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Articles |
1
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5 Canada.
2
Department of Laboratory Medicine and Pathobiology,
University of Toronto, Toronto, Ontario, M5G 1L5 Canada.
3
Institute for Biological Sciences, National Research
Council of Canada, Ottawa, Ontario, K1A 0R9 Canada.
4
Faculty of Pharmacy, Department of Pharmacology,
University of Montreal, Montreal, Quebec, H3C 3J7 Canada.
a Address correspondence to this author at: Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario, M5G 1X5 Canada. Fax 416-586-8628; e-mail
ediamandis{at}mtsinai.on.ca.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Recombinant hK10 protein was produced and purified using a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK10 antisera. A sandwich-type immunofluorometric assay was then developed using these antibodies.
Results: The hK10 immunoassay has a detection limit of 0.05 µg/L. The assay is specific for hK10 and has no detectable cross-reactivity with other homologous kallikrein proteins, such as prostate-specific antigen (hK3), human glandular kallikrein 2 (hK2), and human kallikrein 6 (hK6). The assay was linear from 0 to 20 µg/L with within- and between-run CVs <10%. hK10 is expressed in many tissues, including the salivary glands, skin, and colon and is also detectable in biological fluids, including breast milk, seminal plasma, cerebrospinal fluid, amniotic fluid, and serum.
Conclusions: We report development of the first immunofluorometric assay for hK10 and describe the distribution of hK10 in biological fluids and tissue extracts. This assay can be used to examine the value of hK10 as a disease biomarker.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The normal epithelial cell-specific 1 (NES1) gene is one of
these newly identified genes. With the new nomenclature, which will be
used throughout this report, NES1 is designated as
KLK10 and the encoded protein as hK10. KLK10 was
isolated with subtractive hybridization, by virtue of its
down-regulation in radiation-transformed breast epithelial cells
(8). KLK10 resides on chromosome 19q13.3-q13.4,
spans 
5.5 kb of genomic DNA sequence, and contains six exons (one
untranslated) and five introns (9). KLK10 encodes
for a secreted serine protease whose amino acid sequence has 35–40%
identity and 50–55% similarity with other members of the human
kallikrein gene family, including PSA. The physiological function of
KLK10 is still not clear. Because the KLK10 gene
is down-regulated in breast cancer cell lines, it is considered to be
involved in the regulation of normal cell growth. Further experimental
evidence suggests that KLK10 may encode for a tumor
suppressor gene. When the KLK10 gene was transfected into
the tumorigenic breast cancer cell line MDA-MB-231, its
anchorage-independent growth was reduced, and when this cell line was
inoculated into nude mice, tumor formation was significantly decreased
(10).
Because KLK10 encodes for a secreted serine protease and its expression appears to be altered in some disease states, including breast and prostate cancer (8)(10)(11), we hypothesized that hK10 protein may be secreted into various biological fluids and that its concentration might change during disease initiation and progression. However, no suitable method for measuring hK10 with high sensitivity and specificity has been described to date. Consequently, the concentrations of this protein in biological fluids and tissue extracts have not been reported. In this report, we describe the first immunoassay for hK10 protein, which is suitable for measuring hK10 with high sensitivity and specificity. With this method, we were able to quantify hK10 in serum, breast milk, seminal plasma, cerebrospinal fluid (CSF), and amniotic fluid as well as in various tissue extracts. This newly developed immunoassay is suitable for examining the possible value of hK10 as a biomarker in various human diseases, including cancer.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A using standard
molecular biology techniques (12).
Production of hK10 in yeast.
pPIZA vector containing the
KLK10 cDNA was introduced into the yeast strain X-33, and a
stable clone was selected following the manufacturer’s instructions.
hK10 was produced by growing the stable yeast clone in a medium
containing 10 g/L yeast extract, 20 g/L peptone, 100 mmol/L potassium
phosphate (pH 6.0), 13.4 g/L yeast nitrogen base, 40 mg/L biotin, and 5
mL/L methanol in a 30 °C shaking incubator (250 rpm) for 5 days. The
cells were then spun down, and the supernatant was collected.
