Antibody-coated Magnetic Beads Research Articles

Yoctomole detection of proteins using solid phase binding-induced DNA assembly

A binding-induced DNA assembly (BINDA) strategy is able to covert target-binding to amplifiable DNA assembly, thereby extending advances in DNA amplification to the detection of other non-amplifiable molecules. We describe here the concept of solid phase BINDA and demonstrate an approach of detecting proteins using affinity capture of the specific proteins on magnetic beads followed by BINDA. The antibody-coated magnetic beads are first introduced to capture target proteins from sample solutions. The unbound components are removed by washing. Two affinity probes, each conjugated to a DNA motif, are utilized to bind to the captured protein molecules. The binding of the two probes to the same target molecule induces assembly of the two DNA motifs that are conjugated to affinity ligands. Enzymatic ligation of the two DNA motifs generates a new DNA sequence that is then measured with real-time PCR. The highly sensitive real-time PCR detection of the DNA assembly provides an indirect detection of the target proteins that is responsible for the binding-induced DNA assembly. The incorporation of solid phase affinity capture effectively reduces the potential interferences from complex matrix and further improves the sensitivity of protein detection by pre-concentration. An application of the technique to the determination of prostate specific antigen in dilute serum shows a detection limit of 200 yoctomole (2×10−22mol) from a 20-μL sample. The concentration detection limit (10attomolar) is 10-fold lower than using the homogeneous BINDA. Substantial reduction of background is a main reason for the improvement in detection limits observed with the BINDA assays. Solid phase BINDA complements to homogeneous BINDA for accomplishing ultrasensitive protein detection.

Chemokine (C-C Motif) Ligand 20, a Potential Biomarker for Graves' Disease, Is Regulated by Osteopontin

ContextGraves’ disease (GD) is a common autoimmune disease involving the thyroid gland. The altered balance of pro- and anti-inflammatory cytokines plays an important role in the pathogenesis of GD. Chemokine (C-C motif) ligand 20 (CCL20) is important for interleukin-17 (IL-17) signal activation and a potent chemoattractant for Th17 cells. Meanwhile, Osteopontin (OPN), a broadly expressed pleiotropic cytokine, has been implicated in GD through inducing Th1-involved response to enhance the production of proinflammatory cytokines and chemokines, but little is known about the role of OPN in regulating CCL20 and IL-17 signaling.ObjectiveThis study sought to explore the possibility of CCL20 level as a biomarker for GD, as well as investigate the role of OPN in regulating CCL20 production.MethodsFifty untreated GD patients, fifteen euthyroid GD patients, twelve TRAb-negative GD patients and thirty-five healthy control donors were recruited. OPN, CCL20 and other clinical GD diagnosis parameters were measured. CD4+T cells were isolated from peripheral blood mononuclear cells (PBMCs) using antibody-coated magnetic beads. Enzyme-linked immune-sorbent assay and quantitative polymerase chain reaction were used to determine CCL20 expression level.ResultsWe found that the plasma CCL20 level was enhanced in GD patients and decreased in euthyroid and TRAb-negative GD patients. In addition, CCL20 level correlated with GD clinical diagnostic parameters and plasma OPN level. Moreover, we demonstrated that recombinant OPN and plasma from untreated GD patients increased the expression of CCL20 in CD4+T cells, which could be blocked by OPN antibody. Furthermore, we found that the effect of OPN on CCL20 expression was mediated by β3 integrin receptor, IL-17, NF-κB and MAPK pathways.ConclusionsThese results demonstrated that CCL20 might serve as a biomarker for GD and suggested the possible role of OPN in induction of CCL20 expression.

Abstract 3647: Dose-dependent alteration of CpG methylation in AHRR and GFI1 in mononuclear cell DNA of smokers.

