Molecular Form Differences Between Prostate-Specific Antigen
Prostate-specific antigen (PSA) assays currently used for the detection of prostate cancer (PCa) lack the specificity necessary to differentiate PCa from benign prostatic hyperplasia and have high levels false positives. PSA standards used to create standard curves in these assays are typically purified from seminal plasma and contain many molecular forms (intact PSA and cleaved sub-forms). The purpose of this study was to determine whether the composition of the molecular forms of PSA found in these PSA standards contribute to the unreliability of the PSA test.
To this end, seminal plasma-purified PSA standards from different commercial sources were studied by western blot (WB) and in multiple research-grade PSA ELISAs. The WB results revealed that all PSA standards contained different mass concentrations of intact and cleaved molecular forms. Increased mass concentrations of intact PSA gave higher immunoassay absorbance values, even between lots from the same manufacturer.
Standardization of seminal plasma-derived PSA calibrator molecular form mass concentrations and purification methods will help address gaps in PCa test measurements that require the use of PSA values, such as % PSA free and prostate health index by increasing the accuracy of the calibration curves.
For nearly two decades, immunoassays that measure the serum level of the biomarker PSA have been used for the early detection and therapeutic monitoring of prostate cancer (PCa)1. PSA, discovered independently by several researchers2 and purified in 1979 from prostate tissue3, is an N-linked glycoprotein4 composed of a residue of 237 amino acids (28,400 Daltons (Da)).
The predominant immunoreactive forms of PSA, also known as isoforms, that have been identified in serum include free (uncomplexed) PSA (fPSA) and alpha 1-antichymotrypsin complexed PSA (PSA-ACT) . It has been shown that in men with PCa, the PSA-ACT is elevated5. In contrast, benign prostatic hyperplasia (BPH), a benign enlargement of the prostate, is associated with higher serum levels of non-intact free PSA. High rates of false positives6, problems detecting free and complexed PSA antibodies in equal molar ratios (equimolarity)7, as well as discrepancies in measured PSA values between immunoassay manufacturers8 have been the subject of controversies leading to great debate over whether using the test is beneficial. Identifying the source of inaccuracy in PSA measurements would greatly help to increase the specificity of PCa tests.
The standard curve of a PSA immunoassay plays a critical role in the accurate measurement of an unknown mass concentration of serum PSA. Calibration standards used in clinical and research grade PSA immunoassays are either free PSA, a ratio of free and complexed PSA, or PSA of a recombinant form. Non-recombinant PSA protein standards are purified from seminal plasma. In addition to the precursor forms of PSA, seminal plasma contains free PSA which is composed of enzymatically active intact PSA and enzymatically inactive, cleaved and cleaved (internally cleaved) molecular subforms9.
These free PSA subforms are usually internally cleaved between residues 85–86, 145–146 and 182–18310,11. Since seminal plasma-derived PSA standards are purified from pooled samples (i.e. multiple donors)12, PSA immunoassay standards may potentially contain differences in intact PSA concentrations , as well as molecular subforms of PSA. A possible source of the unreliability of PSA immunoassays may be due, in part, to molecular differences in seminal plasma-purified PSA standards.
The objective of this study was to determine whether differences in the subform composition of intact PSA and cleaved PSA created discrepancies in PSA ELISA mass concentration measurements. In this study, PSA and PSA-ACT calibrators of known mass concentration from different commercial sources were used as control standards (CS) and examined comparatively in several quality total PSA (t-PSA) ELISAs. research.
These immunoassays served as model clinical systems. Two of the test standards, one of which was a recombinant PSA, were also comparatively studied.