Monoclonal Capture Research Articles

Graphene Oxide Signal Reporter Based Multifunctional Immunosensing Platform for Amperometric Profiling of Multiple Cytokines in Serum.

Cytokines are small proteins and form complicated cytokine networks to report the status of our health. Thus, accurate profiling and sensitive quantification of multiple cytokines is essential to have a comprehensive and accurate understanding of the complex physiological and pathological conditions in the body. In this study, we demonstrated a robust electrochemical immunosensor for the simultaneous detection of three cytokines IL-6, IL-1β, and TNF-α. First, graphene oxides (GO) were loaded with redox probes nile blue (NB), methyl blue (MB), and ferrocene (Fc), followed by covalent attachment of anti-cytokine antibodies for IL-6, IL-1β, and TNF-α, respectively, to obtain Ab2-GO-NB, Ab2-GO-MB, and Ab2-GO-Fc, acting as the signal reporters. The sensing interface was fabricated by attachment of mixed layers of 4-carboxylic phenyl and 4-aminophenyl phosphorylcholine (PPC) to glassy carbon surfaces. After that, the capture monoclonal antibody for IL-6, IL-1β, and TNF-α was modified to the carboxylic acid terminated sensing interface. And finally a sandwich assay was developed. The quantitative detection of three cytokines was achieved by observing the change in electrochemical signal from signal reporters Ab2-GO-NB, Ab2-GO-MB, and Ab2-GO-Fc. The designed system has been successfully used for detection of three cytokines (IL-6, IL-1β, and TNF-α) simultaneously with desirable performance in sensitivity, selectivity, and stability, and recovery of 93.6%-105.5% was achieved for determining cytokines spiked in the whole mouse serum.

An Electrochemical Immunosensor for the Detection of B-Type Natriuretic Peptide (BNP) Based on Sandwich ELISA Using Screen Printed Carbon Electrodes

B-type natriuretic peptide (BNP) is a 32-amino acid polypeptide, a cardiac neuro hormone that specifically secreted from heart ventricle as a response towards the increase of volume and pressure in the heart. The determination of BNP concentration in patients blood is one of the method used to diagnose heart failure. An electrochemical immunosensor modified with the streptavidin/biotin system on screen printed carbon electrodes for the detection of the BNP antigen was developed in this study. Monoclonal anti-BNP capture antibody was immobilized on streptavidin-modified SPCEs to give a well oriented of antibody. Furthermore, a biotinylated anti-BNP that conjugated with horseradish peroxidase (HRP) was used as secondary antibody. The electrochemical signal produced by redox activity of substrate 3,3,5,5'-tetramethybezidine dihydrochloride (TMB/H2O2) was measured by differential pulse voltammetry. The BNP immunosensor showed a linear response between 1.0×10-2 and 1.0×102 ng/mL, and the limit of detection was 3.3 ng/mL. BNP immunosensor is a promising technology for the rapid and convenient detection of heart failure.

Improvement of monoclonal antibody–immobilized granulocyte antigen assay for the detection of anti‐HNA‐1 alloantibodies

Currently, the gold standard for the identification of antibodies against human neutrophil antigens (HNAs) is the monoclonal antibody-immobilized granulocyte antigen (MAIGA) assay. However, the handling of this assay is laborious and therefore cumbersome for the rapid screening of neutrophil antibodies. In this study, we simplified the performance of the conventional MAIGA procedure and approved it for the identification of anti-HNA-1 with HNA-1-typed neutrophils and stable transfected (HEK293) cell line expressing HNA-1a, -1b, and -1c. In contrast to the conventional MAIGA, all working steps including the incubation of antibodies with cells, washings, cell lysis, and subsequent antibody detection could be performed on microtiter plates. This modification accelerates the work schedule of MAIGA and reduces pipetting errors. Comparison between both assay performances using neutrophils as target showed concordant results. Subsequently, stable mammalian cell lines were tested. In comparison to neutrophils, cell lines were stable for a longer period of time (>4 weeks) and results obtained with these cell lines showed better interassay precision. Analysis of different FcγRIIIb capture monoclonal antibodies (MoAbs) for the MAIGA assay showed that MoAb LNK16 is superior for the detection of anti-HNA-1a, -1b, and -1c, whereas MoAb 3G8 showed false-negative results, caused by competitive inhibition of anti-HNA-1c alloantibodies. The modification of MAIGA and the use of transfected HEK293 cells improve the detection of anti-HNA-1 alloantibodies that may allow screening analysis in large cohort of samples.

