Peroxidase Labeling Research Articles

Genotyping and phenotyping of Haptoglobin gene polymorphisms and their correlation with cardiovascular disease and biochemical parameters in Iranian patients with type 2 diabetes mellitus

PurposeWe aimed to examine genotyping and phenotyping of Haptoglobin (Hp) gene polymorphisms and their correlation with cardiovascular disease and biochemical parameters in Iranian patients with type 2 diabetes mellitus. MethodsWe analyzed the phenotype (by polyacrylamide gel electrophoresis and peroxidase labeling), genotype (by extracting DNA), and biochemical characteristics to determine the relationship of Hp polymorphisms with premature CVD and its severity in 148 patients with T2DM, 150 patients with both CVD and T2DM, and 130 healthy controls at Afshar Hospital in Yazd city, Iran. ResultsAfter adjustment for age and gender, the Hp21 and Hp2–2 genotypes and phenotypes were related to an elevated risk of CVD in diabetic participants compared to the control group (OR = 10.35, 95% CI: 3.39–31.59, p = 0001and OR = 9.25, 95% CI: 2.29–28.59, p = 0001, respectively). Such an association was significantly observed in patients with diabetes compared to the control group (OR = 1.06, 95% CI: 0.25–4.20, p = 0.956); however, the frequency of Hp2–1and Hp2–2 genotypes in patients with both diabetes and CVD was not significantly different in comparison with diabetic individuals (OR = 1.65, 95% CI: 0.40–6.83, p = 0.484). After adjusting for age, sex, and disease, we also discovered that the Hp polymorphisms are correlated with low density lipoprotein cholesterol (LDL) and triglyceride (TG) levels. ConclusionBased on what we found, the genotypes and phenotypes of Hp2–2 and Hp2–1 are thought to be risk factors for type 2 diabetes. In addition, these polymorphisms might play an important role in increasing LDL and TG levels.

Multiplex signal amplification for ultrasensitive CRP assay via integrated electrochemical biosensor array using MOF-derived carbon material and aptamers

Accurate and precise detection of disease-associated proteins, such as C-reactive protein (CRP), remains a challenge in biosensor development. Herein, we present a novel approach－an integrated disposable aptasensor array－designed for precise, ultra-sensitive, and parallel detection of CRP in plasma samples. This integrated biosensing array platform enables multiplex parallel testing, ensuring the accuracy and reliability in sample analysis. The ultra-sensitivity of this biosensor is achieved through multiplex signal amplification. Leveraging the superior conductivity and extensive surface area of MOF-derived nanoporous carbon material (CMOF), the biosensor enhances recognition elements (aptamers) by catalyzing the horseradish peroxidase (HRP) label enzyme reaction to multiply the number of probe molecules. Optimized conditions yielded exceptional performance, exhibiting high accuracy (relative standard deviation, RSD≤10.0 %), a low detection limit (0.3 pg/mL, S/N = 3), ultra-sensitivity (0.16 μA/ng mL−1 mm−2), and a rapid response (seven parallel tests within 60 min). Importantly, this multi-unit integrated disposable aptasensor array accurately quantified CRP in human serum, demonstrating comparable results to commercial enzyme–linked immunosorbent assay (ELISA). This technology showcases promise for detecting various biomarkers using a unified approach, presenting an appealing strategy for early disease diagnosis and biological analysis.

Graphene as basis of biological sensors for determining markers of neurodegenerative dementia

Objective. To develop technique immobilizing antibodies graphene surface of proteins that play a significant role in pathogenesis Alzheimer's disease.Materials and methods. Graphene films were obtained sublimation surface of SiC substrates. Presence graphene monolayer was confirmed spectroscopy spectra. Graphene surface quality was evaluated cyclic voltammetry. Functionalization by amino groups was carried out method based on sorption pyrene derivatives from a solution and phenylnitrogroups electrochemical method. Graphene was kept in solutions monoclonal antibodies to human beta-amyloid peptide 1–42. Preparations were also kept in solution secondary antibodies labeled with FITZ. Results were evaluated fluorescence microscopy. Additionally, samples were kept in solution antibody with peroxidase label, which was detected chemiluminescence.Results. For attachment specific antibodies surface of graphene, quality its surface is great importance. Optimal working concentration of antibodies of human beta-amyloid 1–42 in solution for subsequent manufacture biological sensors is 15 micrograms per 1 ml. Covalent crosslinking antibodies with glutaraldehyde with amino groups on graphene gives a slight gain in the level fluorescence compared with noncovalent sorption on graphene with nitro groups. Functionalization phenylnitrogroups is optimal for further work related to the identification specific antigens.Conclusions. The technique of immobilization on the graphene surface of specific antibodies to beta-amyloid in concentrations detected by fluorescence microscopy and chemiluminescence is investigated. Amount antibodies sufficient to create a biosensor is immobilized on graphene. It was found that functionalization of phenylnitrogroups allows creating optimal conditions for the attachment of antibodies to the graphene surface, as well as washing resulting antibody-antigenic complexes for further reuse of graphene biosensors.

