Screening And Profiling Protein Expression In Human Cancer Serum Using Antibody Array Technologies

Antibody arrays have been a promising and inexpensive tool for bulk analysis
of protein level changes in human plasma and serum. These analyses have
lead to proteomic profiling of a number of disease states, as well as biomarker
discovery. Here, we show the value of using a series of Panorama® antibody
microarrays comparing normal and cancer serum samples to identify potential
disease biomarkers. The arrays chosen for this work contained antibodies for
proteins with known significance in intracellular processes. These arrays were
used because it is believed that biomarkers exist in the low abundance tissue
leakage proteins that make up approximately one-third to one-half of the
thousands of proteins found in blood. In our study, we have also highlighted
the contribution that depletion gives to the discovery/validation of the low
abundance tissue leakage proteins on the antibody microarrays.
 
Materials and Methods
Serum Samples
Serum samples were obtained through Genomics Collaborative. Cancer samples
were from either hepatocellular carcinoma (36-year old Vietnamese male) or
renal cell carcinoma (66-year old Caucasian male) patients. Normal samples
were from a 51-year old Caucasian male.
Serum Depletion
Depletion was completed using the ProteoPrep® 20 Immunoaffinity Depletion
Kit (PROT20) and following the supplied protocol.
ELISA Analysis
Serum samples were coated onto 96-well ELISA plates. Plates were incubated
with purified primary antibodies corresponding to those on the arrays. Plates
were washed and incubated with HRP-conjugated secondary antibodies. After
a final wash, the plates were visualized using TMB substrate, and stopped with
1 M HCl. The absorbance was measured at 450 nm.
Serum samples were first depleted of twenty of the high abundance proteins.
The depleted samples were then labeled with either Cy3 or Cy5 (Amersham)
and mixed at equal protein amounts to allow for parallel analysis. The labeled
serum samples were incubated on both the Panorama Antibody Array – p53
Pathways (PPAA4) and the Panorama Antibody Array – Cell Signaling (CSAA1)
for 30 minutes. Following incubation, the slides were scanned using a ScanArray
Express (Perkin Elmer) and analyzed using ImaGene 7.0 software (BioDiscovery).
Each Panorama p53 array was incubated with 100 μg of both depleted and
whole (non-depleted) serum conjugated with either Cy3 or Cy5. A dye swap
was performed to confirm results. In the comparisons, the whole serum is green
and the depleted serum is red. Select proteins are identified. Note that only the
top half of the Cell Signaling array slide is shown. As seen in the comparisons
above, more proteins are visible in the depleted serum than in the whole serum.
The depletion has made the less abundant proteins visible.
 
Following depletion, each Panorama p53 array was simultaneously incubated
with a mixture containing equal amounts of normal and cancer serums (Renal
cancer sample for slide A, Liver cancer for slide B) conjugated with either Cy3
or Cy5. A dye swap was performed to validate results. As seen in the slides, a
number of spots were differentially expressed with the cancer samples when
compared to the normal control. In both comparisons above, the normal serums
are labeled red and the cancer serums are labeled green. Therefore, a red spot
would indicate down-regulation in the cancer sample, and a green spot would
indicate up-regulation in the cancer sample. Select proteins are identified.
Similar results were seen using the Cell Signaling array. Additional proteins
found to be significantly different using the Cell Signaling array include, but
are not limited to: alpha Catenin (up-regulated in both cancer samples), MAP
Kinase (Erk1 + Erk2) (up-regulated in both cancer samples), Calmodulin (upregulated
in the liver cancer sample), Cyclin D1 (down-regulated in the liver
cancer sample), DOPA Decarboxylase (down-regulated in both cancer samples),
and Synculein (down-regulated in the renal cancer sample). 
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