LAPORAN PENELITIAN STRATEGIS NASIONAL TAHUN ANGGARAN 2011

Produksi Antibodi Monoklonal Berbasis Protein Adhesi Mycobacterium tuberculosis dan Reseptornya Untuk Bahan Deteksi Dini

Penyakit Tuberkulosis (TB)

Dr. Uun Yanuhar, SPi., MSi

Asep Awaludin, MP

dr. Dwi Yuni Nurhidayati, MKes

Dibiayai Oleh Direktorat Jenderal Pendidikan Tinggi, Kementerian Pendidikan Nasional sesuai dengan Surat Perjanjian Pelaksanaan Hibah Penelitian Strategis Nasional

Nomor : 404/SP2H/PL/Dit. Litabmas/IV/2011 Tanggal 14 April 2011

UNIVERSITAS BRAWIJAYA

NOVEMBER 2011

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 tuberkulosis 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 antibodi monoklonal anti protein adhesi dengan teknik dot blotting dan western blotting maupun pemeriksaan respon antibodi dengan scaning mikroskop elektron (SEM).

Pada penelitian 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 5

haemaglutininya. Pengujian lebih lanjut ditetapkan bahwa antibodi monoklonal dibuat dari protein adhesin pili M. tuberculosis dengan BM 38 kDa.

Penelitian ini juga 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 antibodi 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. Antibodi monoklonal yang dihasilkan dalam penelitian ini adalah antibodi monoklonal dari protein adhesin Tb 38 kDa, yang dihasilkan melalui proses produksi hibridoma dan rekombinan RNA antibodi.

Pada penelitian akhir telah dikembangkan produksi antibodi melalui teknik rekombinan RNA, dan dihasilkan antibodi monoklonal adhesin 38 kDa melalui rekombinan RNA antibodi. 6

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). From this monoclonal antibodies are then carried out the manufacture of recombinant antibody

In this research, It has been yielded protein character of pili protein adhesion 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 7

specifity and its genetic character. Monoclonal antibodies generated in this study is a monoclonal antibody Tb adhesin 38 kDa produced by hybridoma antibody production and recombinant RNA.

At the end of the study, it has developed the production of antibodies through recombinant RNA techniques and produced adhesin monoclonal antibodies 38 kDa through RNA recombinant antibody

DAFTAR PUSTAKA

Abbas, LichmanA. 2003. Cellular and Molecular Immunology. 5th edition. Saunders, Boston

Aditama T Y ,2006. Pedoman Nasional Penanggulangan Tuberkulosis, Edisi 2, Departemen Kesehatan Republik Indonesia.

Allen, S.S and Mac Murray, D.N.2003. Coordinate Cytokine Gene Exprresion In Vivo Following Inductions Tuberculous of Pleurisy in Guenia pigs. Infectios and Immunity, August, 2003. P.4271-4277.

Algood, HMS et al. 2004. TNF influence chemokine expression of macrophages in vitro and that of CD11b cells in vivi during Mycobacterium tuberculosis infection. The Journal of Immunology, 176: 6846-6857.

Alteri Christoper J. 2007. Mycobacterium tuberculosis produces pili during human infection. PNAS

Baratawijaya BG. 2006. Imunologi Dasar, edisi ke tujuh,FKUI, Jakarta Bloom, B.R. dan Murray. 1992. Tuberculosis: Commentary on reemergent killer.

Science: 257, pp.1055-1056. Butler, W.R., Guthertz, L.S. 2001. Mycolic Acid Analysis by High-Performance Liquid

Chromatography for identification of Mycobacterium Species. Clinical Microbiology Reviews, Oct., p. 704-726.

Comolli Janes C., Waite Leslie L., Mostov Keith E., Engel Joanne N., 1999, Pili Bending to Asialo – GM 1 on Epithelial Cells Can Mediate Cytotoxicity or Bacterial Internalisation by Pseudomonas aeruginosa, Infection and Imunity 67 (7) p 3207 – 3214.

