Generation of Human scFv Antibody Libraries: PCR Amplification and Assembly of Light- and Heavy-Chain Coding Sequences

Andris‐Widhopf, Jennifer; Schmitz, Peter; Fuller, Roberta P.; Rader, Christoph; Barbas, Carlos F.

doi:10.1101/pdb.prot065573

Cited by 46 publications

(54 citation statements)

References 11 publications

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“…Degenerative primers that enable heavy-and light-chain variable domain amplification from the species of origin are the key reagents in this common process. Such primers, as well as detailed protocols, are described in the literature for mouse and rat (Breitling and Dübel 1998;Toleikis et al 2004;Siegel 2009;Strebe et al 2010), rabbit (Rader 2009), llama (Harmsen et al 2000), and human (Marks and Bradbury 2004;Ozawa et al 2006;Tiller et al 2008;Andris-Widhopf et al 2011) variable regions. Online tools (http://www.abysis.org/; http://www.imgt.org; http://www.vbase2.org) exist to facilitate alignment with existing immunoglobulin databases and to delineate the CDRs within the variable regions.…”

Section: Complementarity Determining Regions: the Unique Characteristmentioning

confidence: 99%

Characterizing Antibodies

Weis-Garcia¹,

Carnahan²

2017

Cold Spring Harb Protoc

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Perhaps because they are such commonly used tools, many researchers view antibodies one-dimensionally: Antibody Y binds antigen X. Although few techniques require a comprehensive understanding of any particular antibody’s characteristics, well-executed experiments do require a basic appreciation of what is known and, equally as important, what is not known about the antibody being used. Ignorance of the relevant antibody characteristics critical for a particular assay can easily lead to loss of precious resources (time, money, and limiting amounts of sample) and, in worst-case scenarios, erroneous conclusions. Here, we describe various antibody characteristics to provide a more well-rounded perspective of these critical reagents. With this information, it will be easier to make informed decisions on how best to choose and use the available antibodies, as well as knowing when it is essential and how to determine a particular as yet-undefined characteristic.

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Section: Complementarity Determining Regions: the Unique Characteristmentioning

confidence: 99%

Characterizing Antibodies

Weis-Garcia¹,

Carnahan²

2017

Cold Spring Harb Protoc

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“…cDNA was synthesized from 20ug total RNA using the Superscript III first-strand synthesis system (Invitrogen) with a combination of random hexamers and oligodT primers to ensure broad representation of antibody classes. Each V family of variable regions (VH or VL) was amplified by independent PCR, with a total of 45 different reactions according to previously published methods [2]. A PCR-overlapping process was performed to join both V domains.…”

Section: Body Of Workmentioning

confidence: 99%

Marrow-Derived Antibody Library for Treatment of Neuroblastoma

Sholler¹

2013

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)Van Andel Research Institute Grand Rapids, MI 49503 PERFORMING ORGANIZATION REPORT NUMBER SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR'S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 SPONSOR/MONITOR'S REPORT NUMBER(S) DISTRIBUTION / AVAILABILITY STATEMENTApproved for Public Release; Distribution Unlimited SUPPLEMENTARY NOTES ABSTRACTNeuroblastoma (NB) is the most common solid tumor in children, which accounts for 15% of all pediatric cancer deaths in the US. New therapeutic antibodies to treat NB are urgently needed to improve survival. The purpose of this project is to produce patient-specific therapeutic antibodies to treat neuroblastoma. The goal is to develop new methods and strategies to capture the auto-immune response reaction in neuroblastoma patients using phage display and B cell hybridoma technologies.

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“…The first primer sets and conditions (described by AndrisWidhopf et al 20 ) were used to amplify fragments from FR1 to FR 4. An unequal mixture of 19 primers served as the V H sense primer and an equal mixture of four primers as the antisense one.…”

Section: Cell Linesmentioning

confidence: 99%

Identification of two aberrant transcripts derived from a hybridoma with amplification of functional immunoglobulin variable genes

et al. 2010

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Murine monoclonal antibodies (mAbs) are widely used but have limitations if administered in humans. The use of chimeric or humanized mAbs can reduce immunogenicity. The first step in producing such mAbs is to clone murine variable genes from a hybridoma, but it is possible to amplify both functional and aberrant variable genes, as they coexist in the hybridoma. During the development of a murine-human chimeric antibody, we have cloned from a hybridoma the functional heavy chain variable region (V H ) and light chain variable region (V L ) genes of a mAb that blocks the binding of anthrax lethal factor to protective antigen. In this study, we report the detection of two aberrant transcripts from a hybridoma produced using myeloma cell line OUR-1, the development of a method to distinguish between the functional and abundant aberrant V L transcripts, and the origins of these aberrant genes. The aberrant V L gene is derived from OUR-1 cells, while the aberrant V H gene might derive from antibody repertoires in B cells or from gene rearrangement in the hybridoma cells. The aberrant V H and V L genes in this study may facilitate discrimination between the functional and aberrant variable genes from hybridoma cells.

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Generation of Human scFv Antibody Libraries: PCR Amplification and Assembly of Light- and Heavy-Chain Coding Sequences

Cited by 46 publications

References 11 publications

Characterizing Antibodies

Characterizing Antibodies

Marrow-Derived Antibody Library for Treatment of Neuroblastoma

Identification of two aberrant transcripts derived from a hybridoma with amplification of functional immunoglobulin variable genes

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