Integration of Aqueous Two-Phase Extraction as Cell Harvest and Capture Operation in the Manufacturing Process of Monoclonal Antibodies.

Substantial improvements have been made to cell culturing processes (e.g., higher product titer) in recent years by raising cell densities and optimizing cultivation time. However, this has been accompanied by an increase in product-related impurities and therefore greater challenges in subsequent clarification and capture operations. Considering the paradigm shift towards the design of continuously operating dedicated plants at smaller scales—with or without disposable technology—for treating smaller patient populations due to new indications or personalized medicine approaches, the rising need for new, innovative strategies for both clarification and capture technology becomes evident. Aqueous two-phase extraction (ATPE) is now considered to be a feasible unit operation, e.g., for the capture of monoclonal antibodies or recombinant proteins. However, most of the published work so far investigates the applicability of ATPE in antibody-manufacturing processes at the lab-scale and for the most part, only during the capture step. This work shows the integration of ATPE as a combined harvest and capture step into a downstream process. Additionally, a model is applied that allows early prediction of settler dimensions with high prediction accuracy. Finally, a reliable process development concept, which guides through the necessary steps, starting from the definition of the separation task to the final stages of integration and scale-up, is presented.

A regenerable microfluidic device with integrated valves and thin-film photodiodes for rapid optimization of chromatography conditions

The optimization of chromatography operating conditions is a lengthy and demanding task that typically requires large volumes of reagents and costly dedicated equipment. In particular, the use of multimodal chromatography, in which multiple interaction groups co-exist in the same ligand, can make the optimization process even more challenging. Nevertheless, the interest in using this emerging type of ligands has been increasing due to the enhanced selectivity and stability demonstrated by multimodal ligands in the purification of several biopharmaceuticals and their lower cost compared to biological affinity ligands. In this work, we report the development of an integrated, regenerable and portable microfluidic platform for performing a rapid screening of chromatography conditions. This platform was tested for the capture of fluorophore-labeled monoclonal antibodies directly from a cell culture supernatant using a multimodal ligand (Capto MMC). Liquid insertion in the microfluidic device was controlled by pneumatically actuated embedded valves and chromatography cycles were monitored via fluorescence measurements using thin-film a-Si:H photodiodes aligned beneath the device. The regeneration of the chromatography micro-column (70 nL) was performed in the end of each cycle, providing repeatable results over several consecutive cycles. Different buffers were successively evaluated in cycles, each cycle having duration of about 3 min, and the different adsorption and elution kinetics were compared, allowing the rapid optimization of conditions to address the capture of the monoclonal antibody from complex media. Overall, a versatile, rapid and relatively inexpensive platform to perform screening studies was developed, providing a high degree of scalability.

Identification of a common Ara h 3 epitope recognized by both the capture and the detection monoclonal antibodies in an ELISA detection kit.