Purification of hK10 with cation-exchange and reversed-phase
chromatography.
The recombinant hK10 protein was purified from the
yeast culture supernatant by cation-exchange chromatography using
CM-Sepharose fast flow (Pharmacia) and reversed-phase liquid
chromatography using a C4 column (0.45 x 5
cm; Vydac). The presence of hK10 in various fractions was identified
with Western blotting using an anti-hK10 peptide antibody. In brief,
the CM-Sepharose beads previously activated with 1 mol/L KCl were
equilibrated in 10 mmol/L MES buffer (pH 6.5). The yeast
culture supernatant was first adsorbed on CM-Sepharose beads by
incubation at 4 °C overnight under agitation. The beads were then
washed with 10 mmol/L MES buffer (pH 6.5), and hK10 was eluted with 300
mmol/L KCl in 10 mmol/L MES buffer (pH 6.5). Trifluoroacetic
acid as ion-pairing agent was added into this eluate (final
concentration, 10 mL/L), which was then loaded on a
C4 column equilibrated with 1 mL/L
trifluoroacetic acid in water. A linear gradient (1%/min) of
acetonitrile from 15% to 50% in 1 mL/L trifluoroacetic acid was then
performed. hK10 eluted at 42% acetonitrile. The fraction containing
hK10 was then immediately evaporated on a SpeedVac (Savant Instrument).
The purified material was then separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with
Coomassie Blue to assess its purity and its molecular mass. The protein
concentration of the purified hK10 was determined by the bicinchoninic
acid method, which uses bovine serum albumin as a calibrator (Pierce
Chemical).
identification of hK10 BY MASS SPECTROMETRY
Proteolytic digestion.
Polyacrylamide gels were stained with
Coomassie G-250 for visualization, and selected bands were subsequently
excised and destained with 300 mL/L acetonitrile in 100 mmol/L
ammonium bicarbonate. Each band was then reduced (10 mmol/L
dithiothreitol in 50 mmol/L ammonium bicarbonate, pH 8.3) and alkylated
(55 mmol/L iodoacetamide in 50 mmol/L ammonium bicarbonate, pH 8.3)
before overnight trypsin digestion (Promega). Peptide fragments were
then extracted with 50 mL/L acetic acid, evaporated to dryness on a
Savant concentrator, and reconstituted in 10 µL of a solution of
methanol-water-acetic acid (500:495:5 by volume).
Mass spectrometry.
All nanoelectrospray mass
spectrometry experiments were conducted on a Q-Star (PE/Sciex)
hybrid quadrupole/time-of-flight instrument, for high resolution and
online tandem mass spectrometry (MS-MS) experiments (13).
Conventional mass spectra were obtained by operating the quadrupole in
a radiofrequency-only mode while a pusher electrode was pulsed
(frequency 7 kHz) to transfer all ions to the time-of-flight
analyzer. MS-MS experiments on tryptic peptides identified in survey
scans were conducted using a nanoelectrospray source. Precursor ions
were selected by the first quadrupole while a pusher electrode was
pulsed (frequency 7 kHz) to transfer fragment ions formed in the
radiofrequency-only quadrupole cell to the time-of-flight analyzer.
Mass spectral resolution was typically 9000–10 000. A scan duration
of 1 and 2 s was set for conventional and MS-MS mass spectral
acquisition, respectively. Collisional activation was performed using
nitrogen collision gas with typically a 30-V offset between the DC
voltage of the entrance quadrupole and the radiofrequency-only
quadrupole cell. Data were acquired and processed using LC Tune and
Biomultiview programs from PE/Sciex.
production of polyclonal antibodies against hK10
The purified recombinant hK10 was used as an immunogen to immunize
rabbits and mice. hK10 (100 µg) was injected subcutaneously into
female Balb/c mice and New Zealand White rabbits. The protein was
diluted 1:1 in complete Freund’s adjuvant for the first injection and
in incomplete Freund’s adjuvant for the subsequent injections.
