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      How to avoid pitfalls in antibody use

      discussion
      a , 1 , b , 2
      F1000Research
      F1000Research
      antibodies, validation, characterization, target, application, databases, unique identifier

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          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.

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          Most cited references12

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          An assessment of histone-modification antibody quality

          We report testing of the specificity and utility of over 200 antibodies raised against 57 different histone modifications, in Drosophila melanogaster, Caenorhabditis elegans and human cells. While most antibodies performed well, over 25% failed specificity tests by dot blot or western blot. Among specific antibodies, over 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use and provide a website for posting new test results.
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            Simultaneous quantification of five bacterial and plant toxins from complex matrices using a multiplexed fluorescent magnetic suspension assay.

            Proteotoxins such as ricin, abrin, botulinum neurotoxins type A and B (BoNT/A, BoNT/B) and staphylococcal enterotoxin B (SEB) are regarded as potential biological warfare agents which could be used for bioterrorism attacks on the food chain. In this study we used a novel immunisation strategy to generate high-affinity monoclonal and polyclonal antibodies against native ricin, BoNT/A, and BoNT/B. The antibodies were used along with antibodies against SEB and abrin to establish a highly sensitive magnetic and fluorescent multiplex bead array with excellent sensitivities between 2 ng/L and 546 ng/L from a minimal sample volume of 50 microL. The assay was validated using 20 different related analytes and the assay precision was determined. Advancing the existing bead array technology, the novel magnetic and fluorescent microbeads proved amenable to enrichment procedures, by further increasing sensitivity to 0.3-85 ng/L, starting from a sample volume of 500 microL. Furthermore, the method was successfully applied for the simultaneous identification of the target toxins spiked into complex food matrices like milk, baby food and yoghurt. On the basis of our results, the assay appears to be a good tool for large-scale screening of samples from the food supply chain.
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              IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

              Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20°C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).
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                Author and article information

                Journal
                F1000Res
                F1000Res
                F1000Research
                F1000Research
                F1000Research (London, UK )
                2046-1402
                7 September 2015
                2015
                : 4
                : 691
                Affiliations
                [1 ]Department of Ophthalmology, University Hospital Regensburg, Regensburg, 93053, Germany
                [2 ]Department of Immunotechnology, University of Potsdam, Potsdam, 14476, Germany
                [1 ]University of California, San Diego, CA, USA
                [1 ]Department of Biomedicine, Aarhus University, Aarhus, Denmark
                Author notes

                DP conducted survey; DP, KH analyzed and interpreted data; DP, KH draft manuscript. All authors have seen and agreed to the final content of the manuscript.

                Competing interests: No competing interests were disclosed for D. Pauly. K. Hanack is co-founder of new/era/mabs GmbH (Potsdam, Germany), who are generating antibodies. This activity does not interfere with her scientific interests and use of antibodies.

                Competing interests: I run the antibodyregistry.org and the Resource Identification Initiative, which are mentioned in the paper.

                Competing interests: No competing interests were disclosed.

                Article
                10.12688/f1000research.6894.1
                4722690
                26069726
                63fa732f-3ba3-4b79-a5ae-6b40021c0c0c
                Copyright: © 2015 Pauly D and Hanack K

                This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 5 August 2015
                Funding
                The author(s) declared that no grants were involved in supporting this work.
                Categories
                Opinion Article
                Articles
                Antigen Processing & Recognition
                Applied Microbiology
                Immunological Biomarkers

                antibodies,validation,characterization,target,application,databases,unique identifier

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