An Automated Microfluidic Assay for Photonic Crystal Enhanced Detection and Analysis of an Antiviral Antibody Cancer Biomarker in Serum.

We report on the implementation of an automated platform for detecting the presence of an antibody biomarker for human papillomavirus-associated oropharyngeal cancer from a single droplet of serum, in which a nanostructured photonic crystal surface is used to amplify the output of a fluorescence-linked immunosorbent assay. The platform is comprised of a microfluidic cartridge with integrated photonic crystal chips that interfaces with an assay instrument that automates the introduction of reagents, wash steps, and surface drying. Upon assay completion, the cartridge interfaces with a custom laser-scanning instrument that couples light into the photonic crystal at the optimal resonance condition for fluorescence enhancement. The instrument is used to measure the fluorescence intensity values of microarray spots corresponding to the biomarkers of interest, in addition to several experimental controls that verify correct functioning of the assay protocol. In this work, we report both dose-response characterization of the system using anti-E7 antibody introduced at known concentrations into serum and characterization of a set of clinical samples from which results were compared with a conventional enzyme-linked immunosorbent assay (ELISA) performed in microplate format. The demonstrated capability represents a simple, rapid, automated, and high-sensitivity method for multiplexed detection of protein biomarkers from a low-volume test sample.

Susceptibility to quantum dot induced lung inflammation differs widely among the Collaborative Cross founder mouse strains.

Quantum dots (QDs) are engineered semiconductor nanoparticles with unique physicochemical properties that make them potentially useful in clinical, research and industrial settings. However, a growing body of evidence indicates that like other engineered nanomaterials, QDs have the potential to be respiratory hazards, especially in the context of the manufacture of QDs and products containing them, as well as exposures to consumers using these products. The overall goal of this study was to investigate the role of mouse strain in determining susceptibility to QD-induced pulmonary inflammation and toxicity. Male mice from 8 genetically diverse inbred strains (the Collaborative Cross founder strains) were exposed to CdSe-ZnS core-shell QDs stabilized with an amphiphilic polymer. QD treatment resulted in significant increases in the percentage of neutrophils and levels of cytokines present in bronchoalveolar lavage fluid (BALF) obtained from NOD/ShiLtJ and NZO/HlLtJ mice relative to their saline (Sal) treated controls. Cadmium measurements in lung tissue indicated strain-dependent differences in disposition of QDs in the lung. Total glutathione levels in lung tissue were significantly correlated with percent neutrophils in BALF as well as with lung tissue Cd levels. Our findings indicate that QD-induced acute lung inflammation is mouse strain dependent, that it is heritable, and that the choice of mouse strain is an important consideration in planning QD toxicity studies. These data also suggest that formal genetic analyses using additional strains or recombinant inbred strains from these mice could be useful for discovering potential QD-induced inflammation susceptibility loci.

A halotyrosine antibody that detects increased protein modifications in asthma patients.

Airway inflammation has a pathophysiological role in asthma. Eosinophils, which are commonly increased in asthmatic airways, express eosinophil peroxidase and thereby produce hypobromite and bromotyrosine. Bromotyrosine is believed to be a specific marker for eosinophil activity, but developing an antibody against monobromotyrosine, the predominant brominated tyrosine residue found in vivo has proven difficult. We evaluated whether a 3-bromobenozoic acid hapten antigen produced antibodies that recognized halogenated tyrosine residues. Studies with small-molecule inhibitors or brominated or chlorinated protein suggested that a mouse monoclonal antibody (BTK-94C) selectively bound free and protein mono- and dibromotyrosine and, to a lesser degree, chlorotyrosine, and thus was designated a general halotyrosine antibody. We evaluated if this antibody had potential for characterizing human asthma using an enzyme-linked immunosorbent assay (ELISA) microarray platform to examine the halogenation of 23 proteins in three independent sets of sputum samples (52 samples total). In 15 healthy control or asthmatic subjects, ICAM, PDGF and RANTES had greater proportional amounts of halogenation in asthmatic subjects and the halogenation signal was associated with the severity of exercise-induced airway hyperresponsiveness. In 17 severe asthma patients treated with placebo or mepolizumab to suppress eosinophils, drug-related decreases in halogenation were observed with p values ranging from 0.006 to 0.11 for these 3 proteins. Analysis of 20 subjects that either had neutrophilic asthma or were healthy controls demonstrated a broad increase in halotyrosine (possibly chlorotyrosine) in neutrophilic asthmatics. Overall, these results suggest that an ELISA utilizing BTK-94C could prove useful for assessing airway inflammation in asthma patients.