Injections were repeated six times at 3-week intervals. Blood
was drawn from the animals and tested for antibody generation. To test
for production of anti-hK10 polyclonal antibodies, we used the
following immunoassay. Sheep anti-mouse or goat anti-rabbit IgG
(Jackson ImmunoResearch) was immobilized on 96-well white ELISA plates.
The mouse/rabbit serum was then applied to the plates in different
dilutions ranging from 1:500 to 1:50 000. Biotinylated recombinant
hK10 was then added (5–10 ng/well). Finally, alkaline
phosphatase-conjugated streptavidin was added, and the alkaline
phosphatase activity was detected with time-resolved fluorescence (for
more details, see below).
immunofluorometric assay for hK10
Standard assay procedure.
White polystyrene microtiter plates
were coated with sheep anti-mouse IgG, Fc fragment-specific antibody
(Jackson ImmunoResearch) by overnight incubation of 100 µL of coating
antibody solution (containing 500 ng of antibody diluted in 50 mmol/L
Tris buffer, pH 7.80) in each well. The plates were then washed
six times with the washing buffer (9 g/L NaCl and 0.5 g/L Tween 20 in
10 mmol/L Tris buffer, pH 7.40). Mouse anti-hK10 antiserum was diluted
5000-fold in a general diluent [60 g/L bovine serum albumin,
50 mmol/L Tris (pH 7.80), and 0.5 g/L sodium azide], and 100 µL was
applied to each well. After a 1-h incubation, the plates were washed
six times with washing buffer.
hK10 calibrators or samples were pipetted into each well (100 µL/well) and incubated for 1 h with shaking; the plates were then washed with washing buffer six times. Subsequently, 100 µL of rabbit anti-hK10 antiserum diluted 5000-fold in buffer A (containing the components of the general diluent plus 25 mL/L normal mouse serum, 100 mL/L normal goat serum, and 10 g/L bovine IgG) was applied to each well and incubated for 30 min; plates were then washed as above. Finally, 100 µL/well of alkaline phosphatase-conjugated goat anti-rabbit IgG, Fc fragment-specific (Jackson ImmunoResearch), diluted 3000-fold in buffer A was added to each well and incubated for 30 min, and plates were washed as above.
Diflunisal phosphate [100 µL of a 1 mmol/L solution in substrate buffer (0.1 mol/L Tris (pH 9.1), 0.1 mol/L NaCl, and 1 mmol/L MgCl2] was added to each well and incubated for 10 min. Developing solution (100 µL, containing 1 mol/L Tris base, 0.4 mol/L NaOH, 2 mmol/L TbCl3, and 3 mol/L EDTA) was pipetted into each well and mixed for 1 min. The fluorescence was measured with a time-resolved fluorometer, the Cyberfluor 615 Immunoanalyzer (MDS Nordion). The calibration and data reduction were performed automatically, as described in detail elsewhere (14).
Determination of the sensitivity of the hK10 immunoassay.
Recombinant hK10 was used to generate the calibration curve. hK10
calibrators were prepared by diluting the purified recombinant hK10 in
the general diluent. These calibrators were used to define the
detection limit of the assay.
Determination of the specificity of the hK10 immunoassay.
Milk
and seminal plasma samples and recombinant hK10 were used to determine
the specificity of the developed immunoassay. These samples were first
measured by the standard assay procedure described above. The mouse and
rabbit anti-hK10 antisera were then successively replaced with sera
from the same animals, obtained before immunization (preimmune sera).
The samples were then measured again, and the fluorescence counts were
compared with the counts obtained by the standard assay. The
cross-reactivities of other homologous proteins were investigated using
purified recombinant PSA, hK2, and hK6 (available in-house).
