Goat Anti-human Immunoglobulin G Research Articles

Highly-Sensitive Polymer Optical Fiber SPR Sensor for Fast Immunoassay

A new type of human immunoglobulin G (IgG) sensors based on the surface plasmon resonance (SPR) in the low refractive index (RI) plastic optical fiber (POF) and an antibody immobilization method is presented. A 50-nm-thick gold film was formed on the polished D-shaped fiber surface by magnetron sputtering. The RI response of the POF sensor is 30 049.61 nm/RIU, which is 26.5 times higher than that of single mode fiber (SMF) SPR sensors. The proposed SPR biosensor can be developed by simple and rapid modification of the gold film with 11-mercapto undecanoic acid (MUA). Upon immobilization of the goat anti-human IgG antibody, the resonance wavelength shifts by 11.2 nm. The sensor can be used to specifically detect and quantify the human IgG at concentrations down to 245.4 ng/mL with the sensitivity of 1.327 7 nm per µg/mL, which offers an enhancement of 12.5-fold compared to that of the conventional SMF based SPR sensors. The proposed device may find the potential applications in the case of use at the point of care.

Quantitation of total antibody (tAb) from antibody drug conjugate (ADC) PYX-201 in rat and monkey plasma using an enzyme-linked immunosorbent assay (ELISA) and its application in preclinical studies

PYX-201 is an investigative ADC oncology drug composed of a monoclonal human immunoglobulin G (IgG) antibody targeting the extra domain B splice variant of fibronectin (EDB + FN) conjugated to an auristatin payload through a cleavable linker. Effective measurement of PYX-201 tAb is the key to ADC drug PYX-201 preclinical pharmacokinetics (PK) assessment. PYX-201 monoclonal antibody (mAb) was used as the reference standard, goat anti-human IgG polyclonal antibody (pAb) or rabbit anti-human Kappa light chain mAb was employed as the capture antibody, and mouse mAb or goat pAb anti-human IgG the crystallizable fragment (Fc) (horseradish peroxidase (HRP)) was utilized as the detection antibody in this ELISA. This assay was validated with a dynamic range 250 – 10,000 ng/mL and 250 – 6000 ng/mL in rat and monkey K2EDTA plasma, respectively. PYX-201 tAb bioanalytical ELISA assay was reported for the first time in any biological matrix. This is the first time for a bioanalytical method to be validated for a tAb from an ADC drug targeting EDB + FN in any biological matrix.

Method feasibility for cross-species testing, qualification, and validation of the Filovirus Animal Nonclinical Group anti-Ebola virus glycoprotein immunoglobulin G enzyme-linked immunosorbent assay for non-human primate serum samples.

An anti-Zaire Ebola virus (EBOV) glycoprotein (GP) immunoglobulin G (IgG) enzyme linked immunosorbent assay (ELISA) was developed to quantify the serum levels of anti-EBOV IgG in human and non-human primate (NHP) serum following vaccination and/or exposure to EBOV. This method was validated for testing human serum samples as previously reported. However, for direct immunobridging comparability between humans and NHPs, additional testing was warranted. First, method feasibility experiments were performed to assess cross-species reactivity and parallelism between human and NHP serum samples. During these preliminary assessments, the goat anti-human IgG secondary antibody conjugate used in the previous human validation was found to be favorably cross-reactive with NHP samples when tested at the same concentrations previously used in the validated assay for human sample testing. Further, NHP serum samples diluted in parallel with human serum when tested side-by-side in the ELISA. A subsequent NHP matrix qualification and partial validation in the anti-GP IgG ELISA were performed based on ICH and FDA guidance, to characterize assay performance for NHP test samples and supplement the previous validation for human sample testing. Based on our assessments, the anti-EBOV GP IgG ELISA method is considered suitable for the intended use of testing with both human and NHP serum samples in the same assay for immunobridging purposes.

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.

Immunoglobulin G-Encapsulated Gold Nanoclusters as Fluorescent Tags for Dot-Blot Immunoassays.

Few-atom gold nanoclusters (AuNCs) have been fabricated and used for various fields owing to their remarkable optical and photophysical features. However, the rational design for the antibody-mediated synthesis of fluorescent AuNCs for direct antigen-antibody reactions remains unexplored. In this work, immunoglobulin G (IgG)-functionalized AuNCs (IgG-AuNCs) were successfully prepared via a facile and fast biomineralization process. The generated IgG-AuNCs can emit intense red fluorescence with a high photoluminescence quantum yield. Besides strong emission, the bioactivity of IgG on the IgG-AuNCs can be retained. Surface plasmon resonance measurements suggested that IgG-AuNCs can bind to goat anti-human IgG with an affinity constant of 6.21 × 10-8 M. A simple detection method was then developed using a dot-blot immunoassay with IgG-AuNCs as fluorescent tags. Experimental results confirmed that the IgG-AuNC-based fluorescent reporters had many advantages such as low nonspecific adsorption and good photostability, offering immense potential for the development of efficient biosensors. This work can be extended to other specific antibodies to produce multifunctional AuNCs and utilized to detect and monitor targeted analytes and biological events of interest.