Abstract About 45 million (∼19%) of U.S. adults smoke, 46 million are former smokers and >100 million are exposed to environmental tobacco smoke. Tobacco use is associated with many types of cancer, heart disease, stroke, and respiratory disease. While hundreds of tobacco smoke constituents cause DNA damage, many adverse outcomes are not related to DNA damage and an emerging hypothesis is that exposure-induced epigenetic effects may mediate many of these outcomes. Using epigenome-wide association, Joubert et al (Env Health Perspectives, 2012) observed Bonferroni-corrected statistically significant associations (480,000 tests, p<10-7) between maternal smoking and DNA methylation in cord blood at 26 CpG sites in 10 genes. We selected 21 smoking- associated CpGs in 4 genes to test as biomarkers of tobacco smoke exposure in adults. We measured methylation in peripheral blood mononuclear cell DNA of healthy adult smokers (5-70 cigarettes per day, n=43) and nonsmokers (n=19) using Illumina 450K methylation arrays. We used linear regression to assess the relationship between current smoking, pack-years, years smoked, and methylation level (log ratio beta) as the outcome. Blood from smokers and nonsmokers was also fractionated using antibody-coated magnetic beads into granulocytes, B cell, and T cell subpopulations to identify effects in specific hematopoietic lineages. Age and race were not significantly associated with methylation differences. Seven of 21 tested CpGs (AHRR, GFI1 and MYO1G) were strongly associated with both dose-dependent current smoking and years of smoking (Bonferroni, p<0.0023) while 13/21 displayed nominal significance (p<0.05). The greatest changes associated with current smoking were among CpGs located in the Ah Receptor Repressor gene (AHRR, cg05575921, delta beta = 27%, p= 1x10-10; cg23576855, delta beta=21%, p=1.96x10-6) and the GFI1 gene (four CpGs, p <0.005). Of the 21 CpGs tested, 17 displayed methylation differences that were directionally consistent with those observed in cord blood in relation to maternal smoking in pregnancy suggesting these CpGs may be a general hematopoietic biomarker of smoking. Surprisingly CpG methylation levels in the CYP1A1 regulatory region were not influenced by smoking, contrary to the effect in cord blood. The functional meaning of these smoking-induced alterations in CpG methylation is not yet clear but in ongoing work we are examining gene expression and cellular phenotype in the blood of smokers. Citation Format: Xuting Wang, Gary S. Pittman, Dan Su, Kelly N. Adamski, Michelle R. Campbell, Bonnie R. Joubert, Zhiqing Y. Huang, Cathrine Hoyo, Susan K. Murphy, Stephanie A. London, Douglas A. Bell. Dose-dependent alteration of CpG methylation in AHRR and GFI1 in mononuclear cell DNA of smokers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3647. doi:10.1158/1538-7445.AM2013-3647

Attomolar protein detection using a magnetic bead surface coverage assay

We demonstrate a microfluidic method for ultra-sensitive protein detection in serum. First, 'large' (2.8 μm) antibody-functionalized magnetic beads specifically capture antigen from a serum matrix under active microfluidic mixing. Subsequently, the large beads loaded with the antigens are gently exposed to a surface pattern of fixed 'small' (1.0 μm) antibody-coated magnetic beads. During the exposure, attractive magnetic bead dipole-dipole interactions improve the contact between the two bead types and help the antigen-antibody immunocomplex formation, while non-specific large bead adsorption is limited by exploiting viscous drag forces in the microfluidic channel on the small-bead pattern. This efficient antigen-antibody recognition and binding mechanism mimics a biological process of selective recognition of tissue molecules, like is the case when leukocytes roll and slow down on blood vessel walls by selectin-mediated adhesion. After exposure of the large beads to the pattern of small beads, the antigen concentration is detected by simply counting the number of surface pattern-bound large magnetic beads. The new technique allows detection of proteins down to the sub-zeptomole range. In particular, we demonstrate detection of only 200 molecules of Tumor Necrosis Factor-α (TNF-α) in a serum sample volume of 5 μL, corresponding to a concentration of 60 attomolar or 1 fg mL(-1).

Inducible expression of endomorphins in murine dendritic cells.