Oxidatively modified proteins as plasma biomarkers in breast cancer.

BACKGROUND: Post-translational protein modifications (PTMs) are increased in breast tumors. OBJECTIVE: We explored whether PTMs on proteins secreted by the breast could be detected in plasma and potentially used for the early detection of breast cancer. METHODS: We used a custom ELISA microarray platform to measure 4-hydroxynonenal (HNE), glutathione (GSH), nitrotyrosine and halotyrosine adducts in 27 secreted proteins, for a total of 108 candidate biomarkers. Two independent sets of human plasma samples were measured, for a total of 160 samples. The results were analyzed for consistent cancer-associated changes across the two sample sets. Plasma samples for both cases and benign controls were collected at the time of tissue diagnosis after referral from a positive screen (such as mammography). The results from both studies were evaluated using ANOVA and t-tests or receiver operator curves (ROC). RESULTS: Levels of GSH-modified ceruloplasmin and HNE-modified PDGF were significantly altered in plasma samples from cancer patients relative to benign controls. Healthy controls, which were only included in the first set of samples, were similar to the benign controls for both of these markers. A combination of three glutathionylated proteins produced the best area under the ROC curve, with a value of 76%. CONCLUSIONS: Specific PTMs in individual proteins may be useful for distinguishing between women with breast cancer and those with benign breast disease. These oxidative changes in plasma proteins may reflect redox changes in breast cancer. Additional studies on oxidative modifications in individual proteins are warranted.

Diet-induced obesity reprograms the inflammatory response of the murine lung to inhaled endotoxin.

The co-occurrence of environmental factors is common in complex human diseases and, as such, understanding the molecular responses involved is essential to determine risk and susceptibility to disease. We have investigated the key biological pathways that define susceptibility for pulmonary infection during obesity in diet-induced obese (DIO) and regular weight (RW) C57BL/6 mice exposed to inhaled lipopolysaccharide (LPS). LPS induced a strong inflammatory response in all mice as indicated by elevated cell counts of macrophages and neutrophils and levels of proinflammatory cytokines (MDC, MIP-1γ, IL-12, RANTES) in the bronchoalveolar lavage fluid. Additionally, DIO mice exhibited 50% greater macrophage cell counts, but decreased levels of the cytokines, IL-6, TARC, TNF-α, and VEGF relative to RW mice. Microarray analysis of lung tissue showed over half of the LPS-induced expression in DIO mice consisted of genes unique for obese mice, suggesting that obesity reprograms how the lung responds to subsequent insult. In particular, we found that obese animals exposed to LPS have gene signatures showing increased inflammatory and oxidative stress response and decreased antioxidant capacity compared with RW. Because signaling pathways for these responses can be common to various sources of environmentally induced lung damage, we further identified biomarkers that are indicative of specific toxicant exposure by comparing gene signatures after LPS exposure to those from a parallel study with cigarette smoke. These data show obesity may increase sensitivity to further insult and that co-occurrence of environmental stressors result in complex biosignatures that are not predicted from analysis of individual exposures.

Photonic crystal enhanced fluorescence for early breast cancer biomarker detection.

