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Tetua dari semua virus avian influenza adalah itik atau unggas air lainya yang kemudian mengalami mutasi dan adaptasi sehingga menjadi patogen pada ayam atau unggas lainya. Oleh karena itu, penyidikan keberadaan virus influenza pada itik terutama yang dekat dengan peternakan ayam sangat penting. Serum dari 54 ekor itik dan 51 entok yang dipelihara penduduk disekitar peternakan ayam ras petelur komersial di Kabupaten Cianjur dan Sukabumi diambil pada bulan Maret dan April 2014. Indikasi adanya infeksi dilakukan dengan pemeriksaan serologis menggunakan serangkaian alat uji yang meliputi: competitive dan indirect ELISA untuk antibodi nucleoprotein, ELISA MM2e untuk antibodi protein M2e, uji HI, indirect ELISA dan dot blot untuk antibodi haemagglutinin, dan dot blot untuk antibodi neuraminidase. Haemagglutinin rekombinan (H1-H13 dan H15), neuraminidase rekombinan (N1, N2,N7 dan N9) dan rekombinan nucleoprotein virus influenza A digunakan dalam indirect ELISA dan dot blot. Sebanyak 63% dari itik dan 13% dari entok memiliki antibodi terhadap nucleoprotein, dan 62% dari sampel itik yang seropositif nucleoprotein juga memiliki antibodi terhadap M2e. Tingginya seroprevalensi AI pada itik disekitar peternakan ayam ras komersial menunjukkan bahwa penerapan biosekuriti yang ketat pada peternakan ayam komersial masih sangat diperlukan. Berdasarkan hasil pemeriksaan ELISA dan dot blot diduga bahwa pada itik tersebut beredar subtipe H5N2 dan H9N2, selain H5N1. Konfirmasi lebih lanjut dengan isolasi virus perlu dilakukan mengingat subtipe H9N2 dan H5N2 dapat menimbulkan penyakit yang serius pada unggas dan keberadaanya belum pernah diketahui sebelumnya di Indonesia.

Tarigan S, Sumarningsih. 2014. Production and purification of streptavidin with higher biotin-binding activity. JITV 19(3): 231-238. DOI: http://dx.doi.org/10.14334/jitv.v19i3.1086The objective of this study was to develop practical, efficient method for production, purification and assay of binding activity of streptavidin. Streptomyces avidinii was first propagated on agar plates, the bacterial cells on the agar were scrapped and suspended in a defined synthetic media (4.4 ml/cm 2 ). After 7 days agitation on a rotary shaker (200 rpm/min) at room temprature (≈28°C), the bacterial cells in the culture were pelleted. The culture supernatant was concentrated to 1/62 original volume with 75% saturation ammonium sulphate. After intensive dialysis against ammonium carbonate buffer pH 11, the suspension was loaded into an iminobiotin agarose column chromatography. The adsorbed protein (streptavidin) was eluted with sodium acetate buffer, pH 4, and the eluate was concentrated with an ultrafiltration divice and suspended in PBS. The strepatavidin-binding activity was assayed by a competitive ELISA, a competition between streptavidin in the sample and the HRP-streptavidin conjugate for the biotin (biotinyl IgG) immobilised on wells of a microtitre plate. The detection limit of this assay measured 0.16 µg/ml streptavidin. The method developed in this study produced 160 µg/ml streptavidin in the culture supernatant. After concentration with the ammonium sulphate, the streptavidin concentration increased to 4 mg/ml (69% recovery). At the final step of purification, streptavidin with 10 mg/ml concentration was obtained. The purity of the streptavidin was higher (95%) with a recovary of 19%. The purified streptavidin in this study appeared as a dimer core streptavidin on SDS PAGE and its binding activity was twice as high as that of a commercial one. Penelitian ini bertujuan mengembangkan teknik produksi, purifikasi dan pengukuran streptavidin yang efisien dan praktis. Streptomyces avidinii mula-mula dipropagasi dalam lempeng agar kemudian sel bakteri dari agar dipindahkan kedalam media cair sintetik (4,4 ml/cm 2 ). Setelah 7 hari diagitasi diatas rotary shaker 200 rpm/min pada suhu ruangan (≈28°C), sel bakteri dipeletkan, supernatan dikonsentrasikan menjadi 1/62 volume awal dengan amonium sulfat saturasi 75%. Setelah didialisis dalam larutan amonium carbonat pH 11, suspensi protein diadsorbsikan kedalam kolom iminobiotin agarose. Protein (streptavidin) yang teradsorbsi dielusi dengan larutan sodium asetat pH 4, eluat dikonsentrasikan dengan ultrafiltrasi dan disuspensikan dalam PBS. Pengukuran aktivitas binding streptavidin dilakukan dengan ELISA kompetitif, kompetisi antara streptavidin dalam sampel yang diukur dengan konjugat HRP-streptavidin memperebutkan biotin (IgG biotinil) yang diimobilisasi pada microtitre plate. ELISA ini mempunyai limit deteksi 0.16 µg/ml streptavidin. Metode produksi dan purifikasi yang dikembangkan dalam penelitian ini menghasilkan 160 µg/ml pada biakan supernatan. Setelah dikonsentrasik...

The current diagnosis of leptospirosis, micro Agglutination Test (MAT) and isolation, is expensive, impractical and technically demanding. This study was aimed at developing an ELISA based on recombinant LipL32 as a practical, inexpensive test for Leptospirosis. The DNA encoding LipL32 was isolated from Leptospira pomona, inserted into pRSET-C plasmid then expressed in E.coli BL21 as a poly-histidine-tagged protein. The amount of LipL32 protein, which was purified from the supernatant of lysed cells by a Ni-NTA column, was 1mg/l culture. This purified LipL32 was used as the coating antigen at 5µg/ml. The accuracy of ELISA was evaluated based on ROC analysis, by comparing the ELISA and MAT results of 517 bovine sera. Result in this study showed that the area under curve (AUC) was 0.853, which categorised the LipL32 ELISA as a “moderately accurate” test and indicates that the ELISA was able to differentiate positive and negative Leptospirosis serum. The result also showed ELISA LipL32 could detect serum positive MAT to Hardjo, Grippotyphosa, Tarrasovi, Rachmati and Bataviae. The optimal cut off for OD ELISA determined based on ROC curve was 0.504, and it showed sensitivity and specificity of ELISA LipL32 relative to MAT were 86.0% and 69.5%, respectively. Overall, the result in this study showed that ELISA LipL32 can be used as a rapid test for identification of anti-Leptospira antibodies in bovine.