Immunofluorescent Probes Research Articles

Janus-Type Immunofluorescent Probes and a Quantitative Immunoassay System.

To realize highly sensitive immunoassays, high optical density probes conjugated with antibodies for target antigens have been demanded in order to increase the visibility of antigen-antibody complex formation. We herein demonstrate the development of an immunoassay system using magnetic and fluorescent Janus particles as probes in conjunction with an antibody-immobilized microfluidic device. The concentration of the detection limit at which there was a significant difference between SARS-CoV-2 and human coronavirus 229E antigens was 3.1 ng/mL, and the standard deviation of the signal was less than 5%. The immunofluorescent probe and immunoassay system developed in this study are expected to be applicable not only to SARS-CoV-2 but also to the quantitative measurement of various other disease marker proteins and biomolecules.

Hypothermia promotes tunneling nanotube formation and the transfer of astrocytic mitochondria into oxygen-glucose deprivation/reoxygenation-injured neurons

Mitochondrial transfer occurs between cells, and it is important for damaged cells to receive healthy mitochondria to maintain their normal function and protect against cell death. Accumulating evidence suggests that the functional mitochondria of astrocytes are released and transferred to oxygen–glucose deprivation/reoxygenation (OGD/R)-injured neurons. Mild hypothermia (33 °C) is capable of promoting this process, which partially restores the function of damaged neurons. However, the pathways and mechanisms by which mild hypothermia facilitates mitochondrial transfer remain unclear. We are committed to studying the role of mild hypothermia in neuroprotection to provide reliable evidences and insights for the clinical application of mild hypothermia in brain protection. Tunneling nanotubes (TNTs) are considered to be one of the routes through which mitochondria are transferred between cells. In this study, an OGD/R-injured neuronal model was successfully established, and TNTs, mitochondria, neurons and astrocytes were double labeled using immunofluorescent probes. Our results showed that TNTs were present and involved in the transfer of mitochondria between cells in the mixed-culture system of neurons and astrocytes. When neurons were subjected to OGD/R exposure, TNT formation and mitochondrial transportation from astrocytes to injured neurons were facilitated. Further analysis revealed that mild hypothermia increased the quantity of astrocytic mitochondria transferred into damaged neurons through TNTs, raised the mitochondrial membrane potential (MMP), and decreased the neuronal damage and death during OGD/R. Altogether, our data indicate that TNTs play an important role in the endogenous neuroprotection of astrocytic mitochondrial transfer. Furthermore, mild hypothermia enhances astrocytic mitochondrial transfer into OGD/R-injured neurons via TNTs, thereby promoting neuroprotection and neuronal recovery.

A Novel and Rapid Smear Cytomorphology Detection Strategy Based on Upconversion Nanoparticles Immunolabeling Integrated with Wright's Staining for Accurate Diagnosis of Leukemia.

Accurate, sensitive, and rapid identification of leukemia cells in blood and bone marrow is of paramount significance for clinical diagnosis. An integrative technique combining traditional cytomorphology with immunophenotyping was proposed to improve the diagnostic efficiency in leukemia. On account of high photostability, biocompatibility, and signal-to-background ratio, upconversion nanoparticles (UCNPs) as luminescent labels have drawn substantial research scrutiny in immunolabeling. To achieve simultaneous determination, NaYF4:Yb,Er UCNPs were coupled with CD38 antibodies to construct immunofluorescence probes that were developed to bind to diffuse large B cell lymphoma (DLBCL) cells, followed by Wright's staining that has been widely used in clinical work for morphological diagnosis. Further, the experimental conditions were optimized, such as medium, slice-making method, antibody dosage, incubation time, etc. The cell morphology and immunolabeling could be observed simultaneously, and its simple operation rendered it a possibility for clinical diagnosis. The developed immunolabeling assay could achieve DLBCL cell counting with high reproducibility and stability, and the detection limit was as low as 1.54 cell/slice (>3 σ/s). Moreover, the proposed method also realized real blood and bone marrow sample analysis, and the results were consistent with the clinical diagnosis. Overall, this strategy can be carried out after simple laboratory training and has prospective biomedical applications in leukemia classification, diagnosis validation, and differential diagnostics.

