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A combinatory antibody-antigen microarray assay for high-content screening of single-chain fragment variable clones from recombinant libraries

Persson, Nina; Jansson, Bo LU ; Stuhr-Hansen, Nicolai; Kovács, András LU ; Welinder, Charlotte LU ; Danielsson, Lena LU and Blixt, Ola (2016) In PLoS ONE 11(12).
Abstract

We have developed a combinatory antibody-antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a "spot-on-spot" print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse... (More)

We have developed a combinatory antibody-antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a "spot-on-spot" print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcαserine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation.

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published
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in
PLoS ONE
volume
11
issue
12
publisher
Public Library of Science
external identifiers
  • scopus:85007318574
  • wos:000392853100065
ISSN
1932-6203
DOI
10.1371/journal.pone.0168761
language
English
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yes
id
0390c932-550f-4f6a-8504-f19126ad4abd
date added to LUP
2017-01-13 08:57:44
date last changed
2017-11-19 04:36:42
@article{0390c932-550f-4f6a-8504-f19126ad4abd,
  abstract     = {<p>We have developed a combinatory antibody-antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a "spot-on-spot" print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcαserine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation.</p>},
  articleno    = {e0168761},
  author       = {Persson, Nina and Jansson, Bo and Stuhr-Hansen, Nicolai and Kovács, András and Welinder, Charlotte and Danielsson, Lena and Blixt, Ola},
  issn         = {1932-6203},
  language     = {eng},
  month        = {12},
  number       = {12},
  publisher    = {Public Library of Science},
  series       = {PLoS ONE},
  title        = {A combinatory antibody-antigen microarray assay for high-content screening of single-chain fragment variable clones from recombinant libraries},
  url          = {http://dx.doi.org/10.1371/journal.pone.0168761},
  volume       = {11},
  year         = {2016},
}