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Prestige Antibodies® as Tools in Plasma Profiling | Biowire Fall 2011


 

Biowire Fall 2011 — Screening — microRNA Target Identification

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Filling the Need for Cancer Biomarkers

Cancer is a major cause of death in the western world today. Cancer is an all-inclusive term; however, there is considerable heterogeneity among the many types and forms of cancer that exist. Therefore, it is important to classify the type of cancer correctly in order to stratify patients into appropriate treatment programs. However, we lack the targeted strategies necessary to enable physicians to identify markers for the different types, stages, and forms of cancer so they can provide patients individualized care.

There are only a handful of biomarkers used in clinics today. One example is the prostate-specific antigen (PSA). Although not originally intended as a screening marker for prostate cancer, PSA is used as such in some countries. However, only 25–30% of patients with elevated PSA levels can be confirmed to have prostate cancer after biopsy. Thus, there is a high false positive rate for prostate cancer detection.

Prostate cancer is usually a slow-growing disease predominantly occurring in older men. Consequently, many men never have symptoms or undergo therapy, and ultimately die from other causes. One third of prostate cancers are aggressive. The issue stems from the fact that there are no existing markers to distinguish between indolent and more aggressive forms. Therefore, following a positive PSA test, the decision to treat and which treatment to perform is a difficult one. As a result, patients undergo surgery to remove the prostate, even though surgery may not be necessary and often results in negative side effects such as incontinence and impotence.

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Plasma Profiling and the Human Protein Atlas

The Human Protein Atlas (HPA) project was initiated with a goal to generate antibodies covering the whole proteome. This effort resulted in an impressive collection of expression profiles of human proteins in a multitude of normal tissues, cancer cells, and cell lines using the validated Prestige Antibodies as reagents. Today, more than 11,200 human protein-encoding genes have been analyzed, and the data is publicly available through the Human Protein Atlas web portal (www.proteinatlas.org)1,2. The aim of the HPA project is to generate profiles for all human protein-encoded genes by 2015.

This resource of antibodies produced within the HPA now extends into high-throughput biomarker discovery in body fluids. An ongoing effort within the project strives to develop a systematic approach for protein profiling of human plasma in the quest for potential biomarkers. Using antibody suspension bead arrays and heat-induced epitope retrieval, protein profiles of human plasma can be generated with limits of detection into the lower ng/ml ranges3,4,5. The ability of the antibodies to detect their targets in plasma samples was enhanced by heat treatment, most likely due to beneficial exposure of epitopes at elevated temperatures3. The procedure of the antibody suspension bead array is shown in Figure 1.

Prestige Antibodies
Figure 1. Prestige Antibody suspension bead array workflow — Plasma samples are distributed, diluted and directly labeled with amino-reactive biotin A, B). The Prestige Antibodies are normalized to the same concentration and coupled to colorcoded beads (one color-code/Prestige Antibody). The mixed Prestige Antibody bead arrays are formed and then incubated with the heat-treated plasma samples. To determine specific antibodytarget recognition, unbound proteins are washed away C), and a fluorescently labeled reporter molecule (SAPE) is then added for biotin detection. By identifying the color-coded beads via green laser, the reporter fluorescence is excited by a red laser, linking amounts of captured target molecules to a Prestige Antibody D). With this procedure, up to 384 Prestige Antibody-derived protein profiles can be determined in a single sample in parallel and consumes less than 1 μl of undiluted plasma per analysis.

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CNDP1: A Candidate Prostate Cancer Biomarker

Using the plasma profiling procedure described, the protein carnosine dipeptidase 1 (CNDP1) was identified as a potential biomarker. A number of proteins were analyzed in unfractionated human plasma (Figure 2). CNDP1 was further analyzed in plasma from patients with a high and low risk of developing prostate cancer respectively. The level of CNDP1 protein was decreased in plasma from high-risk patients compared to low-risk patients. The differences in CNDP1 protein levels were determined (Figure 3A) from an additional cohort composed of healthy subjects (n=30), patients diagnosed with indolent prostate cancer (n=30), and patients diagnosed with aggressive prostate cancer (n=30). Patients with aggressive prostate cancer had lower plasma levels of the CNDP1 protein than both the healthy controls and the patients diagnosed with the indolent cancer form. Figure 3B shows the difference in CNDP1 level detected in a selection of samples from low- and high-prostate cancer risk patients, as analyzed by a sandwich immunoassay, the preferred clinical methodology for analyzing plasma. Again, there was a lower level of CNDP1 protein in plasma of patients with a high risk for prostate cancer than there was in the low-risk group.

In conclusion, the CNDP1 protein is an interesting candidate marker for prostate cancer that may be used to distinguish between indolent and aggressive forms. Potentially, CNDP1 could be used for screening of aggressive prostate cancer. Differential amounts of CNDP1 can be detected using established techniques utilized in clinical analysis.

Prestige Antibodies
Figure 2. Presence of CNDP1 in human blood was determined by A) immunohistochemical staining of prostate cancer tissue detecting CNDP1 in blood vessels, and by B) Western blot. Lane 1 hosts the marker (230,130,95,72,56,36,28,17,11 kDa), lane 2 RT-4 cell lysate, lane 3 U-251MG sp cell lysate, lane 4 human plasma, lane 5 tonsil lysate and lane 6 liver lysate. In both methods, the Anti-CNDP1 antibody HPA008933 was used for protein detection.

Prestige Antibodies
Figure 3. A) CNDP1 in plasma from three groups of prostate cancer subjects as determined by plasma profiling. The first group represents healthy subjects (n=30), the second group, patients diagnosed with less aggressive prostate cancer (n=30), and the third group, patients diagnosed with aggressive prostate cancer (n=30). B) Results from a sandwich detection assay using the anti-CNDP1 antibody (HPA008933) as capture reagent. Samples 1–3 represent the prostate cancer-free control group (white bars) and samples 4–6 are from patients with an elevated risk for prostate cancer (burgandy bars).

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Summary

For accurate and efficient treatment of cancer patients, there is a great need for novel biomarkers capable of distinguishing between different types, stages, and forms of cancer.

Prestige Antibodies are a great tool in high-throughput plasma profiling efforts.

The Human Protein Atlas and antibody array technologies constitute an excellent platform when searching for new potential cancer biomarkers.

CNDP1 has been found to be an interesting candidate biomarker for prostate cancer that holds the potential to distinguish between indolent and aggressive tumor forms.


Prestige Antibodies
The antibodies developed and characterized within the Human Protein Atlas project are available to the scientific community as Prestige Antibodies. Prestige Antibodies are co-exclusively sold by Sigma-Aldrich and Atlas Antibodies in Europe, and exclusively sold by Sigma-Aldrich outside of Europe.

Discover more at sigma.com/prestige.

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Materials

     

References

  1. Berglund L, et al. A gene-centric human protein atlas for expression profiles based on antibodies. Mol Cell Proteomics. 2008;(10):2019–27.
  2. Uhlén M, et al. Towards a knowledge-based Human Protein Atlas. Nat Biotechnol. 2010;28(12):1248–50.
  3. Schwenk JM, et al. Towards next generation plasma profiling via heat-induced epitope retrieval and arraybased assays. Mol Cell Proteomics. 2010;9(11):2497–507.
  4. Schwenk JM, et al. Comparative protein profiling of serum and plasma using an antibody suspension bead array approach. Proteomics. 2010;10(3):532–40.
  5. Rimini R, et al. Validation of serum protein profiles by a dual antibody array approach. J Proteomics. 2009;73(2):252–66.

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