More than magnetic isolation: Dynabeads as strong Raman reporters toward simultaneous capture and identification of targets

AbstractDynabeads are superparamagnetic particles used for the immunomagnetic purification of cells and biomolecules. Post‐capture, however, target identification relies on tedious culturing, fluorescence staining, and/or target amplification. Raman spectroscopy presents a rapid detection alternative, but current implementations target cells themselves with weak Raman signals. We present antibody‐coated Dynabeads as strong Raman reporter labels whose effect can be considered a Raman parallel of immunofluorescent probes. Recent developments in techniques for separating target‐bound Dynabeads from unbound Dynabeads make such an implementation feasible with high specificity. We deploy Dynabeads anti‐Salmonella to bind and identify Salmonella enterica, a major foodborne pathogen. Dynabeads present major peaks around 1000 and 1600 cm−1 from aliphatic and aromatic C‐C stretching of the polystyrene coating and near 1350 cm−1 from the ɣ‐Fe2O3 and Fe3O4 core, confirmed with electron dispersive X‐ray (EDX) imaging. Minor to no contributions are made from the surface antibodies themselves as confirmed by Raman analysis of surface‐activated, antibody‐free beads. Dynabeads' Raman signature can be measured in dry and liquid samples even at single shot ~30 × 30 μm area imaging using 0.5 s, 7‐mW laser acquisition with single and clustered beads providing a 44‐ and 68‐fold larger Raman intensity compared with the signature from cells. Higher polystyrene and iron oxide content in clusters yields larger signal intensity, and conjugation to bacteria strengthens clustering as a bacterium can bind to more than one bead as observed via transmission electron microscopy (TEM). Our findings shed light on the intrinsic Raman reporter nature of Dynabeads. When combined with emerging techniques for the separation of target‐bound Dynabeads from unbound Dynabeads such as using centrifugation through a density media bilayer, they have the potential to demonstrate their dual function for target isolation and detection without tedious staining steps or unique plasmonic substrate engineering, advancing their applications in heterogeneous samples like food, water, and blood.

Characterization of Terminal Erythroid Differentiation (TED) and Its Prognosis Impact Inpatients with Myelodysplastic Syndrome (MDS)

