Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. May/Jun 2018;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

Free PMC article

When Monoclonal Antibodies Are Not Monospecific: Hybridomas Frequently Express Additional Functional Variable Regions

Andrew R M Bradbury et al. MAbs. .
Free PMC article


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.


Figure 1.
Figure 1.
Additional heavy or light chains secreted by a hybridoma create additional binding sites in a combinatorial way. a, hybridomas with one additional chain (example given here: LC) generate three different IgGs with two different antigen binding sites, while b, ten different IgGs with combinations of four different paratope structures are produced if both one additional LC and HC are present. + correct paratope, X1-X3 additional paratopes.
Figure 2.
Figure 2.
Additional immunoglobulin DNA isolated from hybridomas can originate from many different sources. Note that all identified factors can also be present simultaneously.
Figure 3.
Figure 3.
Recombinant antibodies show greater specificity and sensitivity in immunohistochemistry. Hybridomas expressing antibodies with more than one additional functional chain (3A), an additional functional VL (3B), and antibodies with no additional functional chains (3C) were identified by next-generation sequencing. Antibodies purified from either hybridoma supernatant or after recombinant antibody expression in HEK cells, were tested on adjacent sections at identical concentrations. For each antibody, the lowest concentration of purified hybridoma antibody that provided a signal was used, as well as higher concentrations for ß2 microglobulin, EpCAM and MUC1. Recombinant antibodies show stronger and more specific binding compared to the hybridoma antibodies. All micrographs were taken with identical photographic parameters, with the adjustment of white points to identical levels being the only correction made.

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles


    1. Dove A. Agreeable antibodies: Antibody validation challenges and solutions. Science. 2017;357:1165–7. doi:10.1126/science.357.6356.1165. - DOI
    1. Bradbury A, Plückthun A. Reproducibility: Standardize antibodies used in research. Nature. 2015;518:27–9. doi:10.1038/518027a. PMID:25652980. - DOI - PubMed
    1. Bradbury AR, Plückthun A. Getting to reproducible antibodies: The rationale for sequenced recombinant characterized reagents. Protein Eng Des Sel. 2015;28:303–5. doi:10.1093/protein/gzv051. PMID:26446960. - DOI - PubMed
    1. Andersson S, Sundberg M, Pristovsek N, Ibrahim A, Jonsson P, Katona B, Clausson CM, Zieba A, Ramström M, Söderberg O, et al. Insufficient antibody validation challenges oestrogen receptor beta research. Nat Commun. 2017;8:15840. doi:10.1038/ncomms15840. PMID:28643774. - DOI - PMC - PubMed
    1. Weller MG. Quality Issues of Research Antibodies. Anal Chem Insights. 2016;11:21–7. doi:10.4137/ACI.S31614. PMID:27013861. - DOI - PMC - PubMed

MeSH terms


LinkOut - more resources