Goat Anti-human Immunoglobulin Research Articles

Optofluidic chip with directly printed polymer optical waveguide Mach-Zehnder interferometer sensors for label-free biodetection

Optofluidic devices hold great promise in biomedical diagnostics and testing because of their advantages of miniaturization, high sensitivity, high throughput, and high scalability. However, conventional silicon-based photonic chips suffer from complicated fabrication processes and less flexibility in functionalization, thus hindering their development of cost-effective biomedical diagnostic devices for daily tests and massive applications in responding to public health crises. In this paper, we present an optofluidic chip based on directly printed polymer optical waveguide Mach-Zehnder interferometer (MZI) sensors for label-free biomarker detection. With digital ultraviolet lithography technology, high-sensitivity asymmetric MZI microsensors based on a width-tailored optical waveguide are directly printed and vertically integrated with a microfluidic layer to make an optofluidic chip. Experimental results show that the sensitivity of the directly printed polymer optical waveguide MZI sensor is about 1695.95 nm/RIU. After being modified with capture molecules, i.e., goat anti-human immunoglobulin G (IgG), the polymer optical waveguide MZI sensors can on-chip detect human IgG at the concentration level of 1.78 pM. Such a polymer optical waveguide-based optofluidic chip has the advantages of miniaturization, cost-effectiveness, high sensitivity, and ease in functionalization and thus has great potential in the development of daily available point-of-care diagnostic and testing devices.

High sensitive optical fiber SPR sensor for label-free detection of Staphylococcus aureus

Optical fiber bacterial sensors have attracted attention in many fields due to their unique advantages. However, it is still difficult to promote its practicality which is mainly subject to their low sensitivity, inferior stability, and reliability. This work reported here an optical fiber surface plasmon resonance (SPR) biosensor enhanced by gold nanoparticles (AuNPs)/Au film coupling for label-free detection the Staphylococcus aureus (S. aureus), while both the gold substrates were successfully biofunctionalized with goat anti-human immunoglobulin G (IgG) as the probe to capture and detect the bacteria. In the experiment, the refractive index（RI） sensitivity of the Multimode-Singlemode-Multimode optical fiber (MSMOF) coated with Au film was improved by 19 % after the AuNPs were homogeneously decorated on the Au film surface. To detect S. aureus by the sensor, the SPR wavelength variation in the transmission spectrum was recorded, and its limit of detection (LOD) was demonstrated experimentally to be about 50 CFU/mL and was obtained theoretically to be 1.18 CFU/mL during its concentration range of 50–108 CFU/mL. Also, it shows great linearity in the range of 102-108 CFU/mL and the sensor exhibits excellent specificity and stability. This kind of SPR-enhanced optical fiber biosensor is fully promising for its applications in biomedicine, food safety, pharmaceuticals, etc.

Total Internal Reflection Fluorescence Foldscope Adapter: Design, Fabrication and Applications

Fluorescence microscopy is one of the important instruments used in research and clinical laboratories on an everyday basis due to its wide range of applications ranging from a diagnostic tool to a molecular imaging device. However, the high cost, complex instrumentation, and requirement of sophisticated facilities limit its availability to school and college students, especially in developing and underdeveloped countries. In this work, we report the design and development of a slide-like portable total internal reflection fluorescence (TIRF) adapter, which can be integrated easily with Foldscope (a paper microscope) to observe and capture the fluorescence images using any smartphone. The adapter consists of a battery-powered LED (light source), a small piece of multimode optical fiber, a slide-like 3-D printed waveguide platform to hold the optical fiber and light emitting diode (LED) at an appropriate position. The potential of the adaptor is validated by capturing the fluorescence images of two most commonly used dyes i.e. fluorescein isothiocyanate (FITC) and rhodamine-B, after being immobilized on amine-functionalized optical fiber probe. Further, this adaptor is successfully used to demonstrate (i) Forster resonance energy transfer phenomenon by employing FITC dye as donor and gold nanoparticles as quencher moiety and (ii) fluorescence bioassay by observing antigen-antibody interactions using human immunoglobulin G as the receptor and FITC-tagged goat anti-human immunoglobulin G as analyte. The inexpensive, portable and user-friendly TIRF adapter is a potential alternative to conventionally used fluorescence microscopes, especially for the school and college students and in resource-constrained clinical laboratories.