Impact of bridge heterology on functional parameters of ELISA for 17α-methyltestosterone

In this study, we have developed bridge heterologous ELISA for the detection of 17α- Methyltestosterone by incorporating aromatic spacers between 17α-Methyltestosterone-3-Carboxymethyloxime and Horseradish peroxidase label through N-hydroxysuccinimide mediated carbodiimide reaction method. The immunogen 17α-Methyltestosterone-3-Carboxymethyloxime-Bovine serum albumin used to generate the antibody was also prepared by the N-hydroxysuccinimide mediated carbodiimide reaction without using any spacer. We have studied the impact of bridge/aromatic spacers on functional parameters i.e. sensitivity, affinity and ED50 of the bridge heterologous assay and compared it with homologous assay. The five combinations of bridge heterologous assay using 17α-Methyl testosterone-3-CMO-BSA antiserum and 17α-MT-3-CMO-4,4’-Diaminodiphenyl sulphide-HRP, 17α MT-3-CMO-4,4’-Oxydianiline-HRP, 17α-MT-3-CMO-Benzidine-HRP, 17α- MT-3-CMO-p-Phenylenediamine-HRP and 17α-MT-3-CMO-Dapson-HRP enzyme conjugates were evaluated. Out of these five combinations, the combination 17α-MT-3-CMO-BSA with 17α-MT-3-CMO-Benzidine-HRP showed the best results. Sensitivity, affinity and ED50 were improved and found to be 0.02 ng/mL, 0.086 × 10−8 L/mol and 2.95 ng/mL than homologous assay where Sensitivity, affinity and ED50 were 0.11 ng/mL, 0.02 × 10−8 L/mol and 5.78 ng/mL respectively. The cross-reactivity for this bridge heterologous assay combination was seen with only 4 steroids (6-hydrotestosterone- 6%, Testosterone-5.14%, Danazol-0.9% and Nandrolone-0.85%) instead of eight steroids (6-hydrotestosterone-43.75%, Testosterone-38.3%, Danazol-25.14%, Androstenediol-19.16%, Nandrolone-19%, Metandienone-5%, Androstenedione-3.52%, and 17α dimethyltestosterone-2%) as in homologous assay out of 59 structurally related steroids.Thus, the results of this study conclude that the incorporation of aromatic spacer (bridge) in enzyme conjugate has a crucial role in improving sensitivity, specificity, ED50 and affinity of the developed assay. The assay was then studied for parameters such as recovery (97.4%–108.6%), precision (Inter and Intra-assay coefficient of variation <10%), correlation coefficient (R2 = 0.96) by comparing with the commercial kit and validated by measuring levels of 17α- methyltestosterone in rat serum after administering them.

Abstract 119: Exploring The Role Of Rho-related BTB Domain Containing 1(RhoBTB1) And Phosphodiesterase 5(PDE5) To Identify Potential Proteomic Targets In Cardiovascular Function

The study of macromolecular (protein:protein) interactions has significantly enhanced our comprehension of human impairments, such as cardiovascular diseases (CVDs). We previously identified that RhoBTB1 acts as a key regulator of vascular tone by associating with Cullin-3 (CUL3) to control the activity of phosphodiesterase 5 (PDE5) through the ubiquitination-proteasomal pathway. PDE5 selectively hydrolyzes cGMP thus regulating NO-mediated vasodilation. We hypothesized that the PDE5 binding region on RhoBTB1 could be responsible for binding to other unknown partners in RhoBTB1-CUL3 pathway. We truncated RhoBTB1 into its component domains individually (GTPase, BTB1, BTB2, and CT) and in combination (B1B2C, PB1B2, and B2C) and studied their interaction with PDE5 by co-immunoprecipitation (co-IP) in HEK293 cells. These analyses revealed that the B1B2C domain of RhoBTB1 is the minimal region required to bind PDE5. Interestingly, the CT region is essential as the truncations lacking CT could not participate in PDE5 binding and its subsequent proteasomal degradation. The GTPase and Proline-rich (P) domains were dispensable for binding of RhoBTB1 to PDE5. We identified that Pro 353 and Ser 363 are key amino acid residues in the B1B2C region involved in CUL3 binding to RhoBTB1. Mutation of these amino acids resulted in hampered binding to CUL3. The mutants failed to degrade PDE5 despite their preserved binding to PDE5. We employed ascorbate peroxidase (APEX2) labeling using a B1B2C-APEX2 fusion construct as a bait to identify unknown RhoBTB1 binding partners. As a contemporaneous screen, we used B1B2-APEX2 since it does not bind to PDE5, and therefore is not expected to bind to those unknown partners. We “filtered” the captured proteome for its interaction with B1B2C better than B1B2 (without the CT). SET Domain Containing 2 (SETD2) was among several B1B2C binding partners identified and validated. SETD2 and RhoBTB1 directly interacted, and the level of SETD2 increased in response to RhoBTB1-inhibition with siRNA, inhibition of proteasomal machinery either by pharmacological means or CUL3 editing. This suggests that SETD2 is regulated by the RhoBTB1-CUL3 axis. Future studies may pave a way to determine if SETD2 is important for cardiovascular function.