INTRODUCTION: Culture assays of normal bone marrow (BM) have shown that TED follows a doubling maturation pattern in each erythroid stage from proerythroblast to orthochromatic erythroblast. For a long time, it has been suggested that TED is profoundly abnormal across all myelodysplastic syndrome (MDS) subtypes. Nevertheless, morphologic quantification of cells undergoing TED in BM both from normal and MDS samples has not been systematically assessed, despite the fact morphological analysis still represents the backbone for the diagnosis. The aims of this study were to describe the pattern of TED by conventional morphology in BM of individuals without myeloid malignancies and in patients with MDS, and to compare clinical characteristics and prognostic features between MDS patients with normal and abnormal TED. METHODS: Three independent cytologists from three different institutions performed the morphological analysis of erythroid cells in BM smears in a 500-cell differential count from 74 patients diagnosed with lymphoma without BM involvement, and from 284 patients with MDS at diagnosis. TED stages were conventionally classified into proerythroblast (PE), basophilic (BA), polychromatic (POLYS), and orthochromatic erythroblasts (ORTHOS), and percentages were calculated based on the total cell population. Furthermore, flow cytometry analysis was performed on 11 further lymphoma patients without BM involvement, by using specific immunofluorescent probe patterns detected through the DxFLEX© cytometer. Patients with MDS were categorized as: TED-Normal, when total TED made up ≥15% of total BM cells and followed the expected doubling pattern; TED-Incomplete, when total TED were ≥15% but did not follow a doubling pattern; and TED-absent, when total TED composed <15%. Statistical analysis was performed by R (4.2.0). RESULTS: Morphologic analysis of normal BM confirmed the doubling growth pattern of TED [median PE of 0.8% (0-1.23); BA of 1.8% (1-2.4; ) POLYS of 8% (6.4-10.89) and POLYS of 8% (6.4-10.89)]. Whereas, immunophenotype analysis showed a linear growth pattern [median PE=0.57% (0.54-0.61), BA=1.35% (1.18-1.75), POLYS=2.64% (2.01-3.01), and ORTHOS=3.91% (3.3-5.37), probably explained by the lysis of the late-stage erythroblast cells by the immunophenotype preparation technique. Among the 284 MDS patients, 211 (75.5%) were TED-Normal, 46 (16.5%) TED-Incomplete, and 22 (8%) TED-Absent. Patients' characteristics are detailed in Table 1. MDS patients with TED-Normal presented significantly higher levels of hemoglobin (99 g/L vs. 89.5 g/L), reticulocytes (56 x109/L vs. 39 x109/L), and a lower percentage of ring sideroblast (0% vs. 26%) compared to TED-Incomplete (Table 1). Survival analysis showed that MDS patients with TED-Normal had a median OS of 39.03 months (95% IC 29.73-49.13), whereas it was of 23.77 months (95% IC 12.17-38.7) for TED-Incomplete, and 13.23 months (95% IC 7.17-NA) for TED-Absent; [HR 2.2; p=0.00132]. Of note, no OS differences were found between TED-Incomplete and TED-Absent [HR=1.41; p= 0.246] [Figure 2]. CONCLUSION: Morphological analysis is a suitable methodology for the study of TED maturation. Remarkably, 24.5% of patients with MDS presented with an abnormal TED (incomplete or absent) at diagnosis, this being associated with poorer clinical features and outcome. Finally, with the advent of newer erythroid maturation agents, analysis of TED might become paramount for treatment selection and response monitoring of patients with MDS in the near future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TRPC6 inhibits renal tubular epithelial cell pyroptosis through regulating zinc influx and alleviates renal ischemia-reperfusion injury.

Canonical transient receptor potential-6 (TRPC6) has been reported to be involved in cell damage after ischemia/reperfusion (I/R) injury in target organs. While the effect and of TRPC6 on pyroptosis in renal I/R injury remain unclear. In our study, we first established the renal I/R mouse model and oxygen-glucose deprivation and re-oxygenation (OGD/R) cell model, and investigated the impacts of TRPC6 on the pyroptosis-related proteins using CCK-8, western blot, ELISA, and immunofluorescence probes. Besides, we also explored the mechanism of TRPC6 in pyroptosis of renal tubular epithelial cells through A20 knockdown or overexpression and zinc chloride (ZnCl2 ) or a zinc ion chelator (TPEN) treatment. Our results indicated that I/R injury could cause downregulation of TRPC6 both in vivo and in vitro. In the I/R injury murine model, TRPC6 inhibition exacerbated tissue damage and upregulated NLRP3, ASC, caspase-1, IL-18, and IL-1β, which could be alleviated by the administration of ZnCl2 . In the OGD/R cell model, inhibitor of TRPC6 (SAR7334) reduced zinc ion influx, aggravated cell death and upregulated pyroptosis-related protein. The pyroptosis phenotype also could be alleviated by ZnCl2 and intensified by TPEN. Overexpression of A20 reduced the expression of pyroptosis-related protein, increased cell viability in the sh-TRPC6 and TPEN-treated OGD/R cell models, while A20 deficiency impaired the protective effect of zinc ion. Therefore, our results indicate that TRPC6 could promote zinc ion influx in renal tubular epithelial cells, thereby upregulating intracellular A20, inhibiting the activation of inflammasome NLRP3, and ultimately attenuating renal I/R injury.

