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MAbs. 2018 May/Jun;10(4):539-546. doi: 10.1080/19420862.2018.1445456. Epub 2018 Mar 29.

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions.

Author information

1
a Specifica Inc. , 1512 Pacheco St, Santa Fe , NM , USA.
2
b SwitchGear Genomics , 1914 Palomar Oaks Way, Carlsbad , CA USA.
3
c Miltenyi Biotec GmbH , Friedrich-Ebert-Str. 68, Bergisch Gladbach , Germany.
4
d Absolute Antibody, Wilton Centre , Redcar , Cleveland TS10 4RF , United Kingdom.
5
e NeoBiotechnologies , 2 Union Square, Union City , CA , USA.
6
f International Centre for Genetic Engineering and Biotechnology (ICGEB) , Padriciano 99, Trieste , Italy.
7
g The University of Texas at Austin, Cockrell School of Engineering , McKetta Department of Chemical Engineering , 200 E Dean Keeton St. Stop C0400, Austin , Texas , USA.
8
h Department of Physiology and Membrane Biology , University of California , Davis, One Shields Avenue, Davis , CA , USA.
9
i Institute for Molecular Genetics , University of Heidelberg , Im Neuenheimer Field 260, Heidelberg , Germany.
10
j Stanford University, School of Medicine , Stanford , California , USA.
11
k Department of Urology , Medical Center, University of Freiburg , Breisacher Str. 66, Freiburg , Germany.
12
l Yumab GmbH , Inhoffenstr. 7, Braunschweig , Germany.
13
p Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics , Spielmannstr. 7, Braunschweig , Germany.
14
m A*Star p53 laboratory , 06-06 Immunos, Singapore , Singapore.
15
n IMGT®, the international ImMunoGeneTics information system®, Laboratoire d'ImmunoGénétique Moléculaire LIGM, Institut de Génétique Humaine IGH, UPR CNRS 1142, Montpellier University , Montpellier cedex 5 , France.
16
o Clark Antibodies Ltd , 10 Wellington Street, Cambridge , CB1 1HW , United Kingdom.

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.

KEYWORDS:

hybridoma; monoclonal antibodies; paratope; recombinant antibodies; specificity

PMID:
29485921
PMCID:
PMC5973764
DOI:
10.1080/19420862.2018.1445456
[Indexed for MEDLINE]
Free PMC Article

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