Bone marrow precursor cells were extracted from C57BL/6J mice aged 7–8 weeks, and dendritic cells were purified using anti-CD11c (a specific marker for dendritic cells) antibody-coated magnetic beads. Immunofluorescence staining revealed that the expression levels of endomorphin-1 and endomorphin-2 were upregulated in dendritic cells activated by lipopolysaccharide. An enzyme immunoassay showed that lipopolysaccharide and other Toll-like receptor ligands promoted the secretion of endomorphin-1 and endomorphin-2 from activated dendritic cells. [3H]-thymidine incorporation demonstrated that endomorphin-1 and endomorphin-2 both inhibited the proliferation of T lymphocyte induced by activated dendritic cells. Furthermore, this immunosuppressive effect was blocked by CTOP, a specific antagonist of µ-opioid receptors. Our experimental findings indicate that activated dendritic cells can induce the expression and secretion of endomorphins, and that endomorphins suppress T lymphocyte proliferation through activation of µ-opioid receptors.

Epithelial–Mesenchymal Transition and Stem Cell Markers in Patients with HER2-Positive Metastatic Breast Cancer

Currently, there is extensive information about circulating tumor cells (CTC) and their prognostic value; however, little is known about other characteristics of these cells. In this prospective study, we assessed the gene transcripts of epithelial-to-mesenchymal transition-inducing transcription factors (EMT-TF) and cancer stem cell (CSC) features in patients with HER2(+) metastatic breast cancer (MBC). Epithelial cells were enriched from peripheral blood mononuclear cells (PBMC) using antibody-coated anti-CD326 antibody (CD326(+)) magnetic beads, and the residual CD326(-) PBMCs were further depleted of leukocytes using anti-CD45 antibody-coated magnetic beads (CD326(-)CD45(-)). RNA was extracted from all cell fractions, reverse transcribed to cDNA, and subjected to quantitative reverse transcription PCR to detect EMT-TFs (TWIST1, SNAIL1, ZEB1, and TG2) as a measure of CTCs undergoing EMT (EMT-CTCs). In addition, PBMCs were analyzed using multiparameter flow cytometry for ALDH activity and CSCs that express CD24, CD44, and CD133. Twenty-eight patients were included in this study. At least one EMT-TF mRNA was elevated in the CTCs of 88.2% of patients and in the CD326(-)CD45(-) cell fraction of 60.7% of patients. The CD326(-)CD45(-) fraction of patients with elevated SNAIL1 and ZEB1 transcripts also had a higher percentage of ALDH(+)/CD133(+) cells in their blood than did patients with normal SNAIL1 and ZEB1 expression (P = 0.038). Our data indicate that patients with HER2(+) MBCs have EMT-CTCs. Moreover, an enrichment of CSCs was found in CD326(-)CD45(-) cells. Additional studies are needed to determine whether EMT-CTCs and CSCs have prognostic value in patients with HER2(+) MBCs treated with trastuzumab-based therapy.

Potential application of antibody-mimicking peptides identified by phage display in immuno-magnetic separation of an antigen

Phage display was performed against human IgG (hIgG) through five rounds of ‘biopanning’. Each round consisted of: (1) incubating a library of phage-displayed 12-mer peptides sequences on hIgG-coated magnetic beads, (2) washing the unbound phages, and (3) eluting the bound phages. The eluted phages were either amplified to enrich the pool of positive clones or subjected to the next round without amplification. Through ELISA, four clones (F9, D1, G5, and A10) showing specific binding affinity to hIgG were identified. Among these, F9 had the highest affinity (Kd=6.2nM), only one order of magnitude lower than the native anti-hIgG antibody (0.66nM). Following the DNA sequences of the selected clones, four 12-mer peptides were chemically synthesized. Among them, D1 peptide showed the highest binding affinity to hIgG via SPR biosensor measurements. This peptide was conjugated to biofunctionalized magnetic beads, and its immuno-binding ability was compared with that of the native antibody immobilized to magnetic beads. The mol-to-mol binding efficacy of the peptide-coated magnetic beads was approximately 1000-fold lower than that of the antibody-coated magnetic beads. Our results suggest a feasibility of using antibody-mimicking peptides identified by phage display technique for immuno-magnetic separation of an antigen.

Ovarian hormones increase mitochondrial biogenesis in cerebrovascular endothelial cells

In brain endothelium, mitochondria are critical for maintaining blood‐brain barrier function and transporter activity. We previously found estrogen increases mitochondrial efficiency and decreases ROS production in cerebral blood vessels, but effects on individual cell types remain unclear. To study brain endothelial cells under physiological conditions with endogenous ovarian hormones, cells were isolated from ovariectomized (OVX) and intact female mice using PECAM1 antibody‐coated magnetic beads. Hypothesis: mRNA levels of mitochondrial transcriptional regulators are decreased in brain endothelial cells from OVX mice. We found significant differences (p<0.05) in endothelial mRNA levels between OVX and intact groups. OVX increased nuclear cofactor PGC1α by 148% (0.8 ± 0.1 vs. 0.3 ± 0.02) but decreased PGC1β by 63% (1.0 ± 0.2 vs. 2.8±0.4). Moreover OVX decreased levels of NRF1 (1.2 ±0.1 vs. 1.9 ± 0.2) and TFAM (0.6 ± 0.1 vs. 1.4 ± 0.4). Endothelial mitochondrial DNA content was 29% lower in OVX mice, indicating decreased mitochondrial biogenesis, likely via decreased PGC1β stimulation of NFR1 and TFAM, factors essential in maintenance of oxidative phosphorylation and mitochondrial biogenesis. Thus endogenous ovarian hormones normally influence transcriptional master regulators of mitochondria in cerebral endothelial cells, increasing mitochondrial content and function. [NIH RO1 HL50775]

Measurement of platelet responsiveness using antibody-coated magnetic beads for lab-on-a-chip applications

We investigate novel methods for the quantification of platelet responsiveness that are suited for implementation in lab-on-a-chip devices. Magnetic beads are convenient carriers for rapid capture and manipulation of biological cells in a miniaturized system. In this article, we demonstrate that antibody-coated magnetic beads can be used to quantify platelet responsiveness. We use anti-CD62P coated beads to capture activated platelets from samples stimulated with a PAR-1 specific agonist SFLLRN, also known as thrombin receptor activator peptide. The responsiveness of the platelets is analyzed via the remaining unbound platelets in the solution and compared to a reference method in which the number of activated platelets is analyzed via fluorescent labeling. The effective concentrations for platelet activation are in agreement for the two assay types, proving that platelet responsiveness can be quantified using antibody-coated magnetic beads. We discuss the outlook for application in lab-on-a-chip devices.

Immunologic Studies in Humans.

Immunologic Studies in Humans.

Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay

Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10 5 cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10–10 4 cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10 CFU per mL of water. The assay can be completed in less than 3 h since enrichment is not required, as compared to existing techniques that typically require a 24 h incubation for pre-enrichment, followed by confirmatory tests.

High-throughput Screening Uncovers a Compound That Activates Latent HIV-1 and Acts Cooperatively with a Histone Deacetylase (HDAC) Inhibitor

Current antiretroviral therapy (ART) provides potent suppression of HIV-1 replication. However, ART does not target latent viral reservoirs, so persistent infection remains a challenge. Small molecules with pharmacological properties that allow them to reach and activate viral reservoirs could potentially be utilized to eliminate the latent arm of the infection when used in combination with ART. Here we describe a cell-based system modeling HIV-1 latency that was utilized in a high-throughput screen to identify small molecule antagonists of HIV-1 latency. A more detailed analysis is provided for one of the hit compounds, antiviral 6 (AV6), which required nuclear factor of activated T cells for early mRNA expression while exhibiting RNA-stabilizing activity. It was found that AV6 reproducibly activated latent provirus from different lymphocyte-based clonal cell lines as well as from latently infected primary resting CD4(+) T cells without causing general T cell proliferation or activation. Moreover, AV6 complemented the latency antagonist activity of a previously described histone deacetylase (HDAC) inhibitor. This is a proof of concept showing that a high-throughput screen employing a cell-based model of HIV-1 latency can be utilized to identify new classes of compounds that can be used in concert with other persistent antagonists with the aim of viral clearance.

Micromachine‐Enabled Capture and Isolation of Cancer Cells in Complex Media

Circulating tumor cells (CTCs) are the primary entities responsible for spawning cancer metastasis. Detection of CTCs provides an indicator for the clinical diagnosis and prognosis of various types of cancers. Several approaches, based primarily on flowing the sample through antibody-coated magnetic-beads[1] or microchip[2,3] surfaces have been described for isolating and counting CTCs. However, these approaches require extensive sample preparation and/or complex surface microstructures to detect the extremely low abundance of CTCs in blood.[3,4] In this study we describe a immunomicromachine-based approach for an in-vitro isolation of cancer cells that holds promise for direct CTC detection without sample pre-processing.

