Abstract
Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 539-546 |
| Number of pages | 8 |
| Journal | mAbs |
| Volume | 10 |
| Issue number | 4 |
| DOIs | |
| State | Published - May 19 2018 |
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Keywords
- hybridoma
- monoclonal antibodies
- paratope
- recombinant antibodies
- specificity
ASJC Scopus subject areas
- Immunology and Allergy
- Immunology
Cite this
When monoclonal antibodies are not monospecific : Hybridomas frequently express additional functional variable regions. / Bradbury, Andrew R.M.; Trinklein, Nathan D.; Thie, Holger; Wilkinson, Ian C.; Tandon, Atul K.; Anderson, Stephen; Bladen, Catherine L.; Jones, Brittany; Aldred, Shelley Force; Bestagno, Marco; Burrone, Oscar; Maynard, Jennifer; Ferrara, Fortunato; Trimmer, James; Görnemann, Janina; Glanville, Jacob; Wolf, Philipp; Frenzel, Andre; Wong, Julin; Koh, Xin Yu; Eng, Hui Yan; Lane, David; Lefranc, Marie Paule; Clark, Mike; Dübel, Stefan.
In: mAbs, Vol. 10, No. 4, 19.05.2018, p. 539-546.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - When monoclonal antibodies are not monospecific
T2 - Hybridomas frequently express additional functional variable regions
AU - Bradbury, Andrew R.M.
AU - Trinklein, Nathan D.
AU - Thie, Holger
AU - Wilkinson, Ian C.
AU - Tandon, Atul K.
AU - Anderson, Stephen
AU - Bladen, Catherine L.
AU - Jones, Brittany
AU - Aldred, Shelley Force
AU - Bestagno, Marco
AU - Burrone, Oscar
AU - Maynard, Jennifer
AU - Ferrara, Fortunato
AU - Trimmer, James
AU - Görnemann, Janina
AU - Glanville, Jacob
AU - Wolf, Philipp
AU - Frenzel, Andre
AU - Wong, Julin
AU - Koh, Xin Yu
AU - Eng, Hui Yan
AU - Lane, David
AU - Lefranc, Marie Paule
AU - Clark, Mike
AU - Dübel, Stefan
PY - 2018/5/19
Y1 - 2018/5/19
N2 - Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.
AB - Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.
KW - hybridoma
KW - monoclonal antibodies
KW - paratope
KW - recombinant antibodies
KW - specificity
UR - http://www.scopus.com/inward/record.url?scp=85044542376&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85044542376&partnerID=8YFLogxK
U2 - 10.1080/19420862.2018.1445456
DO - 10.1080/19420862.2018.1445456
M3 - Article
C2 - 29485921
AN - SCOPUS:85044542376
VL - 10
SP - 539
EP - 546
JO - mAbs
JF - mAbs
SN - 1942-0862
IS - 4
ER -