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Development of protocols for high-efficiency cloning for production of bispecific antibodies and soluble single-chain variable fragments

Stiller, Joakim LU (2018) KIMM05 20181
Department of Immunotechnology
Abstract
In the field of molecular biology, the cloning of DNA is a vital process. Through cloning, researchers can design new DNA strands which can be used for a number of applications. One of these applications is the creation of synthetic antibodies for cancer treatments. Alligator Bioscience is developing a high-throughput chain to significantly speed up the process. Traditional cloning techniques rely on restriction enzymes and ligation techniques that are time-consuming and lack the efficiency required for a high-throughput process. To solve this, methods have been developed that are both faster and more reliable. In this project, we evaluated the so-called Type IIs cloning strategy of monoclonal and bispecific antibodies, using a new... (More)
In the field of molecular biology, the cloning of DNA is a vital process. Through cloning, researchers can design new DNA strands which can be used for a number of applications. One of these applications is the creation of synthetic antibodies for cancer treatments. Alligator Bioscience is developing a high-throughput chain to significantly speed up the process. Traditional cloning techniques rely on restriction enzymes and ligation techniques that are time-consuming and lack the efficiency required for a high-throughput process. To solve this, methods have been developed that are both faster and more reliable. In this project, we evaluated the so-called Type IIs cloning strategy of monoclonal and bispecific antibodies, using a new pcDNA3.4 TOPO vector, to clone multiple inserts simultaneously. We also evaluated the Seamless Assembly cloning strategy, to produce up to 96 different soluble scFv in a single cloning procedure. The Type IIs cloning strategy was evaluated on DNA coding for five monoclonal- and three bispecific antibodies, and displayed a near 100% cloning efficiency for all antibodies examined. Additionally, the process took less than 2 hours, and was performed in a single tube making it a good candidate for an automated, high-throughput cloning process. The Seamless cloning strategy was limited by the linearization of the vector, but in the end a cloning efficiency of approximately 80% was achieved. These results indicate that the two evaluated methods are fast and efficient replacements for traditional cloning methods, suitable for an automated process. (Less)
Please use this url to cite or link to this publication:
author
Stiller, Joakim LU
supervisor
organization
course
KIMM05 20181
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
8951598
date added to LUP
2018-06-20 13:21:14
date last changed
2018-06-20 13:21:14
@misc{8951598,
  abstract     = {{In the field of molecular biology, the cloning of DNA is a vital process. Through cloning, researchers can design new DNA strands which can be used for a number of applications. One of these applications is the creation of synthetic antibodies for cancer treatments. Alligator Bioscience is developing a high-throughput chain to significantly speed up the process. Traditional cloning techniques rely on restriction enzymes and ligation techniques that are time-consuming and lack the efficiency required for a high-throughput process. To solve this, methods have been developed that are both faster and more reliable. In this project, we evaluated the so-called Type IIs cloning strategy of monoclonal and bispecific antibodies, using a new pcDNA3.4 TOPO vector, to clone multiple inserts simultaneously. We also evaluated the Seamless Assembly cloning strategy, to produce up to 96 different soluble scFv in a single cloning procedure. The Type IIs cloning strategy was evaluated on DNA coding for five monoclonal- and three bispecific antibodies, and displayed a near 100% cloning efficiency for all antibodies examined. Additionally, the process took less than 2 hours, and was performed in a single tube making it a good candidate for an automated, high-throughput cloning process. The Seamless cloning strategy was limited by the linearization of the vector, but in the end a cloning efficiency of approximately 80% was achieved. These results indicate that the two evaluated methods are fast and efficient replacements for traditional cloning methods, suitable for an automated process.}},
  author       = {{Stiller, Joakim}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Development of protocols for high-efficiency cloning for production of bispecific antibodies and soluble single-chain variable fragments}},
  year         = {{2018}},
}