Port Macquarie, Australia
Port Macquarie, Australia

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You J.,Minomic International Ltd. | You J.,University of New South Wales | Fitzgerald A.,Minomic International Ltd. | Cozzi P.J.,University of New South Wales | And 14 more authors.
Electrophoresis | Year: 2010

This is the first 2-DE study using sequential dyes to analyse phospho-, glyco- and total tear protein profiles (Pro-Q Diamond for phosphoprotein, Pro-Q Emerald for glycoprotein and Sypro Ruby for total protein). This method minimised the gel-gel variations, allowing better comparisons among the three profiles and generated a whole map of PTM profiles of tear protein. A novel tear protein, dermcidin, was identified for the first time in this study. The identification of this antimicrobial protein suggests a new model of defence in tears. In addition, we are able to present the first experimental evidence of the presence of glycosylated lipocalin 1 and cystatin S. Nucleobindin 2 was only detected using phospho staining, suggesting it is only phosphorylated in tears. This study provides the groundwork for understanding the PTM of tear proteins and consequently these methods could be useful in the search for biomarkers in tears. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

Sayyadi N.,Macquarie University | Justiniano I.,Macquarie University | Justiniano I.,Minomic International Ltd. | Connally R.E.,Macquarie University | And 11 more authors.
Analytical Chemistry | Year: 2016

We describe the application of a synthetically developed tetradentate β-diketonate-europium chelate with high quantum yield (39%), for sensitive immunodetection of prostate cancer cells (DU145). MIL38 antibody, a mouse monoclonal antibody against Glypican 1, conjugated directly to the chelate via lysine residues, resulted in soluble (hydrophilic) and stable immunoconjugates. Indirect labeling of the antibody by a europium chelated secondary polyclonal antibody and a streptavidin/biotin pair was also performed. All of these bright luminescent conjugates were used to stain DU145 cells, a prostate cancer cell line, using time gated luminescence microscopy for imaging, and their performances were compared to conventional FITC labeling. For all prepared conjugates, the europium chelate in conjunction with a gated autosynchronous luminescence detector (GALD) completely suppressed the cellular autofluorescence background to allow capture of vivid, high contrast images of immune-stained cancer cells. © 2016 American Chemical Society.

PubMed | Mark Medical, University of New South Wales, Macquarie University, Minomic International Ltd and Queensland University of Technology
Type: | Journal: Analytical biochemistry | Year: 2016

The mass spectrometry technique of multiple reaction monitoring (MRM) was used to quantify and compare the expression level of lactoferrin in tear films among control, prostate cancer (CaP), and benign prostate hyperplasia (BPH) groups. Tear samples from 14 men with CaP, 15 men with BPH, and 14 controls were analyzed in the study. Collected tears (2l) of each sample were digested with trypsin overnight at 37C without any pretreatment, and tear lactoferrin was quantified using a lactoferrin-specific peptide, VPSHAVVAR, both using natural/light and isotopic-labeled/heavy peptides with MRM. The average tear lactoferrin concentration was 1.010.07g/l in control samples, 0.960.07g/l in the BPH group, and 0.980.07g/l in the CaP group. Our study is the first to quantify tear proteins using a total of 43 individual (non-pooled) tear samples and showed that direct digestion of tear samples is suitable for MRM studies. The calculated average lactoferrin concentration in the control group matched that in the published range of human tear lactoferrin concentration measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the lactoferrin was stably expressed across all of the samples, with no significant differences being observed among the control, BPH, and CaP groups.

You J.,University of New South Wales | You J.,Minomic International Ltd | Cozzi P.,University of New South Wales | Cozzi P.,St George Hospital | And 9 more authors.
Critical Reviews in Oncology/Hematology | Year: 2010

The marker currently used for prostate cancer (CaP) detection is an increase in serum prostate-specific antigen (PSA). However, the PSA test which may give false positive or negative information, is not reliable and does not allow the differentiation of benign prostate hyperplasia.(BPH), non-aggressive CaP and aggressive CaP. There is thus an urgent need to search for novel CaP biomarkers to improve the early detection and accuracy of diagnosis, determine the aggressiveness of CaP and to monitor the efficacy of treatment. Proteomic techniques allow for a high-throughput analysis of bio-fluids with the visualization and quantification of thousands of potential protein markers and represent very promising tools in the search for new, improved molecular markers of CaP. In this review, we will summarize conventional CaP biomarkers and focus on novel identified biomarkers for CaP early diagnosis and progression that might be used in the future. © 2009 Elsevier Ireland Ltd.

PubMed | Henan University, Macquarie University and Minomic International Ltd
Type: | Journal: Scientific reports | Year: 2016

Prostate cancer is one of the male killing diseases and early detection of prostate cancer is the key for better treatment and lower cost. However, the number of prostate cancer cells is low at the early stage, so it is very challenging to detect. In this study, we successfully designed and developed upconversion immune-nanohybrids (UINBs) with sustainable stability in a physiological environment, stable optical properties and highly specific targeting capability for early-stage prostate cancer cell detection. The developed UINBs were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and luminescence spectroscopy. The targeting function of the biotinylated antibody nanohybrids were confirmed by immunofluorescence assay and western blot analysis. The UINB system is able to specifically detect prostate cancer cells with stable and background-free luminescent signals for highly sensitive prostate cancer cell detection. This work demonstrates a versatile strategy to develop UCNPs based sustainably stable UINBs for sensitive diseased cell detection.

Fitzgerald A.,Minomic International Ltd. | Walsh B.J.,Minomic International Ltd.
Electrophoresis | Year: 2010

The depth of proteome analysis is severely limited in complex samples with a wide dynamic range of protein abundance such as plasma. Removal of high-abundance proteins should reveal the signal of lower abundance plasma proteins. However, smaller proteins may be part of larger protein complexes and hence the removal of proteins involved in complexes with high-abundance proteins such as albumin may inhibit the search for disease biomarkers. Prefractionation of a sample divides it into fractions of reduced complexity, allowing improved detection of lower abundance proteins. Using a prefractionation device, which employs Gradiflow™ technology, we were able to separate small volume plasma samples into multiple fractions based on the molecular weight and/or charge. The resulting samples of reduced complexity were directly compatible with 2-DE. The use of this prefractionation machine may therefore be useful in the hunt for disease biomarkers. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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