Recombinant hK10 (0.025–2 ng), PSA (20 ng to 1 µg), hK2 (2–10 ng)
and hK6 (11–55 ng) were used as samples and were measured with the
standard procedure described above; their fluorescence counts were then
compared (all specified amounts refer to mass of analyte per assay).
Linearity of the hK10 immunoassay.
To determine the linearity
of the hK10 immunoassay, various clinical samples were serially diluted
2-, 4-, 8-, 16-, and 32-fold in general diluent, and their hK10
concentrations were measured with the standard assay.
human tissue cytosolic extracts and biological fluids
Human tissue cytosolic extracts were prepared as follows: Various
frozen human tissues (0.2 g) were pulverized on dry ice to fine
powders. Extraction buffer [1 mL, containing 50 mmol/L Tris (pH 8.0),
150 mmol/L NaCl, 5 mmol/L EDTA, 10 g/L NP-40 surfactant, 1 mmol/L
phenylmethylsulfonyl fluoride, 1 g/L aprotinin, 1 g/L
leupeptin] was added to the tissue powders, and the mixture was
incubated on ice for 30 min with repeated shaking and vortex-mixing
every 10 min. Mixtures were then centrifuged at 14 000 rpm at
4 °C for 30 min. The supernatants (cytosolic extracts) were then
collected. The biological fluids were leftovers of samples submitted
for routine biochemical testing. All tissue cytosolic extracts and
biological fluids were stored at -80 °C until use.
recovery
Recombinant hK10 was added to the general diluent, serum, CSF,
breast milk, seminal plasma, and amniotic fluid at different
concentrations and measured with the developed hK10 immunoassay.
Recoveries were then calculated after subtraction of the endogenous
concentrations.
fractionation of biological fluids with size-exclusion hplc
Serum, milk, and seminal plasma were fractionated on a
silica-based gel filtration column essentially as described elsewhere
(15). The fractions were collected and analyzed for hK10
with the developed immunoassay.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), and then purified with cation-exchange and reversed-phase
chromatography (Fig. 1B
and Fig. 2
). The sequence of this recombinant protein was verified by mass
spectrometry (Fig. 3
). The nanoelectrospray mass spectrum of the tryptic peptides extracted
from the SDS-PAGE band is presented in Fig. 3A
, and shows several
doubly and triply protonated molecules ([M+H]2+
and [M+H]3+) at m/z
550-1400. The calculated masses derived from the m/z values
were compared with the list of tryptic peptide masses predicted from
the hK10 protein sequence. Tryptic peptides corresponding to a segment
of the protein are indicated as subscripts in Fig. 3A
. Confirmation of
the sequence assignment was achieved using MS-MS on selected precursor
ions observed in Fig. 3A
. For example, the product ion spectrum of the
doubly protonated ion m/z 853.4 (Fig. 3B
) shows a series of
well defined y-type fragment ions corresponding to amide bond cleavage
with charge retention on the C-terminal peptide segment
(16). The partial sequence VGDDHLLLLQGEQLR was
identified from the m/z spacing between adjacent fragment
ions and precisely matched that expected for the T96–110 peptide of
hK10.
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sensitivity, specificity, linearity, and precision of the
hK10 IMMUNOFLUOROMETRIC ASSAY
Sensitivity.
A typical calibration curve for the hK10
immunofluorometric procedure is shown in Fig. 4
. The detection limit, defined as the concentration corresponding to the
fluorescence of the zero calibrator plus 2 SD, was 0.05 µg/L.
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Specificity.