Studies on botulinum neurotoxins type/C1 and mosaic/DC using Endopep-MS and proteomics

Botulinum neurotoxins (BoNTs) are very potent toxins and category A biological threat agents. BoNT serotypes /C1 and /D affect birds and mammals and can be potentially lethal to humans. We have previously described the usefulness of the Endopep-MS method to detect the activity of BoNT A through G. This report was followed by the application of the method to clinical samples. The activity of the BoNT serotypes associated with human disease (/A, /B, /E, and /F) was successfully detected. However, BoNT/C and /D require different conditions for fast substrate cleavage, and a comprehensive description of a method to study BoNT/C and /D has not yet been reported. This work describes a new, optimized version of the Endopep-MS method to detect BoNTs /C1 and /DC either spiked directly in 20 μL of reaction buffer or spiked in a larger volume of buffer and further extracted using antibody-coated magnetic beads. It was found that the incubation temperature at 42 °C was more effective for both toxin serotypes, but each toxin serotype has an optimum cleavage pH. Additionally, we describe for the first time a proteomics study using a fast trypsin digestion method and label-free quantification of these toxin serotypes.

Costimulated, Tumor-Derived Donor Lymphocyte (TDL) Infusion for B-Cell Tumor Relapse After Allogeneic Hematopoietic Stem Cell Transplantation

AbstractAbstract 683Donor lymphocyte infusion (DLI) is the primary treatment for relapse after allogeneic hematopoietic stem cell transplantation (allotransplant), but is effective in less than half of patients and is associated with significant graft-vs-host disease (GVHD). Furthermore, DLI is not available for cord-blood or many unrelated-donor recipients. We theorized that tumor from patients relapsing post-allotransplant would contain infiltrating donor T- lymphocytes with tumor-specificity. If such cells had been “primed”, but inadequately costimulated for tumor cell killing, costimulation ex-vivo could generate a donor lymphocyte product with increased graft-vs-tumor (GVT) potency. Preclinical feasibility was demonstrated in tumor biopsies from patients with tumor progression after allotransplant. Ex-vivo CD3/CD28 costimulation of tumor-infiltrating lymphocytes with antibody-coated magnetic beads yielded donor products, predominently consisting of effector T-cells; tumor specimens of greater than 1 cm reliably produced donor T cell expansions that would permit clinically relevant cell dosing. Based on these preclinical results, we conducted a Phase 1 study of tumor-derived donor lymphocyte (TDL) therapy in allotransplant recipients with B-lymphoid tumor progression despite full-donor T cell chimerism and DLI, or for which DLI was not available. The primary objectives of (1) feasibility of ex-vivo costimulation of lymphocytes from surgically harvested tumor to generate a TDL product and (2) safety of TDL infusion (TDLI) in patients with relapse after allotransplant were met within the first 7 of 15 planned subjects. All 7 patients had chemotherapy-refractory relapse after allotransplant (diffuse large-B cell lymphoma (DLBCL) = 2; Hodgkin's Lymphoma (HL) = 4; chronic lymphocytic leukemia (CLL) = 1). Tumor was surgically resected. Lymphocytes were released from tumor tissue through mechanical dispersion and cultured with CD3/CD28 antibody-coated magnetic beads in media supplemented with IL-2 (100 IU/ml) for a median of 12 days (range: 7– 42 days). T cells comprised from 0.5 – 63.4% of tumor cell suspensions (culture inocula) and 82 – 99% of expanded products (22- to 144-fold expansion). PCR-based chimerism assessment of products demonstrated that the TDL were of donor origin (median 99% donor). During TDL expansion, markers of activation and effector function increased (CD4: CD40L; CD8: CD137, perforin, NKG2D). Percentages of FoxP3+ Treg cells declined and TBet+ Th1/Tc1 effectors increased in the costimulated TDL product. Expression of CD27 and CD28 has been associated with long-term persistence of infused effector T cells; although these markers declined in culture, expression was retained in a significant proportion of cells from the 5 products that were cultured for less than 14 days. TDL products meeting release criteria for tumor contamination (<5%) and sterility were generated for all subjects. TDL were administered to all subjects, with a median of 2.63 × 107 cells/kg (range: 0.93 – 10 × 107 cells/kg) infused. No acute toxicities were seen during 24 hours of observation post-infusion, and there was no evidence of GVHD over a minimum follow-up of 4 weeks. Three subjects succumbed to their cancer prior to disease restaging (2 DLBCL and 1 HL). In 2 of 4 evaluable subjects, tumor-specific effects were observed following TDLI: in the first (HL), FDG-PET uptake by tumor declined at 4 and at 8 weeks after TDLI; in the second (HL), peri-tumoral pulmonary infiltrates developed 4 weeks after TDLI, with a subsequent tumor response upon initiation of systemic steroids. The remaining 2 subjects demonstrated mixed responses, with transient overall disease stabilization. We have demonstrated the feasibility and safety of TDL therapy, with suggestive evidence of tumor-directed biologic effects and potential GVT activity. Incorporating host immune depletion prior to TDLI, as has been required for efficacy in autologous adoptive cellular therapy, may permit more potent and sustainable GVT responses. TDL may provide a therapeutic option for patients without a source of DLI, e.g., cord-blood recipients, or for those whose tumors do not respond to standard DLI. Disclosures:June:N/A: Inventor, Government-held Patent.

