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Immunoglobulin G (IgG) is an antibody used in numerous therapeutic indications. For this reason, high-purity IgG is required, which implies the use of selective adsorption chromatographic purification techniques. Although, before using chromatography, it is highly important to study the interaction between IgG and the adsorbent. Due to its characteristics, biopolymeric adsorbents are widely studied. However, there are no studies in the literature that evaluate IgG adsorption onto fibroin, a natural polymer that can be used as an ion exchange adsorbent. Thus, the aim of this work was to evaluate the adsorption of human IgG on fibroin microparticles. The microparticles were prepared from fibroin solution using the atomization method, with an average diameter of 108.5 μm. Three buffers (MOPS, MES and Tris–HCl), with different pH, were used, and for the best conditions, the influence of ionic strength (NaCl from 0 to 1 mol L−1), temperature (4 °C to 37 °C) and rotation (20 rpm to 40 rpm) were studied. The highest adsorption capacity (906.45 mg g−1) was reached with the MOPS buffer at pH 8.0, without NaCl, 25 °C and 30 rpm, which is higher than typical adsorption capacities found in the literature for other adsorbents. The adsorption capacity reduced with increasing temperature from 25 °C to 37 °C and rotation rate from 30 to 40 rpm. The thermodynamic parameters demonstrated that adsorption is spontaneous and endothermic. These results open up new possibilities of application of fibroin microparticles, considering the medium in which they are inserted, and highlights the improvement of IgG chromatographic purification for uses in pharmaceutical field. [ABSTRACT FROM AUTHOR]

The sensing properties of poly 3‐(3‐carboxypropyl) thiophene‐2,5‐diyl (PT‐COOH) and hydroxylated polythiophene (PT‐OH) as bioreceptor layers were studied and are discussed in this paper. The polymer films cover the channel region of the OECT devices and anti‐human IgG was immobilized on the polymer films. We use threshold voltage (Vth) change as a sensing signal to detect the interaction between anti‐human IgG and human IgG. By adding different concentrations of human IgG, Vth difference can be observed on anti‐human IgG immobilized polymer films, with optimized detection from a blend of the two polymers. Open circuit potential (OCP) measurement was also done on the OECT devices based on the same anti‐human IgG and human IgG interaction pair to help us understand the mechanism behind the antibody functionalization and the interaction between antibody and antigen. Importantly, the observed positive OCP change for the PT‐OH system was self‐consistent with the negative OECT Vth change that was obtained, since the latter is applied to the gate while the former is measured at the channel. [ABSTRACT FROM AUTHOR]

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 (λR) 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 λR 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.

Background & Aims: Antibodies, well-known as immunoglobulins (Igs), are produced by B lymphocytes and specifically defend against pathogens. Igs are glycoproteins and have high diagnostic value in several diseases including infections (1). Igs are composed of light and heavy chains (2, 3). Each chain is comprised of about 110-120 amino acid residues which create immunoglobulin folds named domains (4, 5). Domains play an important role in Igs biological functions. Every domain is included of two anti-parallel β-sheets. B-sheets play an important role in epitope formation because most of the epitopes are located in B-sheets (6, 7). Epitope/ antigenic determinant is a part of antigen recognized by antibody (8). For precise detections of Igs, diagnostic tools such as Igs- epitope specific monoclonal antibodies (MAbs) are needed (11). Therefore B-sheets are valuable tools in detection of proteins epitopes. IgG is the highest Ig in serum and has vital role in defense against infections (12). For optimizing current IgG diagnostic tests, accurate determination of IgG-specific epitopes is extremely important (18). Immunoinformatic is a branch of immunology uses a large amount of computer-generated biological information to solve immunological problems and diagnosis of diseases faster and more accurate (19-21). Immunoinformatic has spread to almost all areas of immunology and has exceedingly facilitated understanding of immune responses and their role in disease and health. Accordingly immunoinformatic has provided novel research opportunities to study the molecular mechanisms of immunological processes and related diseases and thus provide more effective diagnostic and treatment strategies (24-26). As B-sheets play an important role in epitope formation (6, 7) and immunogenic epitopes are very useful tools for production of specific monoclonal antibodies against a molecule (11), we conducted this research for recognition of human IgG B-sheets with immunoinformatic method and study their compatibility with IgG epitopes. Methods: The confirmed amino acid sequence of reference human IgG was determined with PDB data bank in http://www.rcsb.org/pdb/home/home.do web site. The second structure of human IgG was obtained with Phyre2 (Protein Homology/analogy Recognition Engine V 2.0) software available on the http://www.sbg.bio.ic.ac.uk/phyre website. The location of B-sheets and epitopes in IgG was determined by the IEDB (Immune Epitope Database) at www.immuneepitope.org. Website. Results: Figures 1 and 2 show the second structure of light and heavy chains of the human IgG molecule, respectively, obtained by phyre2 software. The IgG light and heavy chains second structure consists of alpha spirals and B-sheets. Figures 3 and 4 show the location of B-sheets in light and heavy chains of the human IgG molecule obtained by IEDB software. Most of the B-sheets in IgG molecules were located in 150 - 175 amino acid sequences of light chains and in 130-140, 160-170, 190-200, 220-245, 375-410 and 420-425 amino acid sequences of heavy chains respectively as was determined by IEDB software. Three epitopes sited to constant domain of IgG light chain (CL) were predicted. These epitopes were located at 150-175 and 180-205 amino acid sequences of light chain. Also three epitopes situated to second and third constant domains of IgG heavy chain (CH2 and CH3) were predicted. These epitopes were located at 200-270, 290-360 and 380-400 amino acid sequences of heavy chain. Based on the results of present study, 66% of human IgG light chain epitopes and 66% of human IgG heavy chain epitopes are situated at the IgG B-sheets locations. Also in present study, 91% of the amino acids forming B-sheets sited in the heavy chains of IgG molecule were located in the CH2 and CH3 domains and 100% of the human IgG heavy chain epitopes were located in the CH2 and CH3 domains Conclusion: According to the results of this study, most of human IgG epitopes are located in regions of the molecule where B-sheets are most likely situated, indicating a relatively high compatibility of B-sheets with IgG epitopes. Given that the B-sheets present in the second structure of proteins play an important role in epitope formation (6, 7), the occurrence of most human IgG epitopes (66%) at the location of B-sheets seems evident. Moreover, according to the findings of the present study, a total of 91% of the amino acids forming B-sheets sited in the heavy chains of IgG molecule were located in the CH2 and CH3 domains and 100% of the human IgG heavy chain epitopes were located in the CH2 and CH3 domains. This again indicates the high compatibility of B-sheets with epitopes located on the heavy chain of the human IgG molecule. The findings of our study are confirmed by Hajighasemi et al. study (31). In study of Hajighasemi et al. (31), about 91% of the linear epitopes located on the heavy chains of the IgG molecule, were situated in the CH2 and CH3 domains. Similarly, in our study 100% of human IgG heavy chain epitopes located on CH2 and CH3 domains. Also the results of present study are confirmed by another research (36) in which all (100% ) of spatial epitopes on fragment of crystallizable [Fc] component (part of heavy chains) of the human IgG molecule were located in CH3 and CH2 domains as were identified by DiscoTope and ElliPro immunoinformatic softwares. Similarly in the present study, 100% of IgG heavy chain epitopes were located in the CH2 and CH3 domains. Moreover the results of another study performed by Rohani et al. (37) support the findings of current study. Rohani et al., reported that most of the areas with highest surface accessibility, hydrophilicity and flexibility, were situated in CH2 and CH3 domains of human IgG heavy chains. (37). According to our results, most of B-sheets are located in the CH2 and CH3 domains of human IgG heavy chains. As B-sheets are regions with high accessibility, hydrophilicity and flexibility (6), presence of most B-sheets in the CH2 and CH3 domains which are reported to be highly accessible, hydrophilic and flexible ( 37), is reasonable. Furthermore, the high immunogenicity of B-sheets (39), once again confirms results of the present study. As immunogenicity plays an important role in epitope development, the high adaptation between the location of B-sheets and epitopes ( shown in our study) which both are highly immunogenic, seems logical. Besides since B-sheets play an important role in epitope formation(6, 7), the high compatibility between location of B-sheets and IgG epitopes ( shown in present study) seems sensible. Overall, presence the most of B-sheets and IgG epitopes in CL, CH2 and CH3 domains of human IgG, shows the high compatibility between location of B-sheets and epitopes in human IgG. These findings are precious tools not only for prediction of IgG immunogenic epitopes in order to producing specific monoclonal anti IgG antibodies to optimize current human IgG diagnostic kits but also for more exact detection of IgG functions. Moreover our results are helpful in producing of similar proteins for diagnostic and therapeutic aims, structure- function relationships and phylogenic studies. [ABSTRACT FROM AUTHOR]