To confirm that our immunoassay measures hK10
specifically, we replaced mouse and rabbit anti-hK10 antisera with
preimmune mouse and rabbit sera, respectively. Milk and seminal plasma
samples and recombinant hK10 were measured. With the standard
procedure, these samples all produced relatively high counts
(>100 000 arbitrary fluorescence units). However, when either mouse
or rabbit anti-hK10 polyclonal antibodies were replaced with preimmune
sera, the fluorescence counts of these samples were reduced almost to
zero (Fig. 5
). Because hK10 is a member of the human kallikrein family, it
shares significant amino acid sequence homology with other members. To
further demonstrate that there is no interference from these homologous
proteins, the cross-reactivities of hK10, PSA, hK2, and hK6 were
examined. As shown in Fig. 5
, when recombinant PSA, hK2, and hK6 were
measured with the developed standard hK10 assay, they produced no
counts even when they were present at amounts 5–500 times higher than
hK10. These results suggest that our immunoassay can efficiently
discriminate hK10 from other homologous proteins and that it measures
hK10 with high specificity.
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Linearity.
To assess the linearity of this assay, various
samples were diluted serially and hK10 was measured. There is excellent
dilution linearity with this assay.
Precision.
Within- and between-run precision was assessed with
various hK10 calibrators and clinical samples. In all cases, the CVs
were 2–9%, consistent with the precision of typical microtiter
plate-based immunoassays (data not shown).
distribution of hK10 IN VARIOUS HUMAN TISSUE EXTRACTS
AND BIOLOGICAL FLUIDS
The distribution of hK10 in various human tissue extracts and
biological fluids was investigated with the developed hK10 immunoassay.
As shown in Fig. 6
, hK10 is present in many tissues, such as salivary gland, skin, colon,
fallopian tube, prostate, testis, pituitary, endometrium, and lung.
hK10 is also detectable in various biological fluids, including milk,
seminal plasma, serum, CSF, and amniotic fluid (Table 1
).
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Using the rabbit anti-hK10 antiserum, we immunohistochemically
localized hK10 in the epithelial cells of fallopian tube (Fig. 7
). hK10 is present in the cytoplasm of epithelial cells lining the
lumen.
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We found that, with the exception of CSF (recovery, 88–100%), recovery from various biological fluids was incomplete: 50–80% in serum, 0–77% in milk, 64–100% in seminal plasma, and 18–53% in amniotic fluid.
fractionation of biological fluids with size-exclusion hplc
To determine the molecular mass of the protein detected in the
biological fluids, three samples were fractionated with gel filtration,
including milk, seminal plasma, and serum. The presence of hK10 in
various fractions was measured with the developed immunoassay. When the
fluorescence counts were plotted against the different fractions, a
single peak (fraction 40), which corresponds to 30 kDa, was detected
(Fig. 8
). No higher molecular mass complexes were found. These results indicate
that the protein detected with the hK10 immunoassay is a single
species, with a molecular mass of 30 kDa, which is consistent with the
molecular mass of free hK10.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although hK10 was predicted to be a secreted protein (8), no methods currently exist for detecting the protein with high sensitivity and specificity. We have thus undertaken the task of developing such a method, based on noncompetitive immunoassay principles. For detection, we used time-resolved fluorometry to achieve high sensitivity (14). Because there is no known natural source of large amounts of hK10, we expressed it in P. pastoris. We then purified the protein to homogeneity by combining ion-exchange and reversed-phase chromatography. This technology ensures complete absence of homologous proteins and other contaminants in the final preparation. The recombinant hK10 protein was positively identified by mass spectrometry. However, we did not attempt to characterize the enzymatic activity of this protein. It appears that our protein has a smaller molecular mass than native hK10 (8), probably because of differences in glycosylation.
The developed assay is highly sensitive, detecting hK10 at
concentrations of 0.05 µg/L or higher. Furthermore, this assay
appears to be very specific: we did not observe any detectable
cross-reactivities with the highly homologous kallikrein proteins PSA
(hK3), hK2, and hK6. In addition, substitution of our antibodies with
preimmune sera from the same animals completely abolished the signal.