A novel method to derive amniotic fluid stem cells for therapeutic purposes

BackgroundHuman amniotic fluid stem (hAFS) cells have become an attractive stem cell source for medical therapy due to both their ability to propagate as stem cells and the lack of ethical debate that comes with the use of embryonic stem cells. Although techniques to derive stem cells from amniotic fluid are available, the techniques have limitations for clinical uses, including a requirement of long periods of time for stem cell production, population heterogeneity and xeno-contamination from using animal antibody-coated magnetic beads. Herein we describe a novel isolation method that fits for hAFS derivation for cell-based therapy.Methods and ResultsWith our method, single hAFS cells generate colonies in a primary culture of amniotic fluid cells. Individual hAFS colonies are then expanded by subculturing in order to make a clonal hAFS cell line. This method allows derivation of a substantial amount of a pure stem cell population within a short period of time. Indeed, 108 cells from a clonal hAFS line can be derived in two weeks using our method, while previous techniques require two months. The resultant hAFS cells show a 2-5 times greater proliferative ability than with previous techniques and a population doubling time of 0.8 days. The hAFS cells exhibit typical hAFS cell characteristics including the ability to differentiate into adipogenic-, osteogenic- and neurogenic lineages, expression of specific stem cell markers including Oct4, SSEA4, CD29, CD44, CD73, CD90, CD105 and CD133, and maintenance of a normal karyotype over long culture periods.ConclusionsWe have created a novel hAFS cell derivation method that can produce a vast amount of high quality stem cells within a short period of time. Our technique makes possibility for providing autogenic fetal stem cells and allogeneic cells for future cell-based therapy.

Isolation and Purification of &lt;em&gt;Drosophila&lt;/em&gt; Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Oral Administration of Carbon-14 Labeled Cyanocobalamin (14C-Cbl) Reveals Variable Degradation of Vitamin B12 in the Gastrointestinal Tract That Impacts Vitamin B12 Absorption and Status.

