Abstract
Monoclonal antibodies (mAbs) have become indispensable tools in biomedical science. 1 They are widely used reagents in research and across a variety of applications. Historically, however, a lack of rigorous standards for the reporting of mAbs has led to reproducibility issues. 2 The scientific community has recognized the broader problem associated with poor antibody validation. To grapple with the problem, the International Working Group for Antibody Validation (IWGAV) 3 has proposed five conceptual pillars for antibody validation: genetic strategies, orthogonal strategies, independent antibody strategies, tagged-protein expression, and immunocapture followed by mass spectrometry. To improve reagent reproducibility, the use of Research Resource Identifiers (RRIDs) provides a mechanism for uniquely identifying antibodies in publications. For animal studies, the ARRIVE 2.0 guidelines 4 reinforce the importance of transparent reporting of experimental design and methods. Building on these foundations, we propose that articles submitted to Monoclonal Antibodies in Immunodiagnosis and Immunotherapy describing new mAbs adhere to the validation principles outlined by the IWGAV and include a minimum set of information sufficient to establish identity, specificity, reproducibility, and utility of the new mAb reagent.
Antibody Identity and Provenance
A key principle of reporting a new mAb reagent is the creation of a detailed description that allows researchers to unambiguously identify the mAb they are working with. The best practice for antibody identification is the assignment of an RRID. As such, authors are highly encouraged to register their newly reported mAbs in the RRID portal. When describing and reporting new mAbs, authors should provide the antibody name, clone designation, laboratory identifier, and any aliases used during development. The provenance of the antibody should be clearly described in the article. Details must include the host species, immunization protocol, immunogen identity, adjuvant, route of immunization, screening strategy, and method of monoclonal derivation. For hybridoma-derived antibodies, the myeloma fusion partner and cloning method should be reported. For recombinant mAbs, authors should state whether the antibody was generated from an immunized animal, naive library, immune library, synthetic library, or single B cell cloning and mention the type of display platform (phage, yeast, mammalian) employed. At a minimum, readers and reviewers should be able to understand where the antibody came from, how it was selected, and what exactly is being reported.
Antigen and Immunogen Description
An important parameter that dictates the specificity and application of an mAb is the details regarding the antigen used in the generation of the antibody. For this reason, it is important that the target antigen be defined precisely in the article. The common practice of simply reporting only a gene or protein name lacks sufficient detail. At a minimum, authors should clearly describe the origin species of the immunizing and screening antigen and report the accession number (e.g., UniProt accession ID). If the information is available, additional details regarding any posttranslational modification or other relevant protein structure information should be included. Although historically native purified antigens were used for immunization, the advent of facile recombinant DNA technology means that many antibodies are generated using recombinant antigens. When reporting recombinant antigens, authors should report the expression system and purification method. For peptide immunogens, the peptide sequence, position within the target protein, and conjugation carrier should be reported. If practical, the authors should also indicate whether the epitope is expected to be linear or conformational in nature. Finally, for cell-based immunization, authors should describe the cell line and the antigen expression method. Providing a detailed record of the immunogen can be invaluable for interpreting antibody biological behavior. There can frequently be differences in the epitope structure and availability in the antigen’s native biological context where the antibody reacts differently with the antigen expressed on the cell compared to an isolated recombinant protein.
Sequence
A key step in ensuring reproducibility from antibody reagents is the availability of publicly available sequence information. 5 The availability of sequence information would facilitate recombinant antibody production and decrease the variability associated with using polyclonal antibodies or antibodies purified from hybridomas. For this reason, we highly encourage that newly developed mAbs have their sequence information deposited in databases such as the ABCD (AntiBodies Chemically Defined) database 6 whenever possible. The sequencing of antibody hybridomas is relatively straightforward and inexpensive 7 and well within the domain of any lab equipped to carry out basic molecular biology. The annotation of the sequence should include heavy-chain and light-chain variable-region sequences, complementarity-determining region definitions, as well as heavy- and light-chain isotypes. If full sequence disclosure is restricted for intellectual property or commercialization reasons, authors should explicitly state this limitation in the article.
