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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.

Monoclonal antibodies were generated against the feline homologue of CD4 (fCD4) by immunisation of mice with solid matrix antigen-antibody complexes of monoclonal antibody Fel7 (anti-fCD4) and formalin-fixed Staphylococcus A (SMAA-fCD4). The resulting fusion produced nine monoclonal antibodies each of which recognised a major population of feline lymphocytes and which immunoprecipitated a 55 kDa ligand from the feline T lymphosarcoma cell line 3201. Epitope mapping of the antibodies against soluble fCD4 by surface plasmon resonance indicated that the antibodies recognised five separate epitopes distinct from that defined by the Fel7 antibody used to prepare the SMAA-fCD4. These data demonstrate that SMAA complexes are an efficient means of generating monoclonal antibodies recognising novel epitopes on an antigen. One monoclonal antibody (vpg39) recognised an epitope that was expressed variably between cats, being either present or completely absent. Analysis of peripheral blood lymphocytes from specific pathogen free cats suggested that failure to react with the vpg39 antibody was an inherited trait.

A method is described for forming monolayers of Staphylococcus aureus Cowan strain A on plastic tissue culture plates (A plates). Bacteria remain bound to such plates even under strong protein denaturing conditions. The specific binding of antibody to A plates provides a rapid method for immune precipitation on a solid matrix. Antibody can be covalently crosslinked to staphylococcus monolayers by fixation with paraformaldehyde without significant loss of specific antigen binding capacity. Furthermore antigen-antibody complexes may be efficiently disrupted with 9 M urea and non-ionic detergent and both antibody and antigen renatured by removal of urea, antibody regaining the ability to bind fresh antigen. In the presence of 9 M urea and 1% Nonidet P-40 fixed antibody remains bound to plates while antigen is released into the urea solution, providing a method for the immunological purification of proteins. Plates with such fixed antibody may be used multiple times to bind antigen. The use of this method is illustrated by the purification of 2 adenovirus structural proteins and a herpes simplex virus glycoprotein, by means of specific monoclonal antibodies to these proteins crosslinked to A plates. A method is also desribed to enrich for functional antibody from immune precipitates by dissociating antibody-antigen complexes with 9 M urea and 1% Nonidet P-40 and isolating the antigen free antibody on Staphylococcus aureus Cowan strain A. A plates have also been used to separate suspension cells on the basis of their cell surface antigens (e.g., lymphocyte subpopulations or cells expressing virus antigens on their surface). Cells were either (1) reacted with specific antisera to cell surface determinants and panned over A plates, cells with antibody on their surface binding to the monolayer, or (2) panned over A plates to which specific antibody had been coupled. Separated cells may be probed directly for certain properties, such as their ability to support virus replication. The staphylococcus monolayer does not prevent the growth of tissue culture cells on the plastic surface and the selected cell population can be examined for cells giving rise to infectious centres by the subsequent addition of permissive cells.