Capture Antibody Research Articles

Ultra-Sensitive Detection of the SARS-CoV-2 Nucleocapsid Protein via a Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a-Mediated Immunoassay.

Tracking trace protein analytes in precision diagnostics is an ongoing challenge. Here, we developed an ultrasensitive detection method for the detection of SARS-CoV-2 nucleocapsid (N) protein by combining enzyme-linked immunosorbent assay (ELISA) with the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system. First, the SARS-CoV-2 N protein bound by the capture antibody adsorbed on the well plate was sequentially coupled with the primary antibody, biotinylated secondary antibody, and streptavidin (SA), followed by biotin primer binding to SA. Subsequently, rolling circle amplification was initiated to generate ssDNA strands, which were targeted by CRISPR/Cas12a to cleave the FAM-ssDNA-BHQ1 probe in trans to generate fluorescence signals. We observed a linear relationship between fluorescence intensity and the logarithm of N protein concentration ranging from 3 fg/mL to 3 × 107 fg/mL. The limit of detection (LOD) was 1 fg/mL, with approximately nine molecules in 1 μL of the sample. This detection sensitivity was 4 orders magnitude higher than that of commercially available ELISA kits (LOD: 5.7 × 104 fg/mL). This method was highly specific and sensitive and could accurately detect SARS-CoV-2 pseudovirus and clinical samples, providing a new approach for ultrasensitive immunoassay of protein biomarkers.

An Unconventional Immunosensor for Biomolecule Detection via Nonspecific Gold Nanoparticle-Antibody Interactions.

Immunogold, that is, gold nanoparticles (AuNPs) conjugated with biomolecules such as antibodies and peptides, have been widely used to construct sandwiched immunosensors for biodetection. Two main challenges in these immunoassays are difficulties in finding and validating a suitable antibody, and the nonspecific interaction between the substrate and immunogold, which lowers the detection sensitivity and even causes false results. To avoid these issues, we took advantage of the nonspecific interaction between AuNPs and capture antibodies and proposed a new sensing mechanism. That is, after the capture of analyte targets by the capture antibodies on the substrate, AuNPs of certain chemical functionality would preferably bind to the free capture antibodies. Consequently, the amount of deposited AuNPs will inversely depend on the concentration of the analytes. As a proof-of-concept, we designed a mass-based sensor where anti-IgG antibodies were coated on a quartz crystal microbalance substrate. After IgG was introduced, tannic acid-capped AuNPs were applied to bind with the free anti-IgG antibody molecules. A frequency change (Δf) of the quartz substrate was induced by the increased mass loading. To further amplify the loading mass, an Ag enhancer solution was added, and Ag growth was catalyzed by the bound AuNPs. The Δf response showed a concentration-dependent decrease when increasing IgG concentration with a detection limit of 2.6 ng/mL. This method relies on the nonspecific interaction between AuNPs and anti-IgG antibodies to realize sensitive detection of IgG and eliminates the use of detection antibodies. The concept is an alternative to many existing immunoassay technologies.

Abstract PO4-20-11: Excellent Response to Trastuzumab Deruxtecan in “HER-2 Low” Leptomeningeal Breast Cancer and Diagnostic and Monitoring Utility of CNSide Assay

Abstract A 72-year-old female presented to the emergency department with sudden onset of imbalance and cognitive decline. Two years ago, she was diagnosed with stage III estrogen receptor (ER) positive, progesterone receptor (PR) negative and human epidermal growth factor receptor 2 (HER2) negative (immunohistochemistry score: 0) invasive breast cancer. She subsequently underwent lumpectomy, adjuvant radiation and chemotherapy. She had been taking adjuvant letrozole at the time of presentation. Magnetic resonance imaging (MRI) of the brain showed obstructive hydrocephalus without any parenchymal lesion or leptomeningeal enhancement. MRI of spine, computerized tomography of chest/abdomen/pelvis and bone scan were unrevealing except a left adrenal mass which was equivocal for metastasis. Cerebrospinal fluid (CSF) analysis showed rare malignant cells. Based on the morphology of malignant cells and the patient’s prior history of breast cancer, the diagnosis of leptomeningeal disease with breast primary was made. There were insufficient cells for additional studies. CNSide, a microfluidic platform that uses antibody capture method to detect CSF tumor cells, showed 4678 tumor cells per milliliter (ml) of CSF. Immunocytochemistry of these cells showed that the cells were ER negative (criterion: < 2.6% ER positive cells) and programmed cell death ligand 1 (PD-L1) negative (criterion: < 3.4% PD-L1 positive cells). Fluorescence in situ hybridization (FISH) testing showed 22% of tumor cells with HER2 amplification (either HER2 to CEP17 ratio >/= 2.0 or HER2 copy number >/= 6). She underwent ventriculoperitoneal (VP) shunt placement with complete recovery of her neurological symptoms. Five weeks later, she was started on trastuzumab deruxtecan (T-DXd) mainly because of the HER2 FISH results from CNSide. She did not receive central nervous system(CNS) irradiation due to complete resolution of neurological symptoms with VP shunt placement. After two cycles of T-DXd, MRI brain showed mild leptomeningeal enhancement along bilateral superior cerebellar sulci. Positron emission tomography/computed tomography(PET/CT) scan showed a new sclerotic rib focus, thought to represent treatment response. It also showed decrease in size of the adrenal mass. The adrenal mass was hypermetabolic on PET, consistent with malignancy. CSF cytology was negative, while CSF tumor cell count significantly reduced to 6.72 cells/ml using CNSide. There were no new neurological symptoms. Despite MRI brain findings, she was continued on T-DXd given overall favorable clinical, radiological and CSF data. After 7th cycle of T-DXd, repeat CSF cytology continued to remain negative, CNSide CSF tumor cell count decreased further to 6.27 cells/ml, MRI brain showed interval resolution of prior leptomeningeal enhancement, CT scan showed further decrease in size of the adrenal mass (baseline 5.4cm to 3cm), stable rib sclerotic lesion and no additional metastatic lesion. To date, she has undergone eight cycles of T-DXd which has effectively controlled her disease for a period of seven months. She has not needed VP shunt revision and continues to do well. To our knowledge, this is the first case demonstrating the activity of T-DXd against CSF tumor cells where a small subset exhibited HER2 amplification. This case also highlights the benefits of using advanced and highly sensitive CSF assays to diagnose and surveil leptomeningeal disease and to assess HER2 signaling. This case is well-timed especially in the era of next-generation, CNS penetrating, potent HER2-directed antibody-drug conjugate like T-DXd which can mount antitumor activity even with minimal HER2 expression in tumors. Citation Format: Sarah Stanford, Zeni Kharel, Lauryn Hemminger, Tyler Schmidt, Sara Hardy, Jason Zittel, Nimish Mohile, Ajay Dhakal. Excellent Response to Trastuzumab Deruxtecan in “HER-2 Low” Leptomeningeal Breast Cancer and Diagnostic and Monitoring Utility of CNSide Assay [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-20-11.