Lectin Blotting of Cell Lysates.

Glycans are composed of branched structures consisting of monosaccharides, such as glucose and galactose linked by glycosidic bonds. Glycans are often bound to proteins and lipids and are localized at the cell surface. They are deeply involved in a wide range of multicellular systems inside and outside the cells, such as the quality control of glycoproteins, cell-cell communication, and various diseases. While western blotting uses antibodies to detect proteins, lectin blotting uses lectins, which are glycan-binding proteins, to detect glycans on glycoconjugates, such as glycoproteins. Lectin blotting was first reported in the early 1980s and has been widely used in life science for several decades. However, it is not straightforward to obtain consistent data using lectin blotting, which tends to show high backgrounds and lab-to-lab variation. Here, we describe the protocol used in our laboratory for lectin blotting following protein separation by SDS-PAGE to detect glycoproteins extracted from cell membrane fractions. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Extraction and quantification of proteins from cell lysate Basic Protocol 2: Lectin peroxidase labeling and lectin blotting.

Immunohistochemistry and Immunofluorescence.

Immunohistochemistry (IHC) is one of the most widely used protein detection techniques. The principle of this technique is based on the binding of a specific antibody to a matching specific antigen in tissue. The bound antigen-antibody complex then is visualized using a range of detection techniques. IHC uses a number of different enzymatic labels, such as peroxidase and alkaline phosphatase, for the detection of the antigens of interest whereas immunofluorescence (IF) uses a fluorescent signal. In this chapter, IHC will be described using the peroxidase label. Both IHC and IF can be used on formalin-fixed paraffin-embedded (FFPE) or appropriately processed fresh tissues. IHC/IF can be multiplexed to detect more than one antigen at a time, or may be sequentially stained to detect multiple targets. These techniques are routinely used in diagnostic pathology laboratories, not just for diagnostic purposes but many biomarkers are used for patient staging, treatment allocation, and prognostication. Immunofluorescence is routinely used for the detection of antibodies and antigens in freshly biopsied tissues, particularly for immune-mediated and vesiculobullous lesions. In this chapter, the principles of IHC are reviewed followed by examples of IHC and IF staining using readily available antibodies. Steps and processes involved in IHC/IF double staining are also described.

An In Situ Ascorbate Peroxidase Labeling in Secreted Exosomes Identifies Oxidative Stress-Induced Exosome Proteome Alteration in Renal Proximal Tubules

An In Situ Ascorbate Peroxidase Labeling in Secreted Exosomes Identifies Oxidative Stress-Induced Exosome Proteome Alteration in Renal Proximal Tubules

