Detection Of Human IgG Research Articles

An Artificial Miniaturized Peroxidase for Signal Amplification in Lateral Flow Immunoassays.

Signal amplification strategies are widely used for improving the sensitivity of lateral flow immunoassays (LFiAs). Herein, the artificial miniaturized peroxidase Fe(III)-MimochromeVI*a (FeMC6*a), immobilized on gold nanoparticles (AuNPs), is used as a strategy to obtain catalytic signal amplification in sandwich immunoassays on lateral flow strips. The assay scheme uses AuNPs decorated with the mini-peroxidase FeMC6*a and anti-human-IgG as a detection antibody (dAb), for the detection of human-IgG, as a model analyte. Recognition of the analyte by the capture and detection antibodies is first evidenced by the appearance of a red color in the test line (TL), due to the accumulation of AuNPs. Subsequent addition of 3,3',5,5'-tetramethylbenzidine (TMB) induces an increase of the test line color, due to the TMB being converted into an insoluble colored product, catalyzed by FeMC6*a. This work shows that FeMC6*a acts as an efficient catalyst in paper, increasing the sensitivity of an LFiA up to four times with respect to a conventional LFiA. Furthermore, FeMC6*a achieves lower limits of detection that are found in control experiments where it is replaced with horseradish peroxidase (HRP), its natural counterpart. This study represents a significant proof-of-concept for the development of more sensitive LFiAs, for different analytes, based on properly designed artificial metalloenzymes.

Biosensors based on DNA-functionalized CdTe quantum dots for the enhanced electrochemical detection of human-IgG.

Electrochemical detection of human-IgG via biosensors is vital in clinical diagnostics, owing to their simple equipment, facile operation, high selectivity, economical, short diagnostic time, fast response, and easy miniaturization, but the need to improve sensitivity for protein detection is still a barrier limiting its wider practical applications. A hypersensitized electrochemical biosensor based on steric effects for IgG detection was developed in this work. The results indicate that IgG-modified sig-DNA attached to CdTe quantum dots (CdTe-sig-DNA) limited the ability of CdTe-sig-DNA or CdTe-sig-DNA-IgG conjugate to hybridize through the captured DNA strand (cap-DNA) immobilized on a chitosan/nitrogen-doped carbon nanocomposite (CS/N-C) modified glassy carbon electrode surface (GCE). The concentration of IgG based on CdTe concentration was detected by differential pulse anode stripping voltammetry (DPASV) on the electrode surface. The efficiency for hybridizing CdTe-sig-DNA with cap-DNA was found to be logarithmically inverse to the concentration of IgG attached. A highly sensitive and selective detection of IgG from 5 pM to 50 μM with a relatively low detection limit of 1.7 pM was achieved. Therefore, the steric hindrance effect of IgG limited the quantity of DNA that could be functionalized on CdTe QDs, significantly improving the signal, and providing a practical strategy for the clinical analysis of IgG.

A Au nanoparticle and polydopamine co-modified biosensor: A strategy for in situ and label-free surface plasmon resonance immunoassays

This article reports a surface plasmon resonance (SPR) strategy capable of label-free yet amplified in situ immunoassays for sensitive and specific detection of human IgG (hIgG), a serum marker that is important for the diagnosis of certain diseases. Primarily, a wavelength-modulated Kretschman configuration SPR analyzer was constructed, and Au film SPR biosensor chips were fabricated. Specifically, based on Au nanoparticles (AuNPs) adsorbed on the surface of the Au film, the AuNP/Au film was coated with polydopamine (PDA) to fix streptavidin (SA), and then the biotinylated antibodies were connected to the surface of the biosensor chip. The SPR analyzer was utilized for in situ real-time monitoring of hIgG. Due to the immunological recognition between the receptor and target, the surface plasmon waves produced by the attenuated total reflection were affected by the changes in the surface of the biosensor chip. The resonance wavelength ( λ R ) of the output spectra gradually redshifted, and the redshift degrees were directly related to the target concentration. The biosensor can realize the in situ detection of hIgG, displaying satisfactory sensitivity, excellent specificity and stability. Briefly, by monitoring the shift in λ R after specific binding, a new SPR immunoassay can be customized for label-free, in situ and amplified hIgG detection. The operating principle of this research could be extended as a common protocol for many other targets of interest.