Allergy to peanuts has become a common and severe problem, especially in westernized countries. In this study, we evaluated the target and epitope specificity of the capture and detection mouse monoclonal antibodies (mAbs) used in a commercial peanut allergen detection platform. We first identified the target of these antibodies as Ara h 3 and then used an overlapping peptide array of Ara h 3 to determine the antibody-binding epitopes. Further amino acids critical for the binding via alanine substitutions at individual amino acid residues within the epitope were mapped. Finally, inhibition ELISA and inhibition immunoblotting using a recombinant Ara h 3 protein were performed to confirm these results. Surprisingly, the capture and detection mAbs showed identical binding characteristics and were presumed to represent two isolates of the same clone, a notion supported by both isoelectric focusing electrophoresis and Liquid chromatography–mass spectrometry experiments. The simultaneous binding of a pair of identical mAbs to an individual allergen such as Ara h3 is attributed to the multivalency of the analyte and has implications for developing diagnostic assays for additional multimeric allergens.

Continuous countercurrent tangential chromatography for mixed mode post-capture operations in monoclonal antibody purification

Continuous Countercurrent Tangential Chromatography (CCTC) has been shown to demonstrate significant advantages over column chromatography including higher productivity, lower operational pressure, disposable flow path, and lower resin use. Previous applications of CCTC have been limited to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest. In this present article, a CCTC system was designed and tested for a post-capture antibody purification step. Mixed mode cation exchange-hydrophobic interaction chromatography resins with two different particle sizes were used to reduce host cell protein (HCP), leached protein A, DNA, and aggregates from a mAb stream after a protein A operation. Product output from CCTC was obtained at a steady-state concentration in sharp contrast to the periodic output of product in multi-column systems. The results show up to 101g of mAb/L of resin/hr productivity, which is 10× higher than in a batch column. A 5% yield increase (95% with CCTC vs. 90% in batch column) resulted from optimizing elution pH within a narrow operational window (pH 4–4.5). Contaminant removal was found to be similar to conventional column performance. Data obtained with the smaller particle size resin showed faster binding kinetics leading to reduced CCTC system volume and increased productivity. Buffer and water usage were modeled to show potential for utilization of in-line mixing and buffer tank volume reduction. The experimental results were used to perform a scale up exercise that predicts a compact CCTC flow path for 500 and 2000L batches using commercially available membranes. These results demonstrate the potential of using CCTC for post-capture operations as an alternative to packed bed chromatography, and provide a framework for the design and development of an integrated continuous bioprocessing platform based on CCTC technology.

Polyclonal and monoclonal IgG binding on protein A resins-Evidence of competitive binding effects.

Protein A (ProA) chromatography is used extensively in the biopharmaceutical industry for the selective capture of both polyclonal and monoclonal antibodies (mAbs). This work provides a comparison of the adsorptive behavior of a highly heterogeneous polyclonal hIgG versus that of a mAb as well as the behavior of their mixtures on representative ProA resins. Both pH gradient elution and frontal loading experiments using human polyclonal IgG (hIgG) reveal a distribution of IgG-ProA binding strengths likely associated with multiple IgG subclasses and the heterogeneity of the variable region. pH gradient analysis of fractions collected along the breakthrough curve demonstrate a clear progression from weaker binding (higher pH eluting) to stronger binding (lower pH eluting) IgG species leaving the column suggesting the possibility of stronger binding species displacing the weaker binding ones. Displacement is directly observed by visualizing the adsorption of fluorescently labeled mAb and hIgG using confocal laser scanning microscopy (CLSM). Here, the displacement of hIgG results in a broad adsorption front compared to the sharp, "shrinking core" behavior typically observed with mAbs. Sequential CLSM adsorption experiments with a mAb and hIgG confirm that stronger or equivalent-binding hIgG species are able to displace and desorb bound mAb molecules. These phenomena are examined using a variety of ProA resins including CaptivA PriMAB, MabSelect, and MabSelect SuRe to understand the effect of different ligand properties on binding strength and competition among different IgG species. The results of these comparisons suggest that the competition kinetics are slower with ligands that have a single-point covalent attachment to the base matrix compared to a multi-point attachment. Biotechnol. Bioeng. 2017;114: 1803-1812. © 2017 Wiley Periodicals, Inc.

