Antibody-conjugated Magnetic Beads Research Articles

Colorimetric immunosensing using liposome encapsulated MnO2 nanozymes for SARS-CoV-2 antigen detection

Development of specific signal reporters with signal amplification effect are highly needed for sensitive and accurate detection of pathogen. Herein, we design a colorimetric immunosensing nanosystem based on liposome encapsulated quantum dots-sized MnO2 nanozyme (MnO2QDs@Lip) as a signal reporter for ultrasensitive and fast detection of SARS-CoV-2 antigen. The pathogenic antigens captured and separated by antibody-conjugated magnetic beads (MBs) are further connected with antibody-modified MnO2QDs@Lip to form a sandwich-like immunocomplex structure. After triggered release, MnO2 QDs efficiently catalyze colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxidized TMB, which can be qualitatively observed by naked eyes and quantitatively analyzed by UV–Vis spectra or smartphone platforms. By taking advantages of immuno-magnetic separation, excellent peroxidase-like catalytic activity of MnO2 QDs, and high encapsulation efficiency of MnO2QDs@Lip, ultrasensitive detection of SARS-CoV-2 antigen ranging from 0.1 pg/mL to 100 ng/mL is achieved within 20 min. The limit of detection (LOD) is calculated to be 65 fg/mL in PBS buffer. Furthermore, real clinical samples of SARS-CoV-2 antigens can be effectively identified by this immunosensing nanosystem with excellent accuracy. This proposed detection nanosystem provides a strategy for simple, rapid and ultrasensitive detection of pathogens and may shed light on the development of new POCT detection platforms for early diagnosis of pathogens and surveillance in public health.

Distinct Lipid Phenotype of Cancer-Associated Fibroblasts (CAFs) Isolated From Overweight/Obese Endometrial Cancer Patients as Assessed Using Raman Spectroscopy.

Obesity is strongly linked with increased risk and poorer prognosis of endometrial cancer (EC). Cancer-associated fibroblasts (CAFs) are activated fibroblasts that form a large component of the tumor microenvironment and undergo metabolic reprogramming to provide critical metabolites for tumor growth. However, it is still unknown how obesity, characterized by a surplus of free fatty acids drives the modifications of CAFs lipid metabolism which may provide the mechanistic link between obesity and EC progression. The present study aims to evaluate the utility of Raman spectroscopy, an emerging nondestructive analytical tool to detect signature changes in lipid metabolites of CAFs from EC patients with varying body mass index. We established primary cultures of fibroblasts from human EC tissues, and CAFs of overweight/obese and nonobese women using antibody-conjugated magnetic beads isolation. These homogeneous fibroblast cultures expressed fibroblast markers, including α-smooth muscle actin and vimentin. Analysis was made in the Raman spectra region best associated with cancer progression biochemical changes in lipids (600-1800 cm-1 and 2800-3200 cm-1). Direct band analysis and ratiometric analysis were conducted to extract information from the Raman spectrum. Present results demonstrated minor shifts in the CH2 symmetric stretch of lipids at 2879 cm-1 and CH3 asymmetric stretching from protein at 2932 cm-1 in the overweight/obese CAFS compared to nonobese CAFs, indicating increased lipid content and a higher degree of lipid saturation. Principal component analysis showed that CAFs from overweight/obese and nonobese EC patients can be clearly distinguished indicating the capability of Raman spectroscopy to detect changes in biochemical components. Our results suggest Raman spectroscopy supported by chemometric analysis is a reliable technique for characterizing metabolic changes in clinical samples, providing an insight into obesity-driven alteration in CAFs, a critical stromal component during EC tumorigenesis.