Photonic crystal surfaces offer a compelling platform for improving the sensitivity of surface-based fluorescent assays used in disease diagnostics. Through the complementary processes of photonic crystal enhanced excitation and enhanced extraction, a periodic dielectric-based nanostructured surface can simultaneously increase the electric field intensity experienced by surface-bound fluorophores and increase the collection efficiency of emitted fluorescent photons. Through the ability to inexpensively fabricate photonic crystal surfaces over substantial surface areas, they are amenable to single-use applications in biological sensing, such as disease biomarker detection in serum. In this review, we will describe the motivation for implementing high-sensitivity, multiplexed biomarker detection in the context of breast cancer diagnosis. We will summarize recent efforts to improve the detection limits of such assays though the use of photonic crystal surfaces. Reduction of detection limits is driven by low autofluorescent substrates for photonic crystal fabrication, and detection instruments that take advantage of their unique features.

Line-scanning detection instrument for photonic crystal enhanced fluorescence.

A laser line-scanning instrument was developed to optimize the near-field enhancement capability of a one-dimensional photonic crystal (PC) for excitation of surface-bound fluorophores. The excitation laser beam is shaped into an 8 µm × 1 mm line that is focused along the direction of the PC grating, while remaining collimated perpendicular to the grating. Such a beam configuration offers high excitation power density while simultaneously providing high resonant coupling efficiency from the laser to the PC surface. Using a panel of 21 immunofluorescence assays on the PC surface in a microarray format, the approach achieves an enhancement factor as high as 90-fold between on-resonance and off-resonance illumination. The instrument provides a capability for sensitive and inexpensive analysis of cancer biomarkers in clinical applications.

High-throughput, multiplexed analysis of 3-nitrotyrosine in individual proteins.

Reactive nitrogen species (RNS) and reactive oxygen species (ROS) are derived as a result of inflammation and oxidative stress and can result in protein modifications. As such, these protein modifications are used as biomarkers for inflammation and oxidative stress. In addition, modifications in single-tissue-associated proteins released into blood can provide insight into the tissue localization of the inflammation or oxidative stress. We have developed an enzyme-linked immunosorbent assay antibody microarray platform to analyze the levels of 3-nitrotyrosine in specific proteins in a variety of biological samples, including human plasma and sputum. Selective-capture antibodies are used to immunoprecipitate individual proteins from samples onto isolated spots on the microarray chips. Then, a monoclonal antibody for 3-nitrotyrosine is used to detect the amount of 3-nitrotyrosine on each spot. Our studies suggest that this approach can be used to detect trace amounts of 3-nitrotyrosine in human plasma and sputum. In this paper, we describe our antibody microarray protocol for detecting 3-nitrotyrosine in specific proteins.

Multiplexed cancer biomarker detection using quartz-based photonic crystal surfaces.

A photonic crystal (PC) surface is demonstrated as a high-sensitivity platform for detection of a panel of 21 cancer biomarker antigens using a sandwich enzyme-linked immunosorbent assay (ELISA) microarray format. A quartz-based PC structure fabricated by nanoimprint lithography, selected for its low autofluorescence, supports two independent optical resonances that simultaneously enable enhancement of fluorescence detection of biomarkers and label-free quantification of the density of antibody capture spots. A detection instrument is demonstrated that supports fluorescence and label-free imaging modalities, with the ability to optimize the fluorescence enhancement factor on a pixel-by-pixel basis throughout the microarray using an angle-scanning approach for the excitation laser that automatically compensates for variability in surface chemistry density and capture spot density. Measurements show that the angle-scanning illumination approach reduces the coefficient of variation of replicate assays by 20-99% compared to ordinary fluorescence microscopy, thus supporting reduction in limits of detectable biomarker concentration. Using the PC resonance, biomarkers in mixed samples were detectable at the lowest concentrations tested (2.1-41 pg/mL), resulting in a three-log range of quantitative detection.

Plasma biomarkers for detecting Hodgkin's lymphoma in HIV patients.

