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"Optimasi uji imunofluoresensi untuk mendeteksi dan membedakan serotipe virus dengue telah dilakukan. Berdasarkan hasil, galur sel Vero76 merupakan sel terbaik untuk visualisasi dengan konsentrasi optimum antibodi primer yaitu 3,5 µg/ml untuk 4G2 (Anti Flavivirus), 3,8 µg/ml untuk 15F3 (Anti DEN1), 4,1 µg/ml untuk 3H5 (Anti DEN2), 4,4 µg/ml untuk 5D4 (Anti DEN3), dan 5,8 µg/ml untuk 1H10 (Anti DEN4), antibodi sekunder FITC sebesar 10 µg/ml, dan 7 µg/ml untuk antibodi dilabel Alexa Fluor. Uji sensitivitas menunjukkan bahwa uji imunofluoresensi mampu mendeteksi virus hingga 10-3 (0,001) plaque forming unit (PFU)/ml. Uji spesifisitas menunjukkan antibodi monoklonal yang diproduksi spesifik terhadap setiap serotipe.

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Optimization of immunofluorescence assay (IFA) for detecting and serotyping dengue virus had been done. The results showed that Vero76 cell line was the best cell for visualization, with optimum concentration for primary antibody was 3.5 µg/ml for 4G2 (Anti Flavivirus), 3.8 µg/ml for 15F3 (Anti DEN1), 4.1 µg/ml for 3H5 (Anti DEN2), 4.4 µg/ml for 5D4 (Anti DEN3), and 5.8 µg/ml for 1H10 (Anti DEN4), while for FITC-labelled secondary antibody was 10 µg/ml, and 7 µg/ml for Alexa Fluor-labeled antibody. The sensitivity showed that IFA were able to detect viruses up to 10-3 PFU/ml. The specificity assay demonstrated that the monoclonal antibodies were specific to each serotype."

"Pembuatan Stable Cell Line sendiri membutuhkan proses pengantaran materi genetik untuk mencapai tahap ekspresi gen rekombinan secara berkelanjutan. Metode ini menggunakan transfeksi untuk membantu mencapai tahapan tersebut. Badan Riset dan Inovasi Nasional (BRIN) Indonesia telah berhasil membuat konstruksi plasmid rekombinan pengekspresi protein Spike SARS-CoV-2. Namun, belum dilakukan penelitian lebih lanjut terkait penggunaan konstruksi plasmid rekombinan tersebut. Oleh karena itu, tujuan penelitian ini adalah untuk memvalidasi ekspresi protein rekombinan dari plasmid rekombinan pengekspresi Spike SARS-CoV-2 yang akan digunakan dalam pembuatan Stable Cell Line pada galur sel mamalia 293T. Validasi ekspresi dari empat protein SARS-CoV-2 (Spike Full, Subunit S1, Subunit S2, dan Receptor Binding Domain) dilakukan melalui metode Immunofluorescence Assay (IFA) dan Western Blot (WB). Hasil menunjukkan bahwa dari empat ragam protein (Spike Full, Subunit S1, Subunit S2, dan Receptor Binding Domain) terbukti fungsional secara ekspresi dan sesuai dengan ukuran protein yang sesuai. Uji IFA menunjukkan bahwa terdapat dua nilai rata-rata Corrected Total Cell Fluorescence (CTCF) yang unggul yaitu pada protein Spike Full dan Subunit S2 (118.813 dan 264.159 CTCF) sel pasca transfeksi yang menandakan bahwa terdapat perbedaan kemampuan ekspresi dari masing-masing protein. Uji western blot telah membuktikan dua protein (Spike Full dan Subunit S2) memiliki ukuran molekul yang sesuai (142,5 dan 66,0 kDa). Sehingga, dapat disimpulkan bahwa plasmid rekombinan yang dikonstruksi oleh BRIN terbukti fungsional dan dapat dilanjutkan ke dalam penggunaannya untuk pembuatan Stable Cell Line.