In Vivo Cross-Linking and Co-Immunoprecipitation Procedure to Analyze Nuclear tRNA Export Complexes in Yeast Cells.

tRNAs are small noncoding RNAs that are predominantly known for their roles in protein synthesis and also participate in numerous other functions ranging from retroviral replication to apoptosis. In eukaryotic cells, all tRNAs move bidirectionally, shuttling between the nucleus and the cytoplasm. Bidirectional nuclear-cytoplasmic tRNA trafficking requires a complex set of conserved proteins. Here, we describe an in vivo biochemical methodology in Saccharomyces cerevisiae to assess the ability of proteins implicated in tRNA nuclear export to form nuclear export complexes with tRNAs. This method employs tagged putative tRNA nuclear exporter proteins and co-immunoprecipitation of tRNA-exporter complexes using antibody-conjugated magnetic beads. Because the interaction between nuclear exporters and tRNAs may be transient, this methodology employs strategies to effectively trap tRNA-protein complexes in vivo. This pull-down method can be used to verify and characterize candidate proteins and their potential interactors implicated in tRNA nuclear-cytoplasmic trafficking.

Antibody-Conjugated Magnetic Beads for Sperm Sexing Using a Multi-Wall Carbon Nanotube Microfluidic Device.

This study proposes a microfluidic device used for X-/Y-sperm separation based on monoclonal antibody-conjugated magnetic beads, which become positively charged in the flow system. Y-sperms were selectively captured via a monoclonal antibody and transferred onto the microfluidic device and were discarded, so that X-sperms can be isolated and commercially exploited for fertilization demands of female cattle in dairy industry. Therefore, the research team used monoclonal antibody-conjugated magnetic beads to increase the force that causes the Y-sperm to be pulled out of the system, leaving only the X-sperm for further use. The experimental design was divided into the following: Model 1, the microfluid system for sorting positive magnetic beads, which yielded 100% separation; Model 2, the sorting of monoclonal antibody-conjugated magnetic beads in the fluid system, yielding 98.84% microcirculation; Model 3, the sorting of monoclonal antibody-conjugated magnetic beads with sperm in the microfluid system, yielding 80.12% microcirculation. Moreover, the fabrication microfluidic system had thin film electrodes created via UV lithography and MWCNTs electrode structure capable of erecting an electrode wall 1500 µm above the floor with a flow channel width of only 100 µm. The system was tested using a constant flow rate of 2 µL/min and X-/Y-sperm were separated using carbon nanotube electrodes at 2.5 V. The structure created with the use of vertical electrodes and monoclonal antibody-conjugated magnetic beads technique produced a higher effective rejection effect and was able to remove a large number of unwanted sperm from the system with 80.12% efficiency.

Dual-color quantum dots nanobeads based suspension microarray for simultaneous detection of dual prostate specific antigens

Featured with high multiplexibility, suspension microarray technology usually involves the conjugation of second-antibody with organic dye based fluorescent proteins, which are inherently unsuitable for multicolor signaling under single wavelength excitation. In addition, application of single QDs-based fluorescent reporter in suspension microarray is dramatically hampered since its preparation often suffers from poor reproducibility and stability. Herein, a novel suspension microarray system based on dual color quantum dots (QDs) nanobead as fluorescence label was developed for simultaneous detection of prostate specific antigen (PSA) biomarkers. When both antigens are present in sera sample, free (f)-PSA antibody-conjugated green and complexed (c)-PSA antibody-conjugated red QDs nanobeads would be both specifically absorbed onto the surface of total PSA antibody-conjugated magnetic beads, leading to the formation of magnetic fluorescent hybrid that can be purified from the mixture by an external magnet prior to flow cytometry analysis. The proposed method demonstrates simultaneous detection ability for f- and c- PSA antigen assay with high detection sensitivity that is comparable to clinical approaches including ELISA and chemiluminescence assay. Furthermore, result of clinical application of the proposed method is consistency with clinical data, demonstrating its potential in suspension microarray for accurate prostate cancer diagnosis.

Rolling Circle Amplification-Assisted Flow Cytometry Approach for Simultaneous Profiling of Exosomal Surface Proteins.

