Reporter Dye Research Articles

Abstract 3065: Targeting of metabolic reprogramming in lung cancer by utilizing patient-derived organoid tissue for precision medicine

Abstract In a preliminary pilot study, we reported elevated activity of indoleamine 2,3-dioxygenase 1 (IDO1) in serum from cisplatin-resistant (CR) lung cancer patients compared to cisplatin-sensitive (CS) patients. This heightened IDO1 activity was associated with increased tryptophan (TRP) catabolism, resulting in the upregulation of kynurenine (KYN) production. The detection of KYN can now be readily achieved through immunohistochemistry (IHC). We demonstrated that this upregulation contributed to increased Treg (regulatory T cell) and MDSC (myeloid-derived suppressor cell) populations resulting in an immunosuppressive tumor microenvironment. To investigate this effect, we used dual inhibition of IDO1/TDO2 (tryptophan2,3-dioxygenase 2) in an orthotopic mouse model of CR-Lewis lung carcinoma. This resulted in a significant reduction in tumor volume compared to LLC-CS (n=7, p<0.01), confirming the role of TRP catabolism in CR tumorigenesis. To determine the relevance of the data derived from this mouse model to clinical settings, we established 50 patient-derived organoid tissues (PDOT) from patients with NSCLC. PDOT is an experimental ex vivo tissue model developed to recapitulate the intricate features of lung architecture, including the lung acinus. Upon optimizing our collection and growth protocols, each PDOT successfully developed into a spherical body within 14 days. Furthermore, through bulk RNA-seq analysis, we showed that PDOT closely mirrors the expression of key molecular markers (PD-L1 and mutations), as observed in the original tumor tissues. PDOT from patients possessing high levels of PD-L1 and KYN were exquisitely sensitive to dual inhibition with 2-3-fold greater sensitivity compared to patients with undetectable KYN expression in growth inhibition assays (GI50=20±5.5μM vs. 65±7.5μM). Increased lipid peroxidation was detected via flow cytometry by accumulation of the C11-BODIPY reporter dye in the dual inhibition treatment group. Moreover, we integrated PDOT with a novel 3D microfluidic culture that can be supplied with patient-matched peripheral blood mononuclear cells (PBMC) to assess immune response. Using CyTOF, we showed that dual inhibitors with Atezolizumab (anti-PD-L1) enhanced immune effector (CD8+, NK) and suppressed immunosuppressive (Treg, MDSC) populations. We believe that our swift progress in PDOT development may bridge the divide between cancer biomarker expression and patient trials by timely complementing conventional drug studies based on cell lines and xenografts to facilitate the design of personalized therapies. As a result, the extensive reporting that we have contributed for KYN expression profiling in CR has the potential to serve as a valuable companion diagnostic tool in the future. This can assist in applying precision oncology by finding the most appropriate patients for immunotherapies. Citation Format: Chunjing Wu, Jonathan D. Nguyen, George Theodore, Estelamari Rodriguez, Sydney Spector, Emily Kim, Taranatee Khan, Pablo Puente, Yujie Wang, Dao M. Nguyen, Cheng-Bang Chen, Diane Lim, Lynn G. Feun, Niramol Savaraj, Medhi Wangpaichitr. Targeting of metabolic reprogramming in lung cancer by utilizing patient-derived organoid tissue for precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3065.

Analysis of individual nanoscale block copolymer vesicles by atomic force microscopy combined with time-resolved fluorescence microscopy

Abstract We report on the analysis of individual dye loaded block copolymer (BCP) vesicles via a combination of confocal laser scanning microscopy (CSLM)/fluorescence lifetime imaging microscopy (FLIM) on the one hand and atomic force microscopy (AFM) on the other hand. AFM measurements on ATTO 647N-loaded poly(styrene-block-poly(acrylic acid)) (PS115-b-PAA15) vesicles were carried out with high spatial resolution and afforded morphology and dimensions of BCP vesicles on solid substrates. By contrast the CSLM and FLIM data are diffraction limited, but from the time resolved fluorescence data information on the local vicinity of the reporter dye can be extracted. In the combined experiment individual dye-loaded vesicles and vesicle aggregates were discerned, analyzed metrologically by AFM and in more detail by CSLM and FLIM. On the basis of FLIM data the partitioning of the reporter dye was analyzed. The dye resides preferentially in the hydrophilic corona inside the vesicles. The dye concentration in the polymersome was about 90 times higher than in the solution used for encapsulation. These results underline that the combination of AFM with sensitive optical techniques, especially FLIM, is a promising approach for obtaining a deeper understanding of molecular interactions and nanoenvironments in supramolecular macromolecular structures and beyond.