Cytokine profile in community-acquired pneumonia in children

Community-acquired pneumonia (CAP) is one of the most common acute infectious diseases. To date, the incidence of CAP among children was decreased significantly worldwide, mainly due to increasing use of effective preventive measures. Nevertheless, CAP remains a common cause of childhood morbidity and mortality. Pneumonia may develop at any age, but most often it occurs in young children, who are more likely to have a more severe course of pneumonia. Currently, early diagnosis and prognosis of the disease severity in children is an urgent issue. It was found that, in most cases, a panel of conventional biomarkers, including the number of leukocytes, procalcitonin, CRP is not sufficient for the diagnosis of pediatric CAP. There is a demand for new biological markers which, along with clinical evaluation, may significantly improve diagnostics and management of CAP in children, thus reducing the risk of adverse outcomes associated with this disease. Such markers could be found among the cytokines, which are active participants in the CAP pathogenesis. The aim of this study was to determine the level of several cytokines in blood serum of children with CAP and to assess changes in the cytokine profile depending on the patient’s age and severity of the disease. The study included 117 children aged 1 to 18 years with a diagnosis of CAP confirmed by X-ray examination. The comparison group included 28 healthy children who did not have CAP or other signs of acute respiratory viral infection at the time of examination, being free of any chronic pathology requiring outpatient observation. A number of cytokines were determined quantitatively in blood serum, i.e., IL-1β, IFNγ, IL-2, IL-4, IL-6, IL-10, TNFα, IFNλ2 (IL-28A), IFNλ3 (IL-28B), IL-8, MCP-1, IL-17AF, GM-CSF using test systems based on the “sandwich” method of solid-phase ELISA using peroxidase labeling. As a result, it was found that the content of IL-6, IL-17AF, IL-1β, IFNγ, MCP-1, IFNλ2 (IL-28A), IFNλ3 (IL-28B), GM-CSF was significantly higher in the group of children with severe community-acquired pneumonia. The levels of certain cytokines, e.g., IL-6, IFNλ2 (IL-28A), IFNλ3 (IL-28B), GM-CSF varied depending on the age of patients, thus, probably, reflecting the degree of immune system activation in the children of different age groups.

Ultra-Low Detection of Infectious Disease Nucleic Acid Biomarkers By a Visual Colorimetric Sensor

The gold standard method for detecting nucleic acid markers is the polymerase chain reaction. However, this method demands high technical skills, labor-intensive, time-consuming, and expensive. Many resource-limited countries around the globe do not have access to such laboratory-based PCR tests. To address the global test needs of virus and other pathogen infections, as well as address the early biomarker detection of diseases. We have developed an affordable nanoparticle colorimetric sensor approach. In our system, the virus RNA marker is captured by the complementary oligonucleotide attached to low fouling gold-coated iron oxide nanoparticles. Further specificity is achieved through a second complementary hybridization probe conjugated with streptavidin-horseradish peroxidase labels. Visual colorimetry is performed using a chromogenic substrate allowing the visual conversion of the colorless solution to a blue color confirming a positive test. The intensity of the color is in direct correlation to the concentration of the virus RNA marker. We have successfully demonstrated attomolar detection in buffer and picomolar detection in real undiluted biofluids such as in neat human serum. Our ongoing efforts focus on saliva-based non-invasive detection with ultra-low capabilities, optimization of the assay, repeatability, and expanding this work for a multiplex detection format

Immunoarray Measurements of Parathyroid Hormone-Related Peptides Combined with Other Biomarkers to Diagnose Aggressive Prostate Cancer.

Parathyroid hormone-related peptide (PTHrP) is related to bone metastasis and hypercalcemia in prostate and breast cancers and should be an excellent biomarker for aggressive forms of these cancers. Current clinical detection protocols for PTHrP are immunoradiometric assay and radioimmunoassay but are not sensitive enough to detect PTHrPs at early stages. We recently evaluated a prostate cancer biomarker panel, including serum monocyte differentiation antigen (CD-14), ETS-related gene protein, pigment epithelial-derived factor, and insulin-like growth factor-1, with promise for identifying aggressive prostate cancers. This panel predicted the need for patient biopsy better than PSA alone. In the present paper, we report an ultrasensitive microfluidic assay for PTHrPs and evaluate their diagnostic value and the value of including them with our prior biomarker panel to diagnose aggressive forms of prostate cancer. The immunoarray features screen-printed carbon sensor electrodes coated with 5 nm glutathione gold nanoparticles with capture antibodies attached. PTHrPs are bound to a secondary antibody attached to a polyhorseradish peroxidase label and delivered to the sensors to provide high sensitivity when activated by H2O2 and a mediator. We obtained an unprecedented 0.3 fg mL-1 limit of detection for PTHrP bioactive moieties PTHrP 1-173 and PTHrP 1-86. We also report the first study of PTHrPs in a large serum pool to identify aggressive malignancies. In assays of 130 human patient serum samples, PTHrP levels distinguished between aggressive and indolent prostate cancers with 83-91% clinical sensitivity and 78-96% specificity. Logistic regression identified the best predictive model as a combination of PTHrP 1-86 and vascular endothelial growth factor-D. PTHrP 1-173 alone also showed a high ability to differentiate aggressive and indolent cancers.

