1. Batch-to-batch variability – a side effect of polyclonal antibody development
There are different factors contributing to the antibody reproducibility crisis. The first comes from the nature of antibody production. In human and animal bodies, antibodies are secreted by B cells in a polyclonal mixture of heterogeneous molecules that recognize and bind to different epitopes of an antigen. In the lab, polyclonal antibodies (pAbs) are produced by injecting a particular antigen into an animal that elicits an immune response. The pAbs secreted are then harvested from the animal and used in different antigen-specific experiments.
The reproducibility crisis of polyclonal-based development stems from batch-to-batch variability that commonly occurs during production. The problem arises when different host animals are injected with the same antigen but then produce pAbs that have different specificities and affinities. This means that even if one was to use a new batch of antibodies, they cannot reproduce the exact same experimental results. Even more importantly, the same animal sometimes may have different immune responses to the same antigen and also exhibit time-dependent variation known as the affinity maturation process.
2. Fragile, unstable hybridomas are endangering monoclonal antibodies
Ever since the breakthrough discovery of hybridoma technology by Köhler and Milstein in 1975, it has remained to be the primary method for scientists to produce monoclonal antibodies (mAbs). The process starts by injecting animals with specific antigen that provokes an immune response. Individual B cells producing antibodies that bind to the injected antigen are then isolated from the animal and fused with myeloma cells to produce a hybrid cell line called a hybridoma. By fusing short-lived antibody-producing B cells with immortal myeloma cells, hybridoma cells are expected to provide a never-ending supply of identical mAbs.
However, in the laboratory, hybridomas are often considered unstable and fragile, so they require high-standard procedural controls to maintain cell line viability. Although researchers spend a significant amount of time and effort on culturing hybridoma cell lines, poor growth or even cell death still occurs regularly. Under normal conditions, antibody contents can decrease continuously during the course of hybridoma cultivation (4). Many other factors can lead to failure of hybridoma survival. Long-time storage, repeated freeze-thaw cycles, improper handling, and contamination can all cause hybridoma death and permanent loss of important antibodies.
In addition, hybridoma cell lines are also known to undergo gene mutations and rearrangements over time, leading to antibody heterogeneity and batch-to-batch variability (5). A recent study of 185 clonal hybridoma cell lines identified nearly a third contain additional heavy or light chain genes, resulting in impaired affinity and specificity to the target antigen. An investigation conducted by Rapid Novor in 2019 showed similar results – among 80 research-purpose mAbs analyzed, a significant portion (14%) were shown to have a second light chain present (Figure 1). In other cases, hybridomas may even lose the chromosome containing antibody genes completely arresting the production of antibodies (6).
3. Lack of standardized validation – when antibodies mislead
Despite the complexity and uncertainty of antibody production, there is no standardized protocol or regulation for the validation of antibodies, including assessing their specificity, affinity, sequences, and applications in different assays.
Among over 2 million commercial antibodies provided by more than 300 vendors, the majority of them are polyclonal. The QC data listed on the product sheets are often obtained from previous batches and no longer valid for the ones delivered to users. With regard to mAbs, there is also no way of knowing if the current hybridoma cell line is characterized as stable and functional. This largely stems from the issue of inconsistent catalog numbers that have not accounted for new culturing environments, production animals or company merges. Lack of a proper validation process leads to the release of a large number of poorly characterized antibodies (“bad antibodies”) to users for research. It was estimated that despite there being 2 million antibodies on the market, only 12.5 – 25% are considered unique ‘core’ antibodies (7).
When it comes to selecting key antibodies, the process varies by user and laboratory. For example, some labs regularly purchase from their trusted vendors, some rely on word of mouth, and some buy a few from different vendors and then test which one works. One similarity tends to exist, many researchers will reference publications in which their antibodies were referenced or will refer to the information provided on the product sheets. However, this still does not guarantee a perfectly safe decision. Take for example the following antibodies that were used to identify therapeutically relevant clinical biomarkers. Despite all of these antibodies being regularly cited in industry publications and journals for their respective use cases, all were shown to exhibit cross-reactivity resulting in significant sums of research resources being lost (Table 1) (3).
|M20 and C20
|12 out of 13
|WDCP, POU2F1, multiple
|2 out of 3
Table 1. Some cross-reactive antibodies erroneously used to identify therapeutically relevant clinical biomarkers caused devastating personal and financial damage to science and medical progress. Adapted from reference (3).
Thus, using antibodies as biomarker tools for detection can be potentially fatal if they have not been fully verified with rigorous scrutiny. The unfortunate reality is, it’s no longer sufficient to rely solely on vendor’s quality assurance protocols or scientific publications, it is necessary to independently assess and verify candidates.