Rapid detection of monkeypox virus and differentiation of West African and Congo Basin strains using endonuclease restriction-mediated real-time PCR-based testing.

The ongoing multi-country outbreak of monkeypox virus (MPXV) has continuously attracted global attention, highlighting the critical need for timely and accurate methods to detect MPXV and differentiate its clades. Herein, we devised a novel multiplex ET-PCR (endonuclease restriction-mediated real-time PCR) assay that integrates PCR amplification, restriction endonuclease cleavage and real-time fluorescence detection to diagnose MPXV infection and distinguish the Congo Basin and West African MPXV strains. In the MPXV ET-PCR system, three sets of specific primers were designed for MPXV, Congo Basin and West African strains. A short sequence, which could be recognized by restriction endonuclease enzyme BstUI, was added to the 5'end of amplification primers. Then, the modified primers were assigned different reporter dyes and corresponding quenching dyes to each of the three targets, enabling real-time fluorescence reporting of the results and multiplex detection. The designed assay enabled the detection of single or three targets in a single tube, with excellent specificity and analytical sensitivity in terms of plasmid and pseudotyped virus. Moreover, the clinical feasibility of our assay was validated using artificially simulated plasma, nasopharyngeal swab and skin swab samples. In conclusion, the multiplex ET-PCR assay devised here had the advantages of simple primer design, cost-effectiveness, low contamination risk, excellent sensitivity, high specificity and multiplex detection, making it a valuable and dependable tool for curbing the extensive spread of MPXV.

Mixed host co-assembled systems for broad-scope analyte sensing.

Here we report a systems chemistry oriented approach for developing information-rich mixed host chemosensors. We show that co-assembling macrocyclic hosts from different classes, DimerDye sulfonatocalix[4]arenes and cucurbit[n]urils, effectively increases the scope of analyte binding interactions and therefore, sensory outputs. This simple dynamic strategy exploits cross-reactive noncovalent host-host complexation interactions while integrating a reporter dye, thereby producing emergent photophysical responses when an analyte interacts with either host. We first demonstrate the advantages of mixed host co-assembled chemosensors through an increased detection range of hydrophobic, cationic, neutral, and anionic drugs. We then implement mixed host sensors in an array-based platform for the differentiation of illicit drugs, including cannabinoids, benzodiazepine analogs, opiates, anesthetics, amphetamine, and common adulterating substances. Finally, the potential of this approach is applied to profiling real-world multi-component illicit street drug samples, proving to be more effective than classical sensor arrays.

Mitochondrial temperature homeostasis resists external metabolic stresses

Based on studies with a fluorescent reporter dye, Mito Thermo Yellow (MTY), and the genetically encoded gTEMP ratiometric fluorescent temperature indicator targeted to mitochondria, the temperature of active mitochondria in four mammalian and one insect cell line was estimated to be up to 15°C above that of the external environment to which the cells were exposed. High mitochondrial temperature was maintained in the face of a variety of metabolic stresses, including substrate starvation or modification, decreased ATP demand due to inhibition of cytosolic protein synthesis, inhibition of the mitochondrial adenine nucleotide transporter and, if an auxiliary pathway for electron transfer was available via the alternative oxidase, even respiratory poisons acting downstream of oxidative phosphorylation (OXPHOS) complex I. We propose that the high temperature of active mitochondria is an inescapable consequence of the biochemistry of OXPHOS and is homeostatically maintained as a primary feature of mitochondrial metabolism.

Mitochondrial temperature homeostasis resists external metabolic stresses.