Effect of Vildagliptin on the Level of P-Glycoprotein Expression in Normal Conditions and Alloxan–Induced Type 2 Diabetes Mellitus

INTRODUCTION: Vildagliptin is one of the drugs of choice in the treatment of type 2 diabetes mellitus (DM). In the literature review, no study has reported the effect of vildagliptin on the expression level of the transporter protein P-glycoprotein (P-gp, ABCB1 protein).&#x0D; AIM: To assess the in vivo effects of vildagliptin on the level of Р-gp expression in normal conditions and experimental alloxan-induced type 2 DM.&#x0D; MATERIALS AND METHODS: Type 2 DM was induced by a single intravenous injection of freshly prepared alloxan monohydrate solution in citrate buffer (pH = 4.0) at a dose of 80 mg/kg. Glucose content (mmol/L) was determined by the glucose oxidase method using Human kit (Germany) and insulin content (U/mL) by radioimmunoassay technique using Immunotech kit (Czech Republic). As an express method for the determination of glucose levels in the whole blood, OneTouch UltraEasy electrochemical glucose monitor (USA) was used. The level of P-gp expression in rabbit tissues (i.e., jejunum, liver, kidney, and cerebral cortex) was evaluated by the indirect histochemical method. After preliminary treatment, the histological material was embedded in paraffin. Antigens were retrieved before the immune-staining reaction. Then, the sections were incubated with primary antibodies to P-gp (ABCB1 antibody, middle region; 0.1 mL [Aviva Systems Biology ARP51326-P050, USА]). For immune staining, a polymer detection system with a peroxidase label (Leica Microsystems, Germany) was used. The cell nuclei were stained with hematoxylin. Photos of the micropreparation were captured using Canon Power Shot G5 digital camera with 400 magnification. Digital images were analyzed and processed using ImageJ and IHC Profiler plugin designed for quantitative immunohistochemical analysis. The results obtained from healthy animals, animals with alloxan-induced DM, healthy animals administered vildagliptin, and animals with modeled DM administered vildagliptin were compared.&#x0D; RESULTS: After day 14 of administration to healthy rabbits and day 5 of vildagliptin discontinuation (5 mg/kg), no statistically significant changes in P-gp expression in the aforementioned tissues and the levels of basal and postprandial insulin and blood glucose were noted. After the 14-day therapy of alloxan-induced type 2 DM with vildagliptin (5 mg/kg), a statistically significant recovery of expression of the P-gp expression in the jejunum, liver, and kidney was reduced due to the underlying pathology. In addition, the values of healthy rabbits and statistically significant normalization of the insulin and blood glucose levels were observed. On day 5 of drug cancellation, significant inhibition of P-gp expression in the jejunum, liver, and kidney was noted again, and there was a significant reduction of postprandial insulin and blood glucose. No significant alterations of ABCB1 protein expression in the hematoencephalic barrier were noted during the studied periods.&#x0D; CONCLUSION: The administration of vildagliptin (5 mg/kg) to rabbits with alloxan-induced type 2 DM promotes the recovery of P-gp expression in the jejunum, liver, and kidney, which were reduced due to the underlying pathology. Meanwhile, its administration to healthy animals had no significant effect on the expression of P-gp.

Ultrastructure of Rat Rostral Nucleus of the Solitary Tract Terminals in the Parabrachial Nucleus and Medullary Reticular Formation.

Neurons in the rostral nucleus of the solitary tract (rNST) receive taste information from the tongue and relay it mainly to the parabrachial nucleus (PBN) and the medullary reticular formation (RF) through two functionally different neural circuits. To help understand how the information from the rNST neurons is transmitted within these brainstem relay nuclei in the taste pathway, we examined the terminals of the rNST neurons in the PBN and RF by use of anterograde horseradish peroxidase (HRP) labeling, postembedding immunogold staining for glutamate, serial section electron microscopy, and quantitative analysis. Most of the anterogradely labeled, glutamate-immunopositive axon terminals made a synaptic contact with only a single postsynaptic element in PBN and RF, suggesting that the sensory information from rNST neurons, at the individual terminal level, is not passed to multiple target cells. Labeled terminals were usually presynaptic to distal dendritic shafts in both target nuclei. However, the frequency of labeled terminals that contacted dendritic spines was significantly higher in the PBN than in the RF, and the frequency of labeled terminals that contacted somata or proximal dendrites was significantly higher in the RF than in the PBN. Labeled terminals receiving axoaxonic synapses, which are a morphological substrate for presynaptic modulation frequently found in primary sensory afferents, were not observed. These findings suggest that the sensory information from rNST neurons is processed in a relatively simple manner in both PBN and RF, but in a distinctly different manner in the PBN as opposed to the RF.

In Situ Peroxidase Labeling Followed by Mass-Spectrometry Reveals TIA1 Interactome.