A Bio-Conjugation Chemistry Strategy to Self-Immobilize Antibodies with ZIF-90-ZnO-MoS2 Nanohybrid for Exosome Markers Analysis

Motivation Cell signaling proteins have been widely regarded as some of the most powerful biomarkers for cancer diagnosis because they are real therapy related molecules. Extracellular vesicles, exosomes, transport proteins to promote tumor growth and are stable in biofluids at room temperature.[1] Hence, detection of exosomal proteins (e.g., cluster of differentiation (CD) 9, CD 63, CD 81) would be easily deployed at the point-of-care (POC) diagnostic. A successful electrochemical biosensor is generally determined by electrodes and their modification as well as specific bio-recognition elements (BREs).Graphene like MoS2 has emerged in biosensing applications owing to their intriguing properties, such as good catalytic properties, interesting metallic-to-semiconducting transition from 1T to 2H phase, and good biocompatibility.[2] However, a particularly vexing problem for MoS2 as the sensing materials is the structural instability that the exfoliated or ultrathin two dimensional (2D) MoS2 is hard to be retained in in their freestanding state. The surface modification technologies, such as growth of building blocks could transfer the 2D to relatively stable 3D nanoarchitectures, in which each component can also exert different function.Herein, we demonstrate a nanostructure consisting of zinc oxide (ZnO) nanowires (NWs) array grown on 2D MoS2forming a new nanoarchitecture (ZnO-MoS2) providing high specific surface area and good electron transport capability. Then, zeolitic imidazolate framework 90 (ZIF-90) thin films are in-situ deposited onto ZnO NWs by using ZnO as precursor to directly react with imidazole-2-carboxyaldehyde (ICA). The integrated hybrid nanostructure, denoted as ZIF-90-ZnO-MoS2, possesses not only effective mass transport and electron transduction capabilities but also covalent bio-conjugation of BREs through the aldehyde group on the bridging ICA ligand in ZIF-90. Of particular note is the ZnO NWs array is an indispensable part of the hybrid nanostructure, since the in-situ growth of ZIF-90 and effective integration with layered MoS2 is still a challenge. Through the cooperation of three different kinds of building blocks, from signal generation, amplification to transduction could be achieved in an integrated nanoarchecture. Results and Discussion Figure 1a displays the scanning electron microscope (SEM) image of obtained ZIF-90-ZnO-MoS2 nanohybrid, from which the ordered NWs array is uniformly grown onto 2D MoS2 material. The higher magnification SEM (Figure 1b) and transmission electron microscopy (TEM) (Figure 1c) images show that the length and diameter of vertical ZnO NWs are 1.5 μm and 50 nm, respectively. Further high resolution TEM (HR-TEM) (Figure 1d) characterization demonstrates that the NWs are consisted of highly crystal ZnO core with the diameter of about 25 nm and ZIF-90 sheath with the thickness of about 12 nm.A FITC-labeled goat anti-human immunoglobulin G (IgG) is employed to verify our hypothesis. The inset of Figure 1e shows the fluorescence microscopy image of microelectrode immobilized with FITC-labeled goat anti-human IgG. It can be seen that only the region deposited with ZIF-90-ZnO-MoS2 nanohybrid visualizes green fluorescence. This result illustrates ZIF-90-ZnO-MoS2 nanohybrid possesses the capability of direct covalent bio-conjugation of antibody. And the results shown Figure 1e further confirm the developed hybrid material could effectively detect signaling protein.We next fabricated a microfluidic electrochemical microsensor chip, and the configuration is shown in Figure 1f. The microsensor chip is composed of a polydimethylsiloxane (PDMS) layer sealed with screen-printed electrodes. The microsensor is consisted of five electrodes, including counter electrode (CE), reference electrode (RE) and three different working electrodes. Among the three working electrodes, electrode 1 and 2 are locally deposited with fabricated ZIF-90-ZnO-MoS2 nanohybrid material as the test electrodes, and electrode 3 is printed with carbon conductive ink for the background signal deduction. The micro-channels on the chip are employed for the simultaneous immobilization of bio-recognition antibodies as well as dual detection of exosome markers (CD63 and CD9). From Figure 1g, our fabricated microsensor chip shows obvious response towards NCI-H 1650, and almost no signal for other cells supernatant (NCI-H 1650: human non-small cell lung cancer cells, 16 HBE: human bronchial epithelial cells, HFL-1: human fetal lung fibroblast-1 and HUVEC: human umbilical vein endothelial cells). Conclusion In summary, the ZIF-90-ZnO-MoS2 nanohybrid integrated with different kinds of building blocks realizes the cooperation of multi-function, including covalent bio-conjugation of antibodies, signal generation and transduction. To further combine with microfluidic electrochemical microsensor chip, the simultaneous immobilization of bio-recognition antibodies and dual detection of exosome markers can also be acquired.