Based on studies with a fluorescent reporter dye, Mito Thermo Yellow (MTY), and the genetically encoded gTEMP ratiometric fluorescent temperature indicator targeted to mitochondria, the temperature of active mitochondria in four mammalian and one insect cell line was estimated to be up to 15°C above that of the external environment to which the cells were exposed. High mitochondrial temperature was maintained in the face of a variety of metabolic stresses, including substrate starvation or modification, decreased ATP demand due to inhibition of cytosolic protein synthesis, inhibition of the mitochondrial adenine nucleotide transporter and, if an auxiliary pathway for electron transfer was available via the alternative oxidase, even respiratory poisons acting downstream of oxidative phosphorylation (OXPHOS) complex I. We propose that the high temperature of active mitochondria is an inescapable consequence of the biochemistry of OXPHOS and is homeostatically maintained as a primary feature of mitochondrial metabolism.

A strip-based assay for detection of CrfA enzyme activity to differentiate Mycobacterium tuberculosis and non-tuberculous mycobacteria

There is an unmet need for tools that permit diagnosis of Tuberculosis (TB) that are affordable, low-tech, and can differentiate Mycobacterium tuberculosis (M.tb) from non-tuberculous mycobacteria (NTM). In this study, we have developed a strip-based assay to detect the activity of a unique Carbapenem Resistance Factor A (CrfA) enzyme present only in M.tb. The strip comprises of PVDF (Polyvinylidene fluoride) membrane that has an immobilized anti-CrfA antibody to capture the CrfA enzyme from M.tb lysate. Lysate of mycobacteria is applied to the strip, washed, and incubated in the presence of chromogenic reporter dye which is a substrate for CrfA. A change in the color of the dye that is readily visible to the naked eye is the readout. We evaluated lysates from M.tb and various NTMs namely, M. abscessus, M. chelonae, M. avium, M. obuense, M. paraintracellulare, M. kansasi, including the patient-derived sputum samples. The strip assay selectively identified only those samples containing M.tb. Based on this evidence, this new assay enables the identification and differentiation of M.tb from NTMs in patient sputum samples. As this tool can be simple to use, therefore has the potential to serve the unmet need for diagnosis of TB and NTM infections in resource-limited settings.

New generation of fluorescent probes for cell-based measurements of caspase Activation and mitochondrial superoxide

Abstract Cell health and stress readouts are critical indicators of altered or impaired function in normal and diseased states of cells, and work has been underway to develop improved small molecule sensor dyes compatible with traditional imaging and High Content Analysis (HCA) interrogation of apoptotic and mitochondrial stress pathways. The CellEvent™ Caspase Green dye effectively reports caspase activation, but suffers complications in assay configuration when attempting to multiplex with the Green Fluorescent Protein (GFP), calcein, or other 488 laser line tools in fluorescence microscopy. Here, we describe the testing and functional characterization of a new candidate molecule for measuring apoptosis in living cells. Our sensor is comprised of a fluorogenic reporter dye that is liberated from a DEVD peptide substrate by caspase activation, with ex/em spectra of 590/610 nm, permitting easy multiplex with GFP or calcein stained neurons in both traditional and HCA microscopy configurations. MitoSOX™ Green that has specificity to superoxide like MitoSOX™ Red dye. In addition, it has better spectral properties and works with GFP filters in microscopy, and allows for measurement of mitochondrial superoxide in RFP-expressing cells. MitoSOX™ Green Mitochondrial Superoxide Indicator also localizes to mitochondria of live cells and selectively reports superoxide generation, while ignoring other ROS and RNS species in ex vivo testing. With an Excitation/Emission profile in the GFP/FITC microscopy channel, a series of comparative studies in immortalized and neural cells are shown, highlighting photostability, specificity and signal amplitude from the dye. These reagents are research use only, not for diagnostic purposes

Abstract 4781: New generation of fluorescent probes for cell-based measurements of caspase activation and mitochondrial superoxide

