Ten Basic Rules of Antibody Validation

Abstract

The quality of research antibodies is an issue for decades. Although several papers have been published to improve the situation, their impact seems to be limited. This publication makes the effort to simplify the description of validation criteria in a way that the occasional antibody user is able to assess the validation level of an immunochemical reagent. A simple, 1-page checklist is supplied for the practical application of these criteria.

Disposable Sensors in Diagnostics, Food, and Environmental Monitoring

“…The basic rules for selecting recognition elements for sensing can be outlined as follows: i) define the target molecule (large entities such as cells and macromolecules or small substances such as drugs and metabolites), ii) outline the requirements of the particular sensing application (like nonphysiological conditions or type of measurement such as single‐point or continuous monitoring), iii) summarize the features of all possible recognition elements (for example, enzymes exhibit low sensitivity compared to antibodies, but allow for long‐term measurements), and finally, iv) select suitable candidates (for instance, enzymes, antibodies or aptamers for antibiotics) and compare their performance to find the best fit. The following issues, however, must be considered in case of affinity (nonenzymatic) sensors: i) binding strength (affinity) of analyte/bioreceptor complex, ii) selectivity (specificity) by determining “cross‐reactivity” with structurally similar compounds, iii) influence of nontarget substances “matrix effect” in complex matrices (such as whole blood or plasma), and iv) stability and storage conditions . For disposable sensors, another obvious criterion is the cost which must be taken into account when choosing bioreceptors.…”

Antibodies: Will Their Star Continue to Rise?

“…“And researchers also need to stay as up-to-date as possible on the best type of antibodies to use, and where possible choose an antibody that has been shown to work in the specific applications and/or species relevant to their own experiments.” Several open platforms are now available on the internet providing researchers the opportunity to both produce and access reviews of antibodies, such as antibodypedia and pAbmAbs. Checklists have also been produced to help users ensure validation of their antibody [8]. …”

Strategy for Development of Site-Specific Ubiquitin Antibodies

“…Validation of antibodies is critical but it has often been overlooked. Fortunately, there are many recent initiatives to improve the guidelines for quality control and thereby the value of the antibodies and the results that are generated with them (Egelhofer et al, 2010;Älgenäs et al, 2014;Bradbury and Plückthun, 2015;Kungulovski et al, 2015;Marcon et al, 2015;Rothbart et al, 2015;Uhlen et al, 2016;Guillemette et al, 2017;Edfors et al, 2018;Venkataraman et al, 2018;Weller, 2018;Marx, 2019). Validation of site-specific Ub-antibodies is very important given the abundance of ubiquitin modifications in the cell and the complexity of the epitope.…”

“…The generation of monoclonal antibodies in mice involves multiple steps: (i) design and synthesis of non-hydrolyzable Ubpeptide conjugates for immunization; (ii) design and synthesis of extended native iso-peptide linked Ub-peptide conjugates for screening; (iii) immunization, and generation and screening of hybridomas; (iv) clone selection and antibody validation in native context (Figure 1). In this manuscript we focus on the steps that are specific for synthesis of ubiquitin-peptide conjugates for the generation of site-specific ubiquitin antibodies (steps i and ii), as detailed excellent general protocols for antibody development (step iii) and validation (step iv) have been described elsewhere (Egelhofer et al, 2010;Yokoyama et al, 2013;Greenfield, 2014;Ossipow and Fischer, 2014;Kungulovski et al, 2015;Marcon et al, 2015;Rothbart et al, 2015;Uhlen et al, 2016;Guillemette et al, 2017;Holzlöhner and Hanack, 2017;Edfors et al, 2018;Venkataraman et al, 2018;Weller, 2018;Marx, 2019). In addition, we discuss the rationale for the design of antigens used for immunization and screening.…”