A dual channel self-compensation optical fiber biosensor based on coupling of surface plasmon polariton

We propose a dual-channel fiber-optic biosensor based on electronic field coupling between surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR). One sensing channel is coated with a bilayer of graphene oxide/Au, whose surface is modified with goat anti-human immunoglobulin G (IgG) to detect human IgG labeled with Au nanoparticles (Au NPs). The graphene oxide film in the region of the coupled electric fields increases the loading of biomolecules on the sensor surface and the immunological response. This sandwich structure can make full use of the coupled electric field excited by Au film and Au NPs, compared to the traditional LSPR sensor. The other sensing channel is coated only with Ag film as a reference channel to eliminate measurement error caused by non-specific binding and temperature cross-sensitivity. We numerically investigate the electric field coupling strength of SPR/LSPR and conducted corresponding experiments. The sensor had a high refractive index sensitivity with 13,592 nm/RIU and limit of detection of human IgG can be reduced to 15 ng/mL, which is 15.3 times lower than that of conventional SPR sensors. The proposed biosensor has sensing superiorities of high sensitivity, accuracy and temperature insensitive.

Relevance of antibodies to myelin oligodendrocyte glycoprotein in CSF of seronegative cases.

To determine the diagnostic relevance of myelin oligodendrocyte glycoprotein antibodies (MOG-Abs) in CSF of seronegative cases by retrospectively analyzing consecutive time-matched CSF of 80 MOG-Ab-seronegative patients with demyelinating disease. The cohort included 44 patients with NMOSD and related disorders and 36 patients with multiple sclerosis (MS). Two independent neurologists blinded to diagnosis analyzed MOG-Abs by live cell-based immunofluorescence assay with goat anti-human immunoglobulin (Ig) G (whole molecule) antibody. Sera were tested at dilutions of 1:20 and 1:40, and a cutoff of 1:160 was considered for serum positivity. CSF specimens were tested undiluted and at 1:2 dilution with further titrations in case of positivity. Anti-IgG-Fc and anti-IgM-µ secondary antibodies were used to confirm the exclusive presence of MOG-IgG in positive cases. CSF of 13 MOG-Abs seropositive cases and 36 patients with neurodegenerative conditions was analyzed as controls. Three seronegative cases had CSF MOG-Abs (4% of the whole cohort or 7% of cases excluding patients with MS, in which MOG-Abs seem to lack diagnostic relevance). In particular, 2 patients with neuromyelitis optica spectrum disorder (NMOSD) and 1 with acute disseminated encephalomyelitis had MOG-Abs in CSF. Analysis with anti-IgG-Fc and anti-IgM confirmed the exclusive presence of MOG-IgG in the CSF of these patients. Among the control group, MOG-Abs were detectable in the CSF of 8 of 13 MOG-Ab-seropositive cases and in none of the patients with neurodegenerative disorders. Although serum is the optimal specimen for MOG-Ab testing, analyzing CSF could improve diagnostic sensitivity in seronegative patients. This observation has relevant diagnostic impact and might provide novel insight into the biological mechanisms of MOG-Ab synthesis.

Fiber optic SPR sensor for human Immunoglobulin G measurement based on the MMF-NCF-MMF structure

Abstract A sensitive, simple fiber optic surface plasmon resonance (SPR) sensor based on the MMF-NCF-MMF structure is proposed in this work. The structure of the sensing part is constructed by a section of noncore fiber (NCF) sandwiched between two multimode fibers (MMF). Gold film with a thickness of 50 nm was uniformly sputtered on the outside surface of the NCF. SPR phenomenon was stimulated and experimentally verified when light propagates at the interface between the dielectric and the gold layers. The length of the NCF section was optimized by experiments and determined to be 12 mm. The refractive index (RI) sensitivity of the SPR sensor is 3475.1 nm/RIU in the RI range of 1.343–1.373. For the realization of human Immunoglobulin G detection, the surface of this SPR sensor needs to be biomodified. A simple process of polyelectrolyte self-assembled multilayers (PSAMs) deposition cycled four times utilizing diallyldimethyl ammonium chloride (PDDA) and styrenesulfonate sodium salt (PSS). And we choose goat anti-human immunoglobulin G-human immunoglobulin G as a bioconjugated pair to examine the bio-sensing effectiveness of this SPR sensor, the maximum sensitivity of Immunoglobulin G (IgG) detection and the limit of detection (LOD) is calculated to be 57.06 nm/(mg/ml) and 1.75 μg/ml, respectively. The proposed sensor is simple, easy to fabricate, low loss, cost-effective and can be used in numerous chemical and biological analyses.