We demonstrate a temperature-insensitive and high-sensitive label-free biosensor for low-concentration Human IgG detection based on S-tapered optical fiber (STF). The sensor is sensitive to local refractive index changes and has high robustness against temperature noise without a temperature control system. The temperature sensitivity of the sensor is −0.02nm/°C in the range of 25°C-45°C. Within 15 minutes at room temperature, the average drift of the center wavelength is 0.04 nm. The calibration experiments demonstrate that STFs for liquid achieved a sensing precision of 0.04 nm and a sensitivity of 538.62 nm/RIU. The STFs are applied for the concentration measurement of multiple diluted ex vivo IgG biomolecule solutions. The results showed that STFs were reliable for low-concentration IgG detection and its lower limit of measurement was demonstrated as 28 ng/ml. With the advantages of temperature-insensitive, compact size, low cost, high precision, real-time detection, fast response, good specificity, this STF sensors are promising for the antigen detection applications.

Enzyme-linked immunosorbent assay (ELISA) is the gold standard technique for measuring protein biomarkers due to its high sensitivity, specificity, and throughput. Despite its success, continuous advancements in ELISA and immunoassay formats are crucial to meet evolving global challenges and to address new analytical needs in diverse applications. To expand the capabilities and applications of immunoassays, we introduce a novel ELISA-like assay that we call Bioluminescent-bacteria-linked immunosorbent assay (BBLISA). BBLISA is an enzyme-free assay that utilizes the inner filter effect between the bioluminescent bacteria Allivibrio fischeri and metallic nanoparticles (gold nanoparticles and gold iridium oxide nanoflowers) as molecular absorbers. Functionalizing these nanoparticles with antibodies induces their accumulation in wells upon binding to molecular targets, forming the classical immune-sandwich complex. Thanks to their ability to adsorb the light emitted by the bacteria, the nanoparticles can suppress the bioluminescence signal, allowing the rapid quantification of the target. To demonstrate the bioanalytical properties of the novel immunoassay platform, as a proof of principle, we detected two clinically relevant biomarkers (human immunoglobulin G and SARS-CoV-2 nucleoprotein) in human serum, achieving the same sensitivity and precision as the classic ELISA. We believe that BBLISA can be a promising alternative to the standard ELISA techniques, offering potential advancements in biomarker detection and analysis by combining nanomaterials with a low-cost, portable bioluminescent platform.

In this work, we report a self-compensating, label-free, and anti-interference surface plasmon resonance (SPR) fiber biosensor based on a cascaded U-shaped multimode fiber and a single-mode fiber and both coated with gold film. In the traditional dual-channel SPR sensor structure, in order to obtain two independent SPR signals on one probe, different metal films need to be coated on two sensing channels. We proposed a U-shaped cascade dual-channel SPR optical fiber sensor, two channels are covered with gold film, and two independent SPR signals can be obtained by controlling the U-bend radius. In order to achieve self-compensating in detection of biomarkers, the U-shaped multimode fiber area of sensing probe is biologically functionalized as a detection channel, and the straight single-mode fiber without functionalized used as a reference channel. During the process of measuring biomarkers concentration, the existence of reference channel can be used to eliminate the effect of impurities’ physical adsorption in the detection channel on the result. This sensor was used to detect the IgG concentration in complex samples, achieving the high precision detection with a limit of detection of $0.104\mu $ g/mL. A small-in-size, simple-to-manufacture, dual-channel SPR sensor with self-compensation was proposed and demonstrated in this article which has an excellent application prospect in the clinical monitoring of various biomarkers. [ABSTRACT FROM AUTHOR]

(1) Background: The validation of biological antigens is the study’s utmost goal in biomedical applications. We evaluated three different probes with single and multiple epitopes through electrochemical detection of specific IgG in serum for human strongyloidiasis diagnosis. (2) Methods: Screen-printed gold electrodes were used and probes consisting of two single-epitope synthetic peptides (D3 and C10) with different sequences, and a multi-epitope antigen [detergent phase (DP)—hydrophobic membrane proteins]. Human serum samples from three populations were used: Strongyloides stercoralis positive, positive for other parasitic infections and negative controls. To test the immobilization of probes onto a screen-printed gold electrode and the serum IgG detection, electrochemical analyses were carried out through differential pulse voltammetry (DPV) and the electrode surface analyses were recorded using atomic force microscopy. (3) Results: The electrochemical response in screen-printed gold electrodes of peptides D3 and C10 when using positive serum was significantly higher than that when using the DP. Our sensor improved sensitivity to detect strongyloidiasis. (4) Conclusions: Probes’ sequences are critical factors for differential electrochemical responses, and the D3 peptide presented the best electrochemical performance for strongyloidiasis detection, and may efficiently substitute whole antigen extracts from parasites for strongyloidiasis diagnosis in electrochemical immunosensors.