Exosomes that carry multiple proteins from the originating cells are known as emerging biomarkers for tumor diagnostics. However, it is still technically challenging to accurately evaluate subtle differences of exosomal membrane proteins. Here, we developed a rolling circle amplification (RCA)-assisted flow cytometry approach (FCA) to simultaneously profile surface proteins and quantify exosomes. In this work, specific anti-CD63 antibody-conjugated magnetic beads were first utilized to capture exosomes. Then, the captured exosomes were bound with DNA primers, which comprise exosomal surface protein-specific recognition aptamers. The RCA reaction generates repeat DNA sequences for fluorescent probe hybridization. Finally, a conventional flow cytometer was introduced to phenotype exosomal protein markers. Such a sensitive RCA-assisted FCA displays an excellent detection limit of 1.3 × 105 exosome/mL. The variable composition of four protein markers on different cell-derived exosomes was sensitively detected through changing the protein-recognition sequence of the DNA primer, which reveals a heterogeneous pattern. Exosomes from different cell sources could be distinguished by the abundance difference of multiple surface proteins. Furthermore, the developed RCA-assisted FCA enabled quantitative analysis of blood samples from lung cancer patients, indicating its potential for early clinical diagnosis and prognosis of cancer.

Membrane Feature-Inspired Profiling of Extracellular Vesicles for Pancreatic Cancer Diagnosis.

Extracellular vesicles (EVs) have recently emerged as a promising tumor biomarker, and EV phenotyping offers many benefits for cancer diagnosis. However, the practicality of EV assays remains a challenge due to macromolecule disturbances, biomarker heterogeneities, and EV abundance limitations. Here, we demonstrate a membrane-based biosensor for precise and sensitive EV identification. The sensor synergistically integrates EV capture and detection by virtue of EV membrane features (membrane protein and lipid bilayer), comprising antibody-conjugated magnetic beads (AbMBs) and duplex-specific nuclease (DSN)-mediated amplification cycles. Bivalent cholesterol (biChol)-modified RNA-DNA duplexes are designed to insert into the EV membrane, transforming EV signals into RNA signals and initiating the signal amplification. The membrane-based signal production pattern eliminates protein interference. By employing four antibodies specific to PCa-related membrane proteins, the AbMB-biChol platform enables the successful differentiation and monitoring of PCa-related EVs and distinguishes PCa patients from healthy donors with improved efficacy, exhibiting superior efficiency over the analyses based on clinically used biomarker CA19-9 and PCa-related proteins. As such, the developed system has great potential for clinical PCa diagnosis.

Detection of Recurrent Genetic Mutations in Intravascular Large B-Cell Lymphoma By Comprehensive Genetic Analyses Using Peripheral Blood Circulating Cell-Free DNA