Simple SummaryT-cell intracellular antigen 1 (TIA1) is a DNA/RNA-binding protein best known for its different roles in RNA metabolism. Currently, little is known about the interacting protein partners of TIA1 in control and stress conditions that could shed light on its multiple context-specific molecular functions. Proximity labeling is a technique in which a labeling enzyme, here APEX2, that is fused to the protein of interest, in this case TIA1, marks the protein’s interacting network in living cells, allowing for subsequent ex vivo analysis using protein identification methods such as mass spectrometry. Hereby, combining these two techniques, it was revealed that the TIA1 interactome has very distinct protein partners in control and unstressed cells and that these partners are involved in not only previously identified processes such as splicing, nucleocytoplasmic transport, and different levels of protein translation control, but also in novel ones such as DNA double-strand break repair and mitochondrial metabolism. Overall, these findings provide a more precise definition of TIA1’s function in cells and pave the way to dissect its role in each of these processes.TIA1 is a broadly expressed DNA/RNA binding protein that regulates multiple aspects of RNA metabolism. It is best known for its role in stress granule assembly during the cellular stress response. Three RNA recognition motifs mediate TIA1 functions along with a prion-like domain that supports multivalent protein-protein interactions that are yet poorly characterized. Here, by fusing the enhanced ascorbate peroxidase 2 (APEX2) biotin-labeling enzyme to TIA1 combined with mass spectrometry, the proteins in the immediate vicinity of TIA1 were defined in situ. Eighty-six and 203 protein partners, mostly associated with ribonucleoprotein complexes, were identified in unstressed control and acute stress conditions, respectively. Remarkably, the repertoire of TIA1 protein partners was highly dissimilar between the two cellular states. Under unstressed control conditions, the biological processes associated with the TIA1 interactome were enriched for cytosolic ontologies related to mRNA metabolism, such as translation initiation, nucleocytoplasmic transport, and RNA catabolism, while the protein identities were primarily represented by RNA binding proteins, ribosomal subunits, and eicosanoid regulators. Under acute stress, TIA1-labeled partners displayed a broader subcellular distribution that included the chromosomes and mitochondria. The enriched biological processes included splicing, translation, and protein synthesis regulation, while the molecular function of the proteins was enriched for RNA binding activity, ribosomal subunits, DNA double-strand break repair, and amide metabolism. Altogether, these data highlight the TIA1 spatial environment with its different partners in diverse cellular states and pave the way to dissect TIA1 role in these processes.

Sensitive electrochemical immunosensor to detect prohibitin 2, a potential blood cancer biomarker

Blood cancers are difficult to cure completely and frequently show a poor prognosis. Recently, prohibitin 2 (PHB2) has been shown to be a potential biomarker for blood cancers. Sandwich ELISA can be used as a reference method for quantitative analysis of PHB2; however, ELISA can be challenging for early diagnosis and continuous monitoring method due to the need for large sample volumes (25 μL <), technical expertise, complex procedure, relative high cost, and non-portability. Thus, this study developed a sensitive and time efficient electrochemical immunosensor for detecting PHB2 from a blood cancer patient. It is a simple and portable platform consisting of a disposable electrode and blood sample volume of 4 μL. The sensor uses a gold nanostructured electrode and square wave voltammetry (SWV) measurement of a horseradish peroxidase (HRP) label to amplify the electrochemical signal. The immunosensor could quantitatively detect PHB2 with high sensitivity (limit of detection [LoD] = 0.04 ng/mL) and satisfactory reproducibility (relative standard deviation [RSD] <5.2%). The sensor achieved an LoD of 0.63 ng/mL with satisfactory recovery (89.1–104.7%) and reproducibility (RSD <6.4%) with PHB2 spiked into white blood cell (WBC) lysates. When the sensor was compared to a reference ELISA to determine the PHB2 concentrations in WBC lysate samples from healthy patients and those with blood cancer, the correlation coefficient (R2) was 0.996. A 3.3-fold difference was detected in the measured PHB2 concentration between blood cancer patients and healthy individuals. Accordingly, this study suggests a sensitive and accurate analytical method for quantitatively detecting the PHB2 in blood samples.

