Bidang ilmu : KESEHATAN
LAPORAN HIBAH PENELITIAN STRATEGIS NASIONAL
BATCH-I TAHUN ANGGARAN 2010
Produksi Antibodi Monoklonal Berbasis Protein Adhesi Mycobacterium tuberculosis dan Reseptornya Untuk Bahan Deteksi Dini Penyakit Tuberkulosis (TB)
Dr. Uun Yanuhar, SPi.M.Si. dr. Dwi Yuni Nurhidayati, M.Kes.
Dibiayai Oleh Direktorat Jenderal Pendidikan Tinggi, Kementerian Pendidikan Nasional sesuai dengan Surat Perjanjian Penugasan Dalam Rangka Pelaksanaan Hibah Penelitian Strategis Nasional Tahun Anggaran 2010 Nomor : 165/SP2H/PP/DP2M/III/2010, tanggal 01 Maret 2010
UNIVERSITAS BRAWIJAYA NOVEMBER 2010
RINGKASAN Produksi Antibodi Monoklonal Berbasis Protein Adhesi Mycobacteria tuberculosis dan Reseptornya Untuk Bahan Deteksi Dini Penyakit Tuberkulosis (TB) Penyakit tuberkulosis masih merupakan masalah kesehatan yang utama dan Indonesia merupakan penyumbang kasus Tuberkulosis (TB) terbesar ketiga setelah didunia setelah Cina dan India. Infeksi yang disebabkan paparan bakteria Mycobacterium tuberkulosis (M. tuberculosis) diperkirakan kurang lebih selama 5 minggu, sehingga diagnosis tuberculosis yang umumnya berdasarkan gejala klinis dan gambaran radiologis disertai konfirmasi dengan pemeriksaan hapusan dan pemeriksaan sputum mengakibatkan adanya kekurang sensitifan pemeriksaan hapusan sputum dan kultur. Lamanya paparan M. Tuberculosis merupakan kesulitan tersendiri dalam mendiagnosa tuberkulosis paru, sehingga diperlukan pemeriksaan imunodiagnosis yang mudah dikerjakan, murah untuk dapat mendiagnosa tuberkulosis paru. Tujuan penelitian ini untuk mendapatkan protein adhesin M. tuberculosis pili (MTp) untuk memproduksi antibodi monoklonal guna deteksi dini tuberculosis secara cepat dan akurat pada penderita tuberkulosis. Pada penelitian ini dilakukan metode penelitian meliputi identifikasi dan karakterisasi molekul protein adhesi, elektroforesis protein, elektroelusi protein adhesi, uji haemaglutinin, serta pembuatan antibodi monoklonal berbasis protein adhesi melalui kultur sel mieloma, fusi dengan sel b dari spleen mencit yang telah diimunisasi oleh protein adhesin pili pembawa antibodi, produksi antibodi monoklonal dan karakter isasi genotipenya, produksi massal antibodi melalui mice, preparasi lebih lanjut sehingga mendapatkan karakter yang terukur baik melalui uji respon imunogenitas antibody monoklonal anti protein adhesi dengan teknik dot blotting dan western blotting maupun pemeriksaan respon antibodi dengan scaning mikroskop elektron (SEM). Hasil penelitian pada Tahap I telah diawali dengan hasil penelitian sebelumnya yakni telah dilakukan identifikasi protein adhesi M. tuberculosis dan diketahui mempunyai berat molekul 63 kDa, yang berasal dari pili bakteria M. tuberculosis, secara lebih mendalam peran protein tersebut akan dikaji apakah protein tersebut juga merupakan protein adhesi yang berhubungan dengan virulensi seperti kemampuan aglutinasi terhadap eritrosit manusia dan juga hewan yakni mencit Balbc/mice. Pada penelitian tahap awal ini telah dihasilkan karakter protein adhesin pili selain berat molekul 63,3 kDa M. tuberculosis yakni juga berat molekul 38 kDa dan 17 kDa. Protein ini setelah diuji merupakan protein adhesin yang mampu mengaglutinasi eritrosit baik mencit Balb/c juga eritrisit dari darah manusia yakni A, AB, B dan O dan bersifat haemaglutinin, akan tetapi konsentrasi yang dihasilkan berbeda-beda tergantung dari konsentrasi pengenceeran haemaglutininya. Pengujian lebih lanjut ditetapkan bahwa antibodi monoklonal dibuat dari protein adhesin pili M. tuberculosis dengan BM 38 kDa. Penelitian ini selanjutnya telah memproduksi antibodi monoklonal yang berbasis protein adhesin M.tuberculosis melalui eksplorasi molekuler kultur sel hibridoma yang difusikan dengan sel b dari mencit Balb/c. Antibodi poliklonal yang dihasilkan dari berat
molekul 38 kDa telah menunjukkan hasil titer posistif dengan pemeriksaan ELISA, dimana protein adhesi ini akan dikembangkan lebih lanjut untuk produksi massal antibody monoklonal untuk pengembangan bahan diagnostik dini penyakit tuberkulosis. Hasil eksplorasi antibodi monoklonal yang dihasilkan dari berat molekul 38 kDa telah menghasilkan respon antibodi dari sel hibridoma yang dihasilkan melalui fusi sel hibridoma, dan respon antibodi ini telah diuji responnya dengan pemeriksaan dot blotting terhadap sampel sputum penderita tuberculosis dan juga sel eritrosit mencit yang diinduksi dengan M. tuberculosis. Kesimpulan penelitian ini adalah telah dihasilkan antibodi monoklonal anti adhesion pili M. tuberculosis dengan berat molekul 38 kDa. Respon antibodi melalui sel hibrid yang dihasilkan dari protein adhesin pili (MTp. M. Tuberculosis 38 kDa) telah diuji dengan sputum penderita dan juga sel darah dari mencit yang diuji dengan paparan M. tuberculosis. Untuk penelitian lanjutan direkomendasikan untuk menguji antibodi monoklonal protein adhesin BM 38 kDa untuk pengembangan lebih jauh diagnosis dini Tuberculosis baik pada stadia awal serangan Tb dan stadia infeksi tuberculosis lebih lanjut.
