A Focused Antibody Library for Improved Hapten Recognition

Persson, Helena; Lantto, Johan; Ohlin, Mats

doi:10.1016/j.jmb.2006.01.004

Cited by 64 publications

(39 citation statements)

References 38 publications

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“…The high-resolution pT231/ pS235_1 co-crystal structure showed that the mechanism of interaction used by the antibody does not fit strictly into any one of the classical protein-, peptide-, or hapten-binding modes. In general terms, anti-protein antibodies tend to use broad flat interfaces with antigen (40), whereas anti-hapten antibodies tend to have more limited interaction through a smaller paratope that is buried deep in the V H -V L interface (39,65). Meanwhile, peptide-binding antibodies are thought to use a "grooved" paratope that is in between the size of the surfaces observed in anti-protein and anti-hapten clones (55).…”

Section: Discussionmentioning

confidence: 99%

“…It may also inform fundamental comparative immunogenetics and antibody structure-function relationships. In the future, these studies could ameliorate the need for animal immunization by providing sufficient data to create designer, fully synthetic, phospho-biased antibody repertoires, as recently described for libraries biased toward the recognition of haptens (39), proteins (40), and peptides (41).…”

mentioning

confidence: 99%

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An Ultra-specific Avian Antibody to Phosphorylated Tau Protein Reveals a Unique Mechanism for Phosphoepitope Recognition

Shih

Cao

et al. 2012

Journal of Biological Chemistry

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Background:Truly phosphospecific antibodies are difficult to generate and are poorly understood. Results: Avian single chain Fv library selections yielded fully phosphospecific anti-phospho-tau antibodies, enabling the generation of a 1.9 Å co-crystal structure. Conclusion: Phosphospecific antibodies were readily generated and can exhibit unique epitope recognition mechanisms. Significance: High-affinity antibody phosphoepitope recognition has been defined, at high resolution, for the first time.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

An Ultra-specific Avian Antibody to Phosphorylated Tau Protein Reveals a Unique Mechanism for Phosphoepitope Recognition

Shih

Cao

et al. 2012

Journal of Biological Chemistry

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“…Rather than using a general library, it may be possible to create specific libraries targeted to recognize post-translational modifications independently of context as has been recently proposed for other target types (77) using this or other generic PTM antibodies as scaffolds. Within this context, the determination of the structure of this antibody in complex with a tyrosine sulfate-containing protein may provide useful information, including the identification of amino acids involved in binding, the mutation of which may allow the selection of antibodies with higher affinities either for the generic modification or specifically modified sites as has been carried out recently with a hapten binding antibody (86).…”

Section: Discussionmentioning

confidence: 99%

Using Phage Display to Select Antibodies Recognizing Post-translational Modifications Independently of Sequence Context

Kehoe

Velappan

Walbolt

et al. 2006

Molecular & Cellular Proteomics

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Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Much protein activity is regulated by post-translational modifications (PTMs), 1 over 200 of which have been described (1), with changes in enzymatic activity, protein interaction partners, subcellular localization, and targeted degradation being some of the most important regulated activities. A number of different approaches have been used to study PTMs, most of which rely on either specific isolation of the protein of interest and identification of the PTMs that affect that particular protein (2) or isolation of all proteins containing a specific PTM of interest and subsequent identification of the isolated proteins. There are two basic methods to isolate or identify all proteins containing a specific PTM: chemical derivatization or specific affinity reagents. The former rely on the specific chemical reactivity of the PTM to chemical modification, usually by the addition of an easily recognized tag, such as the biotinylation of nitrosylated cysteines (3) or phosphorylated serine/threonine residues (4), allowing them to be either purified or identified by virtue of specific mass shifts. The latter, specific affinity reagents that recognize PTMs, comprise a number of different molecule types, including IMAC using iron or gallium to bind proteins containing phosphotyrosines (5-8) or phosphorylated threonines or serines (9, 10); lectins, which recognize glycosylated proteins; and antibodies recognizing tyrosine modifications. These have all been used in proteomics studies (11)(12)(13)(14)(15)(16)(17). Although antibodies are by far the most common specific affinity reagents, there are very few that recognize PTMs independently of the proteins to which they are attached. Nitrosylated tyrosine and phosphorylated tyrosine are two exceptions for which antibodies have been used in proteomics studies (13-17), and antibodies against phosphoserine/threonine have also been identified (18), indicating that the utility of such reagents...

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“…Synthetic diversity bypasses the natural biases and redundancies of antibody repertoires created in vivo and allows control over the genetic makeup of V genes and the introduction of diversity (Hoogenboom & Winter, 1992). A synthetic library has been described that was constructed on the basis of existing information on the structure of the antigenic site of proteins and small molecules (Persson et al, 2006;Sidhu & Fellouse 2006). Semi-synthetic libraries have been constructed by incorporating CDR loops with both natural and synthetic diversity into one or more of the antibody framework regions.…”

Section: Antibodies From Nonimmune Synthetic and Semi-synthetic Libmentioning

confidence: 99%

“…This approach takes advantage of "random" mutations that have been introduced into VH and VL germline genes in vivo. Phage display and yeast display are often used to facilitate the selection of improved binders from these secondary libraries (Lou et al, 2010;Marks, 2004;Persson et al, 2006). This procedure has also been used to increase the affinity of anti-hapten antibodies and the results are reviewed in table 4.…”

Section: Chain Shufflingmentioning

confidence: 99%