Abstract Background: Intravascular large B-cell lymphoma (IVLBCL) is a subtype of diffuse large B-cell lymphoma (DLBCL) characterized by the selective growth of tumor cells in the lumina of small vessels. The characteristics of the disease, such as the lack of remarkable lymphadenopathy and the presence of non-specific clinical symptoms, including documented fever of unknown origin, make precise diagnosis difficult; random organ biopsies from bone marrow, skin, and lung are still performed to detect tumor cells. Recently, many recurrent genetic mutations have been detected, especially in DLBCL, but the genetic background in IVLBCL has not been fully elucidated because of the difficulty in obtaining sufficient live tumor cells from diagnostic specimens for subsequent genetic analyses. Previously, we demonstrated that peripheral blood circulating cell-free DNA (cfDNA) in DLBCL is an alternative tumor DNA (tDNA) source for comprehensive genetic analyses (Suzuki Y, et al. ASH 2014 #1658). In this report, we demonstrate that the concentration of cfDNA is significantly higher in IVLBCL than in DLBCL, and that efficiently accumulating the tDNA in peripheral blood is useful for comprehensive genetic analyses to detect recurrent genetic mutations in IVLBCL. Aims: Confirmation of recurrent genetic mutations in IVLBCL using plasma cfDNA. Methods: The study population included 54 B-cell lymphoma patients (IVLBCL, N=21; DLBCL, N=21; follicular lymphoma, N=12) and healthy volunteer donors (N=14). In IVLBCL patients, primary bone marrow (BM) cells containing tumor cells were transplanted into NOD/Shi- scid IL2Rγnull mice to generate a xenograft model (Shimada K, et al. Leukemia 2016), and the tumor cells from tumors formed in the spleen of the mouse model were purified using anti-CD19 antibody-conjugated magnetic beads to obtain tDNA. For genetic analyses using cfDNA and DNAs from purified tumor cells, whole-exome sequencing (WES) using the HiSeq 2000 (Illumina) and Sanger sequencing for reconfirmation of those mutations were performed. Results: The cfDNA concentrations were significantly higher in DLBCL and IVLBCL than in normal donors (p=0.037 and 0.036, respectively), and the concentration was significantly higher in IVLBCL than in DLBCL (p Discussion: The concentration of cfDNA is significantly higher in IVLBCL than in DLBCL, and it appeared to be sufficient to carry out comprehensive genetic analyses to accumulate tDNA. Moreover, the WES analysis using cfDNA could detect genetic mutations more efficiently than that using corresponding BM-DNA. The profiling of recurrent genetic mutations in IVLBCL is partly similar to that in activated B-cell type DLBCL with mutations in NF-κB pathway-related factors, and, intriguingly, they resemble those in B-cell lymphomas that infiltrate into immunologically sanctuary organs, e.g., primary CNS, testis, and breast lymphomas. The present data suggest that genetic analysis using cfDNA in addition to random organ biopsies may become a powerful, safer, and painless strategy to predict the presence of IVLBCL in the clinical setting. Disclosures Shimada: Kyowa Hakko Kirin Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Eisai Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria. Kusumoto: Chugai: Honoraria, Other: GALLIUM and GOYA are sponsored by F. Hoffmann-La Roche Ltd. Third-party medical writing support, under the direction of Shigeru Kusumoto, was provided by Cheryl Wright of Gardiner-Caldwell Communications, and was funded by F. Hoffmann-La Roche Ltd, Research Funding. Masaki: MSD: Research Funding; Eisai: Research Funding; Astellas Pharma: Research Funding; Kyowa Hakko Kirin: Research Funding; Daiichi Sankyo: Research Funding; Taisho Toyama Pharmaceuticals: Research Funding; Taiho Pharmaceutical: Research Funding; Takeda Pharmaceuticals: Research Funding; Mitsubishi Tanabe Pharmaceutical: Research Funding; Chugai Pharmaceutical: Research Funding; Teijin Pharmaceutical: Research Funding; Japan Blood Products Organization: Research Funding; Pfizer: Research Funding; Asahi Kasei Pharmaceutical: Research Funding; Ono Pharmaceutical: Research Funding; Shinogi Pharmaceutical: Research Funding; Meiji Seika Pharma: Research Funding; Mochida Pharmaceutical: Research Funding. Kiyoi: Meiji Seika Pharma Co.,Ltd.: Research Funding; Phizer Japan Inc.: Honoraria, Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; MSD K.K.: Research Funding; JCR Pharmaceuticals Co.,Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; ONO Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Celgene Corporation: Consultancy, Research Funding; Novartis Pharma K.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

Magneto-controlled fluorescent immunosensor for sensitive determination of biomarker via three-dimensional AuNCs/liposome networks

The three-dimensional liposome networks are fabricated and act as signal amplification modules for the fabrication of a magneto-controlled fluorescent immunosensor, in which antibody-conjugated magnetic bead (MB-Ab), streptavidin-biotin recognition and liposome amplification as the triple signal amplification pathway. The Aptamer-Liposome-AuNCs (Apt-Lip-AuNCs) is employed as a fluorescence signal probe, which can release abundant of AuNCs by Triton-X 100 to produce fluorescence emission signal upon being excited at 369 nm. In the presence of target CEA, the labeled anti-CEA on the MB and the immobilized aptamer on the Apt-Lip-AuNCs sandwich the target CEA. Upon addition of streptavidin and Biotin-Lip-AuNCs, three-dimensional liposome networks are formed by a streptavidin-biotin reaction due to the capture of streptavidin for Biotin-Lip-AuNCs and the residual biotin site of liposome on the Apt-Lip-AuNCs, thereby increasing the loading capacity of AuNCs. CEA can be linearly assayed in the range from 0.05 ng mL−1 to 40 ng mL−1 with a detection limit of 13.2 pg mL−1 (3σ/K). It provided a concise sensing platform for CEA determination and showed a great potential application for on-site testing in early clinical diagnosis.