CAR T Cells Targeted with a High Affinity Scfv Against the HCMV Glycoprotein Gb As Adoptive T Cell Therapy after Hematopoietic Stem Cell Transplantation

Introduction: Reactivation of human cytomegalovirus (HCMV) in immune compromised patients after hematopoietic stem cell transplantation (HSCT) is associated with high morbidity and mortality, particularly after cord blood transplantation (CBT). Adoptive transfer of T cells expanded in vitro is currently used as therapy for drug-refractory HCMV disease. A major limitation of this approach is the requirement of HLA-restricted HCMV-specific memory T cells. An alternative approach exploring HLA-independent T cell recognition was sought. Because the HCMV envelope glycoprotein B (gB) is highly expressed during lytic infection and in latently infected cells, we hypothesized that T cells can be redirected to recognize and kill HCMV-specific cells by means of a gB-specific chimeric antigen receptor (CAR). We have synthesized and tested a gB-specific CAR derived from the SM5-1 monoclonal antibody which binds with high affinity (KD 5.7x1011) to a conserved antigenic and non-glycosylated domain of gB.Methods: We generated two codon-optimized SM5-derived scFvs (VH->VL and VL->VH) and fused with an existing CAR backbone containing an IgG Fc spacer and intracellular signaling domains. CARs containing either CD28.zeta or 4-1BB.zeta were synthesized and expressed in T cells following a standard retroviral transduction protocol yielding 80-90% transduction rate. Expression of the CARs on T cells was confirmed by flow cytometry using goat anti-human immunoglobulin reactive against the IgG Fc region. 293T cells co-expressing gB and dTomato were used for in vitro cytotoxicity assays.Results: T cells expressing gB-CAR/CD28.zeta were cytotoxic against gB+ target cells producing 90% killing of 293T/gB-dTom cells compared with control CD19 CAR/CD28.zeta cells at an effector-to-target ratio 3:1 for 48 h (parental 293T cells were not killed). The cytolytic activity correlated with expansion of CAR T cells and concomitant loss of gB-dTom expression in the remaining viable 293T cells. Sequential co-culture of these gB-CAR T cells with freshly seeded 293T/gB-dTom resulted into further elimination of target cells. We are currently evaluating the effects of different gB-CAR T cell designs in the killing of HCMV-infected cell lines and primary cells using HCMV laboratory strains expressing the GFP and Gaussia Luciferase reporter genes. Pilot experiments indicated that gB-CAR/CD28.zeta cells with the scFv in the VL->VH orientation resulted into more clustering and killing of HepG2 cells infected with HCMV-GFP after 24h of co-culture than a control CD19 CAR/CD28.zeta. Humanized mice transplanted with cord blood CD34+ stem cells and challenged with these HCMV laboratory strains will be used to evaluate the in vivo effectivity of cord blood-derived donor-matched gB-CAR-T cells to eliminate acute and latent HCMV infections.Conclusion: These studies explore a novel approach in preventing HCMV reactivation in immunosuppressed patients by redirecting T cells expressing a high-affinity gB-CAR to eliminate HCMV-infected cells in a TCR/MHC-independent manner. DisclosuresNo relevant conflicts of interest to declare.

An convenient strategy for IgG electrochemical immunosensor: the platform of topological insulator materials Bi2Se3 and ionic liquid

A substantial outstanding challenge in diagnostics and disease monitoring is the ability to assay rapidly and conveniently for protein biomarkers within complex biological media. Bi2Se3, as an important topological insulator (TI) material, was synthesized by a solvothermal method and characterized structurally. Subsequently, the composite of Bi2Se3 and ionic liquid ([BMIm]BF4 IL) was used as a sensing interface to cross-link goat anti-human immunoglobulin G (anti-IgG) via glutaraldehyde (GA) to fabricate an Bi2Se3/IL/GA/anti-IgG-carbon paste electrode (CPE). The nonspecific binding sites were enclosed with bovine serum albumin (BSA) to develop a label-free IgG immunosensor. The result showed that the proposed label-free IgG immunosensor exhibited high specificity with a detection limit of 0.8 ng mL−1 and linear range from 2 to 300, and 300 to 2200 ng mL−1. Besides, the immunosensor exhibited high specificity for IgG detection, acceptable reproducibility, and stability. Thus, the strategy reported here paved a simple way to design a sensitive and cost-effective sensing platform for extension to other disease biomarkers.