Abstract Cell health and stress readouts are critical indicators of altered or impaired function in normal and diseased states of cells, and work has been underway to develop improved small molecule sensor dyes compatible with traditional imaging and High Content Analysis (HCA) interrogation of apoptotic and mitochondrial stress pathways. The CellEvent™ Caspase Green dye effectively reports caspase activation, but suffers complications in assay configuration when attempting to multiplex with the Green Fluorescent Protein (GFP), calcein, or other 488 laser line tools in fluorescence microscopy. Here, we describe the testing and functional characterization of a new candidate molecule for measuring apoptosis in living cells. Our sensor is comprised of a fluorogenic reporter dye that is liberated from a DEVD peptide substrate by caspase activation, but operates in the Texas Red, 590nm excitation band, with an emission peak near 610 nm, permitting easy multiplex with GFP or calcein stained neurons in both traditional and HCA microscopy configurations. Similarly, mitochondrial superoxide accompanying cell stress is probed in microscopy with the MitoSOX™ Red Mitochondrial Superoxide Indicator dye, which localizes to mitochondria and reports superoxide generation, ignoring other Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS). This dye has an unusually long Stokes’ shift, requiring specialized microscopy and HCA filters that excite at 405nm, and capture emission at 610nm for specific superoxide detection. This unconventional spectroscopic profile prevents the dye’s use on many imaging platforms and promotes phototoxicity. To this end, our team has produced a dye with the same level of specificity for superoxide that will operate in one of the traditional fluorescence microscopy channels. Our candidate dye, here named MitoSOX™ Green Mitochondrial Superoxide Indicator also localizes to mitochondria of live cells and selectively reports superoxide generation, while ignoring other ROS and RNS species in ex vivo testing. With an Excitation/Emission profile in the GFP/FITC microscopy channel, a series of comparative studies in immortalized and neural cells are shown, highlighting photostability, specificity and signal amplitude from the dye. These reagents are research use only, not for diagnostic purposes Citation Format: Bhaskar S. Mandavilli, Daniel Beacham, Yi Zhen Hu, Jongtae Yang, Aimei Chen. New generation of fluorescent probes for cell-based measurements of caspase activation and mitochondrial superoxide. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4781.

Biophysical Properties of Bifunctional Phage-Biosensor.

Biosensor research is a swiftly growing field for developing rapid and precise analytical devices for biomedical, pharmaceutical, and industrial use and beyond. Herein, we propose a phage-based biosensor method to develop a sensitive and specific system for biomedical detection. Our method is based on in vitro selected phages and their interaction with the targeted analytes as well as on optical properties that change according to the concentration of the model analyte. The green fluorescent protein (GFP) was chosen as our model analyte as it has its own well-known optical properties. Brilliant green was used as a reporter component for the sensor. Its presence enables a color intensity (absorbance) change when the analyte is present in the solution. Furthermore, the reporter dye functioned as a quencher for an additional lanthanide label in our assay. It mediated the specific phage-derived interference in the signal measured with the time-resolved luminescence. Most importantly, our results confirmed that the presented bifunctional phage with its liquid crystal properties enabled the measurement of GFP in a concentration-dependent, quantitative manner with a limit of detection of 0.24 µg/mL. In the future, our novel method to develop phage-based biosensors may provide highly sensitive and specific biosensors for biomedical or otherwise-relevant targets.

Nile blue as reporter dye in salt aggregation based-colorimetric aptasensors for peptide, small molecule and metal ion detection

Herein, we report a novel approach for the design of a colorimetric aptasensor, relying on a Dye Salt Aggregation-based Colorimetric Oligonucleotide assay (DYSACO assay). This method is based on the use of an intercalating agent, Nile Blue (NB), whose aggregation capacities (and thus modification of its absorption spectrum) are drastically amplified by adding salts to the working solution. The presence of an aptamer could protect NB from such aggregation process due to its intercalation into double-stranded DNA and/or interaction with nucleobases. In response to the addition of the specific ligand, the competition between NB and the target for binding to the aptamer occurs, resulting in an increase in the dye salt aggregation and then in the blue-to-blank color change of the solution. The proof-of-principle was demonstrated by employing the anti-l-tyrosinamide aptamer and the assay was successfully applied to the trace enantiomer detection, allowing the detection of an enantiomeric impurity down to approximately 2% in a non-racemic sample. Through a reversed mechanism based on the increased capture of NB by DNA upon analyte binding, the sensing platform was further demonstrated for the Hg(II) detection. Water samples of different origin were spiked with Hg(II) analyte at final range concentrations comprised between (0.5–15 μM). An excellent overall recovery of 122 ± 14%; 105 ± 14%; 99 ± 9%; was respectively obtained from river, tap and mineral water, suggesting that the sensor can be used under real sample conditions. The assay was also shown to work for sensing the ochratoxin A and d-arginine vasopressin compounds, revealing its simplicity and generalizability potentialities.