ASSOCIATIONS BETWEEN BLOOD CONCENTRATIONS OF CYTOTOXIC CD8+ CELLS AND LYMPHOCYTE APOPTOSIS IN HEALTHY HUMANS

Introduction: Cytotoxic T-lymphocytes play an important role in a specific immune response via a lytic effect in relation to abnormal cells. The number of these cells greatly increases sin pathological states. In addition, an increased cytotoxic activity is a characteristic of the immune response of people living in the Far NorthTherefore, it is important to understand what function cytotoxic T-lymphocytes predominantly perform in healthy people in the North - cytolytic or suppressor? Aim: To assess associations between CD8+ concentration and immune response and apoptotic deaths of the lymphocytes in healthy individuals. Methods: Ninety-three healthy adult residents of the Russian North comprised the sample. Apoptotic lymphocyte death was studied by flow cytometry. FITC-annexine-V and propidium iodide labelled cells were detected. Concentrations of cytokines and apoptosis mediators were assessed by a solid-phase enzyme immunoassay. Neutrophiles, monocytes and phagocytic activity of neutrophils were studied in blood smears stained by Romanowsky's - Giemsa. The level of phenotypic activity of lymphocytes was assessed by double peroxidase labeling using monoclonal antibodies. The data were presented using means, standard deviations, medians, the 1st and the 3rd quartiles. All study participants were divided into two groups: with normal- (0.2-0,4 х109 kl/l) and elevated (more 0,6 х109 kl/l) blood cytotoxic lymphocyte levels. The groups were similar in terms of by age- and gender distribution. Continuous variables were analyzed using Mann-Whitney tests. Results: In individuals with an increased level of cytotoxic CD8+ lymphocytes in peripheral venous blood had greater concentrations of leukocytes (7.4 ± 0.49 х109 cells/l vs. 5.5 ± 0.23 х109, p = 0.003), lymphocytes (2.8 ± 0.17 х109 cells/l vs.1.8 ± 0.07 х109 cells/t p = 0.005), and mature neutrophils (4.1 ± 0.19 х109 cells/l vs. 3.4 ± 0.49 х109 cells/l, p = 0.013). No associations between the level of apoptosis of lymphocytes (AnV+/PI-) and concentrations of sFasL, TRAIL, TNFa, and cytochrome C were observed. Conclusions: Lymphocytes CD8+ in healthy residents of the Russian North perform mainly cytotoxic function, which is not related to apoptotic cellular death.

Prostate Cancer Diagnosis in the Clinic Using an 8-Protein Biomarker Panel.

The inability to distinguish aggressive from indolent prostate cancer is a longstanding clinical problem. Prostate specific antigen (PSA) tests and digital rectal exams cannot differentiate these forms. Because only ∼10% of diagnosed prostate cancer cases are aggressive, existing practice often results in overtreatment including unnecessary surgeries that degrade patients' quality of life. Here, we describe a fast microfluidic immunoarray optimized to determine 8-proteins simultaneously in 5 μL of blood serum for prostate cancer diagnostics. Using polymeric horseradish peroxidase (poly-HRP, 400 HRPs) labels to provide large signal amplification and limits of detection in the sub-fg mL-1 range, a protocol was devised for the optimization of the fast, accurate assays of 100-fold diluted serum samples. Analysis of 130 prostate cancer patient serum samples revealed that some members of the protein panel can distinguish aggressive from indolent cancers. Logistic regression was used to identify a subset of the panel, combining biomarker proteins ETS-related gene protein (ERG), insulin-like growth factor-1 (IGF-1), pigment epithelial-derived factor (PEDF), and serum monocyte differentiation antigen (CD-14) to predict whether a given patient should be referred for biopsy, which gave a much better predictive accuracy than PSA alone. This represents the first prostate cancer blood test that can predict which patients will have a high biopsy Gleason score, a standard pathology score used to grade tumors.

Ultrastructure of the Adult Hematopoietic Stem Cell Niche Defined Using Correlative Light and Electron Microscopy

