How to avoid pitfalls in antibody use

Abstract

Antibody use is ubiquitous in the biomedical sciences. However, determining best research practices has not been trivial. Many commercially available antibodies and antibody-conjugates are poorly characterized and lack proper validation. Uncritical application of such useless tools has contributed to the reproducibility crisis in biomedical research. Despite early initiatives such as MIAPAR or PSI-PAR, a best practice guideline for antibody characterization is still not in prospect. Here, we analyze 24 antibody-related databases and compare their content with regard to validation aspects and coverage. We also provide a flowchart for end-users with all necessary steps to facilitate finding and choosing specific and sensitive antibodies for their experiments. Based on a growing demand for better and standardized validation procedures and characterization guidelines for antibody molecules we have summarized our findings in a five-point plan. We intend to keep the discussion alive and hope that properly used antibodies will remain as central to biomedicine as they are today.

Antibody anarchy: A call to order

“…Data supplied by both users and companies can be sparse, and some projects share data only if they confirm that an antibody works as expected. "Sometimes it seems easier to hire a detective than to order a specific antibody, " concludes an overview of antibody portals 5 .…”

Reagent Validation to Facilitate Experimental Reproducibility

“…The use of these approaches, alone and in combination, to validate antibodies is anticipated to establish best practices in antibody validation making this a potentially robust process. More immediate practical guidelines for preclinical researchers have been proposed by Pauly and Hanack (2015) and Acharya et al (2017). Like the ICLAC advice (ICLAC, 2014a), the Acharya Guidelines, which are shown in Table 3 in annotated form, provide a wealth of useful information on the selection and validation of antibodies, and publication of the data resulting from their use that is generically applicable to the cell line and other areas Uhlen et al, 2016)…”

“…Table reproduced under the Creative Commons Attribution License (CC BY) of research, and is of especial use for readers of the current overview, especially those researchers who note that "I wasn't trained that you had to validate antibodies; I was just trained that you ordered them," (Baker, 2015). A possible, albeit controversial (Pauly and Hanack, 2015) resolution in resolving issues with antibody validation involves cloning and sequencing antibodies from B cells and producing them recombinantly (Bradbury and Pluckthun, 2015). The resultant recombinant antibodies (rAbs) can then be characterized and validated, with their sequence serving as a unique identifier rather than the trial and error approach to raising monoclonal and polyclonal antibodies (Acharya et al, 2017; Table 3).…”

Improved decision making for prioritizing tumor targeting antibodies in human xenografts: Utility of fluorescence imaging to verify tumor target expression, antibody binding and optimization of dosage and application schedule

“…Over the past decades, different antigen retrieval methods were developed to reverse the formalin-fixation effect and restore the antigenicity of proteins. 15,16 Furthermore, approaches that minimize discrepancies in IHC, e.g., fixation media 14,17,18 and fixation time using ultrasound, 19,20 and help with the selection of the right antibody 21,22 to examine human 12 and rodent 23 tissue are being investigated.…”