A molecularly imprinted electrochemical sensor with tunable electrosynthesized Cu-MOFs modification for ultrasensitive detection of human IgG

Human IgG is one of the most important immunoglobulins in the human body. The present study described the fabrication of four kinds of layer-by-layer structures of copper metal-organic frameworks (Cu-MOFs) on the working electrode by electrodeposition, which were then applied as an electrochemical sensor for the sensitive determination of IgG in serum. First, MOFs synthesized using different deposition potentials are expected to have varied morphology and properties. Herein, four copper MOFs (Cu-MOFs) were electrosynthesized by a simple and direct reduction approach. The as-synthesized Cu-MOFs exhibit varied morphology and electrocatalytic behavior. Then, IgG was employed as a template in the electropolymerization of pyrrole-imprinted films on the surface of glassy carbon electrodes. Finally, the template protein was removed to form a molecularly imprinted film with the capability to qualitatively and quantitatively signaling of IgG. Under optimized conditions, the sensor for IgG exhibits a wide detection range of 0.01–10 ng mL−1 with a limit of detection (LOD) of 3 pg mL−1 (S/N = 3). Besides, other parameters including the selectivity, reproducibility (RSD 3.6%), and recovery rate (95.2–102.0%) are all satisfactory. The practicability of the sensor was verified by detecting IgG in human serum samples, which indicated that the sensor was suitable for potential clinical applications.

Compact Surface Plasmon Resonance IgG Sensor Based on H-Shaped Optical Fiber

A compact surface plasmon resonance sensor based on an H-shaped optical fiber is proposed and demonstrated. The H-shaped optical fiber was fabricated experimentally by using hydrofluoric acid to controllably corrode the polarization-maintaining fiber. A satisfactory distance between the outer surface of the fiber and the core can be achieved, and then the surface plasmon resonance effect can be excited by coating a metal film of appropriate thickness on the surface of the fiber. This technology can realize the preparation of multiple samples at one time, compared to the traditional side-polishing technique. The H-shaped optical fiber obtained from corrosion exhibits a high surface quality and short lengths, down to only a few hundred microns. The effects of the proposed H-shaped optical fiber on spectral properties are induced by process parameters, including fiber remaining thickness, coating thickness and fiber length, and were investigated in detail. The prepared sensor was used for the specific detection of human IgG, and the minimum human IgG concentration that the sensor can distinguish is 3.4 μg/mL. Such a compact surface plasmon resonance fiber sensor has the advantages of an easy fabrication, good consistency and low cost, and is expected to be applied in the specific detection of biomarkers.

Development of an immunoassay for the detection of human IgG against hepatitis C virus proteins using magnetic beads and flow cytometry

Hepatitis C is a public health problem worldwide, affecting chronically about 71 million people, or 1% of the world population. The early detection of infection in clinical practice routine and blood donors is still important, being necessary for the development of new specific, sensitive, reliable tests that can improve the screening of HCV infection. Flow cytometry beads assay opens the possibility to detect multiple antigens, decrease cross-reactivity, increase in the sensitivity, with low sample volumes. Here, we describe proof-of-concept of a method for anti-Core and anti-NS5a detection using magnetic beads and flow cytometry. The immunoassay presented 93.33% sensitivity and 100% specificity for rCore magnetic beads (MgBs), and 93.33% sensitivity and 96.67% specificity for rctNS5a MgBs. The accuracy values for the tests using rCore and rctNS5a MgBs were 96.67% and 95%, respectively. The results of the ROC curve were 0.97 and 0.99 for rCore and rctNS5a MgBs, respectively. Therefore, we concluded that the method presented here for the detection of anti-HCV antibodies using magnetic beads analyzed by flow cytometry showed promising results, achieving satisfactory sensitivity and specificity values, with future potential to be an alternative method for screening anti-HCV antibodies in blood banks and blood centers.