Conde Marcus B, Philip Suffys. 2004. Immunoglobulin A and IgG Immune Respon Against P-90 Antigen for Diagnosis of Pulmonary Tuberculosis and Screening for Mycobacterium tuberculosis Infection, Clinical and Laboratory Immunology .p94- 97

Dorbniewski, F.A., Hoffner, S., Rusch-Gerdes, S., Skenders, G., Thompsen, V., and the WHO European Laboratory Strengthening Task Force. 2006. Recomended Strandarts for Modern tuberculosis Laboratory Services in Europe. Eur Respir J. 28:903-909.

Fenton M and Vermeulen M.1996. Immunopathology of Tuberculosis : Roles of Macrophage and Monocytes. Infect immunol 64;3: 683-690.

Lawn, S.D., Butera, S.T., Shinnick, T.M. 2002. Tuberculosis Unleashed : the Impact of Human Immunodeficienncy Virus Infection on the Host Granulomatous Response to Mycobacterium tuberculosis. Elsevier. Microbes and Infection 4. (2002). 635-646.

Mertaniasih, Ni Made dan Widodo, A.D.W. 2007. Mechanism of Multidrug Resistant Tuberculosis (MDR-TB) and Extensively Drug Resistant Tuberculosis (XDR-TB). Proceeding Simposium Nasional TB Update – IV Surabaya 3-4 Maret 2007.

Nagayama, K., Oguchi,T., Arita, M., Honda,T.,1995, Purification and Characterization of A Cell Associated Haemagglutinin of Vibrio parahaemolyticus, Infec.and Immun 63 (5): 1987 – 1992

Nugraha Yusak, 2007, Development of Tuberculosis Laboratory Test In Departement of Clinical Pathology Disampaikan Pada Simposium Nasional TB UpDate IV 2007 di Surabaya

Rom William N. 2004. Tuberculosis, Second Edition, Philadelpia , p117. Salyers Abigail A., Whitt Dixie D., 1994. Bacterial Pathogenesis. Washington DC: ASM

Press, p.31 -35 ; 262 Sandjaja, B. Isolasi Dan identifikasi Mikrobakteria. Balai laboratorium kesehatan

Jayapura. Hal. 1-141. Sandjaya, B. 1992. Isolasi dan Identifikasi Mikobakteria, Widya Medika, Jakarta. Saurez-Mendez R, Garcia – Garcia I,Fernandez Oliveira.2004. Adjuvant interferon

gamma in patient with drug resistant pulmonary tuberculosis: a pilot studi. Infect Dis 4:44.

Schlossberg D, et al. 2006. Tuberculosis and Nontuberculosis Mycobacterium Infection, edisi ke lima, Mc. Grawhill Co. Philadelphia.

Schlichtherle,M. Wahlgren,M, 2000. Methodsin Research. Third edition. Malaria reserch and reference Reagen Resource Center. Manassaa.p46

Senoi G., Humoral Imune Response Against 38 – kDa and 16 –Kda Mycobacterial Antigens in Tuberculosis, Eur J 2007:28:143 -148 Vol 29 Number 1

Soini, H and J.M. Musser, 2001. Molecular Diagnosis of Mycobacteria. Clinical Chemistry, 47:5

Todar K., 2002. Bacterial Structure in Relationship to Pathogenicity, University of Wisconsin – Madison depatement of bacteriology, p 1-5

Todar K., 2002.The Mechanisms of Bacterial Pathogenicity, University of Wisconsin – Madison Departement of Bacteriology,p 1- 6

Winarsih,S., Sumarno, Rokistiningsih, 1997, Kajian Fungsi dan Sifat Imunogenisitas Protein Hemaglutinin 32 kDa & 20 kDa pada Helicobacter pylori,Majalah Kedokteran Universitas Brawijaya Malang Vol,XIII No.3,p.135 141