The lifespan of people with human immunodeficiency virus (HIV) infection has increased as a result of effective antiretroviral therapy, and the incidences of the AIDS-defining cancers, non-Hodgkin's lymphoma and Kaposi sarcoma, have declined. Even so, HIV-infected individuals are now at greater risk of other cancers, including Hodgkin's lymphoma (HL). To identify candidate biomarkers for the early detection of HL, we undertook an accurate mass and elution time tag proteomics analysis of individual plasma samples from either HIV-infected patients without HL (controls; n = 14) and from HIV-infected patient samples with HL (n = 22). This analysis identified 60 proteins that were statistically (p<0.05) altered and at least 1.5-fold different between the two groups. At least three of these proteins have previously been reported to be altered in the blood of HL patients that were not known to be HIV positive, suggesting that these markers may be broadly useful for detecting HL. Ingenuity Pathway Analysis software identified "inflammatory response" and "cancer" as the top two biological functions associated with these proteins. Overall, this study validated three plasma proteins as candidate biomarkers for detecting HL, and identified 57 novel candidate biomarkers that remain to be validated. The relationship of these novel candidate biomarkers with cancer and inflammation suggests that they are truly associated with HL and therefore may be useful for the early detection of this cancer in susceptible populations.

Reactive oxygen species alter autocrine and paracrine signaling.

Cytochrome P450 (P450) 3A4 (CYP3A4) is the most abundant P450 protein in human liver and intestine and is highly inducible by a variety of drugs and other compounds. The P450 catalytic cycle is known to uncouple and release reactive oxygen species (ROS), but the effects of ROS from P450 and other enzymes in the endoplasmic reticulum have been poorly studied from the perspective of effects on cell biology. In this study, we expressed low levels of CYP3A4 in HepG2 cells, a human hepatocarcinoma cell line, and examined effects on intracellular levels of ROS and on the secretion of a variety of growth factors that are important in extracellular communication. Using the redox-sensitive dye RedoxSensor red, we demonstrate that CYP3A4 expression increases levels of ROS in viable cells. A custom ELISA microarray platform was employed to demonstrate that expression of CYP3A4 increased secretion of amphiregulin, intracellular adhesion molecule 1, matrix metalloprotease 2, platelet-derived growth factor (PDGF), and vascular endothelial growth factor, but suppressed secretion of CD14. The antioxidant N-acetylcysteine suppressed all P450-dependent changes in protein secretion except for CD14. Quantitative RT-PCR demonstrated that changes in protein secretion were consistently associated with corresponding changes in gene expression. Inhibition of the NF-κB pathway blocked P450 effects on PDGF secretion. CYP3A4 expression also altered protein secretion in human mammary epithelial cells and C10 mouse lung cells. Overall, these results suggest that increased ROS production in the endoplasmic reticulum alters the secretion of proteins that have key roles in paracrine and autocrine signaling.

Reducing heterophilic antibody interference in immunoassays using single-chain antibodies.

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays can simultaneously quantify the levels of multiple diagnostic targets in a biological sample. However, as with traditional ELISA diagnostics, endogenous antibodies in patient sera can cause interference. We demonstrate here that reducing the diagnostic capture antibody to its minimal functional unit (i.e., a single-chain antibody fragment [scFv]) is an effective strategy for reducing assay interference. Our finding illustrates a source of error introduced by the reliance on immunoglobulin-based capture reagents in sandwich immunoassays with human serum samples. We demonstrate that scFvs can be used in such assays to improve reliability by reducing heterophilic antibody interference, thereby improving biomarker analysis and validation.

Smoking, COPD, and 3-nitrotyrosine levels of plasma proteins.

