed by detecting DEN antigen on the various organs of orally infected Ae. aegypti in the paraffin embedded tissue section, abdomen squash and head squash preparation, furthermore, transstadial infection was demonstrated in the brain of Ae. aegypti after taking blood of DHF patient at incubation period of 8 days and their progeny in the eggs, larvae, and pupae based on immunocytochemical SBPC assay using monoclonal antibody DSSC7 as a primary antibody. Transovarial transmission was also demonstrated by observing dengue antigen on head squash preparation of orally infected Ae. aegypti mosquitoes with DEN-2 and their progeny at imago stage (4) finally, observation of transovarial infection of DEN virus in Ae. aegypti based on ISBPC assay has been conducted during and post an outbreak of DHF 2004 in Yogyakarta. The relationship between the transovarial infection rate and the infection rate of DEN virus in adult of Ae. aegypti caught in November 2006-January 2007 in Bantul District, Sleman District and Yogyakarta Municipality was statistically analyzed using chi square analyses. Result: (1) It has been produced 50 ml of antibody against DEN secreted by DSSC7 hybrid cells in ascites of two Balb/c mice. The monoclonal antibody DSSC7 belongs to IgG Class, IgG1 Sub Class. The monoclonal antibody recognized DEN complex specific epitope and no cross reactivity against Chikungunya antigen based on Western blotting analyses. (2) Evaluation. Positive control. Dengue antigen was detected as brownish color in the cytoplasm of infected cell throughout most fields having brain tissue of infected Ae. aegypti mosquito with DEN-3 strain H87 at incubation period of 11 days under light microscope based on immunocytochemical streptavidin biotin peroxidase assay using mAb against DEN-3 viral E protein (2D3B10) (1:50) as primary antibody. Antigen was also detected as discrete brownish granular deposits throughout most fields having brain tissue of orally infected Ae. aegypti mosquito with DEN-3 strain H87 at incubation period of 8 days under light microscope based on immunocytochemical streptavidin biotin peroxidase assay using commercial mAb as primary antibody. According to the manufacture (Chemicon laboratory) the mAb reacts to DEN-1,DEN-2, DEN-3, DEN-4. Both brownish color in the cytoplasm of infected cells and discrete brownish granular deposits throughout most field having brain tissue of orally infected Ae. aegypti mosquito with DEN-3 strain H87 at incubation period of 11 days were shown under light microscope based on immunocytochemical SBPC assay using mAb DSSC7 (1:50) as primary antibody. The Negative result was shown on head squashes of uninfected Ae. aegypti from non endemic area of DHF and head squashes of Anopheles mosquitoes as blue color throughout most field having brain tissue and no brownish color other than the chitinous mosquito tissues and non specific background distinctly different from specific positive result Positive result was also detected as discrete brownish granular deposits throughout most fields having brain tissue of Ae. aegypti orally infected with DEN-2 at incubation period of 13 days. The agreement (Kappa) value of the immunocychemical assay is very good (0.82) between two observers with sensitivity and specificity of 96% and 97% respectively. (3) The DEN viral antigens were detected in the paraffin embedded tissue section in the mid gut in the cytoplasm cells at incubation period of 1-3 days, in the haemocytes at incubation period of 1-2 days, in the fat (1-2) days, in the ovaries at incubation period of 1-2 days, in the salivary glands at incubation period of 2 days, and in the brain at incubation period of 2 days; DEN viral antigen was detected in oocytes at incubation period of 4 days and 5 days on abdomen squash preparation. The infection rate of abdomen squash and head squash at incubation period of five days were 75% and 33.33% respectively. The result also revealed that the DEN antigen was detected in the brain of Ae. aegypti after taking blood of DHF patient at incubation period of 8 days and their progeny in the eggs, larvae, and pupae stages based on immunocytochemical streptavidin-biotinperoxidase complex ISBPC assay using monoclonal antibody DSSC7 as a primary antibody. The infection rate of orally infected Ae. aegypti with DEN-2 at incubation period of 13 days and their progeny at imago stage were 93.33 % and 82.22% respectively. (4) There were indications that natural transovarial infection rate of Ae aegypti in the bathroom water containers (bak mandi) in Terban and wells in South Klitren, Gondokusuman Sub district was 26.67% post outbreak of DHF 2004. The result indicated that the transovarial infection rates of DEN virus in Ae. aegypti in Bantul, Sleman, and Yogyakarta Municipality were 10.87%, 29.31%, and 16.16% respectively in the end 2006- early 2007. Meanwhile, the infection rates of DEN virus in Ae. aegypti at imago stage in Bantul, Sleman, and Yogyakarta Municipality were 13.64%, 33.77%, and 16.67% respectively. The result also indicated that there was no significantly difference between the TIR of DEN virus in Ae. aegypti and the infection rate of DEN virus at imago stage of Ae. aegypti in Bantul (P>0.05), Sleman (P>0.05), and Yogyakarta Municipality (P>0.05). This finding indicated that the TIR reflects the presence of infectious Ae. aegypti population in nature and it contributes in the outbreak of DHF. This finding indicated that the TIR reflects the presence of infectious Ae. aegypti population in nature and it contributes in the outbreak of DHF. Conclusions: Monoclonal antibody DSSC7 belongs to IgG Class, IgG1 Sub Class, The monoclonal antibody DSSC7 recognized DEN complex specific epitope at molecular weight of 48 kDa (NS1) protein and showing no cross reactivity toward chikungunya antigen. (2) The immunocytochemical streptavidin biotin peroxidase complex procedure using monoclonal antibody DSSC7 as primary antibody is sensitive, specific, valid and reliable for detection DEN infection in Ae. aegypti under light microscope and it was successfully applied for demonstrating : (3) the pathogenesis of infection and the transovarial transmission of Ae. aegypti against DEN in ovaries, and eggs in the ovary and transstadial transmission of DEN virus in eggs, larvae, pupae and imago stages under laboratory condition. The technique is successfully applied for virological surveillance of DEN vector through detection of transovarial infection in DEN vector during inter epidemic of DHF.

Kata Kunci : Penyakit demam berdarah, Teknik imunostikimia, Antibodi monoklonal DSSC7, Patogenesis Infeksi, Transovarial virus dengue, Surveilansi virologis vektor dengue, Nyamuk Aegypti, immunocytochemical test, monoclonal antibody DSSC7, pathogenesis, transovarial t