Evaluation of anti-LipL32 carbon nanotube immunofluorescence probe (carbo-lip) and comparison with MAT, IgM ELISA, IgM spot test and culture for early detection of leptospirosis at local hospital

Leptospirosis is an emerging public health problem affecting people mainly from tropical and subtropical regions. Therefore, there is a need for rapid and sensitive tests for proper and prompt treatment. Recently we have demonstrated Carbo-Lip probe, which was fabricated through immuno recognition method with fluorescent dye functionalized LipL32 monoclonal antibodies, secondary antibody and Leptospira for rapid and accurate diagnosis. In an effort to validate Carbo-Lip, we collected clinical samples from a cohort of 104, consisting of 26 positive, 40 negative and 38 unconfirmed cases of Leptospirosis. Subsequently, the test was also compared and validated with the gold standard method microscopic agglutination test (MAT), IgM ELISA, IgM spot test, and culture. Carbo-Lip exhibited a sensitivity of 75% with specificity of 92.3% for Leptospirosis in comparison with MAT. The fabricated Carbo-Lip sensor could be used as a potential diagnostic tool for early detection of Leptospirosis in patients from endemic areas.

Nanoparticle immuno-fluorescent probes as a method for detection of viable E. coli O157:H7

Development of revolutionary sensitive biosensors for detecting the presence of harmful biological species in the environment is a necessity for countering disease outbreaks. This work examined the interaction of fluorescence-labeled antibody on amine functionalized gold nanoparticles (GNP) as a model system. The synthesized tetramethylrhodamine isothiocyanate (TRITC) labeled antibody-amine functionalized GNP interaction was characterized using UV–Vis spectroscopy and Fluorescent Microscopy imaging. Transmission Electron Microscopy (TEM) was also used to observe the morphology of the GNP. In contrast to TEM, the fluorescence microscopy imaging revealed the coating of the TRITC labeled antibody on the surface of the GNP. The signals were measured using a Photon Technology Inc. fluorometer at excitation of 541 nm and emission at 555 nm to 650 nm. Tests were conducted at near real-time with results obtained using the biosensor assay within 5 min. Results indicated that there was a shift of the wavelength from lower to higher wavelength (blue to red shift) when conjugated GNP (anti-E. coliO157:H7; IgY-TRITC-GNP) are compared to free GNP, a difference of about 28 nm. The GNP demonstrated a quenching capability when compared to the TRITC labeled antibody (degree of labeling of 15.41 mol dye per mole of IgY) using fluorometer. The lower and upper detection range of this method was found to be 103–105 CFU/mL with observed fluorescence of about 42,000 counts per seconds as against 24,000 counts per seconds that was observed when the specificity of the sensor was tested using Salmonella enterica.

A simple fluorescent strategy for liver capillary labeling with carbon quantum dot-lectin nanoprobe.

Taking the hepatic sinusoid (HS) as the main delivery area of liver nutrients and metabolic waste, recognizing its structure is important for a deep understanding of liver function. In this paper, based on lycopersicon esculentum lectin (LEL), with targeting ability for endothelial cells, and carbon quantum dots (CQDs), with high biosafety, an LEL-coupled CQD immunofluorescence probe (CQD@LEL) that can label microvessels is designed and used for the fluorescence labeling and imaging of HS in liver tissue sections. The CQD size is approximately 2 nm. Blue fluorescence is emitted under excitation; its optimal excitation wavelength is 400 nm while the emission is at about 450 nm. Gel electrophoresis and capillary electrophoresis confirm that glutaraldehyde can couple LEL to CQD, and the obtained CQD@LEL retains the fluorescence property and has good stability. Optimization experiments show that its labeling effect is positively correlated with time and probe concentration for dyeing the blood vessels of mouse liver slices. In order to improve the effect further, a probe concentration of 0.17 mg mL-1 and incubation time of 3 h were chosen to label the liver tissue sections. The results show that the liver microvessels are formed by interstitial structures among the hepatic cords, and the HS presents a granular or patchy appearance. H&E and ultrathin section TEM show that the microvascular wall of the liver is composed of discontinuous endothelial cells, and there are Kupffer cells and other cells in the tubes, proving that our probe can clearly label the structure and morphology of liver microvessels. This work is of great significance for the visualization of HS.

Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach.

Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target affinity. Fluorescent antibodies are widely used in flow cytometry for fast and sensitive identification and collection of cells expressing the target surface antigen. Nonetheless, current approaches to fluorescent labeling of antibodies most often use random modification, along with a few rather sophisticated site-specific techniques. The aim of our work was to develop a procedure for fluorescent labeling of immunoglobulin G via periodate oxidation of antibody glycans, followed by oxime ligation with fluorescent oxyamines. Here, we report a novel technique based on an in situ oxime ligation of ethoxyethylidene-protected aminooxy compounds with oxidized antibody glycans. The approach is suitable for easy modification of any immunoglobulin G, while ensuring that antigen-binding domains remain intact, thus revealing various possibilities for fluorescent probe design. The technique was used to label an antibody to PRAME, a cancer-testis protein overexpressed in a number of cancers. A 6H8 monoclonal antibody to the PRAME protein was directly modified with protected-oxyamine derivatives of fluorescein-type dyes (FAM, Alexa488, BDP-FL); the stoichiometry of the resulting conjugates was characterized spectroscopically. The immunofluorescent conjugates obtained were applied to the analysis of bone marrow samples from patients with oncohematological diseases and demonstrated high efficiency in flow cytometry quantification. The approach can be applied for the development of various immunofluorescent probes for detection of diagnostic and prognostic markers, which can be useful in anticancer therapy.

Directionally Modified Fluorophores for Super-Resolution Imaging of Target Enzymes: A Case Study with Carboxylesterases.

In the need for improving the labeling quality of super-resolution imaging, multifarious fluorescent labeling strategies have sprang up. Among them, a small molecule inhibitor-probe (SMI-probe) shows its advancement in fine mapping due to its smaller size and its specific binding to a specific site. Herein, we report a novel protocol of mechanism-guided directional modification of fluorophores into fluorescent inhibitors for enzyme targeting, which could half the size of the SMI-probe. To confirm the feasibility of the strategy, carboxylesterase (hCE) inhibitors are designed and developed. Among the constructed molecule candidates, NIC-4 inhibited both isoforms of hCE1 and hCE2, with IC50 values of 4.56 and 4.11 μM. The CE-targeting specificity of NIC-4 was confirmed by colocalizing with an immunofluorescent probe in fixed-cell confocal imaging. Moreover, NIC-4 was used in live-cell super-resolution microscopy, which indicates dotlike structures instead of the larger staining with the immunofluorescent probe. Moreover, it enables the real-time tracking of dynamic flow of carboxylesterases in live cells.

Application of direct stochastic optical reconstruction microscopy (dSTORM) to the histological analysis of human glomerular disease.

Electron microscopy (EM) following immunofluorescence (IF) imaging is a vital tool for the diagnosis of human glomerular diseases, but the implementation of EM is limited to specialised institutions and it is not available in many countries. Recent progress in fluorescence microscopy now enables conventional widefield fluorescence microscopes to be adapted at modest cost to provide resolution below 50 nm in biological specimens. We show that stochastically switched single‐molecule localisation microscopy can be applied to clinical histological sections stained with standard IF techniques and that such super‐resolved IF may provide an alternative means to resolve ultrastructure to aid the diagnosis of kidney disease where EM is not available. We have implemented the direct stochastic optical reconstruction microscopy technique with human kidney biopsy frozen sections stained with clinically approved immunofluorescent probes for the basal laminae and immunoglobulin G deposits. Using cases of membranous glomerulonephritis, thin basement membrane lesion, and lupus nephritis, we compare this approach to clinical EM images and demonstrate enhanced imaging compared to conventional IF microscopy. With minor modifications in established IF protocols of clinical frozen renal biopsies, we believe the cost‐effective adaptation of conventional widefield microscopes can be widely implemented to provide super‐resolved image information to aid diagnosis of human glomerular disease.