Tutorial: design and fabrication of nanoparticle-based lateral-flow immunoassays

Lateral-flow assays (LFAs) are quick, simple and cheap assays to analyze various samples at the point of care or in the field, making them one of the most widespread biosensors currently available. They have been successfully employed for the detection of a myriad of different targets (ranging from atoms up to whole cells) in all type of samples (including water, blood, foodstuff and environmental samples). Their operation relies on the capillary flow of the sample throughout a series of sequential pads, each with different functionalities aiming to generate a signal to indicate the absence/presence (and, in some cases, the concentration) of the analyte of interest. To have a user-friendly operation, their development requires the optimization of multiple, interconnected parameters that may overwhelm new developers. In this tutorial, we provide the readers with: (i) the basic knowledge to understand the principles governing an LFA and to take informed decisions during lateral flow strip design and fabrication, (ii) a roadmap for optimal LFA development independent of the specific application, (iii) a step-by-step example procedure for the assembly and operation of an LF strip for the detection of human IgG and (iv) an extensive troubleshooting section addressing the most frequent issues in designing, assembling and using LFAs. By changing only the receptors, the provided example procedure can easily be adapted for cost-efficient detection of a broad variety of targets.

Ultrasensitive Label-Free Biosensor Based on the Graphene-Oxide-Coated-U-Bent Long-Period Fiber Grating Inscribed in a Two-Mode Fiber

In this article, we theoretically and experimentally report a newly-designed biosensor, which consists of a particular graphene-oxide (GO)-coated-U-bent long-period fiber grating inscribed in a two-mode fiber (LPFG-TMF). Due to the enhancement of evanescent field, the bent LPFG-TMF is more sensitive to the surrounding refractive index and it allows an extremely high sensitivity. The impact of bent radius on the sensing performance is discussed. The experimental result demonstrates, the sensitivity increases nonlinearly with the surrounding refractive index and the highest sensitivity reaches 6632.32 nm/RIU in the refractive index region from 1.3731 to 1.3830. To optimize the immune-sensing performance, the goat anti-human IgG is immobilized on the sensor via covalent bond through EDC/NHS cross-linker for human IgG recognition. The GO-coated-U-bent LPFG-TMF with bent radius of 15 mm has gained a low limit of detection (23 ng/ml) in the 3-20 μg/ml. In addition, the repeatability of the sensor is also investigated. With its premium ability of easy fabrication, high sensitivity, and compactness, the proposed sensor is adapted for detection of human IgG in clinical diagnostics, pharmaceutical researches, and bioengineering applications.

Development of Rabbit Monoclonal Antibodies for Quantitation of Therapeutic Human Antibodies in Preclinical Non-Human Primate Studies.

During preclinical studies, there is a great need to develop monoclonal antibodies (mAbs) that are specific to human immunoglobulin (IgG), without binding to monkey IgG, to detect therapeutic human mAb in non-human primates. We took advantage of the latest rabbit B cell cloning technology to develop six unique rabbit anti-human IgG mAb clones for this purpose. These clones are capable of binding to both human IgG and Fab with high affinity without nonspecific binding to cynomolgus monkey IgG. These clones have been evaluated as a generic capture reagent for the detection of human IgG and Fab, in the presence of cynomolgus monkey serum, by Gyrolab™ immunoassay. They may be used in singlet or as pairs for the detection of human IgG, in any host animal, to meet the need for therapeutic mAb development in preclinical studies.

Highly Sensitive Fluorescence-Linked Immunosorbent Assay for the Determination of Human IgG in Serum Using Quantum Dot Nanobeads and Magnetic Fe3O4 Nanospheres.

The aim of this study is to establish a new method with high sensitivity, accuracy, and stability for the determination of human IgG and then expand it to analyze severe acute respiratory syndrome corona virus 2 (SARS-CoV-2)-specific IgM and IgG, which is of great significance for the screening and diagnosis of COVID-19. In this study, the magnetic Fe3O4 nanospheres coupled with mouse antihuman IgG (Ab1IgG) were used as an immune capture probe (Fe3O4@Ab1IgG) to capture and separate the target, and rabbit antihuman IgG (Ab2IgG) coupled with highly luminescent quantum dot nanobeads (QBs) as a fluorescence detection probe (QBs@Ab2IgG) was used to realize high sensitivity detection. After the formation of a sandwich immunocomplex, the fluorescence intensity of the precipitate after magnetic separation was measured at the excitation wavelength of 370 nm. Under optimal conditions, a wide linear range varying from 0.005 to 40 ng·mL–1 was obtained for the detection of human IgG with a lower limit of detection at 4 pg·mL–1 (S/N = 3). The recoveries of intra- and interassays were 90.0–101.9 and 96.0–106.6%, respectively, and the relative standard deviations were 6.3–10.2 and 2.6–10.5%, respectively. Furthermore, the proposed method was successfully demonstrated to detect human IgG in serum samples, and the detection results were not statistically different (P > 0.05) from commercial enzyme-linked immunosorbent assay kits. This method is sensitive, fast, and accurate, which could be expanded to detect the specific IgM and IgG antibodies against SARS-CoV-2.

Daratumumab interference in flow cytometric anti-granulocyte antibody testing can be overcome using non-human blocking antibodies.

Daratumumab (DARA), a human IgG1K monoclonal antibody targeting CD38, is used to treat refractory multiple myeloma patients. CD38 is expressed on many cell types (RBCs, granulocytes, lymphocytes, etc.), and thus, DARA can interfere with serological tests. Information regarding how DARA affects anti-granulocyte antibody (AGA) testing and optimal neutralization of DARA will help laboratories perform accurate testing. Screening of AGA was performed by the granulocyte agglutination test (GAT) and the flow cytometric granulocyte immunofluorescence test (Flow-GIFT). Samples were tested from patients on DARA (n=7), non-transfused blood donors (healthy controls, n=7) and AGA reactive samples (positive controls, n=5). Two neutralization experiments, CD38 removal with DTT and DARA epitope blockage with mouse anti-CD38, were evaluated. Positive reactivity of human IgG binding was observed in 5/7 DARA cases when tested by Flow-GIFT; however, all 7 cases had negative GAT agglutination results. Further studies by Flow-GIFT revealed DARA concentrations >0·63μg/ml bound to granulocytes. DARA binding was negated by DTT though a reduced Flow-GIFT sensitivity was observed in positive control samples due to increased background detection of human IgG. Mouse anti-CD38 neutralized the detection of human IgG observed in DARA-treated patient serum without effecting controls. We established that DARA can interfere with AGA testing, leading to false positive Flow-GIFT results without causing GAT agglutination. DTT treatment increased background binding of secondary antibodies causing a decrease in Flow-GIFT sensitivity. In comparison, blockage of the DARA binding epitope using mouse anti-CD38 antibody was effective in neutralizing DARA interference while maintaining Flow-GIFT sensitivity.

Ultra-high sensitivity SPR fiber sensor based on multilayer nanoparticle and Au film coupling enhancement

• Electric field intensity of multilayer of GNPs coupled with gold film was studied. • The RI sensitivity of the double-layer gold nanospheres sensor was 15747 nm/RIU. • The RI sensitivity of the double-layer gold nanorods sensor was 25642 nm/RIU. • The LOD of double-layer gold nanospheres biosensor is 10 ng/mL. • The LOD of double-layer gold nanorods biosensor is 4.6 ng/mL. The main challenge for all biosensors is how to measure the biological samples at very low concentrations. In this paper, a surface plasmon resonance (SPR) fiber biosensor using multi-layer gold nanoparticles (Au NPs)/Au film coupling to enhance sensitivity was proposed. The coupling effect between such longitudinally aligned multilayer nanoparticles increases the intensity of the local electromagnetic field and the depth of propagation to the outside. We used the finite element method to analyze the local enhancement effect of multi-layer Au nanospheres and Au nanorods, and the fabricated double-layer Au NPs (Au nanospheres and Au nanorods)/Au film coupling enhancement sensors. These two sensors’ refractive index sensitivities are 15,747 nm/RIU and 25,642 nm/RIU, respectively. By changing biomolecules immobilized on the sensor’s surface, the highly sensitive double-layer Au nanoparticle/Au film fiber sensor enables high-precision measurement of different biomarkers at low concentration. For detection of human IgG, the detection limit of gold nanosphere sensor is 10 ng/mL and gold nanorod sensor is 4.6 ng/mL.

Nanozyme-linked immunosorbent assay for porcine circovirus type 2 antibody using HAuCl4/H2O2 coloring system

It is of great interest but still a huge challenge to construct an enzyme-free method for the early clinical diagnosis of various diseases with naked eyes. In the traditional enzyme linked immunosorbent assay (ELISA), horseradish peroxidase (HRP) as enzyme label and tetramethylbenzidine (TMB) as coloring agent have been widely used for immunoassays. However, the monochromic changes of TMB and the limited catalytic activity of HRP may confine the performance of ELISA. Herein, a new HAuCl4/H2O2 coloring system was proposed for naked-eye detection of porcine circovirus type 2 (PCV2) antibody using nanozyme, Au-Pt/SiO2 as labels. Be similar to the oxidation capacity of H2O2 towards TMB, HAuCl4 can also be reduced into gold nanoparticles (Au NPs) by H2O2. Based on this finding, antibody grafted Au-Pt/SiO2 nanospheres (APS NPs) were prepared and acted as enzyme labels to catalyze the decomposition of H2O2, thus resulting in a color variation from wine red to light purple with the increasing of analyte. The HAuCl4/H2O2 system was applied to the detection of human IgG and porcine circovirus type 2 (PCV2) antibody, which was comparable with Au-Pt/SiO2 labeled TMB/H2O2 system. This method showed the limit of detection (LOD) of 50 pg mL−1 (n = 3, ±4.8%) for HIgG and a 1:107 dilution detection for PCV2 antibody, two orders of magnitude higher in detection sensitivity compared to conventional ELISA, which revealed its great advantage in developing sensitive enzyme-linked immunoassay.

Smart Hydrogel Grating Immunosensors for Highly Selective and Sensitive Detection of Human-IgG

A smart diffraction grating immunosensor based on antigen-responsive hydrogel with enhanced analyte-induced volume changes is developed for highly selective and sensitive detection of human immunog...

Cigarette smoking differentially affects immunoglobulin class levels in serum and saliva: An investigation and review.

The aim of the present study was to compare concentrations of IgG, IgA, IgM and IgD in both serum and saliva samples from smoking and non-smoking individuals using a protein microarray assay. The findings were also compared to previous studies. Serum and saliva were collected from 48 smoking male individuals and 48 age-matched never-smoker male individuals. The protein microarray assays for detection of human IgG, IgM, IgA and IgD were established and optimized using Ig class-specific affinity-purified goat anti-human Ig-Fc capture antibodies and horseradish peroxidase (HRP)-conjugated goat anti-human Ig-Fc detection antibodies. The Ig class specificity of the microarray assays was verified, and the optimal dilutions of serum and saliva samples were determined for quantification of Ig levels against standard curves. We found that smoking is associated with reduced IgG concentrations and enhanced IgA concentrations in both serum and saliva. By contrast, smoking differentially affected IgM concentrations-causing increased concentrations in serum, but decreased concentrations in saliva. Smoking was associated with decreased IgD concentrations in serum and did not have a significant effect on the very low IgD concentrations in saliva. Thus, cigarette smoking differentially affects the levels of Ig classes systemically and in the oral mucosa. Although there is variation between the results of different published studies, there is a consensus that smokers have significantly reduced levels of IgG in both serum and saliva. A functional antibody deficiency associated with smoking may compromise the body's response to infection and result in a predisposition to the development of autoimmunity.

Hexaammineruthenium (II)/(III) as alternative redox-probe to Hexacyanoferrat (II)/(III) for stable impedimetric biosensing with gold electrodes

Gold electrodes have been used in a wide range of electrochemical biosensors because their functionalization process with thiols has been well described and, in general, they offer good chemical stability. However, one of the most commonly used redox-pairs in electrochemical impedance spectroscopy, Hexacyanoferrate (II)/(III), causes corrosion of the gold electrodes and consequently damages the surface modification. This leads to alterations of the sensing signals, and thus, renders the quantitative and sensitive detection of target molecules virtually impossible. To overcome this problem we introduced the in-situ generation of Hexaammineruthenium (II)/(III) as redox-pair during the impedimetric measurement by applying a DC-bias. This DC-bias was chosen in such a way that it supplied Hexaammineruthenium (II) in a suitable concentration at the electrode surface by reducing Hexaammineruthenium (III). We compared the stability of photolithographically fabricated thin-film and screen-printed gold electrodes in Hexacyanoferrate and Hexaammineruthenium solutions. Further, long-time characterization of the electrochemical properties with cyclic voltammetry and electrochemical impedance spectroscopy revealed that Hexaammineruthenium (II)/(III) was an excellent redox-pair for stable impedimetric measurements with gold electrodes. To demonstrate the suitability of Hexaammineruthenium for biosensing we applied it for the impedimetric detection of human-IgG. This biosensor exhibited a linear range from 11.3 ng/mL to 113 μg/mL, which is a suitable range for diagnostic applications.

