Fine mapping of the binding sites of monoclonal antibodies raised against the Pk tag

C Dunn, AM O'Dowd, Richard Edward Randall

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

Abstract

The monoclonal antibody (mAb) SV5-Pk is used widely in a variety of procedures to detect recombinant proteins tagged with the Pk tag, a 14 amino acid sequence derived from the P and Y proteins of the paramyxovirus Simian Virus 5. Here we report on the isolation and characterisation of four additional SV5-Pk mAbs (termed SV5-Pk2 to 5) that bind the Pk tag. All the SV5-Pk mAbs can detect Pk tagged recombinant proteins in a variety of immunological procedures, including ELISA and immunofluorescence. Using SPOT technology, the minimal binding epitope of each SV5-Pk mAb was defined by one-sided terminal truncation analysis from either the amino- or carboxy-ends of the Pk peptide. Each mAb recognises slightly different epitopes within the Pk tag, ranging from 5 to 9 amino acids in length. The equilibrium dissociation constants (K-d) of the mAbs, as measured by surface plasmon resonance, ranged from approximately 20 to 60 pmol. Cysteine scanner mutations throughout the Pk tag revealed that some amino acids within thr: minimal binding epitopes were critical for mAb binding, while others could readily be substituted with little or no effect on antibody binding. The development of the Pk tag as a spacer arm for site-directed chemical coupling, and the use of the mAbs to monitor purification and coupling procedures, is discussed. (C) 1999 Elsevier Science B.V. All rights reserved.

Original languageEnglish
Pages (from-to)141-150
Number of pages10
JournalJournal of Immunological Methods
Volume224
Issue number1-2
DOIs
Publication statusPublished - 22 Apr 1999

Keywords

  • pk tag
  • SV5-Pk
  • amino acid
  • SIV PROTEINS
  • IN-VITRO
  • KAPPA-B
  • EPITOPE
  • PURIFICATION
  • IDENTIFICATION
  • EXPRESSION
  • COMPLEXES
  • PEPTIDES
  • ANTIGEN

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