Detection and identification of platelet antibodies using a sensitive multiplex assay system-platelet antibody bead array.

Tests for platelet-specific antibodies are important in the diagnosis of immune platelet disorders. Monoclonal antibody glycoprotein capture assays have been the gold standards in platelet antibody detection for almost 30 years. However, such assays are complex and cumbersome to perform, which limits their routine use. We report the performance of a newly developed, easy to perform platelet antibody bead array (PABA) for the detection of platelet-specific antibodies. PABA is the equivalent of the monoclonal antigen capture enzyme-linked immunosorbent assay (ELISA) (MACE) on a bead and instead with fluorescent detection of immunoglobulin (Ig)G platelet antibodies by Luminex. Antibodies against platelet glycoproteins (GP) GPIIb/IIIa, GPIb/IX, GPIa/IIa, GPIV, and class I human leukocyte antigen (HLA) could be detected in a patient's serum simultaneously in a single well of a microplate. Results from 80 patient samples and 20 normal donor samples were compared using PABA, MACE, and a commercial ELISA kit. PABA detected all antibodies, with specificity for human platelet antigens (HPAs) HPA-1a, HPA-1b, HPA-2a, HPA-2b, HPA-3a, HPA-3b, HPA-4a, HPA-4b, HPA-5a, HPA-5b, HPA-15b, and HLA. Overall, compared with MACE, the sensitivity and specificity of PABA were 99% and 97%, respectively, and both were significantly better than those of the commercial ELISA. PABA had greater analytic sensitivity than MACE for HPA-1a, HPA-3a, and HPA-5b antibodies. In addition, PABA detected HPA-5b and HPA-3b antibodies that were missed by MACE. The overall false-positive rate of PABA compared with MACE was 2.7%. The PABA is a rapid, highly sensitive and specific, multiplex bead-based assay for detecting human platelet antibodies. Such a simple yet high-throughput platform will facilitate practical, routine testing for the identification of platelet-specific antibodies.

Development and Validation of Monoclonal Antibody-Based Antigen Capture ELISA for Detection of Group A Porcine Rotavirus.

Porcine rotavirus-A (PoRVA) is one of the common causes of mild to severe dehydrating diarrhea, leading to losses in weaning and postweaning piglets. A rapid, highly specific, and sensitive antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for detection of PoRVA, by using VP6 (a highly conserved and antigenic protein of group-A rotavirus)-directed rabbit polyclonal antibodies (capture antibody) and murine monoclonal antibodies (detector antibody). The detection limit of AC-ELISA was found to be equal to that of conventional reverse transcription-polymerase chain reaction (RT-PCR; about 102.5 TCID50/mL). For validation of the in-house AC-ELISA, 295 porcine fecal/diarrhea samples, collected from different provinces of China, were evaluated and compared with conventional RT-PCR and TaqMan RT-quantitative PCR (qPCR). The sensitivity and specificity of this in-house AC-ELISA relative to RT-qPCR were found to be 91.67% and 100%, respectively, with the strong agreement (kappa = 0.972) between these two techniques. Total detection rate with AC-ELISA, conventional RT-PCR, and RT-qPCR were found to be 11.2%, 11.5%, and 12.2%, respectively, without any statistical significant difference. Moreover, AC-ELISA failed to detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastroenteritis virus, pseudorabies virus, and porcine circovirus-2. These results suggested that our developed method was rapid, highly specific, and sensitive, which may help in large-scale surveillance, timely detection, and preventive control of rotavirus infection in porcine farms.

