Learn about our comprehensive antibody validation methods to ensure monospecificity.  Antibody Validation>>

Why All Monoclonals Are Not Monospecific

There is a growing awareness about monoclonal antibody off-target binding. This concern becomes particularly alarming as the extent of off-target binding in monoclonal antibodies varies across different lots, impacting antibody functionality and experimental results. The origins of off-target binding are multifaceted, stemming from cross-reactive epitopes known as mimotopes to the co-expression of non-target-binding antibody chains during the hybridoma generation process. Additionally, hybridomas exhibit varying chromosome numbers and fusion partner combinations, and the randomness of combining heavy chains (HC) with light chains (LC) in the secretory pathway results in a diverse array of IgG species, particularly when multiple chains are expressed. Previous studies have reported the existence of non-productive aberrant LCs, but little is known about the overall prevalence and impact these additional chains have on monoclonal antibody (mAb) specificity and functionality. Since additional chains pose challenges in research and diagnostic applications, Bradbury et al. (2018) conducted a comprehensive analysis focusing on the issue of hybridomas expressing more than one HC or LC. 

The authors compiled a substantial multicentric dataset from seven laboratories over the past two decades, encompassing 185 hybridomas, most of which commercially produce monoclonal antibodies. This collection of hybridomas was sequenced using various standard methods, such as RACE, V region PCR, and next-generation sequencing (NGS). The correct VH/VL pairs were identified for all 185 hybridomas. However, 31.9% of hybridomas expressed one or more additional productive V genes, which affected the functionality in 28.6% of the analyzed hybridomas.

The dataset also revealed that a mixture of chains compromised functionality by reducing the number of “correct” binding sites per unit of protein and increasing the risk of off-target reactivities. Enzyme-linked immunosorbent assay (ELISA) comparisons between affinity-purified hybridoma IgG and corresponding recombinant IgG from a subset of hybridomas highlighted the functional consequences of additional productive sequences. Remarkably, purified recombinant antibodies consistently exhibited stronger reactivity per unit protein and higher relative specificity than antibodies purified from hybridoma supernatants. Similar results were obtained in immunohistochemistry (IHC) staining using both hybridoma and recombinant antibodies conducted on human tissues. Intriguingly, improved sensitivity was observed even in cases where only a single productive VH and VL gene pair was identified in the hybridoma.

The superior performance of recombinant antibodies, demonstrated through ELISA and IHC assays, emphasizes the importance of rigorous antibody validation and sequencing in ensuring reliable results in research and diagnostics. At Neobiotechnologies, we exhaustively validate all of our hybridoma clones and recombinant antibodies, utilizing human protein arrays (HuProt) that contain a comprehensive selection of >21,000 full-length human proteins (Jeong et al., 2012), including the specific target of interest [consider including a flow diagram or some figure]. Content includes intracellular, membrane (ion channels, transporters, receptors) and secreted proteins, enzymes, transcription factors, cytokines, and immune receptors and checkpoints and coverage for proteins enriched in major tissues of interest. This comprehensive analysis provides critical information about the antibody’s performance, including specificity, binding affinity, and potential cross-reactivity with non-target proteins, which is particularly important in ensuring the accuracy and reliability of downstream experiments. 

It is important to point out that human protein arrays offer several advantages compared to other antibody validation methods. For example, the high-throughput nature and high sensitivity save time and resources compared to traditional methods that may require testing antibodies individually against single targets, and researchers can simultaneously validate antibodies against multiple proteins, reducing false positives and negatives. Additionally, the arrays yield easy-to-visualize quantitative data related to antibody specificity and binding affinity that are often more reliable and less subject to interpretation than traditional validation methods like Western blots. Protein arrays can help standardize the antibody validation process, making it more consistent and trustworthy. 

At Neobiotechnologies, after producing our hybridomas and validating the recombinant antibodies using HuProt and other methods, they are sequenced. Sometimes, multiple LCs and HCs are present, and/or codon optimization is required, so the sequencing is often repeated. Finally, the clone is validated again using HuProt and for other applications. We offer 1000s of antibodies validated using this technology and workflow, providing researchers with dependable tools that contribute to the success of cutting-edge research and the development of innovative biotechnological applications. While this strategy can be time-consuming and rigorous, we are committed to delivering pure functional antibodies with lot-to-lot consistency to our clients.


We hold Exclusive rights to 10,000 recombinant and hybridoma antibody products, available for Licensing or Collaboration.