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

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• 作者：Peter Timmerman ; Evert Van Dijk ; Wouter Puijk ; Wim Schaaper ; Jerry Slootstra ; Stephen J. Carlisle ; John Coley ; Steve Eida ; M. Gani and Tim Hunt ; et al.
• 关键词：affinity measurements ; epitope mapping ; fertility hormones ; peptides
• 刊名：Molecular Diversity
• 出版年：2004
• 出版时间：June 2004
• 年：2004
• 卷：8
• 期：2
• 页码：61-77
• 全文大小：1,326 KB
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Analytical Chemistry
  Polymer Sciences
  Organic Chemistry
  Pharmacy
  
• 出版者：Springer Netherlands
• ISSN：1573-501X

文摘

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- 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 3-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 1-3 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 3-loop, 1-loop, long2-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 3-loop showed measurable binding. Cyclic 3-loop peptide mimics were found to bind significantly stronger (K_d 5 M) than the lineair analogues, in agreement with the fact that the discontinuous epitope is part of a loop structure. Coupling of the lineair 1-peptide ₁₀IAIEKEECRFAI₂₁to the linear 3-peptide*₅₂TFKELVYETVRVPGCAHHADSLYTYPVATQAH₈₃# via disulfide bond formation showed a 2–3 fold increase in K_d, thus conforming participation of the 1-loop in antibody binding for these mAb's.