Simple immunoglobulin G sensor based on thin core single-mode fiber

Abstract In this paper, a simple fiber biosensor (FOB) for immunoglobulin G (IgG) detection is designed and experimentally verified. The FOB is constructed by a 20 mm long thin core single-mode fiber (TCSMF) sandwiched between two single-mode optical fibers (SMFs). First, the refractive index (RI) sensitivity of the fiber structures is calculated by the beam propagation method. The refractive index sensing experiment is performed using different concentrations of glycerol solutions, and the experimental results are mostly consistent with the simulation predictions. The experimental RI sensitivity increases with the surrounding RI and reaches 82.7 nm/RIU. Then the surface of the FOB is functionalized by APTES for covalent bonding. The human IgG and goat anti-human IgG are chosen as a bioconjugated pair to examine the bio-sensing effectiveness of this FOB. The sensitivity of IgG detection is determined to be 10.4 nm/(mg/ml). And the serum IgG concentration in normal adults lies within the range of 6–16 mg/ml (Worsfold et al., 1985), so the sensor is applicable to human IgG monitoring. The specificity of the FOB is also verified by a contrast experiment conducted using rabbit immunoglobulin G. The proposed FOB is simple, low loss, cost-effective, and can be used for various biological and chemical applications.

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.

Micromeritic procedures in assessing antinuclear antibody patterns in immunofluorescent assay.

The aim of this study was to introduce a micromeritic procedure (a statistical approach for small objects) in indirect immunofluorescence assay (IFA) to find objective quantitative parameters of antinuclear antibody (ANA) patterns which could support a diagnosis of auto-immune diseases. Sera of patients with systemic autoimmune diseases, McCoy-Plovdiv serum-free cell line, goat anti-human immunoglobulin-G FITC-conjugate, fluorescent microscope, computer-assisted digital image processing, analysis using a micromeritic procedure, ANOVA. Three ANA fluorescent patterns (homogeneous, rim and speckled) were analyzed by the micromeritic procedure. Parameters for the image brightness of the pixels (pixel grey value) were obtained and discussed as objective characteristics of fluorescent patterns: maximum ANA-linkage volume and surface density were established for the objects with speckled localization pattern. The micromeritic method for getting objective quantitative values of ANA fluorescent patterns in indirect immunofluorescent assay might be a valuable tool aiding in immunological diagnosing if integrated in a laboratory software package.

Silver nanowire-based electrochemical immunoassay for sensing immunoglobulin G with signal amplification using strawberry-like ZnO nanostructures as labels

The quick development of nanoscience and nanotechnology has paved the way for ultrasensitive biosensing and analysis. In this work, an ultrasensitive electrochemical immunosensor was developed for the detection of human immunoglobulin G (IgG) by combining with a newly designed trace tag on a disposable immunosensor array. The array was prepared by immobilizing captured antibodies on ultralong Ag nanowires, whilst the trace tag was prepared by loading horseradish peroxidase (HRP)-labeled goat anti-human IgG (HRP-anti-IgG) on thionine (TH)-doped mesoporous ZnO nanostrawberries (MP-ZnO). With a sandwich-type immunoassay format, mainly due to crystalline framework and high surface area of the mesoporous (MP) materials, as well as the superconductivity of silver nanowires, the electrochemical signal was significantly amplified. The linear range of the developed immunosensor is 0.01–200ngmL−1 and the detection limit is 4pgmL−1 IgG, which make the hierarchically nanostructured composites very promising candidates for the next-generation sandwich-type electrochemical immunoassays.