MiR-17-92 represses PTPROt and PP2A phosphatases and amplifies tonic BCR signaling in DLBCL cells.

B-cell receptor (BCR) signaling plays a pivotal role in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and targeting the BCR pathway is a highly promising therapeutic strategy in this malignancy. The oncogenic microRNA miR-17-92 modulates multiple cellular processes such as survival, proliferation, apoptosis, angiogenesis, and BCR signaling. In the present study, we identified new targets of miR-17-92, PTPROt (protein phosphatase, receptor type O, truncated) and PP2A (protein phosphatase 2A) phosphatases, which regulate the activity of spleen tyrosine kinase (SYK) and AKT, critical components of BCR signal transduction in DLBCL cells. Introduction of miR-17-92 into DLBCL cells dampened the expression of the PTPROt and PP2A regulatory subunits PPP2R2A (protein phosphatase 2, regulatory subunit B, alpha) and PPP2R5E (protein phosphatase 2, regulatory subunit B, epsilon isoform) and increased the magnitude of SYK and AKT phosphorylation upon BCR ligation. Finally, we found that miR-17-92 expression modulates response to inhibitors of BCR signaling because downregulation of miR-17-92 increased SYK inhibitor-mediated toxicity in DLBCL cells. Our study reveals novel posttranscriptional regulatory pathways that contribute to the deregulation of BCR signaling and modulate SYK inhibitor activity in DLBCL.

Label free ultrasensitive optical sensor decorated with polyaniline nanofibers: Characterization and immunosensing application

Conducting polymers have been studied for decades, and the use of such polymers in sensing applications has been explored extensively. Most of these have exploited the fact that protonation or de-protonation of these polymers, have an effect on their conductivity and conformational structure. We demonstrate a phenomenon, i.e. changes to the spectroscopic properties of conducting polymer polyaniline (PAni), without explicit protonation or de-protonation, due to immunological interactions happening on the surface. To explore the reason behind these spectroscopic changes, PAni was analyzed using Hydrogen Nuclear Magnetic Resonance (HNMR), and Fourier Transformed Infrared (FTIR) spectroscopy. It was further characterized using Scanning Electron Microscope (SEM) imaging and Selected Area Electron Diffraction (SAED). These optical alterations of polyaniline have been utilized to develop a simple, cost effective, yet highly sensitive immunosensor. Human Immunoglobulin G (HIgG) was immobilized on the polyaniline coated fiber-optic probe using cross linker molecules, and goat anti-Human Immunoglobulin G (GaHIgG) was used as the analyte. This study fundamentally utilizes optical properties of PAni to develop a simple immunosensor which is able to detect analyte concentrations as low as 37pM.

Highly sensitive covalently functionalized light-addressable potentiometric sensor for determination of biomarker

A biomarker is related to the biological status of a living organism and shows great promise for the early prediction of a related disease. Herein we presented a novel structured light-addressable potentiometric sensor (LAPS) for the determination of a model biomarker, human immunoglobulin G (hIgG). In this system, the goat anti-human immunoglobulin G antibody was used as recognition element and covalently immobilized on the surface of light-addressable potentiometric sensor chip to capture human immunoglobulin G. Due to the light addressable capability of light-addressable potentiometric sensor, human immunoglobulin G dissolved in the supporting electrolyte solution can be detected by monitoring the potential shifts of the sensor. In order to produce a stable photocurrent, the laser diode controlled by field-programmable gate array was used as the light emitter to drive the light-addressable potentiometric sensor. A linear correlation between the potential shift response and the concentration of human immunoglobulin G was achieved and the corresponding regression equation was ΔV (V)=0.00714ChIgG (μg/mL)–0.0147 with a correlation coefficient of 0.9968 over a range 0–150μg/mL. Moreover, the light-addressable potentiometric sensor system also showed acceptable stability and reproducibility. All the results demonstrated that the system was more applicable to detection of disease biomarkers with simple operation, multiple-sample format and might hold great promise in various environmental, food, and clinical applications.