Polyion oligonucleotide-decorated gold nanoparticles with tunable surface charge density for amplified signal output of potentiometric immunosensor

Methods based on nanostructures have been developed for potentiometric immunosensors, but most involve low sensitivity or weak signal output and are unsuitable for routine use in diagnosis. Herein, we devise an in-situ signal-amplification strategy for enhanced electrical readout of potentiometric immunosensor toward target prostate-specific antigen (PSA, one kind of cancer biomarkers), based on polyion oligonucleotide-labeled gold nanoparticles (AuNPs). To decrease the background signal, monoclonal anti-human PSA capture antibody was covalently conjugated onto an activated glassy carbon electrode via typical carbodiimide coupling. AuNPs heavily functionalized with the polyion oligonucleotides and polyclonal anti-PSA detection antibodies (pAb2-AuNP-DNA) were utilized as the signal-generation nanotags. In the presence of target PSA, a sandwich-type immunoreaction was executed between capture antibody and detection antibody on the electrode. The detectable signal derived from the shift in the electric potential as a result of the change in the surface charge before and after the antigen-antibody reaction. With target PSA increased, the captured pAb2-AuNP-DNA to the electrode accompanying detection antibody increased, thereby resulting in the change of the electrode potential. Due to numerous polyion oligonucleotides with the negative charge, the signal readout amplified. Under the optimal conditions, the shift in the output potential was proportional to the logarithm of target PSA concentration and displayed a dynamic linear range from 0.05 to 20 ng mL−1 with a detection limit of 13.6 pg mL−1. An intermediate precision of ≤13.2% was accomplished with the batch-to-batch identification. The selectivity was acceptable. The method accuracy was evaluated for human serum specimens, and gave the consistent results between the potentiometric immunosensor and the referenced enzyme-linked immunosorbent assay (ELISA).

B-Type Natriuretic Peptide (BNP) Detection Using Electrochemical Immunosensor Based on Sandwich ELISA with Horseradish Peroxidase-Tetramethylbenzidine System

A study of B-type natriuretic peptide (BNP) antigen as a good prognostic marker for patients with heart failure antigen detection employing an electrochemical immunosensor is described here. Monoclonal anti-BNP capture antibody was immobilized on streptavidin-modified SPCEs to increase the sensitivity of the assay. An anti-BNP detection antibody conjugated with horseradish peroxidase enzyme (HRP) and 3,3,5,5’-tetramethybezidine dihydrochloride (TMB) was used as a substrate, in connection with voltammetric technique was achieve by measuring the peak current. Incorporation of a streptavidin/biotin system resulted in a well-oriented antibody immobilization of the capture antibody and consequently enhanced the sensitivity of the assay. Several optimizations were performed to reduce background signals; the optimal concentration of anti-BNP detection antibody needed, the time of SPCEs were first blocked with glycine, incubated with various concentrations of BNP and HRP-conjugated anti-BNP detection antibody (0 to 100.0 ng/mL), followed by electrochemical measurement were investigated,. In conclusion, this immunosensor greatly shortened and convenient for detection of heart failure diagnosis in real serum samples.

Optical transformation of a CdTe quantum dot-based paper sensor for a visual fluorescence immunoassay induced by dissolved silver ions.

This work designs a simple low-cost visual fluorescence immunoassay for disease-related biomarkers (carcinoembryonic antigen, CEA, used as a model protein) in biological fluids, based on the structural and optical transformation of CdTe quantum dots (QDs) immobilized on paper induced by dissolved silver ions (Ag+) from silver nanoparticles (AgNPs) via a cation-exchange reaction. A sandwich-type immunoreaction was initially carried out in a removable polystyrene high-binding microplate coated with monoclonal anti-CEA capture antibody by using AgNP-labeled polyclonal anti-CEA antibody as the detection antibody. Thereafter, the carried AgNPs accompanying the sandwiched immunocomplexes were dissolved by acid to release numerous silver ions, which induced the ion-exchange reaction with the immobilized CdTe QDs on the paper (attached onto the microplate lid) for the Cd-to-Ag transformation, thus resulting in the quenching of the visual fluorescence from the CdTe QDs owing to Ag2Te formation. Under optimal conditions, the fluorescence intensity decreased with the increasing CEA concentration from 0.02 to 50 ng mL-1 with a detection limit of 5.6 pg mL-1. Further, a visual assay based on a CdTe QD-based paper sensor was developed for CEA detection, and 5.0 pg mL-1 CEA could be discriminated with the naked eye. In addition, our strategy displayed high specificity, good reproducibility and acceptable accuracy for analyzing human serum specimens with consistent results obtained using the commercialized enzyme-linked immunosorbent assay (ELISA) method.

