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Mapping of a discontinuous and highly conformational binding site on follicle stimulating hormone subunit-β (FSH-β) using domain Scan™ and Matrix Scan™ technology

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Abstract

This paper describes the application of two novel screening technologies, i.e. Domain Scan™ (24- and 30-mer peptides) and Matrix Scan™ (24-mer peptides)technology, in the mapping of a discontinuous epitope on FSH-β for a series of 20 monoclonal antibodies. 11 out of 20 mAb's, mapping of which was not successful by conventional Pepscan™ technology (12-merpeptides), showed selective binding to peptide-constructs corresponding to the β3-loop of FSH in the Domain™ and/or Matrix Scan™. Systematic replacement analysis studies with peptide-construct 57VYETVRVPGCAC-SAc-ADSLYTYPVATQ81 revealed that for most mAb's the amino acids R62, A70,D71, and L73 form the core of the epitope. A DomainScan™ performed in the C-O format showed highly selective binding for mAb's 1 and 2 with only three β1-β3 peptide-constructs covering the residues 60TVRVPGCAHHADSLY74 in combination with 10IAIEKEECRFAI21, while for mAb 10 binding was observed with peptide-constructs containing the C-terminal residues97RGLGPSYCSFGEMKE114 in combination with the residues 10IAIEKEECRFAI21. A Matrix Scan™ of mAb 17 showed that peptides from four different regions on FSH (1st strand β3-loop, α1-loop, longα2-loop, det. loop) showed enhanced binding in combination with several 70ADSL73-containing peptides. BIACORE measurements with mAb's 1, 2, 13, and 17 using a set of 21 different peptide(-construct)s partially confirmed the Domain and MatrixScan™ screening results. Only 24- and 33-mer peptides covering both the 1st and 2nd strand of the β3-loop showed measurable binding. Cyclic β3-loop peptide mimics were found to bind significantly stronger (Kd∼ 5 μM) than the lineair analogues, in agreement with the fact that the discontinuous epitope is part of a loop structure. Coupling of the lineair β1-peptide 10IAIEKEECRFAI21to the linear β3-peptide*52TFKELVYETVRVPGCAHHADSLYTYPVATQAH83# via disulfide bond formation showed a 2–3 fold increase in Kd, thus conforming participation of the β 1-loop in antibody binding for these mAb's.

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Authors and Affiliations

  1. Pepscan Systems B.V., Lelystad, The Netherlands

    Peter Timmerman, Evert Van Dijk, Wouter Puijk, Wim Schaaper, Jerry Slootstra & Rob H. Meloen

  2. Unipath Limited, Priory Business Park, Bedford, United Kingdom

    Stephen J. Carlisle, John Coley, Steve Eida, M. Gani, Tim Hunt, Paul Perry, Gerry Piron & Rob H. Meloen

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  1. Peter Timmerman
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  2. Evert Van Dijk
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  3. Wouter Puijk
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  4. Wim Schaaper
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  5. Jerry Slootstra
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  6. Stephen J. Carlisle
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  7. John Coley
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  8. Steve Eida
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  9. M. Gani
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  10. Tim Hunt
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  13. Rob H. Meloen
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Correspondence to Peter Timmerman.

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Timmerman, P., Van Dijk, E., Puijk, W. et al. Mapping of a discontinuous and highly conformational binding site on follicle stimulating hormone subunit-β (FSH-β) using domain Scan™ and Matrix Scan™ technology. Mol Divers 8, 61–77 (2004). https://doi.org/10.1023/B:MODI.0000025650.94399.bb

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