Evaluation of free psa isoforms, psa complex formation, and specificity of anti‐psa antibodies by hplc and page‐immunoblotting techniques

Abstract

Both high performance liquid chromatographic (HPLC) and polyacrylamide gel electrophoresis-immunoblotting (PAGE-immunoblotting) procedures have been established for the study of isoforms of free prostate-specific antigen (PSA) and the complex formation between free PSA and protease inhibitors, and for the evaluation of the specificities of various anti-PSA antibodies. We found multiple isoforms of free PSA on PAGE, which were all capable of forming complexes with protease inhibitors. The same isoform pattern can be produced from the original seminal fluid. The PSA isoforms differ from each other most likely in charge because they could be converted to one band on SDS-PAGE and to a single peak by gel filtration chromatography. We found it difficult to form large quantities of PSA complex when mixing free PSA from seminal fluid with protease inhibitors, regardless of whether the free PSA or the protease inhibitors were in excess. Except for the PSA-ACT complex, which was consistently detectable by both HPLC and PAGE-immunoblotting techniques after incubation, these two procedures disagreed in their detection of PSA-A2M and PSA-AT complexes. The PSA-A2M complex was usually observable by immunoblotting techniques but barely detectable on HPLC, whereas PSA-AT was totally invisible by immunoblotting but appeared as a peak in the HPLC elution profile. Mixing free PSA with serum clearly resulted in both PSA-ACT and PSA-A2M complexes. However, more PSA-ACT than PSA-A2M was formed; the result was also confirmed by using 125I-PSA for mixing. PSA could be separated into active and inactive PSA by DEAE Sepharose chromatography with a 14-fold difference in protease activity. The difference in enzymatic activity apparently had no effect on complex formation. All the anti-PSA antibodies examined in this study reacted with PSA isoforms, PSA-ACT and PSA-A2M complexes. We conclude that it would be almost impossible to establish an assay to measure all forms of PSA in the serum and to expect to produce precise and accurate PSA values.

Molecular Heterogeneity of Free PSA in Sera of Patients with Benign and Malignant Prostate Tumors

“…Two attempts to partially characterize serum PSA fractions used sera with exceptionally high levels of serum PSA secondary to metastatic disease (>2,000 ng/ml [7, 8]), and applied purification procedures not preventing exclusion of minor small-sized PSA forms.…”

Monitoring percent free PSA in serial specimens: Improvement of test specificity, early detection, and identification of occult tumors

“…At the present time, all PSA assays measure both free PSA (fPSA) and PSA-ACT in the serum and are therefore designated as total PSA (tPSA) assay. Although other PSA complexes, such as PSA-α 2 -macroglobuline, PSA-α 1 -antitrypsin, and PSA-inter-α-trypsin also may exist, they do so either in negligible amounts or are not detectable immunologically (5,9). Practically speaking, free PSA and the PSA-ACT complex together account for the majority of PSA isoforms quantified by the current tPSA assay in the serum.…”

“…Because of the presence of many protease inhibitors in the serum, PSA does not exist in the serum entirely as free molecules, but binds to various protease inhibitors in complex forms (1)(2)(3). Immunologically, only two major forms can be detected in the serum: free PSA (fPSA) and PSA-α1-antichymotrypsin (PSA-ACT) complex (4,5). The percent distribution of these two forms of PSA in serum is not a fixed constant.…”

Development of an immunoassay specific for the PSA-ACT complex without the problem of high background

“…Although there are only two major PSA isoforms detectable immunologically in the serum, other PSA isoforms could also contribute to the value of total PSA determined by the current tPSA assays. In fact, we found low immunoreactivity with PSA-A2M (α2 macroglobulin) in a tPSA assay (14). PSA-AT (antitrypsin) complex could also contribute to the total PSA concentration even though the serum concentration of PSA-AT complex could be very low.…”