A new hybrid resin for stepwise screening of peptide libraries combined with single bead Edman sequencing

Hiemstra, Hoebert S.; Benckhuijsen, Willemien E.; Amons, Reinout; Rapp, W.; Drijfhout, Jan W.

doi:10.1002/(sici)1099-1387(199806)4:4<282::aid-psc145>3.0.co;2-g

Cited by 24 publications

(18 citation statements)

References 14 publications

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“…16 The library was screened using the three-step converging screening system as described. 15 In the initial screening round, two peptide pools induced proliferation (SI of 10 and 20, respectively). The peptide-bearing beads from the pool with the highest induction of proliferation were used in the second round of screening.…”

Section: Resultsmentioning

confidence: 99%

“…Design, synthesis and use of a dedicated synthetic peptide library Hybrid TentaGelH-AM resin (particle size, 90 mm; loading, 100 pmol/bead, 16 pmol acid stabile attached, 84 pmol acid labile attached) 15 was used to synthesize a random onebeadFone-peptide 14-mer peptide library containing a DPw4 binding motif. 16 The hybrid resin allows for a convergent library screening using the acid cleavable part of the peptide material attached to the resin, combined with efficient peptide identification by Edman sequencing or MS-MS mass spectrometryy, using the noncleavable part of the peptide material attached to the resin.…”

Section: Cellsmentioning

confidence: 99%

“…15 Briefly, the library was divided into 384 pools of 20 000 beads. Part (about 25%) of the peptide material was released from the beads for testing in a T-cell proliferation assay (first round of screening).…”

Section: Cellsmentioning

confidence: 99%

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Synaptojanin 2 is recognized by HLA class II-restricted hairy cell leukemia-specific T cells

et al. 2003

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Hairy cell leukemia (HCL) is a chronic mature B-cell leukemia characterized by malignant B cells that have typical hairy protrusions. To characterize possible HCL-associated tumor antigens, we generated an HCL-specific and HLA class II (DPw4)-restricted proliferative CD4 þ T-cell clone. To identify the target antigen of these T cells, we constructed a synthetic peptide library dedicated to bind HLA DPw4, and identified a mimicry epitope recognized by the T-cell clone. With this epitope, the recognition motif of the T-cell clone was deduced and a peptide of human synaptojanin 2 (Syn 2) was identified that stimulated the HCL-reactive T-cell clone. Both Northern and Western blot analyses showed that Syn 2 expression was increased in HCL samples compared to other B cells. Besides, the Syn 2-expressing cell line AML193, with the introduced restrictive HLA-DPw4 molecules, was recognized by the HCLspecific T-cell clone. These results indicate that Syn 2 is a target of autoreactive HCL-specific T cells. Since Syn 2 is a phosphatidylinositol 4,5-biphosphatase involved in cell growth and rearrangement of actin filaments, the increased Syn 2 expression may correlate with the disease etiology or the characteristic morphologic alterations caused by the disease.

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Section: Resultsmentioning

confidence: 99%

Section: Cellsmentioning

confidence: 99%

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Synaptojanin 2 is recognized by HLA class II-restricted hairy cell leukemia-specific T cells

et al. 2003

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“…The beads are microporous and consist of a polystyrene core onto which polyethyleneglycole (PEG) is grafted to a final content of 50 to 70% (w/w). 26 The narrow pore size of TentaGel resins typically prevents fluorescently labeled target proteins, usually larger than 15 kDa, from penetrating into the bead interior within the typical incubation and screening time. This confines the majority of the relevant signal to the bead periphery, i.e.…”

Section: Instrumental Requirementsmentioning

confidence: 99%

Confocal Nanoscanning, Bead Picking (CONA): PickoScreen Microscopes for Automated and Quantitative Screening of One-Bead One-Compound Libraries

