Fine mapping of the binding sites of monoclonal antibodies raised against the Pk tag.
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 V 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 (Kd) 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 the 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.
Dunn
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School of Biomedical Sciences, North Haugh, University of St. Andrews, Fife, UK.
O'Dowd
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A
Randall
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D001665:Binding Sites; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003429:Cross Reactions; D003545:Cysteine; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000276:immunology*; D016601:RNA-Binding Proteins; D014709:Vero Cells; D014764:Viral Proteins / Q000276:immunology*; D015678:Viral Structural Proteins
D000911:Antibodies, Monoclonal; D018985:Epitopes, B-Lymphocyte; C097018:P protein, Simian parainfluenza virus 5; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; C097019:V protein, Simian parainfluenza virus 5; D014764:Viral Proteins; D015678:Viral Structural Proteins; D003545:Cysteine