Effect of protein molecules on the photoluminescence properties and stability of water-soluble CdSe/ZnS core-shell quantum dots

The fact that the photoluminescence properties of quantum dots are always strongly influenced by the environment limits the scope of further progress in the field of QD’ bio-applications. In this paper, the effects of immunoglobulin G (IgG) on the photoluminescence properties and stability of water-soluble CdSe/ZnS core-shell quantum dots coated with amphiphilic poly (acrylic acid) (PAA) are studied. Photoluminescence (PL) spectra, UV-vis spectra and excited state lifetime measurements are used to characterize the influence of different protein molecules, such as IgG (goat anti-human IgG, rabbit anti-human IgG, human IgG, and goat anti-human IgG-human IgG conjugates), avidin and bovine serum albumin (BSA) on the PL properties of QDs. The PL intensity and stability of CdSe/ZnS are largely enhanced compared to that of pure CdSe/ZnS QDs when the IgG molecules are added into the QD solution. The PL intensity increases with increasing the IgG concentration, but there appears no influence on the PL peak and a full width at half maximum (FWHM). The PL evolution of QDs as a function of different protein molecules depends on the structure of protein molecules, which is used as a sensor to recognize human IgG. It is inferred that the interaction between PAA coating layer and IgG molecules results in the enhancement of PL intensity. The study of the effect of pH and ion strength on optical properties of QD-IgG mixed solution, compared with the pure QD solution, suggests that pH value and ion strength do not destroy the interaction between the PAA coating layer and IgG. Excited state lifetime analysis indicates that the PL enhancement comes from the passivation of surface of the QDs with the PAA coating layer. IgG molecules have no effects on the properties of the biological system but can increase the stability and PL intensity of CdSe/ZnS QDs, which will enlarge the application of QDs in biomedicine and other fields.

Label-free and real-time detection of antigen-antibody interactions by Oblique-incidence Reflectivity Difference (OIRD) method

We label-free and real-time detected three interaction processes of antigen-antibodies, Human Immunoglobulin G (IgG), Rabbit IgG, and Mouse IgG as the targets, and Goat Anti-human IgG, Goat Anti-rabbit IgG, and Goat Anti-mouse IgG as the probe, by the Oblique-incidence Reflectivity Difference (OIRD) method. The interaction dynamic curves of the OIRD signal, corresponding to the interaction processes of antigen-antibodies, are generated. The reaction times from beginning to equilibrium state are about 1800, 900, and 1200 s for Human IgG, Rabbit IgG, and Mouse IgG, respectively. The experimental results demonstrate that the OIRD method not only can distinguish biomolecular interactions, but also can be used in real-time detection of interactions and dynamic processes of biomolecules.

Label-Free and Real-Time Detection of Antigen-Antibody Capture Processes Using the Oblique-Incidence Reflectivity Difference Technique

We successfully label-free and real-time detect the capture processes of human immunoglobulin G (IgG)/goat anti-human IgG and mouse IgG/goat anti-mouse IgG antigen-antibody pairs with different concentrations using the oblique-incidence reflectivity difference (OIRD) method, and obtain the interaction kinetics curves and the interaction times. The experimental results prove that the OIRD method is a promising technique for label-free and real-time detection of the biomolecular interaction processes and achieving the quantitative information of interaction kinetics.

Magnetic Bead-Sensing-Platform-Based Chemiluminescence Resonance Energy Transfer and Its Immunoassay Application

A competitive immunoassay based on chemiluminescence resonance energy transfer (CRET) on the magnetic beads (MBs) is developed for the detection of human immunoglobulin G (IgG). In this protocol, carboxyl-modified MBs were conjugated with horseradish peroxidase (HRP)-labeled goat antihuman IgG (HRP-anti-IgG) and incubated with a limited amount of fluorescein isothiocyanate (FITC)-labeled human IgG to immobilize the antibody-antigen immune complex on the surface of the MBs, which was further incubated with the target analyte (human IgG) for competitive immunoreaction and separated magnetically to remove the supernatant. The chemiluminescence (CL) buffer (containing luminol and H(2)O(2)) was then added, and the CRET from donor luminol to acceptor FITC in the immunocomplex on the surface of MBs occured immediately. The present protocol was evaluated for the competitive immunoassay of human IgG, and a linear relationship between CL intensity ratio (R = I(425)/I(525)) and human IgG concentration in the range of 0.2-4.0 nM was obtained with a correlation coefficient of 0.9965. The regression equation was expressed as R = 1.9871C + 2.4616, and a detection limit of 2.9 × 10(-11) M was obtained. The present method was successfully applied for the detection of IgG in human serum. The results indicate that the present protocol is quite promising for the application of CRET in immunoassays. It could also be developed for detection of other antigen-antibody immune complexes by using the corresponding antigens and respective antibodies.

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.