Electrochemiluminescent biosensor with DNA link for selective detection of human IgG based on steric hindrance

A highly selective DNA-based electrochemiluminescence (ECL) based biosensor is described for the detection of human IgG. It is exploiting the effect of steric hindrance that affects the strength of the ECL signal in the presence of IgG. Digoxin-linked signaling DNA was specifically bound to IgG, and this causes steric hindrance which limits the ability of DNA to hybridize with capturing DNA attached to a gold electrode. Europium (II) doped CdSe quantum dots were covalently linked to the DNA in order to generate the ECL signal. Using this steric hindrance hybridization method, the ECL signal of the biosensor were proportional to the concentration of IgG with a wide linear range and a 14 pM detection limit. Conceivably, the method can be expanded to the detection of a wide range of proteins for which homologous recognition elements are available.

Analysis of Multiplexed Nanosensor Arrays Based on Near-Infrared Fluorescent Single-Walled Carbon Nanotubes.

The high-throughput, label-free detection of biomolecules remains an important challenge in analytical chemistry with the potential of nanosensors to significantly increase the ability to multiplex such assays. In this work, we develop an optical sensor array, printable from a single-walled carbon nanotube/chitosan ink and functionalized to enable a divalent ion-based proximity quenching mechanism for transducing binding between a capture protein or an antibody with the target analyte. Arrays of 5 × 6, 200 μm near-infrared (nIR) spots at a density of ≈300 spots/cm2 are conjugated with immunoglobulin-binding proteins (proteins A, G, and L) for the detection of human IgG, mouse IgM, rat IgG2a, and human IgD. Binding kinetics are measured in a parallel, multiplexed fashion from each sensor spot using a custom laser scanning imaging configuration with an nIR photomultiplier tube detector. These arrays are used to examine cross-reactivity, competitive and nonspecific binding of analyte mixtures. We find that protein G and protein L functionalized sensors report selective responses to mouse IgM on the latter, as anticipated. Optically addressable platforms such as the one examined in this work have potential to significantly advance the real-time, multiplexed biomolecular detection of complex mixtures.

Nanoparticle-based biosensing using interfacial electrokinetic transduction

We present a novel label-free electrokinetic method for detecting biomolecular binding on nanoparticles suspended in the vicinity of a laminar microfluidic interface. The sensor is based on the deflection of the liquid interface in an external AC electric field. Biomolecular binding on particles is shown to increase the interfacial electrical conductivity of the suspending electrolyte, which is sensitively transduced as a change in electrokinetic interfacial deflection. Using this approach, we detect the binding of biotin on streptavidin-functionalized nanoparticles at concentrations as low as 500 aM and perform detection of human IgG at clinically relevant concentrations (1.25–12mg/mL) without labels. The interface response is only influenced by specific binding on nanoparticle binding sites and decreases when the particle concentration of reactive particles is reduced. Furthermore, we show that control experiments with both non-reactive particles and lack of a specific target analyte do not produce interfacial deflection. This work provides a promising method for quantifying bead-based binding kinetics for sensitive and specific biosensing in solution without labels.

The selection and optimization of the detection system for self-contained multiplexed dot-immunoassay

ABSTRACTThis study performed a comparative estimation of the detection systems with the use of conjugates based on peroxidase, alkaline phosphatase, colloidal gold, and the amplification system “Super-CARD” for multiplex dot-immunoassay of antibodies. The results of the study show that the sensitivity of the detection system with colloidal gold was approximately 8 times higher than that of the system with amplification “Super-CARD”, 30 times higher than that of the system with conjugate of alkaline phosphatase, and 250 times higher than the sensitivity of the system with the peroxidase conjugate. Gold immunosols limit the direct detection of human IgG of 10 pg with dynamic range of optical signal change from 5 ng to 10 pg. This limit corresponds to a range of concentrations of IgG from 2.5 μg/mL to 5 ng/mL and covers the range of specific IgG concentrations, to which a typical natural immune response leads.The most typical reasons for aggregate formation during obtainment of colloidal gold and the binding of colloidal gold with biocomponents were explored. The method of minimization of particle clusters formation was suggested as well as the method for increasing stability of diluted preparations of probe by means of sedimentation of aggregates from the ready-made product.