Pursuing excitonic energy transfer with programmable DNA-based optical breadboards.

DNA nanotechnology has now enabled the self-assembly of almost any prescribed 3-dimensional nanoscale structure in large numbers and with high fidelity. These structures are also amenable to site-specific modification with a variety of small molecules ranging from drugs to reporter dyes. Beyond obvious application in biotechnology, such DNA structures are being pursued as programmable nanoscale optical breadboards where multiple different/identical fluorophores can be positioned with sub-nanometer resolution in a manner designed to allow them to engage in multistep excitonic energy-transfer (ET) via Förster resonance energy transfer (FRET) or other related processes. Not only is the ability to create such complex optical structures unique, more importantly, the ability to rapidly redesign and prototype almost all structural and optical analogues in a massively parallel format allows for deep insight into the underlying photophysical processes. Dynamic DNA structures further provide the unparalleled capability to reconfigure a DNA scaffold on the fly in situ and thus switch between ET pathways within a given assembly, actively change its properties, and even repeatedly toggle between two states such as on/off. Here, we review progress in developing these composite materials for potential applications that include artificial light harvesting, smart sensors, nanoactuators, optical barcoding, bioprobes, cryptography, computing, charge conversion, and theranostics to even new forms of optical data storage. Along with an introduction into the DNA scaffolding itself, the diverse fluorophores utilized in these structures, their incorporation chemistry, and the photophysical processes they are designed to exploit, we highlight the evolution of DNA architectures implemented in the pursuit of increased transfer efficiency and the key lessons about ET learned from each iteration. We also focus on recent and growing efforts to exploit DNA as a scaffold for assembling molecular dye aggregates that host delocalized excitons as a test bed for creating excitonic circuits and accessing other quantum-like optical phenomena. We conclude with an outlook on what is still required to transition these materials from a research pursuit to application specific prototypes and beyond.

H2S contributed from CSE during cellular senescence suppresses inflammation and nitrosative stress

Aging involves the time-dependent deterioration of physiological functions attributed to various intracellular and extracellular factors. Cellular senescence is akin to aging and involves alteration in redox homeostasis. This is primarily marked by increased reactive oxygen/nitrogen species (ROS/RNS), inflammatory gene expression, and senescence-associated beta-galactosidase activity, all hallmarks of aging. It is proposed that gasotransmitters which include hydrogen sulfide (H2S), carbon monoxide (CO), and nitric oxide (NO), may affect redox homeostasis during senescence. H2S has been independently shown to induce DNA damage and suppress oxidative stress. While an increase in NO levels during aging is well established, the role of H2S has remained controversial. To understand the role of H2S during aging, we evaluated H2S homeostasis in non-senescent and senescent cells, using a combination of direct measurements with a fluorescent reporter dye (WSP-5) and protein sulfhydration analysis. The free intracellular H2S and total protein sulfhydration levels are high during senescence, concomitant to cystathionine gamma-lyase (CSE) expression induction. Using lentiviral shRNA-mediated expression knockdown, we identified that H2S contributed by CSE alters global gene expression, which regulates key inflammatory processes during cellular senescence. We propose that H2S decreases inflammation during cellular senescence by reducing phosphorylation of IκBα and the p65 subunit of nuclear factor kappa B (NF-κB). H2S was also found to reduce NO levels, a significant source of nitrosative stress during cellular senescence. Overall, we establish H2S as a key gasotransmitter molecule that regulates inflammatory phenotype and nitrosative stress during cellular senescence.

Screening Biophysical Sensors and Neurite Outgrowth Actuators in Human Induced-Pluripotent-Stem-Cell-Derived Neurons.

