Alice Ylikoski

Simultaneous Quantification of Human Glandular Kallikrein 2 and Prostate-Specific Antigen mRNAs in Peripheral Blood from Prostate Cancer Patients

We present a multiplexed and internally calibrated quantitative reverse transcription-PCR (QRT-PCR) assay to detect human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) transcripts in blood samples from healthy subjects and prostate cancer (PC) patients. The assay detected 50 copies of hK2 and PSA mRNA, and 1 PSA- and 10 hK2-expressing LNCaP cells in the presence of 2.5 x 10(6) PSA- and hK2-negative cells. In PC patients, 20 of 25 and 19 of 25 gave detectable PSA and hK2 mRNAs, respectively. Number of hK2 mRNA copies was significantly higher than that of PSA mRNA copies in patients with biochemically progressive (P = 0.02) PC, and with locally advanced and metastasized (P = 0.004) PC. Patients with rapidly progressive and hormone refractory PC gave detectable hK2 mRNA only in 2 of 8 and PSA mRNA in 3 of 8 patients. Neither PSA nor hK2 mRNAs were detected in 16 healthy subjects. PSA and hK2 discriminated PC patients with biochemically progressive and advanced disease from the controls and from the aggressive distant metastatic disease. The assay provides a reliable quantification of the number of hK2 and PSA mRNA copies, allows to discriminate PC cases from healthy subjects, and offers a tool for further studies on molecular staging of PC.

A dual-label immunofluorometric assay for human osteocalcin

Circulating human osteocalcin (hOC) has been shown to be comprised of two main forms: the intact 1–49 form and the proteolytic N‐terminal midfragment (N‐mid) spanning amino acid residues 1–43 or 1–44. We used three monoclonal antibodies (MAbs) raised against hOC and bovine osteocalcin in developing a dual‐label assay for the simultaneous measurement of the proportions of the intact and N‐mid forms in serum samples. The assay is based on time‐resolved fluorescence utilizing differently labeled tracer MAbs. Biotinylated MAb 2H9 is used as a capture antibody for both the intact hOC and the N‐mid. Tracer MAb 6F9 labeled with a Europium(III)‐chelate binds to the N‐mid and the intact hOC, whereas tracer MAb 3G8 labeled with a Terbium(III)‐chelate binds to the intact hOC only. The simultaneous binding of the antibodies was tested by comparing full‐length hOC purified from human bone and hOC shortened from the C terminus by four amino acid residues with carboxypeptidase Y. Serum hOC measurements with the dual‐label assay were in agreement with the corresponding single‐label assays (r = 0.96 for intact + N‐mid assays and r = 0.81 for intact assays, n = 91). The lower correlation between the intact assays was attributable to proteolytic susceptibility of the intact form due to one additional freezing and thawing cycle in carrying out the dual‐label assay. As measured with the dual‐label assay, the levels (mean ± SD) of serum intact + N‐mid OC were 6.2 ± 2.1 ng/ml in the premenopausal group (n = 44), 13.9 ± 4.9 ng/ml in the postmenopausal group without hormone replacement therapy (HRT; n = 13), and 7.5 ± 3.4 ng/ml in the postmenopausal group with HRT (n = 13). The levels of intact hOC in the same groups were 4.8 ± 1.4 ng/ml, 9.8 ± 2.9 ng/ml, and 5.3 ± 2.1 ng/ml, respectively. Whether the main forms of OC or their relative proportions in serum can be used for predicting bone diseases or for monitoring the progression and management of diseases awaits further investigations.