Acetaldehyde is a metabolite of ethanol, an important constituent of tobacco pyrolysis and the aldehydic product of lipid peroxidation. Acetaldehyde induced toxicity is mainly due to its binding to cellular macromolecules resulting in the formation of stable adducts accompanied by oxidative stress. The aim of this study was to characterize structural and immunological alterations in human immunoglobulin G (IgG) modified with acetaldehyde in the presence of sodium borohydride, a reducing agent. The IgG modifications were studied by various physicochemical techniques such as fluorescence and CD spectroscopy, free amino group estimation, 2,2‐azobis 2‐amidinopropane (AAPH) induced red blood cell hemolysis as well as transmission electron microscopy. Molecular docking was also employed to predict the preferential binding of acetaldehyde to IgG. The immunogenicity of native and acetaldehyde‐modified IgG was investigated by immunizing female New Zealand white rabbits using native and modified IgG as antigens. Binding specificity and cross reactivity of rabbit antibodies was screened by competitive inhibition ELISA and band shift assays. The modification of human IgG with acetaldehyde results in quenching of the fluorescence of tyrosine residues, decrease in free amino group content, a change in the antioxidant property as well as formation of cross‐linked structures in human IgG. Molecular docking reveals strong binding of IgG to acetaldehyde. Moreover, acetaldehyde modified IgG induced high titer antibodies (>1:12800) in the experimental animals. The antibodies exhibited high specificity in competitive binding assay toward acetaldehyde modified human IgG. The results indicate that acetaldehyde induces alterations in secondary and tertiary structure of IgG molecule that leads to formation of neo‐epitopes on IgG that enhances its immunogenicity. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract In the manuscript, a highly sensitive DNA-based electrochemiluminescence (ECL) biosensor was developed for the detection of human IgG (IgG), which takes advantage of steric hindrance effects to influence the ECL signal in the presence of target protein. The prepared ECL biosensor presented higher sensitivity and could be applied in other proteins with homologous recognition elements. fx1 Highlights • A highly selective DNA-based electrochemiluminescence (ECL) based biosensor is described for the detection of human IgG. • The biosensor takes advantage of steric hindrance effects to influence ECL signal in the presence of IgG. • This method could achieved good results in the analysis of real samples. [ABSTRACT FROM AUTHOR]

The behavior of human immunoglobulin G (IgG) and antigen‐binding fragment (Fab fragment) adsorption onto phospho‐l‐tyrosine immobilized on agarose (P‐Tyr‐agarose) was evaluated by pseudoaffinity chromatography. The effects of buffer systems MES, MOPS, Bis–Tris, Tris–HCl and sodium phosphate (NaP) and pH on IgG adsorption were studied and high purity values were obtained (96%, based on ELISA analysis of albumin, transferrin and immunoglobulins A, G and M) when IgG was purified from human plasma diluted in 10 mmol L−1 NaP buffer at pH 6.0. The capture of IgG by the P‐Tyr‐agarose was also promising, since 91% of the IgG was adsorbed when plasma was diluted in 25 mmol L−1 MES buffer at pH 5.5, recommending its use for IgG depletion from human plasma under this condition. The experimental data on IgG adsorption kinetics were in agreement with the pseudo‐second‐order model. The adsorption isotherm data were well described by the Langmuir–Freundlich model with the value of parameter n being <1 (0.72), indicating negative cooperativity. Selectivity was achieved on P‐Tyr‐agarose from digested human IgG in HEPES 25 mmol L−1 buffer at pH 7.0 where Fab fragments were obtained in eluted fractions without Fc fragments (but with uncleaved IgG) with 86.2% recovery. [ABSTRACT FROM AUTHOR]

Highlights • The RI sensitivity of the GO/silver SPR sensor reaches 3311.47 nm/RIU and 436.11 nm/RIU higher than silver film SPR sensor. • A high sensitivity of 0.4985 nm/(μg/mL) is reached for protein A and goat anti-human IgG modified on the sensor region. • A low limit of detection of human-IgG reaches 40 ng/mL. • The biosensor has advantages of high sensitivity, low limit of detection, inexpensive and simpler fabrication. Abstract A graphene oxide/silver coated polymer cladding silica fiber optical surface plasmon resonance (SPR) human IgG detection biosensor with a high sensitivity and a low limit of detection is presented. Due to the graphene oxide (GO), the intensity of the confined electric field surrounding the sensing layer can be enhanced, the sensitivity of refractive index (RI) of the graphene oxide/silver SPR sensor reached 3311 nm/RIU in the RI range of 1.3334–1.3731 compared with 2875 nm/RIU in the range of 1.3328–1.3739 of the silver film SPR sensor. Afterwards, SPR optical fiber biosensor was developed based on graphene oxide/silver coated optical fiber when staphylococcal protein A and goat anti-human IgG were immobilized on the surface of the graphene oxide film. The sensor can be used to detect different concentrations of human IgG, a high sensitivity of 0.4985 nm/(μg/mL) and a low limit of detection 0.04 μg/mL were observed. Therefore, the graphene oxide/silver film SPR human IgG detection biosensor with high sensitivity and low limit of detection shows a great promise for the future biochemical and biomedical application. [ABSTRACT FROM AUTHOR]

A magnetoelastic (ME) biosensor for wireless detection of analytes in liquid is described. The ME biosensor was tested against human IgG in the range 0–20 μg∙mL−1. The sensing elements, anti-human IgG produced in goat, were immobilized on the surface of the sensor by using a recently introduced photochemical immobilization technique (PIT), whereas a new amplification protocol exploiting gold coated magnetic nanoparticles (core-shell nanoparticles) is demonstrated to significantly enhance the sensitivity. The gold nanoflowers grown on the magnetic core allowed us to tether anti-human IgG to the nanoparticles to exploit the sandwich detection scheme. The experimental results show that the 6 mm × 1 mm × 30 μm ME biosensor with an amplification protocol that uses magnetic nanoparticles has a limit of detection (LOD) lower than 1 nM, works well in water, and has a rapid response time of few minutes. Therefore, the ME biosensor is very promising for real-time wireless detection of pathogens in liquids and for real life diagnostic purpose.

