Reproducibility of recombinant antibodies
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Transitioning to recombinant reagents is essential to increase the reproducibility and reliability of results within the life sciences. Here we demonstrate the reproducibility of our recombinant anti-PD-L1 antibody (ab205921) in 4 different applications.
Contents
Introduction: Overcoming issues with antibody reproducibility by using recombinant antibodies
The ‘reproducibility crisis’ has highlighted the fact that many life science experiments cannot be replicated and that the conclusions based on these studies may be unfounded, with antibodies identified as an essential part of the problem1-7. Across biomedical research, global annual losses from using poorly characterized antibodies have been estimated at $800 million, not counting wasted research time and resources as well as the impact of false conclusions4.
Variability between antibody batches often causes issues with antibody reproducibility, with studies failing to achieve the same results with different antibody batches in the same experimental setup1,8,9. Any proper validation of antibodies must therefore include evidence of robustness from batch to batch. However, validation of commercially available antibodies is not standardized and depends on the vendor, and high variation exists in the quality of the documentation accompanying the batches1.
The method of antibody production influences antibody performance in terms of batch consistency (Table 1). Since polyclonal antibodies are derived from multiple B-cells of the serum of an immunized animal, they contain a heterogeneous mixture of antibodies, recognizing different epitopes on the same antigen, and are therefore prone to high batch-to-batch variability. Compared to polyclonals, monoclonal antibodies produced by hybridoma technology exhibit improved consistency between batches; however, potential genetic drift in hybridoma cell lines can lead to the irreproducibility of different antibody batches. Furthermore, 30% of hybridoma-produced monoclonal antibodies were shown to express additional productive heavy or light chains, rendering these antibodies non-specific10.
Without reproducible reagents, project timelines may be missed due to extensive batch-to-batch standardization procedures or the requirement for new reagents to be developed mid-project. To overcome this reproducibility issue, save millions in research and development costs, and help improve the translation of research to the clinic, affinity reagents must be defined by their sequences and produced as recombinant proteins4.
Recombinant antibodies are developed from a unique set of genes, allowing for controlled and reliable antibody production. Due to their production method, recombinant antibodies overcome batch-to-batch consistency issues of traditional polyclonal and monoclonal antibodies, including limitations of hybridoma-production of monoclonals such as gene loss, gene mutations, and cell-line drift. Engineered using leading-edge technologies to deliver high affinity and specificity, recombinant antibodies allow the use of reduced antibody concentrations compared to conventional antibodies.
Polyclonal | Monoclonal | Recombinant | |
Production method | A heterogeneous mix of antibodies is derived from the immune response of multiple | An antibody-producing B-cell from an immunized animal is fused with a myeloma cell to produce an antibody-producing hybridoma line. | The genetic sequence of the selected antibody is cloned into an expression vector, which is subsequently introduced into expression hosts to provide animal-free antibody production. |
Reproducibility characteristics | Consists of multiple antibodies to various antigen epitopes. Following numerous rounds of serum collection, the animal is dispatched, and the antibody source is no longer available. | A single antibody clone, providing improved batch-to-batch consistency compared to polyclonals. Potential cell-line drift and mutations can impair production and result in antibody changes that affect reproducibility. | Stable production that can be re-generated at any time through the known genetic sequence starting material. |
Table 1. Comparison between polyclonal, monoclonal, and recombinant antibodies in terms of production method and reproducibility.
To provide you with highly specific and reproducible antibodies, we have developed a large portfolio of recombinant versions of our rabbit monoclonal antibodies (Fig. 1), with approximately 1000 new antibodies being launched annually to relevant targets across all applications. We are continuously working hard to improve recognition of the importance of recombinant antibodies in research and help you achieve reproducible results with an uninterrupted supply of antibodies.
Our recombinant antibodies are fully validated for the selected applications listed on their datasheets, and we standardize each aliquot to perform according to the optimized protocols. Validation processes are built to ensure we repeatedly deliver reagents that show the same performance time after time, regardless of the scale of your project.
Figure 1. Summary of recombinant antibody features.
Results: Assessing batch-to-batch reproducibility of recombinant Anti-PD-L1 antibody in different applications
Immunohistochemistry
Across five independently manufactured batches of Anti-PD-L1 antibody [28-8], the observed immunohistochemistry (IHC) results on formalin-fixed paraffin-embedded (FFPE) tissue demonstrate excellent consistency with reproducible, specific staining between batches. Recombinant Anti-PD-L1 antibody [28-8] (ab205921) on FFPE human placenta (Figure 2).