A highly sensitive resonance scattering spectral assay for IgG using Fehling reagent–glucose–immunonanogold reaction

Nanogold exhibits strong catalytic effect on the slow reaction between glucose and Fehling reagent at 70 °C. The production of Cu 2O particles have two stronger resonance scattering (RS) peaks at 390 nm and 505 nm. The catalytic effect of nanogold-labeled goat anti-human IgG (AuIgG) on the reaction was investigated with the RS technique. Coupled the immunoreaction and the immunonanogold catalytic reaction and centrifugal technique, a highly sensitive and selective RS method was developed for the detection of immunoglobulin G (IgG) as a model. With the concentration of IgG increased, the RS intensity at 505 nm decreased. The decreased intensity at 505 nm Δ I 505 nm was proportional to IgG concentration in the range of 0.13–53.3 ng mL −1, with a detection limit of 0.04 ng mL −1 IgG. This new immunonanogold–catalytic Cu 2O–particle RS bioassay was applied to the determination of IgG in serum sample, with high sensitivity, good selectivity, and low cost.

Direct Binding of Human NK Cell Natural Cytotoxicity Receptor NKp44 to the Surfaces of Mycobacteria and Other Bacteria

Our previous studies demonstrated that Mycobacterium bovis bacillus Calmette-Guérin (BCG) can directly interact with human NK cells and induce the proliferation, gamma interferon production, and cytotoxic activity of such cells without the need for accessory cells. Thus, the aim of the present study was to identify the putative receptor(s) responsible for the recognition of BCG by human NK cells and potentially involved in the activation of NK cells. To this end, we first investigated the surface expression of three NK cell-activating receptors belonging to the natural cytoxicity receptor (NCR) family on highly purified human NK cells upon in vitro direct stimulation with BCG. An induction of the surface expression of NKp44, but not of NKp30 or NKp46, was observed after 3 and 4 days of in vitro stimulation with live BCG. The NKp44 induction involved mainly a particular NK cell subset expressing the CD56 marker at high density, CD56(bright). In order to establish whether NKp44 could directly bind to BCG, whole BCG cells were stained with soluble forms of the three NCRs chimeric for the human immunoglobulin G (IgG) Fc fragment (NKp30-Fc, NKp44-Fc, NKp46-Fc), followed by incubation with a phycoerythrin (PE)-conjugated goat anti-human IgG antibody. Analysis by flow cytometry of the complexes revealed a higher PE fluorescence intensity for BCG incubated with NKp44-Fc than for BCG incubated with NKp30-Fc, NKp46-Fc, or negative controls. The binding of NKp44-Fc to the BCG surface was confirmed with immunogold labeling using transmission electron microscopy, suggesting the presence of a putative ligand(s) for human NKp44 on the BCG cell wall. Similar binding assays performed on a number of gram-positive and gram-negative bacteria revealed a pattern of NKp44-Fc binding restricted to members of the genus Mycobacterium, to the mycobacterium-related species Nocardia farcinica, and to Pseudomonas aeruginosa. Altogether, the results obtained indicate, for the first time, that at least one member of the NCR family (NKp44) may be involved in the direct recognition of bacterial pathogens by human NK cells.

A new strategy for electrochemical immunoassay based on enzymatic silver deposition on agarose beads

A novel, sensitive electrochemical immunoassay in a homogeneously dispersed medium is described herein based on the unique features of agarose beads and the special amplified properties of biometallization. The immunochemical recognition event between human immunoglobulin G (IgG) and goat anti-human IgG antibody is chosen as the model system to demonstrate the proposed immunoassay approach. Avidin-agarose beads rapidly react with the biotinylated goat anti-human IgG antibody to form agarose beads-goat anti-human IgG conjugate (agarose bead-Ab). Agarose bead-Ab, alkaline phosphatase conjugated goat anti-human IgG antibody (ALP-Ab) and the human IgG analyte are mixed to form sandwich-type immunocomplex followed by the addition of the enzymatic silver deposition solution to deposit silver onto the surface of proteins and agarose beads. The silver deposited are dissolved and quantified by anodic stripping voltammetry. The influence of relevant experimental variables was examined and optimized. The logarithm of the anodic stripping peak current depended linearly on the logarithm of the concentration of human IgG in the range from 1 to 1000 ng/ml. A detection limit as low as 0.5 ng/ml human IgG was attained by 3 σ-rule. The R.S.D. of the approach is 9.65% for eight times determination of 10 ng/ml human IgG under same conditions. Optical microscope and TEM graphs were also utilized to characterize agarose beads and silver nanoparticles formed.