SUMMARY The Production of Monoclonal Antibody Based on Adhesion Protein of Mycobacterium tuberculosis and Its Receptor for Early Detection Material of Tuberculosis (TB) Tuberculosis still be main health problem and Indonesia is case contributor of Tuberculosis (TB) the biggest third in the world after Chinese and India. Infections caused by exposure to bacteria Mycobacterium tuberculosis (M. tuberculosis) is estimated for about 5 weeks, so the diagnosis of tuberculosis is usually based on clinical symptoms and radiological images accompanied by confirmation by smear examination and sputum examination results in less sensitive examination of sputum smear and culture. The duration of exposure to M. Tuberculosis is a separate difficulty in diagnosing pulmonary tuberculosis, so that the necessary checks done imunodiagnosis an easy, inexpensive to be able to diagnose pulmonary tuberculosis. The purpose of this research is to get protein adhesin M. tuberculosis pili (MTP) to produce monoclonal antibodies for early detection of tuberculosis in a rapid and accurate in patients with tuberculosis. Research methods include the identification and characterization of adhesion molecule protein, protein electrophoresis, Electroelution adhesion proteins, haemaglutinin test, and produce monoclonal antibody-based adhesion proteins through a culture of myeloma cells, fusion with b cells from spleen of mice that have been immunized by a protein adhesin pili carrier antibody, monoclonal antibody production and characterization of genotype, the mass production of antibodies by mice, further preparation so that the gain characteristics measured through response test immunogenicity of monoclonal antibodies anti-adhesion proteins by using dot blotting and western blotting as well as examination of antibody response with the scanning electron microscope (SEM). The results in Phase I has started with results of previous studies that have been done to identify adhesion proteins M. tuberculosis and known to have a molecular weight of 63 kDa, derived from bacterial pili M. tuberculosis, in more depth the role of these proteins will be investigated whether these proteins also an adhesion protein associated with virulence such as the ability agglutination of human erythrocytes and also animals that mice Balb/c (mice). At this early stage of research has produced a character other than pili adhesin protein molecular weight of 63.3 kDa M. tuberculosis that is also the molecular weight of 38 kDa and 17 kDa. This protein is a protein adhesin after the test that is able to agglutinate the erythrocytes both mice Balb/c also erythrocytes from human blood that is A, AB, B and O and are haemaglutinin, but the resulting concentration varies depending on the concentration pengenceeran haemaglutininya. Further testing determined that monoclonal antibodies are made from protein adhesin pili M. tuberculosis with 38 kDa. Further study has produced monoclonal antibody-based protein adhesion M.tuberculosis through the exploration of molecular hybridoma cell cultures fused with b cells from mice Balb / c. Polyclonal antibodies generated from the molecular weight of kDa has shown positive results with ELISA titer. These adhesion proteins will be further developed for mass production of monoclonal antibodies for the development of early diagnostics of tuberculosis disease. Results exploration monoclonal antibodies
generated from the molecular weight of 38 kDa has produced an antibody response from hybridoma cells produced through hybridoma cell fusion, and antibody response was tested response by dot blotting examination of sputum samples of patients with tuberculosis and also erythrocyte of mice induced by M . tuberculosis. Result of research at phase I has been initialed with result of research before by identified adhesion protein of M. tuberculosis and known have weights molecule 63 kDa, is coming from pili of M. tuberculosis. In more deeply, the role of the protein will be studied what the protein also a adhesion protein relating to virulence like ability of agglutination to human erythrocyte and animal mice. At research of this phase I has been yielded protein character of pili 6adhesion besides molecule weight 63,3 kDa M. tuberculosis, also protein with molecule weigh 38 kDa and 17 kDa. This protein after tested is adhesion protein capable to agglutinate erythrocyte and haves the character of haemaglutinin. This research has produced monoclonal antibody based on adhesion protein of M. tuberculosis through molecular exploration of hybridoma cell culture fused with b cell, which will be done furthermore to get monoclonal antibody specifity and its genetic character. Monoclonal antibody yielded from molecule weight 38 kDa already yielded an antibody response of hybrid cell resulted by fused hibridoma cell and its response was tested using dot blott to sputum of tuberculosis patient. The conclusion of this research is to have produced a monoclonal antibody antiadhesin pili M. tuberculosis with a molecular weight of 38 kDa. Antibody response via the hybrid cells resulting from protein adhesin pili (MTp. M. tuberculosis 38 kDa) was tested with patient sputum and blood cells from mice were also tested by exposure to M. tuberculosis. For further research is recommended to test the monoclonal antibody 38 kDa molecular weight adhesin proteins for further development of early diagnosis of tuberculosis in both early stage Tb attack and further stage of tuberculosis infection.
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