A simple method for quantitating confocal fluorescent images

Western blotting (WB), enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FC) have long been used to assess and quantitate relative protein expression in cultured cells and tissue samples. However, WB and ELISA have limited ability to meaningfully quantitate relative protein levels in tissues with complex cell composition, while tissue dissociation followed by FC is not feasible when tissue is limiting and/or cells difficult to isolate. While protein detection in tissue using immunofluorescent (IF) probes has traditionally been considered a qualitative technique, advances in probe stability and confocal imaging allow IF data to be easily quantitated, although reproducible quantitation of relative protein expression requires careful attention to appropriate controls, experiment design, and data collection. Here we describe the methods used to quantify the data presented in Shihan et al. Matrix Biology, 2020 which lays out a workflow where IF data collected on a confocal microscope can be used to quantitate the relative levels of a molecule of interest by measuring mean fluorescent intensity across a region of interest, cell number, and the percentage of cells in a sample “positive” for staining with the fluorescent probe of interest. Overall, this manuscript discusses considerations for collecting quantifiable fluorescent images on a confocal microscope and provides explicit methods for quantitating IF data using FIJI-ImageJ.

PD-L1+ dendritic cells in the tumor microenvironment correlate with good prognosis and CD8+ T cell infiltration in colon cancer.

BackgroundThe prognostic value of tumor‐associated dendritic cells (DC) in colon cancer remains poorly understood. This may be in part due to the interchangeable expression of immunostimulatory and immunoinhibitory molecules on DC. Here we investigated the prognostic impact of CD11c+ DC co–expressing the immunoinhibitory molecule PD‐L1 and their spatial relationship with CD8+ T‐cells in patients treated for stage III colon cancer.MethodsTissue microarrays containing representative cores of central tumor, leading edge, and adjacent normal tissue from 221 patients with stage III colon cancer were immunostained for CD8, CD11c, PD‐L1, and cytokeratin using immunofluorescent probes. Cells were quantified using StrataQuest digital image analysis software, with intratumoral and stromal regions analyzed separately. Kaplan‐Meier estimates and Cox regression were used to assess survival.ResultsIntratumoral CD8+ cell density (HR = .52, 95% confidence interval [CI] .33‐.83, P = .007), stromal CD11c+ cell density (HR = .52, 95% CI .33‐.83, P = .006), intratumoral CD11c+PD‐L1+ cell density (HR = .57, 95% CI .35‐.92, P = .021), and stromal CD11c+PD‐L1+ cell density (HR = .48, 95% CI .30‐.77, P = .003) on leading‐edge cores were all significantly associated with good survival. CD8+ cell density was positively correlated with both CD11c+ cell density and CD11c+PD‐L1+ cell density in tumor epithelium and stromal compartments.ConclusionHere we showed that PD‐L1‐expressing DC in the tumor microenvironment are associated with improved survival in stage III colon cancer and likely reflect an immunologically “hot” tumor microenvironment. Further investigation into the expression of immunomodulatory molecules by tumor‐associated DC may help to further elucidate their prognostic value.

Magnetic particles encoding a suspension probe for ultra-sensitive and quantitative determination of atrazine

As a highly toxic and widely used herbicide, atrazine poses a serious threat to food safety as well as overall environmental and human health. Due to complex matrix interference and the difficulty of signal enrichment, there is an urgent need for a convenient, fast, and ultrasensitive method that detects trace atrazine without concern for matrix effects. Here, we provide the first account of a sensitive and rapid suspension probe based on magnetic microspheres used to detect atrazine in herbs. The self-made magnetic beads featured -COOH groups and were used as the carrier to construct immunofluorescent probes. These probes then conjugated with the atrazine antigen through an activated ester method, ultimately binding to the antibody. Homogeneous detection was ensured using flow cytometry and the microflow optical channel along with allophycocyanin-conjugated goat-anti-mouse secondary antibody (APC-IgG-SecAb) as the fluorescent signal. The magnetic suspension probe allowed for high target enrichment and the inherent two-dimensional selective detection of flow cytometry effectively avoided any matrix interference. This method had good linearity across 1.69−23.19 ng mL−1. The IC50 and LOD values were 4.81 ng mL−1 and 0.95 ng mL−1, respectively; the sensitivity was increased three-fold relative to ELISA. After complete optimization, 2-N-morpholinoeth-anesulfonic acid was used as the coupling solution and maintained good mono-dispersity, stability, and reactivity for the labelled microspheres during the process. The entire experiment was simple, and effectively used reagents; moreover, both the labor required and detection time were greatly reduced. Critically, the strategy presented here greatly reduced interference from complex matrices, and saved preparation for matrix-matched solutions when different herbs were screened. Overall, this strategy was sensitive, rapid, eco-friendly, and labor-saving; collectively, these attributes make it well-suited for on-site screening of atrazine contamination and will allow for increased food safety.

