Effect of prostate specific antigen (PSA) complex formation on PSA assays

Current commercial assays for PSA do not always give the same PSA values. The major reasons relate to the fact that in serum PSA exists primarily complexed to protease inhibitors whereas current assay kits were designed using PSA isolated from seminal plasma (free PSA). The aim of this study was to evaluate the effect various PSA-protease inhibitor complexes have on current PSA assays. Assay kits used in this study: Hybritech Tendem-E PSA, Ciba Corning ACS:180 PSA, Abbott IMx PSA, and Syva Vista PSA and an assay prepared in-house using antibodies from BIoMira and Dako. Antibody from Scripps was also employed. Specimens included; Pooled sera from patients with elevated levels of PSA; Free PSA and PSA complexes isolated from pooled patient sera; PSA purified from seminal plasma and added to pur alph-1-antichymotrypsin (ACT), alpha-1-protease inhibitor (API), alpha-2macroglobulin (A2M) and pooled female sera; Free and complexed fractions isolated from the mixtures of pure PSA and protease inhibitor of HPLC size exclusion chromatography. Free PSA and PSA complexes from serum pool and from PSA-protease inhibitor mixtures were separated using low pressure and HPLC size exclusion chromatography. PSA-protease inhibitor mixtures were evaluated for complex formation using HPLC gel filtration chromatography, inhibition of enzyme activity, PAGE followed by immunoblotting, and immunossays designed in-house for specific complexes. Results confirm that the most abundant complex measured n serum is PDA-ACT. PSA also from complexes with A2M and API. Small amounts of both are detected in serum. PSA-A2M complexes are not well detected using commercial immunoassays. The best method for demonstrating PSA-A2M complex formation is PAGE followed by immunoblotting. All commercial assays detect both free PSA and PSA-ACT complex, but not with equal reactivity. It is very difficult to form complexes between pure PSA and pure inhibitor. It is not know which isoform PSA most readily forms complex. It appears that each commercial assay evaluated is acceptable since each reacts with free PSA and PSA-ACT complex. The results of this study confirm the importance of using the same laboratory and kit while monitoring patients because differences in PSA values obtained using different kits may be clinically significant. This is particularly true for patients being evaluated using rate change of PSA to differentiate BPH from cancer. Different reference rages should be established for current assays, particularly is PSA is to be used as a screening tool. It may be impossible to standardize results form current assays by preparing a universal calibrator, since patient samples may contain both free and complexed PSA in various rations, and the reactivity of the various antibodies with different forms is dissimilar. These results indicate that the use of an immunoassay specific for PSA-ACT complex may eliminate most of the standardization problems and yield accurate results than the current immunoassays.