All living cells maintain a charge distribution across their cell membrane (membrane potential) by carefully controlled ion fluxes. These bioelectric signals regulate cell behavior (such as migration, proliferation, differentiation) as well as higher-level tissue and organ patterning. Thus, voltage gradients represent an important parameter for diagnostics as well as a promising target for therapeutic interventions in birth defects, injury, and cancer. However, despite much progress in cell and molecular biology, little is known about bioelectric states in human stem cells. Here, we present simple methods to simultaneously track ion dynamics, membrane voltage, cell morphology, and cell activity (pH and ROS), using fluorescent reporter dyes in living human neurons derived from induced neural stem cells (hiNSC). We developed and tested functional protocols for manipulating ion fluxes, membrane potential, and cell activity, and tracking neural responses to injury and reinnervation in vitro. Finally, using morphology sensor, we tested and quantified the ability of physiological actuators (neurotransmitters and pH) to manipulate nerve repair and reinnervation. These methods are not specific to a particular cell type and should be broadly applicable to the study of bioelectrical controls across a wide range of combinations of models and endpoints.

CASTING: A Potent Supramolecular Strategy to Cytosolically Deliver STING Agonist for Cancer Immunotherapy and SARS-CoV-2 Vaccination

CASTING: A Potent Supramolecular Strategy to Cytosolically Deliver STING Agonist for Cancer Immunotherapy and SARS-CoV-2 Vaccination

Multiplex qPCR for differentiation of Mycobacterium avium subspecies paratuberculosis in active and passive infection of goats.

Mycobacterium avium paratuberculosis (MAP) is causative agent of Johne's disease (JD) in domestic animals and has broad host range. JD infected animals shed viable MAP in their milk, feces, blood, and tissues which get transmitted to human beings directly or indirectly by consumption of animal products, through contact, animal handling and through contaminated environment, aerosols. In this current study, we developed hydrolysis probe based TaqMan® real-time PCR assay where samples were investigated by targeting IS900 mRNA and ModD gene to differentiate live MAP shedders from inactive/dead MAP bacilli shedding animals. The IS900 mRNA and ModD gene primers were designed using discontiguous unique conserved sequences of IS900 more towards the 3' end and fibronectin attachment protein (FAP) genes, respectively. Two different reporter dyes Cy5 and TexasRed, with compatible quenchers BHQ-1 and BHQ-2, respectively, were used for probe designing of IS900 and ModD genes. Triplex PCR assay was developed by using serially diluted positive MAP culture in log10 dilution and probe and template titration. TaqMan® probe real-time PCR targeting IS900 mRNA and ModD gene detects the MAP infection at early stage with high sensitivity and specificity. The specificity of developed TaqMan probe real-time PCR was found to be high while validated by using Escherichia coli and Staphylococcus aureus in addition to the MAP culture as there is no non-specific signal from other microbes. The sensitivity of developed TaqMan® probe real-time PCR was computed based on copy numbers ranged from 4.14 × 1011 to 4.14 × 104 for IS900 (FAM), 1.27 × 1011 to 1.27 × 104 for IS900 mRNA (Cy5), and 3.68 × 1010 to 3.68 × 104 for ModD (TexasRed), and lowest limit to detect MAP was 4.14 × 104, 1.27 × 104, and 3.68 × 104 copies for respective genes. This assay would be of great aid to contain the MAP infection in the large herd, where silent shedders spread active infection can be differentiated from passive shedding by non-infected animals. This test would also be equivalent to culture test in terms of specificity and hence can be able to be undertaken in molecular epidemiological studies to represent the actual disease prevalence in the future. KEY POINTS: • Multiplex mRNA-based qPCR was developed to identify the actively infective MAP bacilli from passive ones. • ModD and IS900 used as targets to assess active MAP bacilli in fecal samples of suspected animals. • The LOD was computed using copy numbers with 4.14 × 104 and 3.68 × 104 copies for IS900 and ModD, respectively.

Development of a rapid, internally controlled, two target, real-time RT-PCR for detection of rubella virus

Rubella surveillance in elimination setting relies on rapid molecular detection of the virus. In this study a multiplex real-time RT-PCR assay for the detection of rubella virus was validated. The assay includes three independent probes with unique reporter dyes for the simultaneous detection of the rubella viral coding regions for envelope glycoprotein E1 and non-structural p150 protein, and an endogenous control (human RNaseP). Using dilution series of synthetic RNAs, the limits of detection were determined to be at least 50 copies of rubella RNA. The assay is reproducible with low intra-assay and inter-assay coefficients of variation for both the E1 and the p150 targets. After testing 62 confirmed rubella positive and 165 rubella negative archival clinical samples, the sensitivity and specificity of the multiplex assay were 98.4 and 100 %, respectively. No cross reactivity was identified with clinical specimens positive for eleven other viruses. This multiplex assay successfully detected nine viral genotypes including the predominant genotypes 1E, 1G, 1J, and 2B as well as the 1a vaccine genotype.