Evanescent Wave Absorption Based S-shaped Fiber-optic Biosensor for Immunosensing Applications

In this study, S-shaped multimode fiber-optic probe have been employed to improve sensitivity of evanescent wave absorption (EWA) based fiber-optic biosensor. Immunosensing was performed using Human immunoglobulin (HIgG) and fluorescein isothiocyanate (FITC) tagged Goat anti-human immunoglobulin (GaHIgG) antigen-antibody pair. The immunosensing was carried out in direct ELISA format, with the exception of replacing enzyme with FITC as reporter molecule. In an earlier study, we have observed higher EWA sensitivity and refractive index (RI) sensitivity for S-shaped compared to U-shaped fiber-optic probe [1]. The fabricated S-shaped geometry improved the lowest measurable concentration of GaHIgG biomolecule to 1.7 nM. The proposed immunosensor showed negligible non-specific binding on the sensor surface, thereby improving the detection accuracy.

Fluorescence resonance energy transfer based immunosensing of human IgG by using quantum dot/GIgG-gold nanoparticles/IgG conjugation.

A novel immunosensor of human immune globulin (IgG) was fabricated based on the fluorescence transfer between luminescent semiconductor quantum dots (QDs) and gold nanoparticles (AuNPs). AuNPs and CdSe/ZnS QDs were respectively labeled with immune reaction pair:IgG and goat anti-human immunoglobulin (GIgG), by optimizing the conditions including pH value and protein amount. In the assembled QD-GIgG-IgG-AuNP fluorescence resonance energy transfer (FRET) immunocomplex system, the presence of AuNP-IgG directly reduced the fluorescence intensity of the GIgG conjugated QDs. As a result, the concentration of AuNP-IgG had a linear relationship with the fluorescence decrease in a range of 0-1.57 microg/mL. Furthermore, the mechanism of the QDs' fluorescence decay has also been discussed and attributed to the light-induced photobleaching. This novel sensing method achieves quantitative detection of trace proteins, suggesting the potential of biomolecule-AuNPs conjugation based analytical methods in further application.

Surface modification of hydrophobic NaYF4:Yb,Er upconversion nanophosphors and their applications for immunoassay

β-NaYF4:Yb,Er upconversion nanophosphor (UCNP) is known as one of the most efficient NIR-to-visible upconversion materials, which shows great potential in bioanalytical chemistry and bioimaging. However, its applications are greatly limited due to its low water dispersibility and thus poor biocompatibility. In this paper, poly(acrylic acid) (PAA)-based ligand exchange strategies are carried out to modify oleic acid-capped hydrophobic β-NaYF4:Yb,Er UCNPs into hydrophilic ones. After efficient surface modification, the presence of free carboxylic acid groups on the surfaces of UCNPs results in high solubility in water, and also allows further conjugation with NH2-containing biomolecules. Facilitated by the covalent linkage between the-COOH groups on UCNPs surfaces and-NH2 groups in antigen/antibody, a sensitive immunosensor is constructed by using PAA-functionalized β-NaYF4:Yb,Er UCNPs as biolabels. Through monitoring the upconversion fluorescence intensity or fluorescent imaging of the final immunocomplexes, high sensitivity is achieved for the proposed immunoassay and as low as 0.1 ng/mL goat anti-human immunoglobulin G (IgG) can be detected, which suggests that PAA-modified UCNPs may serve as an ideal candidate for use as bioanalysis and bioimaging probes.

Easy Test for Visceral Leishmaniasis and Post-Kala-azar Dermal Leishmaniasis

Easy Test for Visceral Leishmaniasis and Post-Kala-azar Dermal Leishmaniasis

Establishment and clinical application of flow cytometric bead assay in detecting platelet-specific autoantibodies

