Lim , Bee Nar
(2015)
Generation Of A Naïve Human Scfv Antibody Library For The Production Of Human Monoclonal Antibodies By Using Phage Display Technology.
Masters thesis, Universiti Sains Malaysia.
Abstract
Perpustakaan antibodi naïf boleh mengatasi batasan kaedah tradisional hibridoma untuk menghasilkan antibodi monoklonal. Pendulangan pameran faj ialah sejenis teknik yang lebih kos efektif and menjimatkan masa untuk menghasilkan antibodi. Kepelbagaian perpustakaan antibodi naïf yang dibina amat penting bagi memastikan kejayaan dalam memperkaya antibodi terhadap pelbagai jenis sasaran antigen. Strategi baru untuk meningkatkan afiniti pengikatan antibodi diperkenalkan juga. Perpustakaan antibodi naïf scFv dibina daripada 90 orang penderma yang terdiri daripada kumpulan etnik yang berlainan (Melayu, Cina and India) dengan distribusi jantina yang sama. Semua kemungkinan V-gen antibodi diperkayakan dengan PCR konvensional kemudiannya diklonkan dalam vektor fajmid. Dua cara yang berbeza telah digunakan untuk menjana perpustakaan antibodi, iaitu perakitan PCR dan pengklonan dua langkah. Kedua-dua perpustakaan antibodi yang dibina mempunyai saiz anggaran 2x109. Walau bagaimanapun, cara perakitan PCR menunjukkan kadar kemasukan sebanyak 67 % sahaja manakala pengklonan dua langkah mencapai 80 %. Perpustakaan antibodi disaring untuk mendapatkan antibodi monoklonal terhadap tiga jenis antigen, iaitu ubiquitin, hemolysin E dan HIV matriks protein p17 (MAp17).
Naive antibody libraries are able to overcome the limitation of traditional hybridoma method in producing monoclonal antibodies. Phage display biopanning is a more cost effective and less time consuming technology for antibody production. Construction of a naive antibody library with a large diversity is crucial to ensure successful enrichment of antibodies against various target antigens. A naïve scFv antibody library was constructed from 90 donors of different ethnic groups (Malay, Chinese and Indian) with an equal gender distribution. All possible antibody V-genes were amplified by conventional PCR method and cloned into a phagemid vector. Two different approaches were used to generate the antibody library, namely PCR assembly and two step cloning. Both antibody libraries constructed had an estimated size of 2x109. However, the PCR assembly method showed an insert rate of 67 % only while two step cloning reached 80 %. The antibody library was used to screen for monoclonal antibodies against three different types of antigens, ubiquitin, hemolysin E and HIV matrix protein p17 (MAp17).
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