Acetaldehyde is a reactive aldehyde produced as an intermediate of alcohol metabolism and tobacco pyrolysis. It has the potential to interact with different biomolecules in various tissues which results in the formation of stable, unstable and covalent adducts. This causes structural and functional modifications that may lead to severe complications such as cancer. This study has probed the structural modifications in human immunoglobulin G (IgG) as a function of different concentrations of acetaldehyde in the presence of reducing agent, sodium borohydride. Acetaldehyde mediated modifications in IgG have been characterised by various physicochemical techniques. UV-spectrophotometry showed that acetaldehyde modified IgG exhibited marked increase in hyperchromicity. Fluorescence studies revealed a significant quenching of tryptophan fluorescence which resulted in loss of β-sheet secondary structure that was confirmed by circular dichroic analysis. Gross structural changes in the morphology of IgG were confirmed by increase in mass and hydrodynamic radius of this glycoprotein along with the appearance of fibrillar structures in modified IgG, when compared to the granular structure of the native form of IgG observed by scanning electron microscope. The results indicate that acetaldehyde causes alterations in the secondary and tertiary structure of the protein leading to diminution of normal function of IgG molecule. [ABSTRACT FROM AUTHOR]

The use of highly purified immunoglobulin became among the most powerful adopted strategies in therapeutic trials nowadays. Their role as immunomodulatory and anti-inflammatory agents has widened their scope of use. A novel continuous supermacroporous monolithic cryogels embedded with histidine-epoxy-activated-sepharose beads were synthetized as a new monolithic adsorbents for the separation of immunoglobulin G from human serum. The histidine-epoxy-activated-sepharose beads were embedded into the 2-hydroxyethyl methacrylate (HEMA) cryogels present in frozen aqueous solution inside a plastic syringe. The microstructure morphology of the cryogels was characterized by swelling measurement and scanning electron microscopy. The adsorption of human IgG on the histidine-epoxy-activated-sepharose beads pHEMA cryogels appeared to follow the Langmuir–Freundlich adsorption isotherm model. The maximum IgG adsorption was observed at 4°C and pH 7.4 and was found to be 26.95 mg/g of cryogel which is close to that obtained experimentally (24.49 mg/g). The cryogels were used for several adsorption-desorption cycles without any negligible decrease in their adsorption capacity. [ABSTRACT FROM PUBLISHER]

We have observed the unique enhancement of fluorescence signal of surface plasmon coupled emission (SPCE) by graphene oxide (GO) on gold film. Assembling ultrathin GO by electrostatic adsorption between gold film and fluorophore layer (about 30 nm), the enhancement of fluorescence signal of SPCE reached 7 and 25 times respectively as compared to that of the regular SPCE and free space emission (FSE) without GO. The enhancement factors were studied through modifying different concentrations of GO and thicknesses of fluorophore layer, and the possible enhancement mechanisms were investigated. This GO-assisted SPCE was designed as an immunosensor to detect human IgG with a detection limit of 0.006 ng/mL, which was much lower than the detection limit of immunosensor based on regular SPCE. This strategy combing GO and SPCE to enhance the fluorescence signal provides a simple way to improve detection sensitivity in fluorescence-based sensing platforms. [ABSTRACT FROM AUTHOR]

The strategy of bifunctional electrochemistry platform based on heterogeneous composite ZnO@ZIF-8 and ionic liquid (IL) composite film for nitrite (NO 2 − ) and immunoassay human IgG biosensors was proposed. On the one hand, the electrocatalytic ability of myoglobin (Mb) modified electrode (ZnO@ZIF-8/IL/Mb-CPE) to NO 2 − was studied, and a linear response from 10 to 833 μM with a detection limit of 3.5 μΜ was achieved. On the other, a label-free immunosensor for the determination of human IgG was proposed using ZnO@ZIF-8/IL composite film as immobilization matrix. The differential pulse voltammetry (DPV) response of the developed immunosensor was found to be proportional to logarithm of human IgG concentrations in the two ranges of 0.1-10 and 10–400 ng/mL. The lower detection limit was calculated as 0.03 ng/mL. The excellent properties of ZnO@ZIF-8 were attributed to the synergistic effects of ZnO nanorods with good conductivity, biocompatibility and ZIF-8 with high porosity. Meanwhile, IL not only prevented the aggregation of ZnO@ZIF-8 nanorods, but also displayed excellent ability in facilitating the electron transfer. Both biosensors exhibited good selectivity, reproducibility and stability, indicating ZnO@ZIF-8 composite film could be a promising matrix in electrochemical biosensor design. [ABSTRACT FROM AUTHOR]

Humanized mouse models present an important tool for preclinical evaluation of new vaccines and therapeutics. Here we show the human variable repertoire of antibody sequences cloned from a previously described human immune system (HIS) mouse model that possesses functional human CD4+ T cells and B cells, namely HIS-CD4/B mice. We sequenced variable IgG genes from single memory B-cell and plasma-cell sorted from splenocytes or whole blood lymphocytes of HIS-CD4/B mice that were vaccinated with a human plasmodial antigen, a recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP). We demonstrate that rPfCSP immunization triggers a diverse B-cell IgG repertoire composed of various human VH family genes and distinct V(D)J recombinations that constitute diverse CDR3 sequences similar to humans, although low hypermutated sequences were generated. These results demonstrate the substantial genetic diversity of responding human B cells of HIS-CD4/B mice and their capacity to mount human IgG class-switched antibody response upon vaccination. [ABSTRACT FROM AUTHOR]

Rapid diagnostic tests can be developed using ELISA for detection of diseases in emergency conditions. Conventional ELISA takes 1–2 days, making it unsuitable for rapid diagnostics. Here, we report the effect of reagents mixing via shaking or vortexing on the assay timing of ELISA. A 48-min protocol of ELISA involving 12-min incubations with reagent mixing at 750 rpm for every step was optimized. Contrary to this, time-optimized control ELISA performed without mixing produced similar results in 8 h, leaving a time gain of 7 h using the developed protocol. Collectively, the findings suggest the development of ELISA-based rapid diagnostics. [ABSTRACT FROM AUTHOR]

Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. Results: In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Conclusion: Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases.