Objective To establish a novel method to detect autoantibodies against platelatespecific receptors by flow cytometric bead assay and study its clinical application. Methods The beads were coated with monoclonal antibodies SZ2, SZ22, SZ21 and 7E3 against platelet GP Ⅰ b, GP Ⅱ b, GP Ⅲa and GP Ⅱ b/Ⅲ a, respectively. Captured platelet glycoprotein and beads complex was detected by FITC labeled polyclonal goat antihuman immunoglobulin using flow cytometer. The platelet samples that reacted with antibodies (SZ2, SZ22, SZ21 and 7E3) negatively and positively were tested, respectively. Each sample was repeated 20 times to generate intra-day CV for the MFI and once a day for 8 days to generate inter-day CV values. The 85 ITP patients, 17 NITP patients and 50 controls from the First Affiliated Hospital of Soochow University during March 2006 to December 2008 were included in the studies. The sensitivity and specificity of these four platelet antibodies to diagnose ITP were analyzed using ROC curve. The results were compared with MAIPA. Results The CV of the intra-day-assay for samples negative to antibody SZ2, SZ22,SZ21 and 7E3 were 3.26%, 2. 86%, 1.65% and 4. 94%, respectively; While the CV of the intra-day-assay for samples positive to antibody SZ2, SZ22, SZ21 and 7E3 were 6. 16%, 4. 88%, 5.20% and 5. 85%,respectively. The CV of the inter-day-assay for samples negative to antibody SZ2, SZ22, SZ21 and 7E3 were 5. 86%, 4. 74%, 5.69% and 7.56%, respectively; While the CV of the inter-day-assay for samples positive to antibody SZ2, SZ22, SZ21 and 7E3 were 7.53%, 5.49%, 7.11% and 6.25%,respectively. The MFI for SZ2 in ITP group, NITP group and healthy control group were 1.49(0. 88-16. 24),1.12(1.00-1.33), 1.01 (0. 83-1.37), respectively, which showed significant differences (H = 36.89,P＜0.01). The MFI for SZ22 in the three groups were 1.55 (0.84-11.30), 1.13(1.03-1.29), 0.98(0. 85-1.24), respectively (H=28.41, P ＜0.01). The MFI of SZ21 were 1.50 (0.87-11.04), 1.13(0.97-1.32), 1.05 (0.85-1.48), respectively (H=54.42, P＜0. 01). The MFI for7E3 were 1.51(0. 84-9.81), 1.05(0.86-1.13), 1.03 (0.74-1.28), respectively (H =31.97, P ＜0.01). Based on ROC analysis, with cut-off values of 1.37, 1. 24, 1.48 and 1.28 for SZ2, SZ22, SZ21 and 7E3,respectively, the AUC were 0. 86, 0.90, 0. 87 and 0. 84, respectively. The sensitivities of the assays were 58. 82% (50/85), 52. 94% (45/85), 52.94% (45/85) and 51.76% (44/85), respectively. When all four antibodies were used, the sensitivity was increased to 74. 12% (63/85), which was higher than that of MAIPA [ 50. 59% (43/85) ,χ2 = 6. 78, P ＜ 0. 05) ]. Conclusion Flow cytometric bead assay can be used to detect four platelet-specific autoantibodies simultaneously, and may be a useful method to aid in the diagnosis of ITP. Key words: Flow cytometry; Autoantibodies; Glycoprotein; Thrombocytopenia

High-dose dexamethasone therapy for severe thrombocytopenia and neutropenia induced by EBV infectious mononucleosis

Immune thrombocytopenic purpura (ITP) is a hematologic disorder characterized by autoimmune-mediated platelet destruction. Although prednisone (or prednisolone) is generally used for treatment of ITP, high-dose dexamethasone therapy is also effective and leads to rapid recovery of platelet count [1]. In contrast, optimal management of secondary ITP has not been well established. Here, we present a case of significant thrombocytopenia and neutropenia accompanied with Epstein–Barr virus (EBV)induced infectious mononucleosis (IM). A 36-year-old male visited our hospital with 2-week history of sore throat and fever. Physical examination showed cervical lymphadenopathy without hepatosplenomegaly. Peripheral blood findings were as follows: hemoglobin 13.5 g/dL, white blood cell count (WBC) 7.1 9 10/L (22.5% neutrophils, 1.5% monocytes, 6.0% lymphocytes, and 70.0% atypical lymphocytes), platelet count 71 9 10/L, aspartate aminotransferase 177 IU/L, alanine aminotransferase 240 IU/L, and lactate dehydrogenase 564 IU/L. The serological examination for EBV infection revealed the following results: the antibodies for viral capsid antigen (VCA) immunoglobulin (Ig) G was 940, VCA IgM and anti-EB virus nuclear antigen (EBNA) IgG were negative. Also, serum EBV DNA was increased to 4.0 9 10 copies/L. He was diagnosed as EBV-induced IM. His symptom disappeared in a week. However, platelet count continued to decrease, followed by decreased WBC, as shown in Fig. 1. About 60 days after the first symptom appeared, peripheral blood counts were WBC 1.4 9 10/L with 30% neutrophils and platelet count 12 9 10/L. Reticulated platelets were increased to 12.6%. The patient’s serum was positive for anti-neutrophil antibody and platelet-associated IgG (PAIgG) was increased. Antineutrophil antibody was detected by flow cytometry method. Neutrophils from normal donors were incubated with serum from the patient or pooled sera from normal volunteers as a negative control. Then, membrane-bound IgG and IgM antibodies were detected using fluorescein isothiocyanate (FITC)-labeled goat anti-human immunoglobulin. The fluorescence intensity was plotted and judged for positivity. Furthermore, bone marrow was slightly hypercellular, with increased megakaryocytes. Based on these findings, a diagnosis of autoimmune neutropenia and ITP secondary to EBV infection was done. Because thrombocytopenia prolonged and gradually decreased for about 2 months (minimum platelet count of 12 9 10/L), we started treatment with high-dose dexamethasone therapy (40 mg/day for consecutive 4 days orally) and then stopped without tapering. His platelet count increased gradually to 55 9 10/L in 2 weeks after the treatment. Also, neutrophil count improved rapidly and exceeded above 2.0 9 10/L in 3 days. Currently, 4 months after the therapy, he is well with platelet count 100 9 10/L and neutrophil count about 1.0 9 10/L. No adverse effects were noted during and after the therapy. Our patient developed typical symptoms suggestive of IM. Although negative VCA IgM antibody is inconsistent with the present infection, it is reported that VCA IgM antibody by fluorescent antibody test might be negative in 30% of patients with initial EBV infection [2]. In this case, negative EBNA IgG and positive EBV DNA in peripheral blood strongly suggest primary EBV infection. EBV-induced IM is sometimes accompanied by mild Y. Kagoya A. Hangaishi T. Takahashi Y. Imai M. Kurokawa (&) Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan e-mail: kurokawa-tky@umin.ac.jp

