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Journal : Indonesian Journal of Biotechnology

Combination PT-PCR with in vitro Transcription-Translation System: Rapid and Simple Approach for Monoclonal Antibody Production Ali, Muhamad
Indonesian Journal of Biotechnology Vol 14, No 2 (2009)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (428.952 KB)

Abstract

We have developed a simple and efficient system for screening and generation of monoclonal antibodies, which can bypass conventional monoclonal antibody production system that includes time-consuming, labor-intensive establishment and cultivation. Our method consist of: (1) cDNA synthesis from single B cells of immunized mouse, followed by (ii) PCR amplification of the Lc and Hc (Fd portion) cDNAs separately using low concentration (0,05 ìM) of the respective cDNA-specific primers with 5’ homotags in the presence of the homotag-specific primer (0,5 ìM), (iii) overlapping PCR of the amplified Lc and Hc cDNAs with the cassettes for the T7 promoter (T7P) and T7 terminator (T7T) to construct the following expression units: T7P-Lc-T7T and T7P-Hc-T7T, and (iv) in vitro expression of these units using an Escherichia coli S30 extract followed by ELISA screening without</div><div>purification. Light-and heavy-chain cDNA were amplified and expressed using in vitro system, thus avoiding time consuming steps of cloning and bacterial expression. Having successfully amplified and expressed Lc and Hc genes from single B cells in rapid, simple, versatile, labor-saving, and cost effective, this method seem to be useful</div><div>and applicable for the high-throughput monoclonal antibody generation. 
Combination PT-PCR with in vitro Transcription-Translation System: Rapid and Simple Approach for Monoclonal Antibody Production Ali, Muhamad
Indonesian Journal of Biotechnology Vol 14, No 2 (2009)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (428.952 KB) | DOI: 10.22146/ijbiotech.7810

Abstract

We have developed a simple and efficient system for screening and generation of monoclonal antibodies, which can bypass conventional monoclonal antibody production system that includes time-consuming, labor-intensive establishment and cultivation. Our method consist of: (1) cDNA synthesis from single B cells of immunized mouse, followed by (ii) PCR amplification of the Lc and Hc (Fd portion) cDNAs separately using low concentration (0,05 &igrave;M) of the respective cDNA-specific primers with 5&rsquo; homotags in the presence of the homotag-specific primer (0,5 &igrave;M), (iii) overlapping PCR of the amplified Lc and Hc cDNAs with the cassettes for the T7 promoter (T7P) and T7 terminator (T7T) to construct the following expression units: T7P-Lc-T7T and T7P-Hc-T7T, and (iv) in vitro expression of these units using an Escherichia coli S30 extract followed by ELISA screening without</div><div>purification. Light-and heavy-chain cDNA were amplified and expressed using in vitro system, thus avoiding time consuming steps of cloning and bacterial expression. Having successfully amplified and expressed Lc and Hc genes from single B cells in rapid, simple, versatile, labor-saving, and cost effective, this method seem to be useful</div><div>and applicable for the high-throughput monoclonal antibody generation. 
Co-Authors . Adwia . Eliya, . . Juliana . Jumini . Mira, . . NURHAYATI . Nurkholifah, . . Setiawati, . Akbar, Jihad Akbar, Mahfud Alorinda Maruli, Alorinda Ana, Nurul Anggraeni , Novia Anwar Rosyidi Aprianti, Rika Aprilia Novitasari Ardiana Ekawanti, Ardiana Aryana F64112029) Ashari, Muhamad Aswandi . Atlanta, Tatiek Busri Endang Dara Puspita Anggraeni, Dara Puspita Desi Rahmawati Desni Yuniarn, Desni Desni Yuniarni Dian Miranda Dian Utami Dewi Dinamella Wahjuningrum Dwi Khairunnisyah, Dwi Dwi Retnowati Dwiyanti, Devi Ekawati (F54209006) Erawati, Yeni Ernie, Marni Juliarty Evy Eugenie, Olivia Esty Fadillah . Fadillah Fadillah Fatimah, Dayang Srilia Flora, Elina Fransiska RZ FUKUBA, Takako Gita Deviana, Gita Gusti Jhoni Putra, Gusti Jhoni Gustiana, Della Halida . Halida Halida Hanifah . Haris, Ali Henny Noviyeni Hilalia himmawan, Didik ica, Stevania Ichsan, Muhamad Iis Suyantini Ima Arum Lestarini, Ima Arum Indri Astuti Irtia, Fitri Khairil Anwar Khurrotul Akyunin Kiki Amelia, Kiki Kiki Aryanita, Kiki Koprita, Yusela Kurniati F54209020 Kusmiati (F54209021) Leni Dahlia Lilis Sumarni Linda Yulianti Lukmanulhakim ., Lukmanulhakim Lukmanulhakim, Lukmanulhakim Lulu Arafani, Lulu Lusina, Lusina M. Syukri M. Syukri . M. Thamrin Made Sriasih, Made Marianty, . Marisa Dwi Sukmawati, Marisa Dwi Marmawi R Marmawi R. Marmawi, Marmawi Mas ud, Aan Masluyah Suib Merry, Adriana Miranti Eka Pratiwi MIRNAWATI SUDARWANTO Morsek, Wita Kristina Muhamad Amin Muhammad Yusuf Muhammad Zubair Mursal Ghazali, Mursal NAKANO, Hideo Narsi (F54010014) Nasution, Raeni Sariyanti Nefi Andriana Fajri, Nefi Andriana Nefi Andriana, Nefi Neni Endang Kusniarti Ningsih, Sri Rahayu Nony Amelia Noviana, Desi Noviani, Dwi Putri Nurbaiti Nurbaiti Nurdiah . Nurlinayati ., Nurlinayati Nurul Hasanah Nurul Yuliasari Oczalina, Bella Olifia, Nonita PURWANTI PURWANTI Puteri, Sylvana RA Maheswari, Rarah Rahayu, Umi Puji Ratnasari, Erma Restiana, . Resty, Polina Retnosari, Desi Rita, Helena Rotin, Rotin Rukmana, Hajar Rupini, Rupini Rustari, Lilis Sahrul Alim Sari, Wiwik Sartika Selvianita, Selvianita Setyono, Bagus Dwi Hari Sinta, Sinta Siti Salmah Slamet Riyadi Sri Lestari Sri Wahyuni Sulaiman N. Depamede, Sulaiman N. Sulasmi, Neni Suriati, Ira Suryadi, Muhammad Aidil Fitriyan Fadjar Susilawati Susilawati Sutarmanto . Ulfa, Arafatul Ulfa, Vizza Novian Veronika Leni, Veronika Wahyuni, Yuni Wariata, I Wayan Wariata, Wayan Wintia, Sisilia Lilik yarso, Elinawati Yuline . Yuniarti, Desni Yunita Sabrina, Yunita Zariah, . Zulfina, Septi