Size-exclusion HPLC further indicated that the three biological fluids
tested (serum, milk, and seminal plasma) contain only one
immunoreactive peak of the expected molecular mass (30 kDa). These
data suggest that our immunoassay detects the free fraction of hK10 in
these biological fluids. We cannot exclude the possibility that hK10,
like PSA, may be partially complexed to proteinase inhibitors
(17)(18)(19)(20).
We detected hK10 in various tissue extracts but predominantly in
salivary glands, skin, and colon. We further identified relatively high
concentrations of hK10 in many biological fluids, including milk of
lactating women, seminal plasma, amniotic fluid, male and female serum,
and CSF (Table 1
). Because hK10 is a secreted protein, we speculate
that the concentration of this biomarker in serum may change during
initiation and progression of various diseases, including cancer. The
availability of the highly sensitive hK10 immunoassay will facilitate
such studies in the near future. Currently, there is no report in the
literature associating expression of hK10 with any human disease with
the exception of a report on KLK10 down-regulation
in breast cancer cell lines (8) and an abstract describing
down-regulation of KLK10 mRNA in aggressive forms of
prostate cancer (11).
Immunohistochemical localization of hK10 in the fallopian tube indicated that this antigen is produced by the luminal epithelial cells lining the fallopian tube. The immunoreactivity was detected in the cytoplasm and lumen, further suggesting that this protease is secreted. The biological function of this protease in the fallopian tube and other glandular tissues merits further investigation. Furthermore, the presence of high concentrations of hK10 in milk, seminal plasma, amniotic fluid, and CSF suggests that this protein is actively secreted by epithelial cells of the breast, male reproductive organs, the fetoplacental unit, and brain cells. More studies will be necessary to precisely localize the production of hK10 in these tissues.
The recoveries of hK10 from the biological fluids tested were
incomplete, with the exception of CSF. In serum, the recovery was
50–82%, a situation similar to the recovery of PSA (15).
In seminal plasma, the recovery was 64–100%. The lowest recoveries
were seen with milk and amniotic fluid. We speculate that the low
recoveries could be attributable either to sequestration of hK10 by
proteinase inhibitors (e.g., 2-macroglobulin),
as happens with PSA (21), or to uptake of hK10 in
lipids present in milk and amniotic fluid.
In conclusion, we describe here the first highly sensitive and specific immunofluorometric assay for measuring hK10 in biological fluids and tissue extracts. This assay will facilitate further studies on this interesting serine protease, to examine the possibility that hK10 may represent a new biomarker of human disease, similarly to other members of this family, which have already found important applications in prostate cancer diagnostics (PSA and hK2). These studies are now in progress.
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Acknowledgments |
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Footnotes |
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References |
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1-antichymotrypsin is the major form of prostate-specific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Res 1991;51:222-226.1-antichymotrypsin. Clin Chem 1991;37:1618-1627.2-macroglobulin. Clin Chem 1998;44:2471-2479.The following articles in journals at HighWire Press have cited this article:
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  V. Kulasingam and E. P. Diamandis Proteomics Analysis of Conditioned Media from Three Breast Cancer Cell Lines: A Mine for Biomarkers and Therapeutic Targets Mol. Cell. Proteomics, November 1, 2007; 6(11): 1997 - 2011. [Abstract] [Full Text] [PDF]
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 J. L.V. Shaw and E. P. Diamandis Distribution of 15 Human Kallikreins in Tissues and Biological Fluids Clin. Chem., August 1, 2007; 53(8): 1423 - 1432. [Abstract] [Full Text] [PDF]
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 J. Dorn, M. Schmitt, R. Kates, B. Schmalfeldt, M. Kiechle, A. Scorilas, E. P. Diamandis, and N. Harbeck Primary Tumor Levels of Human Tissue Kallikreins Affect Surgical Success and Survival in Ovarian Cancer Patients Clin. Cancer Res., March 15, 2007; 13(6): 1742 - 1748. [Abstract] [Full Text] [PDF]
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 C. A. Borgono, I. P. Michael, J. L. V. Shaw, L.-Y. Luo, M. C. Ghosh, A. Soosaipillai, L. Grass, D. Katsaros, and E. P. Diamandis Expression and Functional Characterization of the Cancer-related Serine Protease, Human Tissue Kallikrein 14 J. Biol. Chem., January 26, 2007; 282(4): 2405 - 2422. [Abstract] [Full Text] [PDF]
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M. H. Slagter, A. Scorilas, L. J.G. Gooren, W. de Ronde, A. Soosaipillai, E. J. Giltay, M. Paliouras, and E. P. Diamandis Effect of Testosterone Administration on Serum and Urine Kallikrein Concentrations in Female-to-Male Transsexuals Clin. Chem., August 1, 2006; 52(8): 1546 - 1551. [Abstract] [Full Text] [PDF]
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M. H. Slagter, L. J.G. Gooren, W. de Ronde, A. Soosaipillai, A. Scorilas, E. J. Giltay, M. Paliouras, and E. P. Diamandis Serum and Urine Tissue Kallikrein Concentrations in Male-to-Female Transsexuals Treated with Antiandrogens and Estrogens Clin. Chem., July 1, 2006; 52(7): 1356 - 1365. [Abstract] [Full Text] [PDF]
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C. V. Obiezu, S. J.C. Shan, A. Soosaipillai, L.-Y. Luo, L. Grass, G. Sotiropoulou, C. D. Petraki, P. A. Papanastasiou, M. A. Levesque, and E. P. Diamandis Human Kallikrein 4: Quantitative Study in Tissues and Evidence for Its Secretion into Biological Fluids Clin. Chem., August 1, 2005; 51(8): 1432 - 1442. [Abstract] [Full Text] [PDF]
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I. P. Michael, G. Sotiropoulou, G. Pampalakis, A. Magklara, M. Ghosh, G. Wasney, and E. P. Diamandis Biochemical and Enzymatic Characterization of Human Kallikrein 5 (hK5), a Novel Serine Protease Potentially Involved in Cancer Progression J. Biol. Chem., April 15, 2005; 280(15): 14628 - 14635. [Abstract] [Full Text] [PDF]
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 C. A. Borgono, I. P. Michael, and E. P. Diamandis Human Tissue Kallikreins: Physiologic Roles and Applications in Cancer Mol. Cancer Res., May 1, 2004; 2(5): 257 - 280. [Abstract] [Full Text] [PDF]
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T. Kishi, A. Soosaipillai, L. Grass, S. P. Little, E. M. Johnstone, and E. P. Diamandis Development of an Immunofluorometric Assay and Quantification of Human Kallikrein 7 in Tissue Extracts and Biological Fluids Clin. Chem., April 1, 2004; 50(4): 709 - 716. [Abstract] [Full Text] [PDF]
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 P. C. Papageorgiou, A. Pourdjabbar, A. A. Amfilochiadis, E. P. Diamandis, F. Boomsma, and D. H. Osmond Are cardiovascular and sympathoadrenal effects of human "new pressor protein" preparations attributable to human coagulation {beta}-FXIIa? Am J Physiol Heart Circ Physiol, March 1, 2004; 286(3): H837 - H846. [Abstract] [Full Text] [PDF]