BACKGROUND: Nitric oxide is a physiological regulator of endothelial function and hemodynamics. Oxidized products of nitric oxide can form nitrotyrosine, which is a marker of nitrative stress. Cigarette smoking decreases exhaled nitric oxide, and the underlying mechanism may be important in the cardiovascular toxicity of smoking. Even so, it is unclear if this effect results from decreased nitric oxide production or increased oxidative degradation of nitric oxide to reactive nitrating species. These two processes would be expected to have opposite effects on nitrotyrosine levels, a marker of nitrative stress. OBJECTIVE: In this study, we evaluated associations of cigarette smoking and chronic obstructive pulmonary disease (COPD) with nitrotyrosine modifications of specific plasma proteins to gain insight into the processes regulating nitrotyrosine formation. METHODS: A custom antibody microarray platform was developed to analyze the levels of 3-nitrotyrosine modifications on 24 proteins in plasma. In a cross-sectional study, plasma samples from 458 individuals were analyzed. RESULTS: Average nitrotyrosine levels in plasma proteins were consistently lower in smokers and former smokers than in never smokers but increased in smokers with COPD compared with smokers who had normal lung-function tests. CONCLUSIONS: Smoking is associated with a broad decrease in 3-nitrotyrosine levels of plasma proteins, consistent with an inhibitory effect of cigarette smoke on endothelial nitric oxide production. In contrast, we observed higher nitrotyrosine levels in smokers with COPD than in smokers without COPD. This finding is consistent with increased nitration associated with inflammatory processes. This study provides insight into a mechanism through which smoking could induce endothelial dysfunction and increase the risk of cardiovascular disease.

Plasma biomarker profiles differ depending on breast cancer subtype but RANTES is consistently increased.

BACKGROUND: Current biomarkers for breast cancer have little potential for detection. We determined whether breast cancer subtypes influence circulating protein biomarkers. METHODS: A sandwich ELISA microarray platform was used to evaluate 23 candidate biomarkers in plasma samples that were obtained from subjects with either benign breast disease or invasive breast cancer. All plasma samples were collected at the time of biopsy, after a referral due to a suspicious screen (e.g., mammography). Cancer samples were evaluated on the basis of breast cancer subtypes, as defined by the HER2 and estrogen receptor statuses. RESULTS: Ten proteins were statistically altered in at least one breast cancer subtype, including four epidermal growth factor receptor ligands, two matrix metalloproteases, two cytokines, and two angiogenic factors. Only one cytokine, RANTES, was significantly increased (P < 0.01 for each analysis) in all four subtypes, with areas under the curve (AUC) for receiver operating characteristic values that ranged from 0.76 to 0.82, depending on cancer subtype. The best AUC values were observed for analyses that combined data from multiple biomarkers, with values ranging from 0.70 to 0.99, depending on the cancer subtype. Although the results for RANTES are consistent with previous publications, the multi-assay results need to be validated in independent sample sets. CONCLUSIONS: Different breast cancer subtypes produce distinct biomarker profiles, and circulating protein biomarkers have potential to differentiate between true- and false-positive screens for breast cancer. IMPACT: Subtype-specific biomarker panels may be useful for detecting breast cancer or as an adjunct assay to improve the accuracy of current screening methods.

Development of a fibrinogen-specific sandwich enzyme-linked immunosorbent assay microarray assay for distinguishing between blood plasma and serum samples.

We have developed a fibrinogen-specific sandwich enzyme-linked immunosorbent assay (ELISA) microarray assay for use in qualitatively distinguishing between blood plasma and serum samples. Three capture antibodies (49D2, HPA001900, and F8512) were evaluated in conjunction with 1D6 as the detection antibody. The data show that 49D2 and (to a lesser extent) F8512 successfully identify previously unknown plasma and serum samples based on approximately a 28-fold difference in signal intensity between the sample types. This assay has utility in rapidly identifying previously archived clinical samples with incomplete annotation in a high-throughput manner prior to proteomic analyses.

Protein secretion in human mammary epithelial cells following HER1 receptor activation: influence of HER2 and HER3 expression.

