A proposal for validation of antibodies

Uhlén, Mathias; Bandrowski, Anita; Carr, Steven A.; Edwards, A.M.; Ellenberg, Jan; Lundberg, Emma; Rimm, David L.; Rodriguez, Henry; Hiltke, Tara; Snyder, M; Yamamoto, Tadashi

doi:10.1038/nmeth.3995

Cited by 508 publications

(472 citation statements)

References 20 publications

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“…Non-specific antibodies would be a major factor in irreproducibility in biological experiments. Ongoing efforts to standardize ways to quantify and document specificity of binding reagents (70) need to consider these aspects of antibody specificity.…”

Section: Discussionmentioning

confidence: 99%

Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

Wang

Maziuk

et al. 2018

Journal of Biological Chemistry

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Antibodies are essential biochemical reagents for detecting protein post-translational modifications (PTMs) in complex samples. However, recent efforts in developing PTMtargeting antibodies have reported frequent nonspecific binding and limited affinity of such antibodies. To address these challenges, we investigated whether directed evolution could be applied to improve the affinity of a high-specificity antibody targeting phospho-threonine 231 (pT231) of the human microtubule-associated protein tau. On the basis of existing structural information, we hypothesized that improving antibody affinity may come at the cost of loss in specificity. To test this hypothesis, we developed a novel approach using yeast surface display to quantify the specificity of PTM-targeting antibodies. When we affinitymatured the single-chain variable antibody fragment through directed evolution, we found that its affinity can be improved > 20-fold over that of the wild-type antibody, reaching a picomolar range. We also discovered that most of the high-affinity variants exhibit cross-reactivity towards the nonphosphorylated target site, but not to the phosphorylation site with a scrambled sequence. However, systematic quantification of the specificity revealed that such a tradeoff between the affinity and specificity did not apply to all variants and led to the identification of a picomolar-affinity variant that has a matching high specificity of the original phospho-tau antibody. In cell-and tissueimaging experiments, the high-affinity variant gave significantly improved signal intensity while having no detectable nonspecific binding. These results demonstrate that directed evolution is a viable approach for obtaining high-affinity PTMspecific antibodies, and highlight the importance of assessing the specificity in the antibody engineering process.

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Section: Discussionmentioning

confidence: 99%

Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

Wang

Maziuk

et al. 2018

Journal of Biological Chemistry

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“…For instance, Antibody Registry [40] is an antibody reagent portal listing commercial antibodies carrying unique identification code, which we employed in this study. However, in general most common applications listed in antibody databases are western blot assays, IHC and ICC [50]. All the antibodies used in the study except one were listed in the database [40], but only the Ab2 was linked to references in literature of which all considered IHC or ICC applications.…”

Section: Discussionmentioning

confidence: 99%

“…With commercial BSA-his conjugate the dynamic range was narrower (4-38 µg/mL), as well as with HRP(kit)-his (4-89 µg/mL) ( Table 2). Using IC 50 as an index for assay sensitivity, the most sensitive detection was achieved with commercial BSA-his (14 µg/mL), however the sensitivity with HRP(kit)-his (20 µg/mL) and HRP-EDC-his (28 µg/mL) were in similar level. When using glutaraldehyde-conjugates sensitivity was lost: with BSA-GA-his the IC 50 was 70 µg/mL and HRP-GA-his 85 µg/mL, respectively.…”

Section: Binding Inhibition Assay Using Ab1mentioning

confidence: 99%

“…A competitive assay was based on the immobilized anti-histamine antiserum and histamine-HRP conjugate as a tracer, which was prepared by reacting histamine towards HRP oxidized with sodium periodate. The assay was specific for intact histamine with a limit of detection of 230 ng/mL (2.1 µM) and IC 50 -value of 520 ng/mL (4.7 µM), whereas cross-reactivity for histidine and other tested compounds related to histamine was less than 1%. These antibodies were later used in a competitive enzyme-linked immunosorbent assay (ELISA) for determination of histamine in cheese [17].…”

Section: Introductionmentioning

confidence: 97%

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Challenges in Developing a Biochip for Intact Histamine Using Commercial Antibodies

2017

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This study describes the development and the challenges in the development of an on-chip immunoassay for histamine using commercially available antibodies. Histamine can be used as an indicator of food freshness and quality, but it is also a relevant marker in clinical diagnostics. Due to its low molecular weight, simple structure and thus low immunogenicity production of high specificity and affinity antibodies is difficult. From six commercial anti-histamine antibodies tested, only two bound the histamine free in the solution. A fluorescent on-chip immunoassay for histamine was established with a dynamic range of 8-111 µg/mL using polyclonal anti-histamine antibody H7403 from Sigma (Mendota Heights, MN, USA). The anti-histamine antibodies described and used in published literature are thoroughly reviewed and the quality of commercial antibodies and their traceability and quality issues are highlighted and extensively discussed.

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“…Relevant to consider that antigenic targets recognition by the use of antibodies can show some differences according to the method used. Regarding the conformational epitope structure, the performance of antibodies recognizing a given epitope by WB can represent nothing about the same antibody performance in ELISA with the same antigen [49]. IgY antibody shows advantages comparing to the mammal IgG use in immunoassay.…”

Section: Immunoassaymentioning

confidence: 99%

IgY-Technology Applied to Studies of Toxoplasma gondii Infection

Júnior¹,

Santos²,

Bassi³

et al. 2017

Toxoplasmosis

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In this chapter, we describe relevant aspects of immunoglobulin Y (IgY) technology for Toxoplasma gondii applications, including comparison of avian IgY antibody with mammalian IgG antibody, egg yolk IgY production and isolation procedures, important applications for IgY antibody, and state of the art and perspectives for IgY-technology in T. gondii studies. T. gondii is a worldwide public health problem. IgY-technology provides an alternative antibody (IgY) to mammalian Immunoglobulin G (IgG) antibody. IgY-technology involves the chicken immunization, yolk IgY isolation, antibody characterization, and purified IgY application to several kinds of methods. Immunized chicken transfers a specific IgY from blood to egg yolk. Phylogenetic distance between chickens and mammals influences the generation of antibody repertoires recognizing an antigen profile. IgY is not bound to rheumatoid factor or mammalian complement protein and thus avoids the false-positive results. Yolk IgY isolation is carried out by simple procedures that are accessible for any laboratory and, also, for IgY isolation at large-scale production. IgY-technology provides antibodies for proteomic studies, diagnostic assays, and immunotherapy. Although IgY-technology is promising, there is a reduced number of investigations with IgY and T. gondii. Future perspectives involve the use of IgY-technology for the screening of new T. gondii antigens for diagnostics, therapy, or vaccine, development of innovative techniques for toxoplasmosis diagnostics and may be an immunotherapy for toxoplasmosis.

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A proposal for validation of antibodies

Cited by 508 publications

References 20 publications

Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

Challenges in Developing a Biochip for Intact Histamine Using Commercial Antibodies

IgY-Technology Applied to Studies of Toxoplasma gondii Infection

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