Abstract Interaction forces between biological molecules such as antigen and antibody play important roles in many biological processes, but probing these forces remains technically challenging. Here, we investigated the specific interaction and unbinding forces between human IgG and rat anti-human IgG using self assembled monolayer (SAM) method for sample preparation and atomic force microscopy (AFM) for interaction force measurement. The specific interaction force between human IgG and rat anti-human IgG was found to be 0.6–1.0 nN, and the force required for unbinding a single pair of human IgG and rat anti-human IgG was calculated to be 144 ± 11 pN. The results are consistent with those reported in the literatures. Therefore, SAM for sample preparation combined with AFM for interaction measurement is a relatively simple, sensitive and reliable technique to probe specific interactions between biological molecules such as antigen and antibody.

The binding of protein to carboxylated single-walled carbon nanotubes (SWCNTs) was believed to play an important role in the biological effects of nanotubes. However, the effects of protein–SWCNTs interactions on the oxidative degradation of nanotubes were not stressed enough. Here, we investigated the binding of human immunoglobulin G (IgG) to SWCNTs, and found that the preferred binding site was located in the F c region of IgG. The hydrophobic and electrostatic interactions might be the crucial factors in stabilizing the binding of SWCNTs with IgG. Through the competitive binding of IgG and myeloperoxidase (MPO) to nanotube surfaces, the binding of IgG could impair MPO-induced SWCNTs biodegradation in vitro. However, both SWCNTs and IgG-SWCNTs were significantly degraded in zymosan-stimulated neutrophils, and the degradation degree was more for IgG-SWCNTs. These results suggest that the binding of IgG may be an important determinant for MPO-mediated SWCNTs biodegradation in activated human inflammatory cells. [ABSTRACT FROM AUTHOR]

A novel electrochemical immunosensor based on a magnetic glassy carbon electrode ( MGCE) was developed for the quantitative determination of human immunoglobulin G ( IgG). The immunosensing interface was fabricated by initially depositing silver nanoparticles on the MGCE surface and then immobilizing anti-human IgG antibodies via the magnetic force between MGCE and Fe3O4 nanoparticles. The antibodies were covalently bonded to the amine-functionalized Fe3O4 nanoparticles. Under optimal conditions, the magnetism-assisted immunosensor exhibited a wide linear range from 0.1 pg/ mL to 1.0 µg/ mL with the detection limit of 0.05 pg/ mL. Furthermore, the immunosensor displayed the advantages of good reproducibility and satisfactory stability. [ABSTRACT FROM AUTHOR]

Improving the performance of conventional enzyme-linked immunosorbent assay (ELISA) is of great importance to meet the demand of early clinical diagnosis of various diseases. Herein, we report a feasible enzyme-free immunosorbent assay (EFISA) system using antibody conjugated Au-Pt/SiO 2 nanocomposites (APS NCs) as labels. In this system, Au-Pt/SiO 2 nanospheres (APS NPs) were first synthesized by wet chemical method and exhibited intrinsic peroxidase and catalase-like activity with excellent water-solubility. Then APS NCs were utilized as labels to replace HRP conjugated antibody, and Fe 3 O 4 magnetic beads (MBs) to entrap the analyte. To discuss the performance of EFISA system, Human IgG was served as a model analyte, and porcine circovirus type 2 (PCV2) serums as real samples. The system boosted the detection limit of HIgG to 75 pg mL −1 with a RSD below 5%, a 264-fold improvement as compared with conventional ELISA. This is the first time that APS NCs have been used and successfully optimized for the sensitive dilution detection of PCV2 antibody (5:10 7 ) in ELISA. Besides, APS NCs have advantages related to low cost, easy preparation, good stability and tunable catalytic activity, which make them a potent enzyme mimetic candidate and may find potential applications in bioassays and clinical diagnostics. [ABSTRACT FROM AUTHOR]

IgG is an important defence protein. To exhibit optimum function the molecule must maintain its native structure. Peroxynitrite is a potent oxidizing and nitrating agent produced in vivo under pathophysiological conditions. It can oxidize and/or nitrate various amino acids causing changes in the structure and function of proteins. Such proteins may be involved in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis. In the present work, peroxynitrite-induced structural changes in IgG have been studied by UV–visible, fluorescence, CD, FT-IR, DLS spectroscopy and DSC as well as by SDS–PAGE. Peroxynitrite-modified IgG exhibited hyperchromicity at 280 nm, quenching of tryptophan fluorescence, increase in ANS fluorescence, loss of β-sheet, shift in the positions of amide I and amide II bands, appearance of new peak in FT-IR, attachment of nitro residues and increase in melting temperature, compared to native IgG. Furthermore, peroxynitrite-modified IgG exhibited an additional peak at 420 nm, quenching in tyrosine fluorescence and enhancement in dityrosine fluorescence compared to native IgG. Generation of nitrotyrosine, dityrosine and nitrotryptophan was also observed in peroxynitrite-modified IgG. Gross structural changes in IgG caused by peroxynitrite and observed in vitro may favour autoantibodies induction in vivo under similar conditions. [ABSTRACT FROM AUTHOR]

A sensitive metalloimmunoassay based on copper nanoparticle labeling was demonstrated with atomic absorption spectrometry. Stable nanosized CuO particles of 5–10 nm diameter were synthesized by an alcothermal method, and conjugated to antibody under moderate conditions. The immunoassay conditions were optimized. After immunoreaction, the copper atoms were released with diluted nitric acid and measured by electro-thermal atomic absorption spectrometry. The atomic absorbance signal had a good linearity with logarithm of the C IgG (concentration of IgG, g/mL). The detection limit was calculated to be 0.19 ng/mL and the assay was linear in the range of 10 − 9 –10 − 5 g/mL human IgG. The relative standard deviation (RSD) for 10 ng/mL human IgG was 3.0%. The human IgG results obtained by the proposed method correlated well with those obtained by inductively coupled plasma-based immunoassay. [ABSTRACT FROM AUTHOR]