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Making a Stable Cell Line requires a process of delivering genetic material to reach the continuous recombinant gene expression stage. This method uses transfection to help achieve this stage. The National Research and Innovation Agency of Indonesia (BRIN) has successfully constructed a recombinant plasmid expressing the SARS-CoV-2 Spike protein. However, no further research has been carried out regarding using these recombinant plasmid constructs. Therefore, this study aimed to validate the expression of recombinant proteins from the SARS-CoV-2 Spike-expressing recombinant plasmid to manufacture Stable Cell Line in mammalian cell line 293T. Validation of the expression of four SARS-CoV-2 proteins (Spike Full, Subunit S1, Subunit S2, and Receptor Binding Domain) was carried out using Immunofluorescence Assay (IFA) and Western Blot (WB) methods. The results showed that the four protein variants (Spike Full, S1 Subunit, S2 Subunit, and Receptor Binding Domain) were functional in expression and according to the appropriate protein size. The IFA test showed two superior Corrected Total Cell Fluorescence (CTCF) values: the Spike Full protein and the S2 Subunit (118.813 and 264159 CTCF) of post-transfection cells, which indicated that there were differences in the expression ability of each protein. The Western Blot test has proven that two proteins (Spike Full and Subunit S2) have the appropriate molecular size (142.5 and 66.0 kDa). Thus, it can be concluded that the recombinant plasmid constructed by BRIN is proven to be functional and can be used to manufacture Stable Cell Lines."

"Kanker kolorektal merupakan salah satu kanker yang memiliki angka penderita yang tinggi di dunia. EPCAM adalah salah satu gen yang diekspresikan pada karsinoma dan ditemukan dalam circulating tumour cell (CTC) yang umum digunakan sebagai penanda kanker. Peripheral blood mononuclear cell (PBMC) adalah sel darah perifer yang juga diduga mengekspresikan gen EPCAM, dan perlu diteliti lebih lanjut sebab kelimpahan PBMC di dalam tubuh lebih banyak dibandingkan CTC. Penelitian dilakukan dengan tujuan untuk mendeteksi ekspresi gen EPCAM pada CTC dan PBMC kanker kolorektal menggunakan metode semi-quantitative RT-PCR dan direct immunofluorescence sehingga potensi EPCAM sebagai biomarker untuk diagnosis kanker kolorektal diketahui. Isolat CTC dan PBMC yang berasal dari delapan sampel darah penderita kanker kolorektal diteliti melalui isolasi dan kuantifikasi RNA, kemudian dilanjutkan dengan amplifikasi cDNA, dan pewarnaan antibodi. Hasil penelitian menunjukkan tidak adanya ekspresi gen EPCAM pada CTC dan PBMC menggunakan metode semi-quantitative RT-PCR. Hasil pengamatan dengan metode direct immunofluorescence menunjukkan pada salah satu sampel PBMC terdapat protein yang belum dapat dipastikan sebagai EpCAM, namun pada CTC ekspresi gen EPCAM tidak terdeteksi. Berdasarkan hasil penelitian, dapat disimpulkan bahwa ekspresi gen EPCAM tidak terdeteksi pada CTC dan PBMC dengan metode semi-quantitative RT-PCR. Penelitian lebih lanjut diperlukan untuk mengkonfirmasi protein yang terdeteksi pada PBMC adalah EpCAM.

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Colorectal cancer is one of the most common cancers in the world. EPCAM is one of the genes expressed in carcinoma and found in circulating tumor cells (CTC) which is commonly used as a cancer marker. Peripheral blood mononuclear cell (PBMC) is a peripheral blood cell that is also thought to express the EPCAM gene and needs to be investigated further because PBMC is more abundant in the body than CTC. The research was conducted to detect the expression of the EPCAM gene in CTC and PBMC of colorectal cancer using semi-quantitative RT-PCR and direct immunofluorescence methods so that EPCAM potential as a biomarker for colorectal cancer diagnosis is known. CTC and PBMC isolated from eight blood samples of colorectal cancer patients were evaluated through RNA isolation and quantification, followed by cDNA amplification, and antibody staining. The results showed that EPCAM gene expression was not detected both in CTC and PBMC using the semi-quantitative RT-PCR method. Furthermore, the direct immunofluorescence method showed that EPCAM gene expression was not detected in CTC. However, a protein that has not been confirmed as EpCAM was detected in PBMC. Based on the results of the study, it can be concluded that the EPCAM gene expression was not detected both in CTC as well as in PBMC by semi-quantitative RT-PCR method. Further research is required to confirm that the protein detected in PBMC is EpCAM.CTC, direct immunofluorescence, ekspresi gen, EPCAM, kanker kolorektal, PBMC, semi-quantitative RT-PCR."