6 Immunological aspects of ovarian follicle ovulation and corpus luteum formation in cattle

Ovarian follicle ovulation and subsequent luteinization have been described as a controlled inflammatory event, comprising tissue damage and repair. To elaborate this further in cattle, the contribution of immune cells to dominant follicle luteinization, ovulation, and corpus luteum formation was investigated. Ovulation in beef heifers was synchronized using an 8-day progesterone-based synchronization program. Heifers were slaughtered at a local abattoir at 5 timepoints (T): (T1) 24h before ovulation (n=10); (T2) 2h before ovulation (n=9); (T3) 6h after ovulation (n=10); (T4) 24h after ovulation (n=10); (T5) 72h after ovulation (n=10), and ovarian tissue was collected and returned to the laboratory on ice. Follicular fluid, theca, granulosa, and corpus luteum (CL) tissues were recovered by dissection and processed for analysis. The concentrations of a panel of cytokines were measured using an antibody-conjugated magnetic bead immunoassay. The abundance of T-lymphocytes, mast cells, neutrophils, eosinophils, monocytes, macrophages, and dendritic cells was determined by immunohistochemistry. The mRNA relative abundance of candidate genes, including angiogenic growth factors, adhesion factors, chemokines and cytokines, was determined by quantitative real-time PCR analysis. The resulting datasets were analysed using the linear mixed model procedure of SAS and data are presented as least squares means; reported differences were deemed significant at P≤0.05. The cytokines IFNy, IP-10, IL-10, IL-36RA, MCP-1, MIP-1a, MIP-1b, and VEGF-A were detected in follicular fluid. The concentrations (pg mL−1) of IL-10 and VEGF-A were significantly higher in T1 follicular fluid samples compared with T2 (7.70 vs. 0.86 and 2193.33 vs. 293.93, respectively). Although dendritic cells were the most abundant cells in bovine ovulatory follicular and early corpus luteum tissue at all time points (P<0.05), their numbers peaked in ovulatory (T2) thecal tissue (261.5 cells/mm2). The greatest number of neutrophils was identified in thecal tissue at T1 (45/mm2); thereafter, their numbers declined to 1.1/mm2 in CL tissue by T5. Similarly, the numbers of T-lymphocytes, mast cells, monocytes, and macrophages declined in CL tissue at T4 and T5. Candidate gene mRNA expression profiles appeared to be time- and tissue-specific; for example, IFNA was highest in the preovulatory granulosa tissue (T1), IL8 was highest in peri-ovulatory thecal tissue (T2), VEGFA and MMP9 were highest in the early CL tissue (T4 and T5), MMP1, TIMP1, and VCAM1 expression was highest in theca, granulosa, and CL tissue collected on or after ovulation (T2, T4, T5), expression of the prostaglandin-related genes PTGES and PTGS2 was lowest in CL tissue, and that of PTGIS was highest. The current findings support the hypothesis that ovulation in heifers is characterised by an initial proinflammatory cascade followed by a dramatic switch to tissue repair, growth, and remodelling, all occurring within a 72-h period and commencing with the LH surge. Our results highlight the roles of neutrophils, dendritic cells, and macrophages as the key actors in this process.

Development of a Multiplex Bead-Based Method for the Microquantitation of δ-Catenin.

