Reaktivitas antibodi sera subyek Dispepsia positif Helicobacter pylori Yogyakarta dan Mataram terhadap antigen fraksi protein Helicobacter pylori isolat Mataram

TRIWIKATMANI, Catharina, drh. Widya Asmara, SU.,PhD

2004 | Tesis | S2 Ilmu Kedokteran Tropis

Kit diagnostik serologi untuk Helicobacter pylori (H. pylori) dapat memberikan hasil yang berbeda bila digunakan di tempat lain pada populasi yang berbeda, kemungkinan disebabkan perbedaan strain H. pylori antar tempat yang dapat menimbulkan respon imun penderita yang berbeda. Unit Riset Biomedis (URB) RSU Mataram, Lombok, NTB telah mengembangkan dua macam kit diagnostik serologi H. pylori menggunakan isolat lokal, tetapi reaksi sera penderita H. pylori dari Mataram terhadap antigen H. pylori isolat Mataram belum diketahui. Selain itu Mataram dan Yogyakarta berbeda secara geografis dengan kemungkinan perbedaan strain H. pylori, sehingga juga perlu diketahui reaksi antibodi sera Subyek Yogyakarta terhadap antigen fraksi protein H. pylori isolat Mataram. Penelitian dilakukan secara seri kasus dengan melihat reaktivitas antibodi sera subyek dispepsia yang positif H. pylori dari Yogyakarta dan Mataram terhadap antigen H. pylori isolat Mataram. Subyek terdiri dari 4 penderita Yogyakarta dan 5 penderita Mataram. Lima isolat H. pylori didapatkan dari keempat subyek Mataram tersebut dan dari seorang penderita Mataram yang tak diikutsertakan dalam hasil penelitian. Satu serum kontrol positif yang digunakan untuk pemeriksaan serologi di URB-RSU Mataram diikutsertakan dalam penelitian. Identifikasi H. pylori dilakukan dari pemeriksaan histopatologi untuk Subyek Yogyakarta atau smear mukus jaringan biopsi untuk Subyek Mataram. Isolat H. pylori diperoleh dari kultur jaringan biopsi Subyek Mataram. Preparasi antigen dilakukan untuk mendapatkan protein membran, sitosol, dan sekresi. Fraksi protein dipisahkan dengan SDS-PAGE 11%, selanjutnya protein dipindah ke membran PVDF. Imunobloting dilakukan antara tiap serum dengan tiap protein dari tiap isolat H. pylori, serta dideteksi dengan AP-conjugated anti-human IgG Fab specific dan indikator NBT-BCIP. BM antigen yang bereaksi ditentukan dengan bantuan kurva protein marker yang dibuat dengan plotting log BM terhadap mobilitas relatif protein, sedangkan penghitungan menggunakan Linest dari Microsoft Excel 2000. Penentuan kuatnya reaksi dilakukan dengan pengamatan langsung berdasarkan tebalnya pita imunohibridisasi. Isolat H. pylori Mataram 1, 2, 3, 4, dan 5 yang diteliti merupakan isolat yang berbeda satu sama lain. Antigen utama memiliki BM 86, 74, 42, dan 37 kDa, sedangkan antigen lain yang selalu menimbulkan respon imun memiliki BM 98, 91, 71, 66, 60, 54, 46, 30, 25, 23, dan 22 kDa. Antigen 101 kDa menimbulkan respon antibodi hanya pada Subyek Mataram, tidak pada Subyek Yogyakarta. Antigen utama 86, 74, 42, dan 37 kDa mungkin dapat digunakan sebagai bahan sarana diagnostik bagi penderita H. pylori dari Yogyakarta dan Mataram. Diperlukan uji reaksi silang dengan protein bakteri enterik lain untuk antigen 42 dan 37 kDa yang merupakan protein MMMP.

The serological diagnostic kit for Helicobacter pylori (H. pylori) can give different results if it is used in different population. These differences were probably due to variability of the H. pylori strains among places. The URB-RSU Mataram, Lombok, NTB, has developed two H. pylori serological diagnostic kits using local isolates, but reactivity of sera from Mataram patients against H. pylori antigens of local isolates has not yet been revealed. In fact, Mataram and Yogyakarta is geographically separated with a possibility of H. pylori strain difference. So, it is worthy to know the reactions between the antibodies of Yogyakarta subjects against the antigens of H. pylori Mataram isolates. This research is a case series of reactivities of antibodies from H. pyloripositive dyspeptic subjects sera from Yogyakarta and Mataram against antigens of H. pylori Mataram isolates. The subjects were 4 patients from Yogyakarta and 5 from Mataram. Five isolates of H. pylori were obtained from 5 patients of Mataram, which 1 of them was not involved in the result. In this research, it was attached one serum used to be positive control for serological examination at the URB-RSU Mataram. Infection status of H. pylori was assesed from histopathology examination for the Yogyakarta Subjects, and from mucous smear examination for the Mataram ones. The H. pylori isolates were cultured from biopsy tissues of the Mataram Subjects. Therefore, membrane, sitosol and secreted proteins were prepared from subcultures. Proteins were separated by SDS-PAGE 11% then they were transfered to PVDF membrane. Next, immunoblotting was done between each serum and each protein from each H. pylori isolate, detected by AP-conjugated anti-human IgG Fab specific and NBTBCIP indicator. The molecular masses of reacted antigens was determined by the curve of protein marker created by plotting log of molecular masses for the relative mobility of the proteins, calculated by Linest of the Microsoft Excel 2000. The strength of the reactions were held by direct observation relied upon how bold of the immunohibridization bands. The Mataram isolates of H. pylori 1, 2, 3, 4, and 5 differ each other. The main antigens has molecular masses 86, 74, 42 and 37 kDa, while others which always induce immune responses have molecular masses 98, 91, 71, 66, 60, 54, 46, 30, 25, 23, and 22 kDa. Specifically, the antigen 101 kDa induces antibody responses only of the Mataram Subjects, not of the Yogyakarta ones. The main antigen 86, 74, 42 and 37 kDa perhaps can be used as a diagnostic tool for Mataram and Yogyakarta population. As a result, it is needed a cross reaction test using other enteric bacteria proteins for the antigen 42 and 37 kDa which belong to the MMMP proteins.

Kata Kunci : Kedokteran Tropis,Antibodi Sera,Dispepsia