Early detection of leptospirosis using Anti-LipL32 carbon nanotube immunofluorescence probe

Leptospirosis is a widespread zoonosis and an emerging public health problem. Leptospirosis symptoms are often confused or misdiagnosed with other febrile illness like malaria, viral hepatitis, influenza, dengue, typhoid, melioidosis, and scrub typhus as the clinical manifestations are almost similar. Therefore, early and accurate diagnosis of leptospirosis is indeed critical for proper and prompt treatment. Herein, we report the development of single-walled carbon nanotubes based immunofluorescence probe (Carbo-Lip) for the detection of leptospirosis at an early phase by utilising major outer membrane protein, LipL32 of Leptospira. The Carbo-Lip probe was fabricated through immuno recognition method with fluorescent dye functionalized LipL32 monoclonal antibodies (mAbs), secondary antibody and Leptospira. Surface characterization studies such as Fourier transform infrared spectroscopy with the attenuated total reflectance, scanning electron microscopy, transmission electron microscopy, Zeta potential, and X-ray photoelectron spectroscopy techniques were used to demonstrate the successful fabrication of Carbo-Lip probe. The sensor probe was capable of detecting the presence of leptospires at a lower concentration of 103/ml, and could detect 102 leptospires in 100μL of sample within 3h of the test conditions, and was stable up to 2 weeks. This Carbo-Lip probe was further tested and validated for its capacity to detect Leptospira in clinical samples, which exhibited high selectivity and specificity towards Leptospira even in the presence of malaria and dengue. Our results were consistent with microscopic agglutination test, which is known as gold standard, immunoglobulin M (IgM) enzyme-linked immunoassay (ELISA), IgM spot test, and culture tests for the diagnosis of Leptospira infection.

Clearing, immunofluorescence, and confocal microscopy for the three-dimensional imaging of murine testes and study of testis biology

Optical clearing techniques provide unprecedented opportunities to study large tissue samples at histological resolution, eliminating the need for physical sectioning while preserving the three-dimensional structure of intact biological systems. There is significant potential for applying optical clearing to reproductive tissues. In testicular biology, for example, the study of spermatogenesis and the use of spermatogonial stem cells offer high-impact applications in fertility medicine and reproductive biotechnology. The objective of our study is to apply optical clearing, immunofluorescence, and confocal microscopy to testicular tissue in order to reconstruct its three-dimensional microstructure in intact samples. We used Triton-X/DMSO clearing in combination with refractive index matching to achieve optical transparency of fixed mouse testes. An antibody against smooth muscle actin was used to label peritubular myoid cells of seminiferous tubules while an antibody against ubiquitin C-terminal hydrolase was used to label Sertoli cells and spermatogonia in the seminiferous epithelium. Specimens were then imaged using confocal fluorescence microscopy. We were able to successfully clear testicular tissue and utilize immunofluorescent probes. Additionally, we successfully visualized the histological compartments of testicular tissue in three-dimensional reconstructions. Optical clearing combined with immunofluorescence and confocal imaging offers a powerful new method to analyze the cytoarchitecture of testicular tissue at histological resolution while maintaining the macro-scale perspective of the intact system. Considering the importance of the murine model, our developed method represents a significant contribution to the field of male reproductive biology, enabling the study of testicular function.

