Accurate determination of adjuvant-associated protein or peptide by ninhydrin assay

Modern peptide and subunit vaccines are increasingly having to rely on the use of immunological adjuvants to achieve effective immunity. However, the only adjuvant currently approved for use in humans is aluminium hydroxide, although many adjuvants are currently under preclinical development. Determining immunogen concentration in the presence of adjuvants such as aluminium hydroxide gel, liposomes or NISV has proved to be problematic. One approach has been to use radiolabelled antigens to extrapolate concentration to a preparation using native immunogen. However, the use of a colorimetric assay would allow greater flexibility in terms of immunogen used and would reduce costs and remove safety problems. Of the colorimetric methods we have examined thus far, only the manual ninhydrin assay has produced consistent results with detection of microgram quantities of protein or peptide in the presence of NISV or Alhydrogel, but not liposomes. As the assay relies on the detection of free amino groups after protein hydrolysis, peptides as well as proteins may be effectively determined irrespective of amino acid composition, a considerable advantage over other colorimetric assay systems.