The immobilized metal-affinity chromatography (IMAC) has gained significant interest as a widespread separation and purification tool for therapeutic proteins, nucleic acids and other biological molecules. The enormous potential of IMAC for proteins with natural surface exposed-histidine residues and for recombinant proteins with histidine clusters. Cryogels as monolithic materials have recently been proposed as promising chromatographic adsorbents for the separation of biomolecules in downstream processing. In the present study, IMAC cryogels have been synthesized and utilized for the adsorption and separation of immunoglobulin G (IgG) from IgG solution and whole human plasma. For this purpose, Cu(II)-ions were coupled to poly(hydroxyethyl methacrylate) PHEMA using poly(ethylene imine) (PEI) as the chelating ligand. In this study the cryogels formation optimized by the varied proportion of PEI from 1% to 15% along with different amounts of Cu (II) as chelating metal. The prepared cryogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The [PHEMA/PEI]–Cu(II) cryogels were assayed for their capability to bind the human IgG from aqueous solutions. The IMAC cryogels were found to have high affinity toward human IgG. The adsorption of human IgG was investigated onto the PHEMA/PEI cryogels with (10% PEI) and the concentration of Cu (II) varied as 10, 50, 100 and 150 mg/L. The separation of human IgG was achieved in one purification step at pH 7.4. The maximum adsorption capacity was observed at the [PHEMA/PEI]–Cu(II) (10% PEI) with 72.28 mg/g of human IgG. The purification efficiency and human IgG purity were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). [ABSTRACT FROM AUTHOR]

We report a new method for detecting human IgG (hIgG) in serum on integrated-optical Mach-Zehnder interferometer biosensors realized in a high index contrast polymer material system. In the linear range of the sensor (5-200 nM) we observed excellent signal recoveries (95-110%) in buffer and serum samples, which indicate the absence of matrix effects. Signal enhancement was reached by using secondary anti-human IgG antibodies, which bind to immobilized target IgGs and allow detecting concentrations down to 100 pM. This polymer based optical sensor is fully compatible with cost-efficient mass production technologies, which makes it an attractive alternative to inorganic optical sensors. [ABSTRACT FROM AUTHOR]

A naked-eye sensitive ELISA-like assay was developed based on gold-enhanced peroxidase-like activity of gold nanoparticles (AuNPs). Using human IgG (H-IgG) as an analytical model, goat anti-human IgG antibody (anti-IgG) adsorbed on microtiter plate and AuNPs-labeled anti-IgG acted as capture antibody and detection antibody, respectively. Because the surfaces of AuNPs were blocked by protein molecules, the peroxidase-like activity of AuNPs was almost inhibited, evaluated by the catalytic oxidation of peroxidase enzyme substrate 3,3′,5,5′-tetramethylbenzidine (TMB), which could produce a bright blue color in the presence of HO. Fortunately, the catalytic ability of AuNPs was dramatically increased by the deposition of gold due to the formation of a new gold shell on immunogold. Under optimal reaction conditions, the colorimetric immunoassay presented a good linear relationship in the range of 0.7-100 ng/mL and the limit of detection (LOD) of 0.3 ng/mL calculated by 3σ/S for UV-vis detection, and obtained LOD of 5 ng/mL for naked-eye detection. The obtained results were competitive with conventional sandwich ELISA with the LOD of 1.6 ng/mL. Furthermore, this developed colorimetric immunoassay was successfully applied to diluted human serum and fetal bovine serum samples, and predicted a broad prospect for the use of peroxidase-like activity involving nanomaterials in bioassay and diagnostics. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

A polydimethylsiloxane (PDMS) microfluidic chemiluminescence (CL) immunodevice for sensitive detection of human immunoglobin G (IgG) with the signal amplification strategy was developed in this work. The immunodevice was prepared by covalently immobilizing capture antibodies (Abs) on the silanized microchannel of microfluidic chip. Gold nanoparticles (AuNPs) functionalized with a high molar ratio of horseradish peroxidase (HRP) were used as an Ab label for signal amplification. Using a sandwich immunoassay, the multi-HRP conjugated AuNPs can catalyze the luminol-H 2 O 2 CL system to achieve the high sensitivity. In addition, the double spiral flow-channel was adopted here, which can still contribute to the high sensitivity. Based on signal amplification strategy, the performance of human IgG tests revealed a lower detection limit (DL) of 0.03 ng/mL and showed an increase of 7.4-fold in detection sensitivity compared to a commercial Ab-HRP conjugation. This microfluidic immunodevice can provide an alternative approach for sensitive detection of human IgG in the field of clinic diagnostic and therapeutic. [ABSTRACT FROM AUTHOR]

Herein, we describe an enzyme-linked immunosorbent assay (ELISA) technique carried out in a polypropylene microcentrifuge tube preactivated by a photolinker, 1-fluoro-2-nitro-4-azidobenzene (FNAB) by a photochemical reaction. Immobilization of capture molecule (antibody) and BSA- blocking are carried out in the tube by ultrasound waves in a sonicator bath in 10 min for each step. Around one drop (100 μl) of blood is directly collected into the tube. By the time, the sample reaches laboratory (nearly 1 h), the analyte present in blood binds to the capture molecule already present in the tube. That means only one step (antibody- conjugate binding step) has to be carried out requiring only 10 min by ultrasound waves. The advantage of the method is that sample collection and testing can be done in the same tube and more importantly without the need of getting serum by a time consuming process. Also, time is saved for Ag-Ab binding step making the procedure more rapid. [ABSTRACT FROM AUTHOR]

We describe the preparation and properties of mixed valence manganite nanoparticles (NPs) of the type LaSrMnO for use in electrochemical immunoassays. The NPs were synthesized using the reverse micelle method and their surface was then functionalized with silica nanoparticles. The resulting NPs (LaSrMnO@SiO) were characterized by X-ray diffraction, scanning electron microscopy and physical property measurements. Their typical diameter is ~200 nm, and the magnetic saturation occurs at 75 emu⋅g‾, and both coercive force and hysteresis loop remain at almost zero. The silica NPs immobilized on the LaSrMnO NPs have a diameter of ~20 nm. The LaSrMnO@SiO NPs were used to develop an electrochemical immunoassay for human IgG. Antibody (anti-human IgG) was immobilized on the silica NPs on the magnetic core, and this process was monitored by cyclic voltammetry and square-wave voltammetry. The formation of the immunocomplex (Ab/Ag) on the surface of the electrode was monitored using hexacyanoferrate redox system as the redox marker. The assay has a linear working range up to an IgG concentration of 5 ng⋅mL‾, and the detection limit is 0.6 ng⋅mL‾. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

