Tesis

Pengembangan Uji Diagnostik Recombinant Immunoblot Assay (RIBA) HIV Berbasis Subtipe HIV-1 yang Ditemukan di Indonesia = Development of Recombinant Immunoblot Assay (RIBA) Diagnostic Test Based on Circulating Subtypes of HIV-1 in Indonesia.

Uji Western blot masih menjadi gold standard untuk konfirmasi diagnosis infeksi HIV yang memerlukan ketiga protein utama HIV, yaitu env, pol, dan gag. Kekurangan pada uji ini yaitu kemungkinan adanya kontaminasi dengan protein selular manusia serta pembuatannya yang relatif mahal. Selain itu, diversitas HIV1 yang tinggi menyebabkan uji western blot menjadi kurang sensiftif. Penggunaa n antigen rekombinan yang imunodominan dan lestari menjadi alternatif lain. Uji RIBA (Recombinant Immunoblot Assay) pada penelitian ini menggunakan antigen rekombinan dari keempat subtipe HIV-1 yang dominan di Indonesia, yaitu subtipe CRF01_AE, B, CRF02_AG, dan C. Antigen rekombinan Gag (p24), Pol (IDR), Env (gp41 IDR) diekspresikan pada sistem ekspresi E.coli dan dipurifikas i menggunakan kromatografi Ni-NTA. Antigen rekombinan yang telah dimurnika n dilihat reaktivitasnya terhadap sampel serum pasien dengan HIV-AIDS sebanyak 50 sampel dan non HIV-AIDS 45 sampel. Sebanyak 21 sampel HIV-AIDS dan 3 sampel non HIV-AIDS dilakukan uji menggunakan kit Western blot MP Diagnostics HIV blot 2.2 sebagai perbandingan terhadap uji RIBA yang menggunakan metode Western blot. Hasil perbandingan memperlihatkan hasil uji RIBA memiliki reaktivitas yang lebih baik dibandingkan dengan hasil uji kit MP Diagnostics HIV Blot 2.2 dengan persentase reaktivitas terhadap protein p24 95,2% (20/21), protein Pol 85,7% (18/21), dan protein gp41 100% (21/21). Pada uji RIBA, 5 sampel tidak menunjukkan reaktivitas terhadap antigen rekombinan Pol (IDR) dan 4 sampel tidak menunjukkan reaktivitas terhadap antigen rekombinan Gag (p24). Seluruh sampel menunjukkan reaktivitasnya terhadap antigen rekombinan Env (gp41 IDR). Penelitian mengenai uji RIBA ini dapat dikembangkan untuk uji diagnostik HIV-1 dengan subtipe-subtipe HIV-1 yang banyak ditemukan di Indonesia.
Kata kunci : HIV-1, RIBA, diagnostik, imunodominan


The Western blot test is still the gold standard to confirm the diagnosis of HIV-1 infection. This test requires three core of HIV proteins, i.e., env, pol, and gag. Nevertheless, this test has several disadvantages, mainly in possibility of contamination with human cellular proteins as well as production cost is relatively expensive. In addition, the high diversity of HIV-1 may causes Western blot test to be less sensitive. Another method that can be used to overcome these obstacles is the use of immunodominant region and conserved region of recombinant antigen in the assay, also known as RIBA (Recombinant Immunoblot Assay). In this research, recombinant antigens were derived from the four subtypes of HIV-1 that are dominant in Indonesia, which are CRF01_AE subtype, B subtype, CRF02_AG subtype, and C subtype. The recombinant antigens comprises Gag (p24), Pol (IDR), Env (gp41 IDR). Each of antigens was expressed in E. coli expression system and purified using Ni-NTA chromatography. Reactivity test of purified antigen was done against a group consist 50 serum samples with HIV-AIDS and 45 serum samples without HIV-AIDS. Twenty-one samples with HIV-AIDS and 3 samples without non HIV-AIDS test were done using Western blot kit MP Diagnostics HIV blot 2.2 too as a comparison toward the RIBA test that using Western blot method. The results showed that RIBA test had better reactivity than kit test with reactivit y percentage toward p24 95,2% (20/21), Pol 85,7% (18/21), and gp41 100% (21/21). RIBA test results performed 5 samples with negative reactivity toward recombinant antigens Pol (IDR) and 4 samples with negative reactivity toward recombinant Gag antigen (p24). All the samples had positive reactivity toward recombinan recombinant antigen Env (gp41 IDR). As diagnostic kit, this RIBA test shows broad possibility for development in diagnosis HIV-1 infection especially with HIV-1 subtypes that circulate in Indonesia.
Key words: HIV-1, RIBA, diagnostic, immunodominant