Liposome-Enhanced Lateral-Flow Assays for Clinical Analyses.

Clinical and environmental analyses frequently necessitate rapid, simple, and inexpensive point-of-care or field tests. These semiquantitative tests may be later followed up by confirmatory laboratory-based assays, but provide an initial scenario assessment important for resource mobilization and threat confinement. Lateral-flow assays (LFAs) and dip-stick assays, which are typically antibody-based and yield a visually detectable signal, provide an assay format suiting these applications extremely well. Signal generation is commonly obtained through the use of colloidal gold or latex beads, which yield a colored band either directly proportional or inversely proportional to the concentration of the analyte of interest. Here, dye-encapsulating liposomes as a highly visible alternative are discussed. The semiquantitative LFA biosensor described in this chapter relies on a sandwich immunoassay for the detection of myoglobin in whole blood. After an acute myocardial infarction (AMI) event, several cardiac markers are released into the blood, the most common of which are troponin, creatine kinase MB, C-reactive protein, and myoglobin. Due to its early release, myoglobin has value as an indicator of a recent heart attack amongst conditions which present with similar symptoms and its lack of elevation can effectively rule out a heart attack (Brogan et al., Ann Emerg Med 24:665-671, 1994). The assay described within relies on sandwich complex formation between a membrane immobilized capture monoclonal antibody against myoglobin, a detector biotinylated monoclonal antibody against a different epitope on myoglobin, and streptavidin-conjugated visible dye (sulforhodamine B)-encapsulating liposomes to allow for signal generation.

High-Throughput Nanoliter-Scale Analysis and Optimization of Multimodal Chromatography for the Capture of Monoclonal Antibodies.

Multimodal ligands are synthetic molecules comprising multiple types of interactions that have been increasingly used for the capture of different biopharmaceutical compounds within complex biological mixtures. For monoclonal antibodies (mAbs) in particular, these ligands have shown the possibility of direct capture from cell culture supernatants in native conditions, as well as enhanced selectivity and affinity compared to traditional single-mode ligands. However, performing the capture of a target mAb using multimodal chromatography comes with the need for extensive optimization of the operating conditions, due to the multitude of interactions that can be promoted in parallel. In this work, a high-throughput microfluidic platform was developed for the optimization of chromatographic conditions regarding the capture of an anti-interleukin 8 mAb, using a multimodal ligand (2-benzamido-4-mercaptobutanoic acid), under a wide range of buffer pH and conductivities. The interaction of the ligand with the fluorescently labeled target mAb was also analyzed with respect to the individual contribution of the hydrophobic (phenyl) and electrostatic (carboxyl) moieties using fluorescence microscopy. The results were further validated at the macroscale using prepacked columns in standard chromatography assays, and recovery yield values of 94.6% ± 5.2% and 97.7% ± 1.5% were obtained under optimal conditions for the miniaturized and conventional approaches, respectively. In summary, this study highlights that a microfluidic-based approach is a powerful analytical tool to expedite the optimization process while using reduced reagent volumes (<50 μL), less resin (∼70 nL), and delivering results in less than 1 min per assay condition.

Sampling Error: Impact on the Quantitative Analysis of Nanoparticle-Based Surface-Enhanced Raman Scattering Immunoassays.