δ-Catenin is overexpressed in human cancers, including prostate, breast, lung, and ovarian cancers. Therefore, detection of δ-catenin level in patient specimens can be used as a diagnostic marker for the cancer screening. In laboratories, δ-catenin levels have been analyzed by western blot, which requires multiple procedures and is incapable of multiplex analysis of a target protein from a single reaction. In this study, we aimed to develop δ-catenin antibody-conjugated magnetic beads that can be used for quantitation of δ-catenin by bead-based multiplex assay. δ-catenin level from HEK293T and CWR22Rv-1 cell lysates was quantified to determine whether the antibody-conjugatedmagnetic bead can be used for the detection of trace amounts of δ-catenin. Our results indicate that analysis with 1 μg of CWR22Rv-1 and HEK293T cell lysates showed 918.67±103.8 and 874.33±37.21 MFI values, respectively. In conclusion, the results suggest that the procedure for detection of δ-catenin level through the antibody-conjugated magnetic beads provides a sensitive and costeffective solution for screening from patient specimens.

Recapitulation of prostate tissue cell type-specific transcriptomes by an in vivo primary prostate tissue xenograft model.

Studies of the normal functions and diseases of the prostate request in vivo models that maintain the tissue architecture and the multiple-cell type compartments of human origin in order to recapitulate reliably the interactions of different cell types. Cell type-specific transcriptomes are critical to reveal the roles of each cell type in the functions and diseases of the prostate. A primary prostate tissue xenograft model was developed using fresh human prostate tissue specimens transplanted onto male mice that were castrated surgically and implanted with a device to maintain circulating testosterone levels comparable to adult human males. Endothelial cells and epithelial cells were isolated from 7 fresh human prostate tissue specimens and from primary tissue xenografts established from 9 fresh human prostate tissue specimens, using antibody-conjugated magnetic beads specific to human CD31 and human EpCAM, respectively. Transcriptomes of endothelial, epithelial and stromal cell fractions were obtained using RNA-Seq. Global and function-specific gene expression profiles were compared in inter-cell type and inter-tissue type manners. Gene expression profiles in the individual cell types isolated from xenografts were similar to those of cells isolated from fresh tissue, demonstrating the value of the primary tissue xenograft model for studies of the inter-relationships between prostatic cell types and the role of such inter-relationships in organ development, disease progression, and response to drug treatments.

A Multiplexed Microarray Platform for Targeted Proteomics

A Multiplexed Microarray Platform for Targeted Proteomics

Open Surface Droplet Microfluidic Magnetosensor for Microcystin-LR Monitoring in Reservoir.

Establishing rapid, simple, and in situ detection of microcystin-LR (MC-LR) in drinking water sources is of significant importance for human health. To ease the situation that current methods cannot address, an open surface droplet microfluidic magnetosensor was designed and validated to quantify MC-LR in reservoir water, which is capable of (1) MC-LR isolation via MC-LR antibody-conjugated magnetic beads, (2) parallel and multistep analytical procedures in 15-array power-free and reusable active droplet microfluidic chips, (3) immunoassay incubation and fluorescence excitation within a miniaturized multifunctional 3D-printing optosensing accessory, and (4) signal read-out and data analysis by a user-friendly Android app. The proposed smartphone-based fluorimetric magnetosensor exhibited a low limit of detection of 1.2 × 10-5 μg/L in the range of 10-4 μg/L to 100 μg/L. This integrated and high throughput platform was utilized to draw an MC-LR contamination map for six reservoirs distributed in the Pearl River delta, Guangdong Province. It promises to be a simple and successful quantification method for MC-LR field detection, bringing many benefits to rapid on-site screening.

