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Immunology 3: Immune Diagnosis and Monitoring

Development of a novel immunoassay for human kallikrein 14, a putative ovarian and breast cancer biomarker

Carla A. Borgoño, Antoninus Soosaipillai, Linda Grass and Eleftherios P. Diamandis
Carla A. Borgoño
Department of Laboratory Medicine and Pathobiology, University of Toronto and Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON, Canada
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Antoninus Soosaipillai
Department of Laboratory Medicine and Pathobiology, University of Toronto and Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON, Canada
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Linda Grass
Department of Laboratory Medicine and Pathobiology, University of Toronto and Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON, Canada
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Eleftherios P. Diamandis
Department of Laboratory Medicine and Pathobiology, University of Toronto and Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON, Canada
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DOI:  Published May 2005
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Proc Amer Assoc Cancer Res, Volume 46, 2005

Abstract

1547

Human kallikrein 14 (hK14; encoded by the KLK14 gene) is a recently identified secreted serine protease, belonging to a family of potential cancer biomarkers which includes prostate-specific antigen (PSA). Our previous studies have demonstrated that the KLK14 gene is steroid hormone-regulated, differentially expressed in breast, ovarian, prostate and testicular cancers and has prognostic value for breast and ovarian cancer patients at the mRNA level. We have recently discovered that hK14 protein levels are elevated in the serum of a proportion of breast and ovarian cancer patients and in cancerous ovarian tissues, suggesting that hK14 may have also diagnostic value. In order to further examine the potential clinical utility of hK14 as a cancer biomarker for endocrine-related malignancies, we have developed a novel, highly sensitive enzyme linked immunosorbent assay (ELISA). Recombinant hK14 was expressed in P. pastoris as the mature enzyme and purified to homogeneity from the culture supernatant after 6 days of methanol induction by cation exchange chromatography. Its identity was verified by tandem mass spectrometry. Purified recombinant hK14 was then used to generate polyclonal and monoclonal antibodies. A sandwich-type ELISA with a monoclonal-polyclonal antibody configuration was developed, coupled with a time-resolved fluorometric detection technique. The hK14-ELISA is sensitive (detection limit of 0.05 μg/L), specific for hK14 (<0.1% cross-reactivity with other kallikreins), and linear from 0.05 to 10 μg/L with between-run and within-run coefficients of variation of <10%. The ELISA was used to quantify hK14 levels with 36 normal tissue extracts, 9 biological fluids and conditioned media from 1 astrocytoma, 11 breast cancer, 1 cervical cancer, 1 colon cancer, 3 neuroblastoma, 2 osteosarcoma, 6 ovarian cancer, 1 pancreatic cancer and 8 prostatic cancer cell lines. The highest levels of hK14 were found in skin (245 ng hK14/g total protein), breast (224 ng hK14/g total protein) and prostatic (131 ng hK14/g total protein) tissue extracts. Among the cancer cell lines studied, hK14 was only found in the conditioned media of a subset of breast cancer (MCF7, MDA453), ovarian cancer (TOV21G, MDAH2774, OV-90, TOV112D), osteosarcoma (Saos) and neuroblastoma (SK-N-MC) cell lines. hK14 was also present in amniotic fluid (7.3±4.4μg/L), seminal plasma (6.3±3.1μg/L), saliva (4.4±2.1μg/L), follicular fluid (1.3±2.1μg/L), breast milk (1.2±0.4μg/L), ascites fluid from metastatic ovarian cancer patients (0.34±0.7μg/L), the serum of prostate cancer patients (0.3±0.9μg/L) and breast tumour cytosolic extracts (0.13±0.09μg/L), but not in cerebrospinal fluid. This novel ELISA will facilitate further studies on the role of hK14 as a cancer biomarker, which may ultimately aid in the diagnosis, prognosis and/or monitoring of ovarian, breast and prostate carcinoma.

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Cancer Research: 65 (9 Supplement)
May 2005
Volume 65, Issue 9 Supplement
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Development of a novel immunoassay for human kallikrein 14, a putative ovarian and breast cancer biomarker
Carla A. Borgoño, Antoninus Soosaipillai, Linda Grass and Eleftherios P. Diamandis
Cancer Res May 1 2005 (65) (9 Supplement) 363;

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Development of a novel immunoassay for human kallikrein 14, a putative ovarian and breast cancer biomarker
Carla A. Borgoño, Antoninus Soosaipillai, Linda Grass and Eleftherios P. Diamandis
Cancer Res May 1 2005 (65) (9 Supplement) 363;
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