Dataset Information

Raman nanoparticle probes for antibody-based protein detection in tissues.

ABSTRACT: Surface-enhanced Raman scattering (SERS) nanoparticles are emerging as a new approach for optical detection of biomolecules. In a model assay in formalin-fixed paraffin-embedded (FFPE) prostate tissue sections, we detect prostate-specific antigen (PSA) using antibody (Ab) conjugated to composite organic-inorganic nanoparticles (COINs), and we use identical staining protocols to compare COIN-Ab and Alexa-Ab conjugates in adjacent tissue sections. Spectral analysis illustrates the fundamental difference between fluorescence and Raman signatures and accurately extracts COIN probe signals from background autofluorescence. Probe signals are used to generate images of PSA expression on the tissue, and quality measures are presented to characterize the performance of the COIN assay in comparison to Alexa. Staining accuracy (ability to correctly identify PSA expression in epithelial cells) is somewhat less for COIN than Alexa, which is attributed to an elevated false negative rate of the COIN. However, COIN provided signal intensities comparable to Alexa, and good intra-, inter-, and lot-to-lot consistencies. Overall, COIN and Alexa detection reagents possess similar performance with FFPE tissues, supporting the further development of Raman probes for this application. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

SUBMITTER: Lutz B

PROVIDER: S-EPMC2326103 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Raman nanoparticle probes for antibody-based protein detection in tissues.

Lutz Barry B Dentinger Claire C Sun Lei L Nguyen Lienchi L Zhang Jingwu J Chmura Aj A Allen April A Chan Selena S Knudsen Beatrice B

The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 20071210 4

Surface-enhanced Raman scattering (SERS) nanoparticles are emerging as a new approach for optical detection of biomolecules. In a model assay in formalin-fixed paraffin-embedded (FFPE) prostate tissue sections, we detect prostate-specific antigen (PSA) using antibody (Ab) conjugated to composite organic-inorganic nanoparticles (COINs), and we use identical staining protocols to compare COIN-Ab and Alexa-Ab conjugates in adjacent tissue sections. Spectral analysis illustrates the fundamental diff ...[more]

PMID: 18071064

Similar Datasets

Project description:Accurate antigen detection is imperative for clinicians to diagnose disease, assess treatment success, and predict patient prognosis. The most common technique used for the detection of disease-associated biomarkers is the enzyme linked immunosorbent assay (ELISA). In an ELISA, primary antibodies are incubated with biological samples containing the biomarker of interest. Then, detectible secondary antibodies conjugated with horseradish peroxidase (HRP) bind the primary antibodies. Upon addition of a color-changing substrate, the samples provide a colorimetric signal that directly correlates to the targeted biomarker concentration. While ELISAs are effective for analyzing samples with high biomarker content, they lack the sensitivity required to analyze samples with low antigen levels. We hypothesized that the sensitivity of ELISAs could be enhanced by replacing freely delivered primary antibodies with antibody-nanoparticle conjugates that provide excess binding sites for detectible secondary antibodies, ultimately leading to increased signal. Here, we investigated the use of nanoshells (NS) decorated with antibodies specific to epidermal growth factor receptor (EGFR) as a model system (EGFR-NS). We incubated one healthy and two breast cancer cell lines, each expressing different levels of EGFR, with EGFR-NS, untargeted NS, or unconjugated EGFR antibodies, as well as detectable secondary antibodies. We found that EGFR-NS consistently increased signal intensity relative to unconjugated EGFR antibodies, with a substantial 13-fold enhancement from cells expressing high levels of EGFR. Additionally, 40x more unconjugated antibodies were required to detect EGFR compared to those conjugated to NS. Our results demonstrate that antibody-nanoparticle conjugates lower the detection limit of traditional ELISAs and support further investigation of this strategy with other antibodies and nanoparticles. Owing to their enhanced sensitivity, we anticipate that nanoparticle-modified ELISAs can be used to detect low levels of biomarkers found in various diseases, such as cancers, tuberculosis, and rheumatoid arthritis, and may ultimately enable earlier diagnosis, better prognostication, and improved treatment monitoring.

Project description:OBJECTIVES:There is growing evidence to suggest that human endogenous retroviruses (HERVs) have contributed to human evolution, being expressed in development, normal physiology and disease. A key difficulty in the scientific evaluation of this potential viral contribution is the accurate demonstration of virally expressed protein in specific human cells and tissues. In this study, we have adopted the endogenous retrovirus, ERV3, as our test model in developing a reliable high-capacity methodology for the expression of such endogenous retrovirus-coded protein. DESIGN:Two affinity-purified polyclonal antibodies to ERV3 Env-encoded protein were generated to detect the corresponding protein expression pattern in specific human cells, tissues and organs. PARTICIPANTS:Sampling included normal tissues from 144 individuals ranging from childhood to old age. This included more than forty different tissues and organs and some 216 different cancer tissues representing the twenty commonest forms of human cancer. SETTING:The Rudbeck Laboratory, Uppsala University and Uppsala University Hospital, Uppsala, Sweden. MAIN OUTCOME MEASURES:The potential expression at likely physiological level of the ERV3Env encoded protein in a wide range of human cells, tissues and organs. RESULTS:We found that ERV3 encoded Env protein is expressed at substantive levels in placenta, testis, adrenal gland, corpus luteum, Fallopian tubes, sebaceous glands, astrocytes, bronchial epithelium and the ducts of the salivary glands. Substantive expression was also seen in a variety of epithelial cells as well as cells known to undergo fusion in inflammation and in normal physiology, including fused macrophages, myocardium and striated muscle. This contrasted strongly with the low levels expressed in other tissues types. These findings suggest that this virus plays a significant role in human physiology and may also play a possible role in disease. CONCLUSION:This technique can now be extended to the study of other HERV genomes within the human chromosomes that may have contributed to human evolution, physiology and disease.

Dataset Information

Raman nanoparticle probes for antibody-based protein detection in tissues.

Publications

Raman nanoparticle probes for antibody-based protein detection in tissues.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure