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.