This paper examines the impact of the sampling error caused by the small size of the focused laser spot when using surface-enhanced Raman scattering (SERS) as a quantitative readout tool to analyze a sandwich immunoassay. The assay consists of a thin-film gold substrate that is modified with a layer of capture monoclonal antibodies (mAbs) and extrinsic Raman labels (ERLs) that consist of gold nanoparticle cores (60 nm diameter) coated with a monolayer of a Raman reporter molecule and a layer of human IgG mAbs to tag the captured antigen. The contribution of sampling error to the measurement is delineated first by constructing and analyzing an antigenic random accumulation model; this is followed by an experimental study of the analysis of an assay substrate using two different laser spot sizes. Both sets of findings indicate that the analysis with a small laser spot can lead to a sampling error (i.e., undersampling) much like that found when the size of a measured soil sample fails to accurately match that of a larger, more representative sample. That is, the smaller the laser spot size, the larger probable deviation in the accuracy of the measurement and the greater the imprecision of the measurement. Possible implications of these results with respect to the general application of SERS for quantitative measurements are also briefly discussed.

An amperometric nanobiosensor using a biocompatible conjugate for early detection of metastatic cancer cells in biological fluid

Metastasis is the major cause of cancer-associated death in humans, and its early diagnosis will help clinicians to develop suitable therapeutic strategies which may save life of cancer patients. In this direction, we designed an amperometric biosensor using a biocompatible conjugate to diagnose cancer metastasis by detecting epithelial cell adhesion molecule expressing metastatic cancer cells (Ep-MCCs). The sensor probe is fabricated by immobilizing monoclonal capture antibody (CapAnti) on the gold nanoparticles (AuNPs)/conducting polymer composite layer. The detection relies on a sandwich-type approach using a bioconjugate composed of reporter antibody (RepAnti), nanostructured collagen (nCOL), AuNPs, and hydrazine (Hyd) which served as a nonenzymatic electrocatalyst for the reduction of H2O2. The binding of Ep-MCCs with the sensor probe was confirmed using electrochemical impedance spectroscopy, cyclic voltammetry, and chronoamperometry. A dynamic range for the Ep-MCCs detection is determined between 45 and 100,000 Ep-MCCs/mL with the detection limit of 28±3 Ep-MCCs/mL. The proposed immunosensor is successfully applied to detect Ep-MCCs in serum and mixed cell samples and interferences due to nontarget cells and molecules present in the real sample matrix are also examined. The early stage of Ep-MCCs was examined by fluorescence-activated cell sorting assay, which confirms that the developed biosensor has detected Ep-MCCs in its early stage.

Nanogold-functionalized g-C3N4 nanohybrids for sensitive impedimetric immunoassay of prostate-specific antigen using enzymatic biocatalytic precipitation

This work reports on a new impedimetric immunosensing strategy for sensitive detection of prostate-specific antigen (PSA) in biological fluids. The assay was carried out on monoclonal anti-PSA capture antibody-modified glassy carbon electrode with a sandwich-type detection format. Gold nanoparticles-decorated g-C3N4 nanosheets (AuNP/g-C3N4), synthesized by the wet-chemistry method, were utilized for the labeling of polyclonal anti-PSA detection antibody and horseradish peroxidase (HRP). Upon target PSA introduction, the sandwiched immunocomplex could be formed between capture antibody and detection antibody. Followed by the AuNP/g-C3N4, the labeled HRP could catalyze 4-choloro-1-naphthol into benzo-4-chlorohexadienone. The as-generated insoluble product was coated on the electrode surface, thus increasing the Faradaic impedance of Fe(CN)64−/3− indicator between the solution and the base electrode. Under the optimal conditions, the impedance increased with the increasing target PSA in the sample, and exhibited a wide linear range from 10pgmL−1 and 30ngmL−1 with a detection limit of 5.2pgmL−1. A repeatability and intermediate precision of <14% was accomplished. The specificity and method accuracy in comparison with commercial PSA ELISA kit for analysis of human serum specimens were relatively satisfactory.