TMIC-36. ALDH1A2 AS A NOVEL PUTATIVE MARKER OF MACROPHAGE DIFFERENTIATION IN GBM

Abstract Tumor-associated macrophages (TAM) are abundant in glioblastoma (GBM), composing up to 30% of the total tumor mass. However, their genotype/phenotype and exact role in tumor progression and immune suppression are not fully understood. Macrophages are believed to be polarized along a spectrum spanning from M0, M1, and M2 states. M1 TAMs are proinflammatory while M2 TAMs are associated with anti-inflammatory, pro-tumor responses. For example, using gelatin zymography we saw an increase in MMP 2 and 9 activities in co-culture of GBM cells and M2 polarized macrophages. In search for factors determining M2 type TAMs, we found Aldehyde Dehydrogenase 1 family A2 (ALDH1A2) to be highly over-expressed in M2 polarized THP1 cells using a gene expression array. We also performed single cell sequencing on CD45+ cells isolated from GBM tumors using antibody-conjugated magnetic beads. Monocytic/macrophage cells were found in 2 clusters and ALDH1A2 expression was increased in 2.1% of CD68+/CD163+ cells in Cluster 1 and 2.7% of CD68+/CD163+ cells in Cluster 2. Notably, ALDH1A2 was not detected in peripheral blood mononuclear cells. Analysis of the single cell sequencing data has led to identification of several genes whose expression is increased in ALDH1A2+ cells compared to ALDH1A2- cells. These genes are associated with lipid metabolism, regulation of neoplastic transformation, and insulin growth factor signaling. To further validate these results, we co-stained GBM tumor sections for CD163 and ALDH1A2 and observed that ALDH1A2 is co-localized in M2 macrophages. We have noticed a propensity of ALDH1A2+ cells to be associated with tumor neovasculature. Being that ALDH1A2 is the main enzyme in retinoic acid (RA) synthesis, it is plausible that this could represent another possible function of these enzyme-positive TAMs. Taken together these data suggest a role for ALDH1A2 as a novel putative marker of a subset of M2 TAM phenotype and function in GBM.

Highly specific and sensitive point-of-care detection of rare circulating tumor cells in whole blood via a dual recognition strategy

Despite the fact that the identification and detection of circulating tumor cells (CTCs) plays a critical role in cancer monitoring and diagnosis, it remains a major challenge to isolate and detect these cells, due to their extreme scarcity in peripheral blood. In this work, by coupling a dual recognition strategy and the commercial personal glucose meter, we established a point-of-care approach for detecting rare CTCs in whole blood with high sensitivity and selectivity. The antibody-conjugated magnetic beads lead to the capture and isolation of the CTCs while the enzyme- and second antibody-modified microspheres yield the signal for detection. Because of the dual recognition format, the developed method is highly selective, and a low detection limit of 7 cells can be realized as well, owing to the great signal amplification through the enzyme-loaded microbead labels. More importantly, the detection of CTCs in whole blood can be achieved in a point-of-care fashion with the using of the glucose meter transducer, offering our method a convenient and attractive alternative to traditional biopsy for the diagnosis of various cancers.

MALDI-TOF mass spectrometry-based quantification of C-peptide in diabetes patients.

Serum C-peptide concentrations reflect insulin secretion and beta cell function and can be used to diagnose and distinguish type-1 and type-2 diabetes. C-peptide is a more accurate indicator of insulin status than direct insulin measurement for monitoring patients with diabetes. However, the current methods available for C-peptide quantification exhibit poor reproducibility, are costly, and require highly trained laboratory personnel. Here, we have developed and evaluated a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based assay to standardize C-peptide measurements, providing highly accurate and comparable results across testing systems and laboratories. C-peptide from human serum was enriched using antibody-conjugated magnetic beads. The eluted isolates were further modified with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) to enhance the ionization of naturally acidic C-peptide. After desalting with ZipTips, the samples were subjected to MALDI-TOF MS analysis. Recombinant human C-peptide was used to develop the assay, and a heavy isotope labeled human C-peptide was used as an internal standard for quantification. The MALDI-TOF MS method was validated in accordance with the restrictions of the device, with a limit of quantitation of 25 pmol/L. A correlation between the MAL-DI-TOF MS assay and a reference method was conducted using patient samples. The resulting regression revealed good agreement. A simple, high-throughput, cost effective and quantitative MALDI-TOF MS C-peptide assay has been successfully developed and validated in clinical serum samples.

An Ex-Vivo Culture System of Ovarian Cancer Faithfully Recapitulating the Pathological Features of Primary Tumors.