Abstract 3018Poster Board II-994Recent evidence from our laboratory and others suggests that a variable portion of ingested cobalamin (Cbl), either crystalline or from food, is degraded in the gastrointestinal tract. We have developed a biosynthetic method to incorporate 14C into the lower axial ligand of cobalamin that has made it possible to study the fate of this vitamin during its passage through the gastrointestinal tract and to assess the presence of Cbl or its breakdown products in biological samples. Following oral administration of an aqueous physiological tracer dose of 14C-Cbl (1.3 μg, 50 nCi), blood, urine, and feces are analyzed for 14C by accelerator mass spectrometry. In 9 subjects, the plasma response was consistent with the expected behavior of peroral Cbl: 14C-Cbl first appeared in the plasma 3h post-dose reaching a peak level within 6-8h. Confirmation that this dose appears bound to the physiological transport protein transcobalamin (TC) was obtained in a subset of subjects by an immunoaffinity method using anti-human TC antibody-coated magnetic beads which selectively bound 95-98% of plasma 14C. Urinary excretion of 14C was maximal in the first 24h, with 14C first appearing in urine as early as 1.5h after dosing. Fecal excretion occurred variably over several days. The amount of 14C found in the urine (10-50% of the dose) was 100-fold greater than in previous reports using 57Co-labeled cyanocobalamin (0.1-0.5%), and fecal excretion was lower than expected (10-20% vs 30-70%). Urinary excretion of 14C was inversely correlated with the peak plasma level of 14C attained (r2=0.610; p<0.001). The bulk of urinary 14C was not associated with intact Cbl and first appeared in the urine before peak 14C levels were attained in the plasma. The peak plasma level of 14C attained also showed a strong positive correlation with plasma holotranscobalamin concentration measured before administration of the 14C-Cbl (r2=0.571; p<0.001). No such correlation was found with total plasma Cbl. In additional experiments on normal volunteers using eggs endogenously labeled with 14C Cbl following intramuscular injection of hens with 14C Cbl, comparably high urinary excretion of 14C was also observed. We conclude that a variable fraction of ingested Cbl is degraded in the gastrointestinal tract of normal individuals. This may be an important determinant of the amount of Cbl absorbed from food or supplement sources. Additionally, our findings suggest that the concentration of holoTC in the plasma reflects absorptive capacity and may therefore be a good surrogate for Cbl absorptive status. Our findings also have implications regarding the bioavailability of Cbl and may inform pending considerations to fortify food supplies with Cbl in order to mitigate the incidence of Cbl deficiency, particularly among the elderly. Intestinal degradation, either microbial or through the action of digestive enzymes, may also be a source of Cbl analogues that have previously been detected in the plasma and tissues. Cbl analogues may interfere with the physiological function of cobalamin, resulting in some of the manifestations of cobalamin deficiency. Disclosures:Green:Vitalea Science: Research Funding. Miller:Vitalea Science: Research Funding. Lee:Vitalea Science: Research Funding. Sutter:Vitalea Science: Research Funding. Allen:Vitalea Science: Research Funding. Dueker:Vitalea Science: Employment.

Electrochemical immunoassay using magnetic beads for the determination of zearalenone in baby food: An anticipated analytical tool for food safety

In this work, electrochemical immunoassay involving magnetic beads to determine zearalenone in selected food samples has been developed. The immunoassay scheme has been based on a direct competitive immunoassay method in which antibody-coated magnetic beads were employed as the immobilisation support and horseradish peroxidase (HRP) was used as enzymatic label. Amperometric detection has been achieved through the addition of hydrogen peroxide substrate and hydroquinone as mediator. Analytical performance of the electrochemical immunoassay has been evaluated by analysis of maize certified reference material (CRM) and selected baby food samples. A detection limit (LOD) of 0.011 μg L −1 and EC 50 0.079 μg L −1 were obtained allowing the assessment of the detection of zearalenone mycotoxin. In addition, an excellent accuracy with a high recovery yield ranging between 95 and 108% has been obtained. The analytical features have shown the proposed electrochemical immunoassay to be a very powerful and timely screening tool for the food safety scene.

Isolation of prostate cancer cell subpopulations of functional interest by use of an on-chip magnetic bead-based cell separator

This work presents the design, fabrication and characterization of a modular magnetic bead-based cell separation device developed for the sequential sorting of a heterogeneous prostate cancer (CaP) cell population. The chief aim is cell sorting carried out on the basis of surface marker expression, serially selecting cellular subpopulations for capture by the use of antibody-coated magnetic beads. The markers of interest, prostate specific membrane antigen (PSMA) and CD10 were selected for their relevance to ongoing CaP development research. The separation device was fabricated out of plastic, by the use of cyclic olefin copolymer (COC) injection molding, nickel–iron electroplating and thermoplastic fusion bonding. Effective depletion and enrichment of cell subsets based on multiple surface markers was achieved. Various flow rates and incubation times were tested for optimizing the sorting procedure.