BACKGROUND: Protein secretion by mammary cells results in autocrine and paracrine signaling that defines cell growth, migration and the extracellular environment. Even so, we have a limited understanding of the cellular processes that regulate protein secretion. METHODS: In this study, we utilize human epithelial mammary cell (HMEC) lines that were engineered to express different levels of HER1, HER2 and HER3. Using an ELISA microarray platform, we evaluate the effects of epidermal growth factor family receptor (HER) expression on protein secretion in the HMEC lines upon initiation of HER1 receptor activation. The secreted proteins include three HER1 ligands, interleukins 1α and 18, RANTES, vascular-endothelial and platelet-derived growth factors, matrix metalloproteases 1, 2 and 9, and the extracellular portion of the HER1 and HER2 proteins. In addition, we investigate whether MAPK/Erk and PI3K/Akt signaling regulate protein secretion in these cell lines and if so, whether the involvement of HER2 or HER3 receptor alters their response to MAPK/Erk and PI3K/Akt signal pathway inhibition in terms of protein secretion. RESULTS: Differential expression of HER2 and HER3 receptors alters the secretion of a variety of growth factors, cytokines, and proteases. Some alterations in protein secretion are still observed when MAPK/Erk or PI3K/Akt signaling is inhibited. CONCLUSION: This study suggests that HER overexpression orchestrates broad changes in the tumor microenvironment by altering the secretion of a diverse variety of biologically active proteins.

Application of photonic crystal enhanced fluorescence to cancer biomarker microarrays.

We report on the use of photonic crystal surfaces as a high-sensitivity platform for detection of a panel of cancer biomarkers in a protein microarray format. The photonic crystal surface is designed to provide an optical resonance at the excitation wavelength of cyanine-5 (Cy5), thus providing an increase in fluorescent intensity for Cy5-labeled analytes measured with a confocal microarray scanner, compared to a glass surface. The sandwich enzyme-linked immunosorbent assay (ELISA) is undertaken on a microarray platform to undertake a simultaneous, multiplex analysis of 24 antigens on a single chip. Our results show that the resonant excitation effect increases the signal-to-noise ratio by 3.8- to 6.6-fold, resulting in a decrease in detection limits of 6-89%, with the exact enhancement dependent upon the antibody-antigen interaction. Dose-response characterization of the photonic crystal antibody microarrays shows the capability to detect common cancer biomarkers in the <2 pg/mL concentration range within a mixed sample.

Cellular recognition and trafficking of amorphous silica nanoparticles by macrophage scavenger receptor A.

The cellular uptake of engineered nanoparticles (ENPs) is known to involve active transport mechanisms, yet the biological molecules involved are poorly understood. We demonstrate that the uptake of amorphous silica ENPs by macrophage cells, and the secretion of proinflammatory cytokines, is strongly inhibited by silencing expression of scavenger receptor A (SR-A). Conversely, ENP uptake is augmented by introducing SR-A expression into human cells that are normally non-phagocytic. Confocal microscopy analyses show that the majority of single or small clusters of silica ENPs co-localize with SR-A and are internalized through a pathway characteristic of clathrin-dependent endocytosis. In contrast, larger silica ENP agglomerates (>500 nm) are poorly co-localized with the receptor, suggesting that the physical agglomeration state of an ENP influences its cellular trafficking. As SR-A is expressed in macrophages throughout the reticulo-endothelial system, this pathway is likely an important determinant of the biological response to ENPs.

Analysis of high-throughput ELISA microarray data.

Our research group develops analytical methods and software for the high-throughput analysis of quantitative enzyme-linked immunosorbent assay (ELISA) microarrays. ELISA microarrays differ from DNA microarrays in several fundamental aspects and most algorithms for analysis of DNA microarray data are not applicable to ELISA microarrays. In this review, we provide an overview of the steps involved in ELISA microarray data analysis and how the statistically sound algorithms we have developed provide an integrated software suite to address the needs of each data-processing step. The algorithms discussed are available in a set of open-source software tools (http://www.pnl.gov/statistics/ProMAT).

Antibody microarrays for high-throughput, multianalyte analysis.

Enzyme-linked immunosorbent assay (ELISA) microarray technology promises to be a powerful tool for detecting and validating protein biomarkers, especially panels of biomarkers. ELISA microarrays are capable of high-throughput analysis of multiple proteins using small sample volumes. In this chapter we review the literature on the use of antibody microarrays for biomarker discovery and validation. We also described the methodologies we employ to obtain high-quality data through protocol optimization and data calibration.