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 C. A. Borgono, L. Grass, A. Soosaipillai, G. M. Yousef, C. D. Petraki, D. H. C. Howarth, S. Fracchioli, D. Katsaros, and E. P. Diamandis Human Kallikrein 14: A New Potential Biomarker for Ovarian and Breast Cancer Cancer Res., December 15, 2003; 63(24): 9032 - 9041. [Abstract] [Full Text] [PDF]
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G. M. Yousef, M.-E. Polymeris, L. Grass, A. Soosaipillai, P.-C. Chan, A. Scorilas, C. Borgono, N. Harbeck, B. Schmalfeldt, J. Dorn, et al. Human Kallikrein 5: A Potential Novel Serum Biomarker for Breast and Ovarian Cancer Cancer Res., July 15, 2003; 63(14): 3958 - 3965. [Abstract] [Full Text] [PDF]
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G. M. Yousef, M.-E. Polymeris, G. M. Yacoub, A. Scorilas, A. Soosaipillai, C. Popalis, S. Fracchioli, D. Katsaros, and E. P. Diamandis Parallel Overexpression of Seven Kallikrein Genes in Ovarian Cancer Cancer Res., May 1, 2003; 63(9): 2223 - 2227. [Abstract] [Full Text] [PDF]
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 C. D. Petraki, V. N. Karavana, and E. P. Diamandis Human Kallikrein 13 Expression in Normal Tissues: An Immunohistochemical Study J. Histochem. Cytochem., April 1, 2003; 51(4): 493 - 501. [Abstract] [Full Text] [PDF]
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L.-Y. Luo, D. Katsaros, A. Scorilas, S. Fracchioli, R. Bellino, M. van Gramberen, H. de Bruijn, A. Henrik, U.-H. Stenman, M. Massobrio, et al. The Serum Concentration of Human Kallikrein 10 Represents a Novel Biomarker for Ovarian Cancer Diagnosis and Prognosis Cancer Res., February 15, 2003; 63(4): 807 - 811. [Abstract] [Full Text] [PDF]
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C. Kapadia, A. Chang, G. Sotiropoulou, G. M. Yousef, L. Grass, A. Soosaipillai, X. Xing, D. H.C. Howarth, and E. P. Diamandis Human Kallikrein 13: Production and Purification of Recombinant Protein and Monoclonal and Polyclonal Antibodies, and Development of a Sensitive and Specific Immunofluorometric Assay Clin. Chem., January 1, 2003; 49(1): 77 - 86. [Abstract] [Full Text] [PDF]
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T. Kishi, L. Grass, A. Soosaipillai, C. Shimizu-Okabe, and E. P. Diamandis Human Kallikrein 8: Immunoassay Development and Identification in Tissue Extracts and Biological Fluids Clin. Chem., January 1, 2003; 49(1): 87 - 96. [Abstract] [Full Text] [PDF]
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C. D. Petraki, V. N. Karavana, L.-Y. Luo, and E. P. Diamandis Human Kallikrein 10 Expression in Normal Tissues by Immunohistochemistry J. Histochem. Cytochem., September 1, 2002; 50(9): 1247 - 1261. [Abstract] [Full Text] [PDF]
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C. V. Obiezu, A. Soosaipillai, K. Jung, C. Stephan, A. Scorilas, D. H. C. Howarth, and E. P. Diamandis Detection of Human Kallikrein 4 in Healthy and Cancerous Prostatic Tissues by Immunofluorometry and Immunohistochemistry Clin. Chem., August 1, 2002; 48(8): 1232 - 1240. [Abstract] [Full Text] [PDF]
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 D. A. Groneberg, F. Doring, S. Theis, M. Nickolaus, A. Fischer, and H. Daniel Peptide transport in the mammary gland: expression and distribution of PEPT2 mRNA and protein Am J Physiol Endocrinol Metab, May 1, 2002; 282(5): E1172 - E1179. [Abstract] [Full Text] [PDF]
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E. P. Diamandis, A. Okui, S. Mitsui, L.-Y. Luo, A. Soosaipillai, L. Grass, T. Nakamura, D. J. C. Howarth, and N. Yamaguchi Human Kallikrein 11: A New Biomarker of Prostate and Ovarian Carcinoma Cancer Res., January 1, 2002; 62(1): 295 - 300. [Abstract] [Full Text] |
), seminal plasma (•), and
serum (
) with size-exclusion HPLC.