The creation of immunofluorescence probes based on monoclonal antibodies series ICO and phycoerythrin - fluorophore of phycobiliprotein group for analysis of cell populations by flow cytometry

Introduction . Fluorescent probes based on monoclonal antibodies (MAb) are widely used in scientific and clinical research in the field of oncology, hematology, immunology, epidemiology. The unique spectral properties of the natural protein, phycoerythrin (PE), made it the dominant dye widely used for the preparation of fluorescent probes based on MAb. Objective . To create of fluorescent probes based on the MAb and the fluorescent dye PE by two alternative methods of protein synthesis. Materials and methods . MAb to T lymphocyte antigen (clone ICO-86, clone ICO-31), fluorescent dye PE and bifunctional agents SPDP and SMCC were used in the work. Liquid chromatography methods were used for isolation and purification of MAb: in the first step, affinity isolation of immunoglobulins on the immobilized protein G or anion exchange column chromatography. Gelfiltration on a PD-10 column was used to purify the conjugates (immunofluorescent probes, IFP) in the second step, the concentration and labeling density of the IFP were determined spectrophotometrically. Results . Conjugation of MAb with PE was carried out at a molar ratio of components of 1: 2. For this both components of the conjugation reaction (PE and MAb) were separately modified. For the creation of IFP on the basis of the MAb of the ICO series, both methods of conjugation of Mab with PE are applicable, but method II, in which the chemical modification by the bifunctional SMCC agent is exposed only to the PE molecule by its free amino groups, and the immunoglobulin molecule is conserved in the maximally native state is preferred. Conclusion . The methods described in the article allow to obtain immunofluorescent probes based on antibodies of the ICO series that are comparable in their analytical characteristics to foreign commercial analogs.

Detection of &lt;i&gt;Escherichia coli&lt;/i&gt; O157:H7 with Antibody Conjugated Amino-Functionalized Graphene Quantum Dots as Immunofluorescence Probes

Escherichia coli O157: H7 (E. coli O157: H7) is a foodborne pathogenic bacterium which can cause fever, diarrhoea and vomiting in humans. Thus, a rapid, simple, and specific bioprobe for pathogen detection in contaminated foods has been attracted more and more attention. In this work, the strong fluorescent amino-functionalized graphene quantum dots (af-GQDs) were prepared by hydrothermal method. The microtopographic height, surface morphology and spectroscopic properties of af-GQDs are characterized by the high resolution transmission electron microscope (HRTEM), atomic force microscope (AFM), UV-vis, fluorescence, Raman spectroscopic techniques. All the results showed that the af-GQDs can be effectively applied in the preparation of biocompatible immunofluorescence probe and in the detection of E. coli O157: H7. The minimum detection limit is 100 cfu/mL. It is a simple, rapid, sensitive, low-cost and easy to be popularized method, which provides a feasible way to monitor E. coli O157: H7 in food safety.

Analysis of PKC-ζ protein levels in normal and malignant breast tissue subtypes.

It is estimated that breast cancer will be the second leading cause of cancer-associated mortality in women in 2018. Previous research has demonstrated that the atypical protein kinase C-ζ (PKC-ζ) is a component of numerous dysregulated pathways in breast cancer, including cellular proliferation, survival, and cell cycle upregulation. The present study investigated the PKC-ζ protein in breast tissue to evaluate its potential as a biomarker for breast cancer invasion, and demonstrated that an overexpression of PKC-ζ protein can be indicative of carcinogenesis. The present study analyzed the expression of PKC-ζ in individuals with no tumor complications and malignant female human breast tissue samples (lobular carcinoma in situ, invasive lobular carcinoma, ductal carcinoma in situ and invasive ductal carcinoma) with the use of western blot analysis, immunohistochemistry and statistical analysis (83 samples). The present study also evaluated the invasive behavior of MDA-MB-231 breast cancer cells following the knockdown of PKC-ζ with a Transwell invasion assay and an immunofluorescent probe for filamentous actin (F-actin) organization. The data demonstrated that PKC-ζ expression was identified to be higher in invading tissues when compared with non-invading tissues. The results also suggest that PKC-ζ is more abundant in ductal tissues when compared with lobular tissues. In addition, the protein studies also suggest that PKC-ζ is a component for invasive behavior through the Ras-related C3 botulinum toxin substrate 1 (Rac1) and Ras homolog gene family member A (RhoA) pathway, and PKC-ζ is required for the F-actin reorganization in invasive cells. Therefore, PKC-ζ should be considered to be a biomarker in the development of breast cancer as well as an indicator of invading tumor cells.