Hematopoietic stem and progenitor cells (HSPCs) that originate from the hemogenic endothelium in the dorsal aorta finally home and engraft within the adult niche, the fetal bone marrow. Within the bone marrow, HSPCs are retained in a complex microenvironment surrounded by niche cells such as megakaryocytes, peripheral nerves, endothelial and mesenchymal stromal cells. While it is known that niche cells secrete hematopoietic factors that are essential for HSPCs to self-renew and repopulate the blood lineages, the ultrastructure of this niche is not well defined as current imaging technology does not allow direct visualization of the fetal bone marrow niche. Zebrafish share a similar hematopoietic ontogeny to mammals, and because the embryos are transparent, intrinsic HSPC interactions with the niche can be directly visualized. To precisely locate rare HSPCs within the large dense kidney marrow, the presumptive adult niche, we genetically tagged endogenous HSPCs to track them live using lightsheet microscopy, followed by high-resolution serial block-face scanning electron microscopy (SBEM) (XY = 10.8 nm/pixel, Z = 70 nm/pixel). Using this technique, we could visually track single mCherry+ HSPCs, then confirm their exact location in the SBEM dataset with high contrast APEX2 peroxidase label. We found HSPC clusters within vessel lumens, as well as single HSPC in a novel perivascular niche. In this perivascular site, a single HSPC simultaneously contacted one mesenchymal stromal cell, multiple endothelial cells, a glial-like cell, and other hematopoietic cells. After extensive tracing of the glial-like cells within the 3D SBEM data, we identified that these cells extended as a long chain and formed contact with HSPCs. Our search for candidate structures in the kidney marrow region suggested that the glial-like cells could be dopamine beta-hydroxylase positive (dbh+) cells. Through fluorescence imaging, we observed cytoplasmic projections extending from dbh+ cells into Runx+ HSPC clusters. To confirm the identity of these glial-like cells, we performed fluorescence imaging of dbh+ transgenic larvae followed by SBEM. By correlating the two datasets, we identified dbh+ glial-like cell in contact with a single HSPC in the niche. Furthermore, treatment with a neurotoxin, 6-hydroxydopamine, led to a significant reduction in HSPC numbers within the niche highlighting the biological significance of dbh+ cells. In summary, through this approach, we could identify a previously unknown cell type within the zebrafish adult hematopoietic niche that is pivotal in maintaining the HSPC pool. Our finding validates the importance of having multiple niche cells that concurrently regulate HSPCs within their niche. Importantly, our approach can be applied to characterize the ultrastructure of rare cells, such as other stem cells, and their niche by using multiple imaging modalities and can also lead to the identification of previously uncharacterized cell types. Further, we can now identify novel intercellular structures that form between an unperturbed HSPC in its endogenous perivascular niche. Disclosures No relevant conflicts of interest to declare.

3047 – THE ADULT HEMATOPOIETIC STEM CELL NICHE IN ZEBRAFISH DEFINED USING CORRELATIVE LIGHT AND ELECTRON MICROSCOPY

Hematopoietic stem and progenitor cells (HSPCs) that originate from the hemogenic endothelium in the dorsal aorta to finally home and engraft within the adult niche, the fetal bone marrow (BM), become surrounded by niche cells in a complex microenvironment. The ultrastructure of this niche is not well defined as current imaging technology does not allow direct visualization of the fetal BM niche. Zebrafish have a similar hematopoietic ontogeny to mammals, and because the embryos are transparent, intrinsic HSPC interactions with the niche can be directly visualized. To track HSPCs during colonization of the presumptive adult niche, the kidney marrow (KM), we used our previously validated HSPC-specific transgenic reporter lines. In 5 days post fertilization fixed larvae, we detected ∼100 HSPCs/larva. To precisely locate these rare HSPCs within the larger dense KM, we genetically tagged endogenous HSPCs to track them live using lightsheet microscopy, followed by high resolution serial block-face scanning electron microscopy (SBEM). Using this technique, we could visually track single mCherry+ HSPCs, then confirm their exact location in a SBEM dataset with high contrast APEX2 peroxidase label. We found HSPC clusters within vessel lumens, as well as single HSPC in a novel perivascular niche. In this perivascular site, a single HSPC was seen to simultaneously contact one mesenchymal stromal cell, multiple endothelial cells, a glial-like cell and other hematopoietic cells. The glial-like cells were dopamine beta hydroxylase positive (dbh+) cells and treatment with small molecules inhibitor of dopamine led to significantly reduced HSPC numbers within the niche. Our approach can be used to identify the ultrastructure of single rare cells within dense tissues by using multiple imaging modalities and can also lead to the identification of previously uncharacterized cell types.

ELISA method to detect ABO blood group in external secretion fluids

Background: Usually it takes a large number of volume sample to determine blood group from external secretion fluids. But, in certain condition, samples are only available in very small amount. The objective of this study is to detect the presence of ABO blood group substances in mucosal fluid using ELISA technique, thus only requires small amount of samples.&#x0D; Objective: To develop an ELISA technique using the current anti-ABO antibodies for determination of blood group by hemagglutination technique and second peroxidase label antibody specific for mouse IgG, originally used for another ELISA technique.&#x0D; Methods: 100 μl of diluted human intestinal mucosal fluid were incubated overnight in 4oC in ELISA microplate wells, followed by addition anti-ABO antibodies. Then after incubation, a second revealing antibody anti mouse IgG labeled with peroxidase was added. After a brief incubation, substrate H2O2 and chromogenic TMB were added.&#x0D; Results: Positive reaction is marked by development of blue colour, which, on termination enzymatic reaction by addition 100 μl H2SO4 change to yellow.&#x0D; Conclusion: An ELISA method for detecting ABO substance in mucosal fluid can be developed from antibodies not specifically made for this technique, but specific only for the target.