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Polymemse chain reaction using mixed primers. Cloning of human monoclonal antibody variable region genes from single hybridoma cells

Larrick, James W; Danielsson, Lena; Brenner, Carol A; Wallace, Ellen F; Abrahamson, Magnus LU ; Fry, Kirk E and Borrebaeck, Carl LU (1989) In Nature Biotechnology 7(9). p.934-938
Abstract
We describe a general approach to rapidly obtain the DNA sequence encoding the variable region of any immunoglobulin chain using the polymerase chain reaction and a mixture of upstream primers corresponding to the leader sequence, and one downstream primer designed from the conserved nucleotide sequence of the constant region. The approach was applied to five different hybridomas producing human monoclonal antibodies and variable regions for both bold gamma and mu heavy chain and kappa and lambda light chain genes were successfully cloned. cDNA encoding variable regions could be amplified from single hybridoma cells isolated by micromanipulation. This approach will permit analysis of B cell clonal ontogeny, antibody diversity and lymphoma... (More)
We describe a general approach to rapidly obtain the DNA sequence encoding the variable region of any immunoglobulin chain using the polymerase chain reaction and a mixture of upstream primers corresponding to the leader sequence, and one downstream primer designed from the conserved nucleotide sequence of the constant region. The approach was applied to five different hybridomas producing human monoclonal antibodies and variable regions for both bold gamma and mu heavy chain and kappa and lambda light chain genes were successfully cloned. cDNA encoding variable regions could be amplified from single hybridoma cells isolated by micromanipulation. This approach will permit analysis of B cell clonal ontogeny, antibody diversity and lymphoma cell progression and heterogeneity. It will also facilitate structural and functional studies of immunoglobulins as well as the rapid construction of chimeric antibodies. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Nature Biotechnology
volume
7
issue
9
pages
934 - 938
publisher
Nature Publishing Group
external identifiers
  • scopus:0024386188
ISSN
1546-1696
DOI
10.1038/nbt0989-934
language
English
LU publication?
yes
id
d967b92c-0b29-43bd-a682-71dc908898d9 (old id 1104909)
date added to LUP
2008-08-07 08:42:59
date last changed
2017-08-27 05:16:39
@article{d967b92c-0b29-43bd-a682-71dc908898d9,
  abstract     = {We describe a general approach to rapidly obtain the DNA sequence encoding the variable region of any immunoglobulin chain using the polymerase chain reaction and a mixture of upstream primers corresponding to the leader sequence, and one downstream primer designed from the conserved nucleotide sequence of the constant region. The approach was applied to five different hybridomas producing human monoclonal antibodies and variable regions for both bold gamma and mu heavy chain and kappa and lambda light chain genes were successfully cloned. cDNA encoding variable regions could be amplified from single hybridoma cells isolated by micromanipulation. This approach will permit analysis of B cell clonal ontogeny, antibody diversity and lymphoma cell progression and heterogeneity. It will also facilitate structural and functional studies of immunoglobulins as well as the rapid construction of chimeric antibodies.},
  author       = {Larrick, James W and Danielsson, Lena and Brenner, Carol A and Wallace, Ellen F and Abrahamson, Magnus and Fry, Kirk E and Borrebaeck, Carl},
  issn         = {1546-1696},
  language     = {eng},
  number       = {9},
  pages        = {934--938},
  publisher    = {Nature Publishing Group},
  series       = {Nature Biotechnology},
  title        = {Polymemse chain reaction using mixed primers. Cloning of human monoclonal antibody variable region genes from single hybridoma cells},
  url          = {http://dx.doi.org/10.1038/nbt0989-934},
  volume       = {7},
  year         = {1989},
}