Immunoassay of Goat Antihuman Immunoglobulin G Antibody Based on Luminescence Resonance Energy Transfer between Near-Infrared Responsive NaYF4:Yb, Er Upconversion Fluorescent Nanoparticles and Gold Nanoparticles

Near-infrared (NIR) light can penetrate biological samples and even tissues without causing sample damage and avoid autofluorescence from biological samples in fluorescence detection. Thus, a luminescence resonance energy transfer (LRET)-based immunoassay that can be excited by NIR irradiation is a promising approach to the analysis of biological samples. Here we demonstrate the use of NIR-to-visible upconversion nanoparticles (UCNPs) as an energy donor, which can emit a visible light upon the NIR irradiation, and gold nanoparticles (Au NPs) as an energy acceptor, which can absorb the visible light emitted from the donor, to develop a sandwich-type LRET-based immunoassay for the detection of goat antihuman immunoglobulin G (IgG). Amino-functionalized NaYF(4):Yb, Er UCNPs and Au NPs were first prepared and then conjugated with the human IgG and rabbit antigoat IgG, respectively. The NIR-excited fluorescence emission band of human IgG-conjugated NaYF(4):Yb, Er UCNPs (lambda(max) = 542 nm) partially overlaps with the visible absorption band of the rabbit antigoat IgG-conjugated colloidal Au NPs (lambda(max) = 530 nm), satisfying the requirement of spectral overlap between donors and acceptors for LRET. A LRET system was then formed when goat antihuman IgG was added to a mixture of human IgG-modified NaYF(4):Yb, Er UCNPs (donor) and rabbit antigoat IgG-modified Au NPs (acceptor). The sandwich-type immunoreactions between the added goat antihuman IgG (primary antibody) and the two different proteins (antigen and secondary antibody on the surface of the donors and acceptors, respectively) cross-bridge the donors and acceptors and thus shorten their spacing, leading to the occurrence of LRET from UCNPs to Au NPs upon NIR irradiation. As a result, the quenching of the NIR-excited fluorescence of the UCNPs is linearly correlated to the concentration of the goat antihuman IgG (in the range of 3-67 microg x mL(-1)) present in the system, enabling the detection and quantification of the antibody. Such sandwich-type LRET-based approach can reach a very low detection limit of goat antihuman IgG (0.88 microg x mL(-1)), indicating that this method is applicable for the trace protein detection. This approach is expected to be extended to the detection of other biological molecules once the donor and acceptor nanoparticles are modified by proper molecules that can recognize the target biomolecules.

Fluorescence cross-correlation spectroscopy using single wavelength laser.

In this paper, we first introduced the basic principle of fluorescence cross-correlation spectroscopy (FCCS) and then established an FCCS setup using a single wavelength laser. We systematically optimized the setup, and the detection volume reached about 0.7 fL. The homebuilt setup was successfully applied for the study of the binding reaction of human immunoglobulin G with goat antihuman immunoglobulin G. Using quantum dots (745 nm emission wavelength) and Rhodamine B (580 nm emission wavelength) as labeling probes and 532 nm laser beam as an excitation source, the cross-talk effect was almost completely suppressed. The molecule numbers in a highly focused volume, the concentration, and the diffusion time and hydrodynamic radii of the reaction products can be determined by FCCS system.