Multiplexed autoantibody (AA) profiling of patients (pts) with metastatic urothelial carcinoma (mUC) receiving immune checkpoint inhibitors or platinum-based chemotherapy.

558 Background: The AA profile may be altered in malignancies and provide insights into tumor biology and the immune state. We hypothesized that the longitudinal AA profiling of mUC pts receiving an immune checkpoint inhibitor (ICI) may provide insights into the immune response, which may be associated with immune events and help discover new therapeutic targets. Methods: We utilized serum from mUC pts receiving an ICI or platinum-based chemotherapy (PBC) at the Dana-Farber Cancer Institute. Age and sex matched healthy controls were also studied. The SeroTag immuno-oncology discovery array (Oncimmune) was utilized, with quantification of the AA reactivity towards 1150 antigens. Bound autoantibodies were detected using an anti-IgG-specific detection antibody conjugated to the fluorescent reporter dye phycoerythrin. The AA reactivity was reported as the median fluorescence intensity (MFI) for each color and sample using a Luminex FlexMAP3D analyzer. A significance analysis of microarrays was performed to identify AAs with elevated levels in bladder cancer compared to matched healthy controls (HCs). AAs with > 1.5 increase between pre- and post-treatment were reported. Scatter and box-whisker plots were reported for all pts and antigens, respectively. Results: Pre- (n = 66) and post treatment (n = 65) serum samples were available from mUC pts receiving pembrolizumab (n = 25), atezolizumab (n = 21), nivolumab (n = 5), avelumab (n = 1), durvalumab + tremelimumab (n = 1), nivolumab plus vaccine (n = 1), and 12 pts who received PBC (cisplatin n = 8, carboplatin n = 4). The median duration between the pre- and post-therapy samples was 6 months, median age was 67.7 years (range 40-91) with 51 men (77.3%). Overall, significant heterogeneity of AAs between pts was observed with 37 AAs showing higher reactivity in pre-treatment mUC pts vs. 47 HCs, notably anti-CTAG1 (NY-ESO-1), CTAG2 (NY-ESO-2), MAGE B-18, KRAS, GRB2, RARRES2, HSP72 and FGFR3 (all p < 0.05). Pre- and post-therapy AA profiles were similar with unique changes seen in each patient. Notably, 3 pts receiving an ICI developed AAs to NY-ESO-1. Pts receiving PBC less frequently developed new AAs, although pts treated with cisplatin appeared to develop AAs more frequently compared to carboplatin-treated pts. Conclusions: This is the first report of a comprehensive AA profile using a novel platform in mUC pts. The study identified multiple elevated AAs in mUC pts vs. HCs, most notably NY-ESO-1, which also developed in some pts following ICIs. Pts treated with PBC did not develop new AAs frequently, although there appeared to be a difference between cisplatin and carboplatin-based chemotherapy. Further development of this platform is warranted to provide data that is orthogonal to genomic/transcriptomic profiling and shed insights on potential therapeutically actionable antigens.

Nile Blue as Reporter Dye in Salt Aggregation Based-Colorimetric Aptasensors for Peptide, Small Molecule and Metal Ion Detection

Nile Blue as Reporter Dye in Salt Aggregation Based-Colorimetric Aptasensors for Peptide, Small Molecule and Metal Ion Detection

Real-Time Detection of Viroids Using Singleplex and Multiplex Quantitative Polymerase Chain Reaction.

Multiplex quantitative polymerase chain reaction (multiplex qPCR) enables the amplification of more than one target in a single reaction using different reporter dyes with distinct fluorescent spectra. The number of reporter fluorophores is typically restricted to three or four, depending upon the capability of the real-time PCR platform and software used. Each target is amplified by a different set of primers and a uniquely labeled probe that distinguishes each PCR amplicon. Thus, the levels of several targets of interest can be quantified in real time. By combining several reactions in a single tube, multiplex qPCR reduces the quantity, and cost of reagents needed to screen a sample for multiple targets. Specificity and efficiency are not affected by the inclusion of the three assays in a multiplex reaction.