Diagnóstico de leptospirosis: evaluación de un enzimoinmunoensayo en fase sólida en diferentes etapas de la enfermedad

To develop a solid-phase enzyme immunoassay (ELISA) for genus-specific immunoglobulin G (IgG) determination with leptospirosis and to evaluate the ELISA in different stages of the disease. A total of 1,077 serum samples from 812 patients with suspected leptospirosis were analyzed. The samples had come from diagnoses done in the laboratory of the National Institute of Respiratory Diseases (Instituto Nacional de Enfermedades Respiratorias), in the city of Santa Fe, Argentina, between 1999 and 2005. Included in the study were 182 confirmed cases (267 samples), 167 negative cases (293 samples), and 40 probable cases (60 samples) (based on case definitions based on the results from the microscopic agglutination test (MAT), leukocyte counts, and neutrophilia values). Each sample was classified, according to the days of the natural history of disease, into one of three stages: first (< 10 days), second (10-25 days), or third (> 25 days). The antigen used in the ELISA was an extract of a mixture of pyrogenes and tarassovi serovars cultivated in a liquid medium, treated with ultrasound, and immobilized by adsorption on polystyrene plates. As a secondary antibody, a peroxidase-conjugated goat anti-human IgG monoclonal antibody was used. The cutoff value, sensitivity, and specificity of the ELISA were determined using the definitions of confirmed cases and of negatives cases as the standard. In order to determine the optimal cutoff value, the area under the receiver operating characteristic curve was calculated. The sensitivity of the evaluated test was much higher in the second stage (93.2%) than in either the first stage (68.1%) or the third stage (78.8%). The specificity increased gradually from 96.3% in the first stage to 100% in the third stage. Our results indicate that this ELISA test can be a very useful complement to the MAT for the diagnosis of leptospirosis in all the stages and, in particular, in order to diagnose acute disease sooner.

Cyclic accumulation of nanoparticles: A new strategy for electrochemical immunoassay based on the reversible reaction between dethiobiotin and avidin

A new method based on cyclic accumulation of gold nanoparticles has been proposed for determinating human immunoglobulin G (IgG) by anodic stripping voltammetry. The dissociation reaction between dethiobiotin and avidin in the presence of biotin provides efficient means for the cyclic accumulation of gold nanoparticles used for the final analytical quantification. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreaction. Goat anti-human IgG is immobilized on the wells of microtiter plates. The human IgG analyte is first captured by the primary antibody and then sandwiched by secondary antibody labeled with dethiobiotin. Avidin-Au solution was introduced to react with dethiobiotinylated antibody followed by adding of bioitin solution to wash it down. The dissociation of avidin-Au from dethiobiotinylated antibody was caused by displacing function of biotin to dethiobiotin. Making use of the ability of dethiobiotinylated antibody keeping its activity after the association/dissociation reaction, the alternatively treatment with avidin-Au and biotin solutions could realize cyclic accumulation of gold nanoparticles for ASV quantification. The anodic peak current increases gradually with the increasing accumulation cycles. Five cycles of accumulation are sufficient for the assay. The experiment conducted with a blank solution without addition of the analyte showed an extremely low background even with the number of accumulation cycles reached 10. The low background of the proposed method is a distinguished advantage providing a possibility for determination of at least 0.1 ng/ml human IgG. The RSD of the method is 9.57% for eight times determination of 1 ng/ml human IgG under the same conditions.

A novel method for simultaneous analysis of specific platelet antibodies: SASPA

Glycoprotein (GP)-specific platelet antibodies can cause allo-immune and auto-immune thrombocytopenia. The specific detection of relevant antibodies is a prerequisite for diagnosis and treatment. Here, we describe an improved method based on simultaneous detection of various platelet-specific immunoglobulin G (IgG) and IgM antibodies. Bead populations with distinct fluorescence intensities, coated with monoclonal antibodies specific for mouse heavy chain isotypes, were used for the simultaneous immobilization of platelet-GP [IIb/IIIa, Ib/IX, human leucocyte antigen (HLA) class I, or Ia/IIa, CD32, GPIV or CD109, Ib/IX, HLA class I]. In order to detect human IgG and IgM antibodies simultaneously, phycoerythrin- and fluorescein isotiocyanate-conjugated goat anti-human IgG and IgM were added. On this basis, the abundance of six distinct antibodies (three anti-GP, each with subclasses IgG and IgM) were simultaneously analysed without cross-reaction by flow cytometry. For evaluation, sera and platelets from 169 patients with platelet-binding and/or platelet-associated antibodies were investigated. The monoclonal antibody-specific immobilization of platelet antigen (MAIPA) assay was performed in parallel as reference test. The simultaneous analysis of platelet-specific antibodies (SASPA) assay was able to detect all platelet-specific IgG and IgM that were also recognized by MAIPA with a comparable sensitivity. SASPA proved to be a rapid and reliable assay that required less platelets than other methods. This method has the potential to pave the way for new investigations of platelet-specific antibodies.