Figure 2. Batch-to-batch reproducibility in the localization of PD-L1 in the FFPE human placenta across five batches of Anti-PD-L1 antibody [28-8] (ab205921). IHC performed manually using a Biocare Decloaking Device and Universal HIER antigen retrieval reagent (ab208572). The primary antibody was incubated overnight at 4°C, followed by Goat anti-rabbit secondary antibody and HRP-linked anti-goat polymer antibody (ab209101). DAB was used as a chromogen with a Hematoxylin counterstain.
Immunocytochemistry
Four separate batches of Anti-PD-L1 antibody [28-8] were used to localize PD-L1 expression in CHO-S cells stably transfected with the immune checkpoint target. Immunolabeling with each of the different batches of antibody identified the same predicted membranous staining pattern of equal intensity in CHO-PD-L1 with negative staining in CHO-S.
Figure 3. Reproducible immunocytochemistry labeling across four batches of Anti-PD-L1 antibody [28-8] (ab205921). CHO-S and CHO-S stably transfected with PD-L1 were fixed in 4% PFA, permeabilized with 0.1% Triton X-100 and blocked with goat serum. The primary antibody was incubated overnight at 4°C, followed by Alexa Fluor® 488-conjugated Goat anti-Rabbit secondary antibody (ab150077) at 1/1000 dilution at RT for 30 min. Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] (Ab195889) was used as a counterstain at 1/200 dilution at RT for 30 min. Nuclei were visualized using DAPI.
Flow cytometry
Four batches of Anti-PD-L1 antibody [28-8] were used to label PD-L1 in stably transfected CHO-S cells. Flow cytometric analysis showed consistent signal intensity across replicate antibody batches, with identical, distinct peaks delineating positive staining in viable CHO-PD-L1 cells and negative staining in viable null CHO-S cells.
Figure 4. Batch-to-batch consistency in flow cytometric analysis across four batches of Anti-PD-L1 antibody [28-8] (ab205921). CHO-S (blue line) and CHO stably transfected with PD-L1 cells (red line) were blocked with 10% goat serum for 1h at 4°C. The primary antibody was incubated for 30 min at 4°C, followed by Alexa Fluor® 488-conjugated goat anti-rabbit secondary antibody (ab150077) at 1/2000 dilution at 4°C for 30 min. 7-AAD was incubated for 5 min at 4°C prior to data acquisition via flow cytometer.
Western blot
Five independent antibody batches reveal equivalent, specific bands for the PD-L1 both in CHO-PD-L1 and U-87 MG cells via western blot. No non-specific signal or cross-reactive bands were observed, displaying a high level of batch-to-batch consistency across all assayed batches of recombinant antibody.
Figure 5. Batch-to-batch reproducibility in western blot detection of PD-L1 across five batches of Anti-PD-L1 antibody (ab205921) in CHO-PD-L1 cells. CHO-S and CHO-PD-L1 cells were washed with cold 1 x PBS prior to rotating at 4°C for 45 min in RIPA lysis buffer. Cells were sonicated for 3 secs for 5–15 times with intervals of 10 secs at 40 kW until clear. Lysates were subject to SDS-PAGE at 150V for 1 h before transferring to PVDF. Blotting was performed manually using goat anti-rabbit IgG H&L (HRP) secondary antibody (ab97051) at 1:20,000 dilution for 1 h at RT prior to developing with ECL.
Figure 6. Batch-to-batch reproducibility in western blot detection of PD-L1 across five batches of Anti-PD-L1 antibody (ab205921) in U-87 MG cells. U-87 MG cells were washed with cold 1 x PBS prior to incubating on ice for 15 min in RIPA lysis buffer. Cells were sonicated for 3 secs for 5–15 times with intervals of 10 secs at 40 kW until clear. Lysates were subject to SDS-PAGE at 150V for 1 h before transferring to PVDF. Blotting was performed manually using goat anti-rabbit IgG H&L (HRP) secondary antibody (ab97051) at 1:20,000 dilution for 1 h at RT prior to developing with ECL.
Summary
The demand for sequence-defined recombinant antibodies continues to grow, particularly as part of various solutions to address issues associated with data reproducibility within scientific research3-5. While many researchers have adopted the use of monoclonal antibodies for increased target specificity11, transitioning to the use of recombinant reagents is essential to increase the reproducibility and reliability of results within the life sciences3-6.
Recombinant antibody production removes the variability associated with different antibody production batches and thus allows for the generation of reproducible results for research longevity. This long-term assurance of recombinant reagent performance could aid in the translation of research discoveries to diagnostic or drug development pipelines while protecting against any potential delays in time or cost, arising due to batch-to-batch reproducibility.
References
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