The success rate of establishing human cancer cell lines is not satisfactory and the established cell lines often do not preserve the molecular and histological features of the original tissues. In this study, we developed a novel culture method which can support proliferation of almost all primary epithelial ovarian cancer cells, as well as primary normal human oviductal epithelial cells. Cancer cells from fresh or frozen specimens were enriched by the anti-EpCAM antibody-conjugated magnetic beads, plated on Matrigel-coated plate and cultivated under the optimized culture conditions. Seventeen newly established ovarian cancer cell lines, which included all four major histotypes of ovarian cancer, were confirmed to express histotype-specific markers in vitro. Some of the cell lines from all the four histotypes, except mucinous type, generated tumors in immune-deficient mice and the xenograft tumor tissues recapitulated the corresponding original tissues faithfully. Furthermore, with poorly tumorigenic cell lines including mucinous type, we developed a novel xenograft model which could reconstruct the original tissue architecture through forced expression of a set of oncogenes followed by its silencing. With combination of the novel culture method and cell-derived xenograft system, virtually every epithelial ovarian cancer can be reconstituted in mice in a timely fashion.

Improving Sensitivity and Specificity of Amyloid-β Peptides and Tau Protein Detection with Antibiofouling Magnetic Nanoparticles for Liquid Biopsy of Alzheimer's Disease.

Alzheimer's disease (AD) is a growing global healthcare burden affecting the aging population and society. Given the lack of effective treatment to AD, early detection at the prodromal stage and timely monitoring of changes during progression are considered the best approach to control and intervene in disease progression. "Liquid biopsy" of AD biomarkers amyloid-β peptides (Aβs) and tau proteins in the cerebrospinal fluid (CSF) or blood samples holds great promises for cost-effective, widely accessible, and easy-administrated noninvasive detection and follow-up of AD. However, current in vitro detection methods have not yet demonstrated sufficient sensitivity and specificity using neither Aβs nor tau proteins biomarkers. One major challenge of accurate detection and measurement of biomarker levels in biofluidic samples is the biofouling effect with nonspecific adsorption of unwanted biomolecules, such as various serum proteins, on the surface of targeted detecting agents or devices, causing false-positive and false-negative findings. In this study, antibiofouling polymer polyethylene glycol-block-allyl glycidyl ether (PEG-b-AGE) coated magnetic iron oxide nanoparticles (IONPs) capable of suppressing the nonspecific interactions with biomolecules, especially proteins, were investigated for the immunomagnetic capturing of Aβ40 and Aβ42 peptides and tau protein spiked in CSF- and serum-mimicking samples using corresponding antibodies conjugated as targeting ligands. Antibody-conjugated antibiofouling IONPs demonstrated improved specificity (>90%) and sensitivity (>95%) over those of antibody-conjugated magnetic micron beads (Dynabeads, ∼50% specificity and 30-40% sensitivity) widely used as magnetic separating agents under the same experimental conditions with the presence of nontargeted interfering proteins. The antibody-conjugated IONPs also exhibited significantly higher sensitivities (80-90%) and better performance of capturing Aβs and tau protein from the human whole blood samples than antibody-conjugated Dynabeads (∼20%).

Microquantitation of Van Gogh-like Protein 1 by Using Antibody-Conjugated Magnetic Beads

Van Gogh-like protein 1 (VANGL1) is an integral membrane protein that has a decisive effect on invasion, migration, and metastasis of tumor cells in various cancers including colorectal cancer. Elevation of VANGL1 level in cancer tissue is often observed with progressive and metastatic colorectal cancer. Therefore, detection of VANGL1 level in patient specimens can be used as a diagnostic marker for colorectal cancer screening. In this study, we developed magnetic beads that conjugate the VANGL1 antibody, which can be used for quantitative analysis of VANGL1. The procedure for bead preparation was optimized, and detection analysis was validated by using Caco2 and HT29 colorectal cancer cell lysates. Through the procedure, VANGL1 level from cell lysates of Caco2 and HT29 cells were quantified to estimate whether the antibody-conjugated magnetic bead can be used to trace amounts of VANGL1. Results from VANGL1 level obtained by using the antibody-conjugated magnetic beads suggest that the procedure has high precision and sensitivity in analyzing VANGL1. In conclusion, the results indicate that the method is appropriate for microquantitation of VANGL1 from patient specimens.