A novel electrochemical immunoassay was developed based on the signal amplification strategy of silver deposition directed by gold nanorods (AuNRs), which was in-situ assembled on the sandwich immunocomplex. The superstructure formed by the self-assembly of AuNRs provided abundant active sites for the nucleation of silver nanoparticles. In this pathway, the stripping current of silver was greatly enhanced. Using human immunoglobulin G (HIgG) as a model analyte, the ultrasensitive immunoassay showed a wide linear range of six orders of magnitude from 0.1 fg mL −1 to 100 pg mL −1 , with the low detection limit down to 0.08 fg mL −1 . The practicality of this electrochemical immunoassay for detection of HIgG in serum was validated with the average recovery of 93.9%. In addition, this enzyme-free immunoassay also has the advantages of acceptable reproducibility and specificity, and thus this immunosensing protocol can be extended to the detection of other low-abundant protein biomarkers. [ABSTRACT FROM AUTHOR]

In this study, we developed human immune system (HIS) mice that possess functional human CD4 + T cells and B cells, named HIS-CD4/B mice. HIS-CD4/B mice were generated by first introducing HLA class II genes, including DR1 and DR4, along with genes encoding various human cytokines and human B cell activation factor (BAFF) to NSG mice by adeno-associated virus serotype 9 (AAV9) vectors, followed by engrafting human hematopoietic stem cells (HSCs). HIS-CD4/B mice, in which the reconstitution of human CD4 + T and B cells resembles to that of humans, produced a significant level of human IgG against Plasmodium falciparum circumsporozoite (PfCS) protein upon immunization. CD4 + T cells in HIS-CD4/B mice, which possess central and effector memory phenotypes like those in humans, are functional, since PfCS protein-specific human CD4 + T cells secreting IFN-γ and IL-2 were detected in immunized HIS-CD4/B mice. Lastly, PfCS protein-immunized HIS-CD4/B mice were protected from in vivo challenge with transgenic P. berghei sporozoites expressing the PfCS protein. The immune sera collected from protected HIS-CD4/B mice reacted against transgenic P. berghei sporozoites expressing the PfCS protein and also inhibited the parasite invasion into hepatocytes in vitro. Taken together, these studies show that our HIS-CD4/B mice could mount protective human anti-malaria immunity, consisting of human IgG and human CD4 + T cell responses both specific for a human malaria antigen. [ABSTRACT FROM AUTHOR]

To achieve the potent therapeutic effects of human immunoglobulin G (IgG), highly concentrated formulations are required. However, the stabilization for highly concentrated human IgG is laborious work. In the present study, to investigate the potentials of polypseudorotaxane (PPRX) hydrogels consisting of polyethylene glycol (PEG) and α- or γ-cyclodextrin (α- or γ-CyD) as pharmaceutical materials for highly concentrated human IgG, we designed the PPRX hydrogels including human IgG and evaluated their pharmaceutical properties. The α- and γ-CyDs formed PPRX hydrogels with PEG (M.W. 20,000) even in the presence of highly concentrated human IgG (>100 mg/mL). According to the results of H-NMR, powder X-ray diffraction, and Raman microscopy, the formation of human IgG/CyD PPRX hydrogels was based on physical cross-linking arising from their columnar structures. The release profiles of human IgG from the hydrogels were in accordance with the non-Fickian diffusion model. Importantly, the stabilities of human IgG included into the hydrogels against thermal and shaking stresses were markedly improved. These findings suggest that PEG/CyD PPRX hydrogels are useful to prepare the formulation for highly concentrated human IgG. [ABSTRACT FROM AUTHOR]

We developed new measurement configurations based on organic electrochemical transistors (OECTs). Three types of COOH-functionalized bioreceptor layers were deposited on indium tin oxide (ITO) electrodes on poly(ethylene terephthalate) (PET) substrates and their performance was tested using single gate functionalization organic electrochemical transistor (S-OECT) and dual gate functionalization organic electrochemical transistor (D-OECT) configurations. The three layers included one p-type semiconductor, one insulator, and one self-assembled layer, and the dual gates were connected in series through buffer solutions, so the solution-electrode interfaces had the opposite polarities. We investigated the sensitivities of these systems using the human IgG antigen-human IgG antibody receptor pair for main experiments, and drifts of antibody-functionalized gates without analytes as control experiments. Drifts without analyte can obscure the real sensitivity. We show that the D-OECT has the capability to cancel the drifts, and is also beneficial for showing the sensitivity more exactly. This configuration has the ability to increase the accuracy of antibody-antigen interaction detection, and further decrease or eliminate the effect of ions in the buffer solution. We also prove that the D-OECT can work well with different bioreceptor materials, which indicates that the system can be further applied to different conditions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Many pathological conditions are accompanied with changes in the concentration of the total IgG or some of its fraction. For this reason there is great interest in the production of reagents specific for IgG. In this paper, the binding characteristics of two new murine monoclonal antibodies (MoAb), assigned MoAb 15 and MoAb 22, are reported. These MoAbs were produced by hybridoma technology. By performing ELISAs and Western blots analyzes, it was demonstrated that both MoAbs interact specifically with human IgG. Cross reactivity with other sera proteins was not observed. In order to precisely localize the epitopes recognized by MoAb 15 and MoAb 22, the Western blots interactions of these MoAbs with electrophoreticaly separated IgG-fragments, obtained by the action of proteolytic enzymes (papain, pepsin, trypsin), were analyzed. According to the results of these experiments, both MoAbs interacted with epitopes in the Cg3 domain. The affinity constants, calculated from Scatchard plots of binding of MoAb15 and MoAb22 to human IgG, were Ka15 = 1.71×106 M-1 and Ka22 = 2.15×109 M-1. According to all these findings, MoAb 15 and MoAb 22 could be used in standard immunochemical techniques. However, the experiments showed that both MoAbs had bad immunoprecipitating properties. In solid phase techniques (ELISAs, Western blot, dot-blot, etc.), their application gave excellent results that highly recommended them for use in these types of analyzes.

Many pathological conditions are accompanied with changes in the concentration of the total IgG or some of its fraction. For this reason there is great interest in the production of reagents specific for IgG. In this paper, the binding characteristics of two new murine monoclonal antibodies (MoAb), assigned MoAb 15 and MoAb 22, are reported. These MoAbs were produced by hybridoma technology. By performing ELISAs and Western blots analyzes, it was demonstrated that both MoAbs interact specifically with human IgG. Cross reactivity with other sera proteins was not observed. In order to precisely localize the epitopes recognized by MoAb 15 and MoAb 22, the Western blots interactions of these MoAbs with electrophoreticaly separated IgG-fragments, obtained by the action of proteolytic enzymes (papain, pepsin, trypsin), were analyzed. According to the results of these experiments, both MoAbs interacted with epitopes in the C 3 domain. The affinity constants, calculated from Scatchard plots of binding ofMoAb15 and MoAb22 to human IgG, wereKa15 = 1.71 106M-1 andKa22 = 2.15 109M-1. According to all these findings,MoAb 15 and MoAb 22 could be used in standard immunochemical techniques. However, the experiments showed that both MoAbs had bad immunoprecipitating properties. In solid phase techniques (ELISAs, Western blot, dot-blot, etc.), their application gave excellent results that highly recommended them for use in these types of analyzes.