Changes in plasma steroids and cytokines levels in betel chewing patients in Taiwan

Betel nut is the second largest economic food product in Taiwan. In Southeast Asia, the habit of chewing betel nut seems to be highly correlated with oral submucous fibrosis and oral squamous cell carcinoma. Oral submucous fibrosis is characterized by abnormal accumulation of oral submucous collagen fibers and limitation of mouth opening. Although the mechanism responsible for tissue damage is still unknown, prolonged irritation caused by betel nut and tobacco is considered to be a major factor contributing to the pathogenesis of oral submucous fibrosis. The effect of betel nut chewing on immune system remains unknown. Present study aims to investigate the change of plasma hormones including cortisol, testosterone, and inflammatory cytokine concentrations in male chewing betel nut compared with normal subjects. Heparinized blood was obtained from control group (normal young+mid-aged individuals), betel nut-chewing, and oral cancer male subjects. The study was approved by the Ethics Committee of the Chang-Gung Memorial Hospital. Written informed consent was granted by the patients. Plasma cortisol and testosterone concentrations were detected by commercial enzyme-linked immunosorbent assay (ELISA). The inflammatory cytokines, including interleukin-1β (IL-1β), IL-15, tumor necrosis factor-α (TNF-α), were analyzed by ELISA with commercial monoclonal capture antibodies and polyclonal detection antibodies. The median concentrations of plasma IL-1β, IL-15, and TNF-α were 3.14pg/ml, 3.14pg/ml, and 6.85pg/ml, respectively, in patients with oral cancer, compared with median plasma IL-1β, IL-15, and TNF-α concentration of 2.64pg/ml, 5.86pg/ml, and 5.38pg/ml, respectively, in patients with betel nut-chewing habit. In contrast, the median concentrations of plasma IL-1β, IL-15, and TNF-α in mid-aged males (aged 30–50) were 7.00pg/ml, 10.64pg/ml, and 31.73pg/ml, respectively, compared with median plasma concentration of IL-1β, IL-15, and TNF-α of 4.48pg/ml, 33.36pg/ml, and 97.77pg/ml in young males (aged 20–22), respectively. Also, significantly elevated plasma cortisol concentration was noted in betel nut-chewing (median 727.2ng/ml) and oral cancer patients (561.9ng/ml) compared to the mid-aged (176.8ng/ml) and young males (173.4ng/ml), respectively. In addition, lower plasma testosterone concentrations were found in betel nut-chewing subjects compared with young males (2.6±3.3ng/ml vs 6.2±2.9ng/ml). To summarize, the inflammatory cytokines and steroid hormones may reflect the degree of inflammation in betel nut-chewing males and the oral cancer subjects. The above findings suggest that betel nut-chewing or oral cancer inhibits plasma cytokines and regulates steroid hormones concentrations compared to mid-aged or young normal subjects. It is also indicated that betel nut-chewing causes decreased inflammatory cytokines as the same levels as in oral cancer subjects.

Phenylboronic acid chromatography provides a rapid, reproducible and easy scalable multimodal process for the capture of monoclonal antibodies

Abstract This work addresses the feasibility of scaling-up phenylboronic acid (PBA) chromatography for the purification of human mAbs directly from Chinese Hamster Ovary cell culture supernatants. Column performance optimization regarding superficial velocity and feed volume was performed with the best results being achieved with superficial velocities of 5.1, 15.3 and 25 cm/min in the adsorption, wash and elution steps, respectively. The column volume was scale-up 100 times from 0.4 cm 3 (laboratory scale) to 4, 16 and 40 cm 3 (preparative scale). This 100-fold scale-up was successfully achieved with a recovery yield of 97.7% and a protein purity of 82.6%. Following purification, purified IgG fractions were characterized in terms of isoelectric point, size, CHO proteins and genomic DNA content. Overall results suggest that using ProSep®-PB for mAbs purification is simple, reproducible, robust and scalable without compromising the target molecule integrity and purity.