Hintersteiner¹,

Buehler²,

Uhl

et al. 2009

J. Comb. Chem.

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Solid phase combinatorial chemistry provides fast and cost-effective access to large bead based libraries with compound numbers easily exceeding tens of thousands of compounds. Incubating one-bead one-compound library beads with fluorescently labeled target proteins and identifying and isolating the beads which contain a bound target protein, potentially represents one of the most powerful generic primary high throughput screening formats. On-bead screening (OBS) based on this detection principle can be carried out with limited automation. Often hit bead detection, i.e. recognizing beads with a fluorescently labeled protein bound to the compound on the bead, relies on eye-inspection under a wide-field microscope. Using low resolution detection techniques, the identification of hit beads and their ranking is limited by a low fluorescence signal intensity and varying levels of the library beads' autofluorescence. To exploit the full potential of an OBS process, reliable methods for both automated quantitative detection of hit beads and their subsequent isolation are needed. In a joint collaborative effort with Evotec Technologies (now Perkin-Elmer Cellular Technologies Germany GmbH), we have built two confocal bead scanner and picker platforms PS02 and a high-speed variant PS04 dedicated to automated high resolution OBS. The PS0X instruments combine fully automated confocal large area scanning of a bead monolayer at the bottom of standard MTP plates with semiautomated isolation of individual hit beads via hydraulic-driven picker capillaries. The quantification of fluorescence intensities with high spatial resolution in the equatorial plane of each bead allows for a reliable discrimination between entirely bright autofluorescent beads and real hit beads which exhibit an increased fluorescence signal at the outer few micrometers of the bead. The achieved screening speed of up to 200,000 bead assayed in less than 7 h and the picking time of approximately 1 bead/min allow exploitation of one-bead one-compound libraries with high sensitivity, accuracy, and speed.

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“…One such resin is TentaGel, a graft copolymer of a crosslinked polystyrene matrix and polyethylene glycol [21]. TentaGel beads have been used extensively in biological assays with combinatorial peptide libraries and show very low non-specific protein adsorption [9, 11]. In this study, we demonstrate a process that combines phage display discovery of mimetope peptides with solid phase immunoassay methods for the quantification of mAb levels in biologic samples.…”

Section: Introductionmentioning

confidence: 99%

A general process for the development of peptide-based immunoassays for monoclonal antibodies

Sánchez

Nguyen

Dema-Ala

et al. 2010

Cancer Chemother Pharmacol

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PurposeMonoclonal antibodies (mAb) are an important and growing class of cancer therapeutics, but pharmacokinetic analyses have in many cases been constrained by the lack of standard and robust pharmacologic assays. The goal of this project was to develop a general method for the production of immunoassays that can measure the levels of therapeutic monoclonal antibodies in biologic samples at relevant concentrations.MethodsAlemtuzumab and rituximab are monoclonal approved for the treatment of B-cell malignancies and were used as a model system. Phage-displayed peptide libraries were screened for peptide sequences recognized by alemtuzumab (anti-CD52) or rituximab (anti-CD20). Synthetic biotinylated peptides were used in enzyme-linked immunosorbent assays (ELISA). Peptides directly synthesized on polymer resin beads were used in an immunofluorescent-based assay.ResultsPeptide mimetope sequences were recovered for both mAb and confirmed by competitive staining and kinetic measurements. A peptide-based ELISA method was developed for each. The assay for rituximab had a limit of detection of 4 μg/ml, and the assay for alemtuzumab had a limit of detection of 1 μg/ml. Antibody-specific staining of peptide conjugated beads could be seen in a dose-dependent manner.ConclusionPhage-displayed peptide libraries can be a source of highly specific mimetopes for therapeutic mAb. The biotinylated forms of those peptides are compatible with conventional ELISA methods with sensitivities comparable to other assay methods and sufficient for pharmacological studies of those mAb given at high dose. The process outlined here can be applied to any mAb to enable improved pharmacokinetic analysis during the development and clinical use of this class of therapies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00280-009-1240-1) contains supplementary material, which is available to authorized users.

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A new hybrid resin for stepwise screening of peptide libraries combined with single bead Edman sequencing

Cited by 24 publications

References 14 publications

Synaptojanin 2 is recognized by HLA class II-restricted hairy cell leukemia-specific T cells

Synaptojanin 2 is recognized by HLA class II-restricted hairy cell leukemia-specific T cells

Confocal Nanoscanning, Bead Picking (CONA): PickoScreen Microscopes for Automated and Quantitative Screening of One-Bead One-Compound Libraries

A general process for the development of peptide-based immunoassays for monoclonal antibodies

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