Study of biophysical interactions have been carried out using specific combination of proteins such as human IgG (as antigen) and anti-human IgG (as complementary antibody; raised in rabbit). Bovine serum albumin (BSA) was used to block any nonspecific interaction between antigen and antibody as BSA has been reported to bind to several sites non-specifically. Optimization of BSA concentration was done in order to make the antigen-antibody interaction relatively more pronounced and specific. We have used gold electrode in order to provide a relatively inert platform for adsorption/immobilization of protein molecules. The interaction between the antigen and antibody caused an increase in the charge transfer resistance (parallel resistance in Randles cell model) for an indicator molecule (hexacyanoferrate) and this was monitored by impedance spectroscopy. Detection limit for the antigen was found to be about 50 ng/mL. The approach presented is general and versatile and can be used for any antigen-antibody pair without any significant modification. [ABSTRACT FROM AUTHOR]

In this work, a novel sulfur-containing compound with aldehyde groups was synthesized, which was used to immobilize antibodies through the covalent bonding of the aldehyde groups with amino groups, in which no additional chemical cross-linker is required. Human IgG was used as a model analyte to fabricate the electrochemical immunosensor. Using the proposed immunosensor, IgG was detected within the range from 0.01 to 25 ng mL −1 with a detection limit of 0.003 ng mL −1 obtained by 3 S/N. The simple electrochemical immunosensor had a good specificity, stability and reproducibility. [ABSTRACT FROM AUTHOR]

Immunoglobulins are proteins produced by the immune system, which bind specifically to the antigen that induced their formation and target it for destruction. Highly purified human immunoglobulins are commonly used in research laboratories for several applications, such as in vitro to obtain hybridomas and in vivo animal immunisation. Several affinity purification methods are used to purify immunoglobulins from human serum, such as protein A/G Sepharose beads, polyethylene glycol, and caprylic acid ammonium sulphate precipitation. Here, we provide a detailed protocol for purification of high-quality IgG from human serum, using affinity chromatography with protein G. The protocol is divided into four main steps (column preparation, serum running, wash, and elution) for IgG purification, and two extra steps (protein dialysis and sucrose concentration) that should be performed when buffer exchange and protein concentration are required. Several IgG affinity purification methods using protein A or G are available in the literature, but protein A has a higher affinity for rabbit, pig, dog, and cat IgG, while protein G has a higher affinity for mouse and human IgG. This affinity-based purification protocol uses protein G for a highly specific purification of human IgG for animal immunization, and it is particularly useful to purify large amounts of human IgG. This protocol was validated in: Pain (2019), DOI: 10.1097/j.pain.0000000000001662 Graphical abstract IgG purification protocol. The IgG purification protocol consists of four main steps (column preparation, serum running, wash, and elution) and two extra steps (protein dialysis and concentration). a. Diluted serum is added to the protein G beads and IgG binds to the Fc receptors on protein G beads. b. Beads are washed in Hartman's solution to fully remove the complex protein mixture (multicolour shapes, as depicted in the graphical abstract). c. IgG (orange triangles, as depicted in the graphical abstract) are removed from protein G with glycine and collected in Tris buffer. d. The IgG is transferred into a semi-permeable membrane ('snake skin') and allowed to dialyse overnight for buffer exchange with a physiological solution (Hartmann's)., Competing Interests: Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

• This sensor detects IgG at pg mL−1 level for the first time. • Tunable electrochemistry of Cu-MOFs for the MIP sensor is reported. • This method has good analytical performance and broad application prospects in clinical testing. 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. [ABSTRACT FROM AUTHOR]

Over the past decade, immobilized metal-affinity adsorbents have attracted increasing interest for purification of natural and recombinant immunoglobulin G (IgG). In this work, nickel and cobalt metal ions complexed with CM-Asp (carboxymethylaspartate) immobilized on poly(ethylenevinyl alcohol) (PEVA) hollow fiber membranes were evaluated for purification of human IgG from serum. The buffer system and NaCl had important effects on human serum protein adsorption in both adsorbents. Efficient purification of IgG was accomplished in sodium phosphate buffer without NaCl at pH 7.0. Under this condition, the electrostatic interactions are important for adsorption. The Ni(II)-CM-Asp-PEVA had a protein adsorption capacity of 17.5 mg of IgG mL fiber when human serum diluted was loaded in crossflow filtration mode and the eluted IgG had a purity of 82.6 % (based on total protein and IgG, IgM, HSA, and Trf nephelometric analysis). Fitting the experimental IgG adsorption data to the Langmuir and Langmuir-Freundlich models showed that Ni(II)-CM-Asp and Co(II)-CM-Asp had Langmuirean and non-Langmuirean behavior, respectively, with positive cooperativity for IgG-Co(II)-CM-Asp binding, probably due to multipoint interactions ( n = 2.12 ± 0.31). Thus, these membranes can be considered as alternative adsorbents for the purification or depletion of IgG from human serum. [ABSTRACT FROM AUTHOR]

Nonenzymatic glycosylation of proteins finally generates advanced glycation end products (AGEs). The Schiff's base and Amadori adduct are stages of early glycation. AGE-modified IgG may undergo conformational alterations and the final entity of the process may be involved in the pathogenesis of Rheumatoid Arthritis (RA). In this study, glycation of human IgG was carried out with varying concentrations of glucose. Effect of incubation period on glycation of IgG has also been studied. Amadori adduct was detected by nitroblue tetrazolium (NBT) dye. The glucose mediated structural alterations in IgG were studied by UV, fluorescence, CD, FT-IR, DLS and DSC spectroscopy, and SDS-PAGE. Glycation-induced aggregation in AGE-IgG was reported in the form of binding of thioflavin T and congo red. Furthermore, AGE-modified IgG exhibited hyperchromicity, decrease of tryptophan fluorescence accompanied by increase in AGE specific fluorescence, loss of β-sheet, appearance of new peak in FT-IR, increase in hydrodynamic size and melting temperature. SDS-PAGE results showed decrease in the band intensity of glycosylated-IgG compared to native IgG. Glycation-induced modifications and aggregation of IgG might be important in the pathogenesis of RA. [ABSTRACT FROM AUTHOR]