Epitope mapping by phage display: Random versus gene-fragment libraries

Fack, Fred; Hügle-Dörr, Barbara; Song, Danying; Queitsch, Iris; Petersen, Gabriele; Bautz, Ekkehard K. F.

doi:10.1016/s0022-1759(97)00083-5

Cited by 83 publications

(56 citation statements)

References 29 publications

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“…Fragment libraries, as described above, have the advantage that if the fragments are big enough, and span the appropriate region, conformational epitopes can also be identified. In fact, when general synthetic peptide libraries have been compared with gene-specific libraries, epitopes were more easily and accurately identified using the gene fragment approach for a number of different antibodies (Fack et al 1997;Matthews et al 2002). However, this approach has two problems: First, the vast majority of clones are nonfunctional when making gene-specific display libraries because of the problem of correct frame; and second, new libraries need to be made for each experiment.…”

Section: Discussionmentioning

confidence: 99%

Selecting Open Reading Frames From DNA

Zacchi

Sblattero²,

Florian³

et al. 2003

Genome Res.

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We describe a method to select DNA encoding functional open reading frames (ORFs) from noncoding DNA within the context of a specific vector. Phage display has been used as an example, but any system requiring DNA encoding protein fragments, for example, the yeast two-hybrid system, could be used. By cloning DNA fragments upstream of a fusion gene, consisting of the β-lactamase gene flanked by lox recombination sites, which is, in turn, upstream of gene 3 from fd phage, only those clones containing DNA fragments encoding ORFs confer ampicillin resistance and survive. After selection, the β-lactamase gene can be removed by Cre recombinase, leaving a standard phage display vector with ORFs fused to gene 3. This vector has been tested on a plasmid containing tissue transglutaminase. All surviving clones analyzed by sequencing were found to contain ORFs, of which 83% were localized to known genes, and at least 80% produced immunologically detectable polypeptides. Use of a specific anti-tTG monoclonal antibody allowed the identification of clones containing the correct epitope. This approach could be applicable to the efficient selection of random ORFs representing the coding potential of whole organisms, and their subsequent downstream use in a number of different systems

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Section: Discussionmentioning

confidence: 99%

Selecting Open Reading Frames From DNA

Zacchi

Sblattero²,

Florian³

et al. 2003

Genome Res.

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“…Our observation is in agreement with that of Orlandi et al (57), who defined a linear subregion of a large conformational epitope of an antiErbB-2 mAb using random peptide libraries. In this regard, gene fragment libraries, by displaying on the surface of phage a collection of ErbB-2 peptides of different sizes (up to 100 aa) in their native structural context (58), were shown to be superior to synthetic peptides and random peptide libraries for identifying large and conformational epitope regions. The fine epitope mapping of anti-ErbB-2 Abs will be greatly facilitated once the three-dimensional structure of ErbB-2 is available.…”

Section: Figurementioning

confidence: 99%

Identification of Epitope Regions Recognized by Tumor Inhibitory and Stimulatory Anti-ErbB-2 Monoclonal Antibodies: Implications for Vaccine Design

Yip

Smith

Koch

et al. 2001

The Journal of Immunology

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The self-oncoprotein ErbB-2 is overexpressed in a number of malignancies. The presence of endogenous anti-ErbB-2 Ab and T cell immune responses to this protein in cancer patients has made ErbB-2 an attractive target for active immunization. However, the finding that murine anti-ErbB-2 Abs can have stimulatory, inhibitory, or no effects on cancer cell growth suggests that an inappropriately induced immune response may have an adverse effect. To ensure the induction of a beneficial Ab response, it is important to identify the epitopes recognized by these Abs. In this study we have used phage-displayed ErbB-2 gene fragment libraries and synthetic peptides to epitope-map a panel of anti-ErbB-2 mAbs. The epitopes of three mAbs, N12, N28, and L87, were successfully located to C531-A586, T216-C235, and C220-C235 of ErbB-2, respectively. It was found that while N12 inhibited tumor cell proliferation, N28 stimulated the proliferation of a subset of breast cancer cell lines overexpressing ErbB-2. The peptide region recognized by N12, (C531-A586; EP531), was used as an immunogen to selectively induce an inhibitory immune response in mice. Mice immunized with the GST fusion peptide (GST-EP531) recognized the peptide region EP531 as well as native ErbB-2. More importantly, Igs purified from mouse sera were able to inhibit up to 85% of tumor cell proliferation. In conclusion, our study provides direct evidence of the function-epitope relationship of anti-ErbB-2 Abs and also emphasizes the value of inducing a potent tumor inhibitory polyclonal Ab response by rationally selecting regions of ErbB-2 used for immunization.

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“…A 6mer random library was screened with the monoclonal antibody GDO5 raised against the Hantaan virus glycoprotein G2. After three rounds of panning, the mimotope obtained had the sequence LEYPWH, which was very similar to the template sequence 94YEYPWH99, implying the site where GDO5 bound (Fack, et al, 1997). The Ph.D.-7 random phage library was panned using the anti-SEB monoclonal antibody ab53981 (Urushibata, et al, 2010).…”

Section: Manual Sequence Analysis With Visual Inspectionmentioning

confidence: 87%