DETERMINATION OF PROSTATE-SPECIFIC ANTIGEN IN SERUM AND A REFERENCE MATERIAL BY ON-CHIP IMMUNOAFFINITY CHROMATOGRAPHY

摘要

Glycoprotein tumor markers are striking examples of heterogeneous analytes. The complexity of their structural forms in biological fluids is generally not reflected in reference materials. Therefore, they are not specified to consist of a distinct form, but rather to contain a mixture of molecular species. In this study, the question of the heterogeneity of free prostate-specific antigen (free PSA) is addressed in reference materials to define the immunoreactive molecular species and compare them to those in clinical serum. The reference material for free PSA and serum samples of subjects with benign prostatic hyperplasia and prostate cancer was examined for immunoreactivity to epitope I-specific anti-free PSA antibody using on-chip immunoaffinity chromatography in combination with mass spectrometry for the determination of bound forms. The mass spectra of the reference material for free PSA and clinical serum, obtained by on-chip immunoaffinity chromatography, were similar. The cluster of major free PSA-immunoreactive peaks at 28–29 kDa corresponding to the mature glycosylated PSA molecule overlapped in both analytes. However, the reference material displayed a more restricted pattern of low molecular mass species corresponding to nicked PSA fragments or PSA degradation products. The PSA concentration in clinical serum seems to consist of more species than equivalent concentrations of reference material. Regarding analysis of heterogeneous proteins, immunoaffinity capture combined with mass-specific detection represents a rapid means for selective detection of distinct molecular species, exceeding the analytical performance of current formats of immunoassays.