Binding Affinity, Kinetics, and Epitope Characterization
Regardless of the specific application, the key to an mAb’s function is its interaction with the antigen. As such, when reporting a new mAb, the authors should detail a rigorous and complete description of binding to the antigen. Where possible, the use of biolayer interferometry or surface plasmon resonance to determine kinetic parameters (association and dissociation rates), along with equilibrium, and the dissociation constant is preferred. If the antibody is intended for interaction with cell-based systems, the addition of apparent affinity determined using flow cytometry-based binding can be informative. We have previously issued guidance to authors on the experimental determination of antibody affinity. 8 When reporting affinity, authors should clearly distinguish between true equilibrium affinity and apparent avidity. The nature of the antibody epitope is important information, particularly for deciphering the mechanism of action of therapeutic antibodies. There are a wide variety of approaches available for epitope mapping, 9 which can provide differing levels of resolution. Epitope mapping should be included in the article if available, especially when describing new therapeutic applications, although we acknowledge that this may represent a significant technical challenge depending on the nature of the particular antigen.
Specificity and Validation
Specificity is one of the most important attributes of an mAb, and underreporting of specificity can result in overinterpretation of biological findings. Authors should provide evidence that the antibody recognizes the intended target and does not cross-react with unrelated proteins or homologous family members. Ideally, the validation should be matched to the intended use of the antibody. For example, an antibody intended for flow cytometry should be validated in flow cytometry, whereas an antibody intended for immunohistochemistry should be validated in fixed tissue under the stated antigen-retrieval conditions. The five-pillar framework proposed by the IWGAV 3 remains a helpful conceptual model when validating a new antibody. Useful validation strategies include knockout or knockdown controls, overexpression controls, orthogonal comparison with independent methods, use of independent antibodies against the same target, peptide blocking, mass spectrometry confirmation, and testing across positive and negative cell lines or tissues. Providing the research community with a well-reported, rigorous validation scheme ensures research reproducibility for the newly described mAb reagent.
Cross-Reactivity and Species Reactivity
Although the concept of “exquisite specificity” predominates the view of the casual mAb user, the reality is that many mAbs display a degree of cross-reactivity. To better understand antibody cross-reactivity, it is important that authors specifically report the species of the antigen’s origin and homologs tested. For antibodies intended to support translational or preclinical research, cross-reactivity with human, mouse, rat, nonhuman primate, or other relevant species may determine whether the antibody can be used in specific disease models. Cross-reactivity should also be evaluated against related protein family members when relevant. In the context of cross-reactivity, negative data are also valuable. If the antibody does not recognize the mouse ortholog, the denatured antigen, fixed antigen, or a closely related family member, that information should be reported.
Production, Purification, and Quality Attributes
Consistent with basic concepts of scientific reproducibility, the method of antibody production should be described with enough detail to allow interpretation of performance and reproducibility. Authors should report whether the antibody was produced from hybridoma supernatant, ascites, transient transfection, stable cell line, or other expression system. Purification methods should be stated, including Protein A/G/L purification, and any additional or alternative chromatographic steps. The implementation of additional biophysical characterization of purified mAb samples is also encouraged, especially the determination of monodispersity (using size-exclusion chromatography and/or light scattering).
Availability of Reagents and Data
In cases where an mAb is being reported for research purposes, authors should have a clear plan to make the reagent available to the broader scientific community. Where possible, authors should make the antibody, hybridoma, plasmid, or cell line available through a public repository. A number of such repositories are available including the UC Davis NeuroMab Facility, the Development Studies Hybridoma Bank, and Addgene. 10 If a public repository is not appropriate, the authors should indicate in the article whether the antibody is available through a material transfer agreement or is distributed commercially. Reagent availability is central to reproducibility, and we encourage all authors reporting new mAbs to report a clear path to making their new reagents available to the public.
Conclusion
The purpose of rigorous antibody reporting guidelines is to ensure antibody research is reproducible and leads to the advancement of science. As the importance of mAbs continues to expand across research and biomedicine, journals have an important role in setting expectations. The editors of Monoclonal Antibodies in Immunodiagnosis and Immunotherapy believe that clear reporting standards will help authors present stronger studies and allow the research community to use published antibodies with greater confidence. We encourage authors submitting articles that describe new mAbs to include sufficient characterization to allow the antibody to be identified, evaluated, and reproduced. Better reporting will strengthen individual articles and the reliability of the antibody field as a whole.