Optical biosensing of monkeypox virus using novel recombinant silica-binding proteins for site-directed antibody immobilization

The efficient immobilization of capture antibodies is crucial for timely pathogen detection during global pandemic outbreaks. Therefore, we proposed a silica-binding protein featuring core functional domains (cSP). It comprises a peptide with a silica-binding tag designed to adhere to silica surfaces and tandem protein G fragments for effective antibody capture. This innovation facilitates precise site-directed immobilization of antibodies onto silica surfaces. We applied cSP to silica-coated optical fibers, creating a fiber-optic biolayer interferometer (FO-BLI) biosensor capable of monitoring the Monkeypox Virus (MPXV) Protein A29L in spiked clinical samples to rapidly detect the MPXV. The cSP-based FO-BLI biosensor for MPXV demonstrated a limit of detection (LOD) of 0.62 ng/mL in buffer, comparable to the 0.52 ng/mL LOD achieved using a conventional streptavidin-based FO-BLI biosensor. Furthermore, it achieved LODs of 0.77 ng/mL in spiked serum and 0.80 ng/mL in spiked saliva, exhibiting no cross-reactivity with other viral antigens. The MPXV detection process was completed within 14 min. We further proposed a cSP-based multi-virus biosensor strategy capable of detecting various pandemic strains, such as MPXV, the latest coronavirus disease (COVID) variants, and influenza A protein, to extend its versatility. The proposed cSP-modified FO-BLI biosensor has a high potential for rapidly and accurately detecting MPXV antigens, making valuable contributions to epidemiological studies.

Exploring Gluten Assessment in Marketed Products through a Sandwich ELISA Methodology Based on Novel Recombinant Antibodies.

This study presents the development of a sandwich ELISA method for gluten detection in foods, using recombinant Fab antibody fragments against gliadin. The Fabs were chemically biotinylated and immobilized on streptavidin-coated plates as capture antibodies, while alkaline phosphatase-conjugated Fabs were used as detection antibodies. Four different gliadin-binding Fabs were tested and the Fab pair Fab8E-4 and Fab-C showed the best compatibility. An indirect sandwich immunoassay, using unmodified Fab8E-4 for capture and Fab-C as the detection antibody, achieved a detection limit of 26 ng/mL of gliadin, corresponding to 10 mg/kg of gluten in foods. No cross-reactivity was observed against 60 gluten-free species commonly used in the food industry. Analysis of 50 commercial products demonstrated consistent results compared to the standard method for gluten detection. The complete lack of cross-reactivity of the developed immunoassay with oat products potentially provides an advantage over other gluten detection systems.

Development of an immunochromatographic test strip to detect pneumolysin by monoclonal antibody capture method

BackgroundThe currently available methods for the detection of Streptococcus pneumoniae are labour-intensive, and involve time-consuming steps needing skilled technicians to perform the assays. Therefore, we developed a rapid lateral flow based immunochromatographic test strip for detection of S. pneumoniae using pneumolysin using monoclonal antibody capture approach. Methodology − The strip was designed by assembling a sample pad, conjugation pad, nitrocellulose membrane and adsorbent pad supported by a plastic backing card. Optimization of control spot was done by using three different concentrations of monoclonal antibody (0.1 µg, 0.25 g and 0.5 µg). Monoclonal antibody coupled with gold nanoparticles was immobilized on conjugate pad. Recombinant pneumolysin at three different concentrations (10 ng/ml, 50 ng/ml and 100 ng/ml) was used. Diluted pneumolysin was poured onto the sample pad which migrated through the conjugate pad containing gold antibody conjugate and the antigen–antibody complex migrated to the test spot to bind to the polyclonal antibody. The remaining free antibody bound to the control spot to visualize a control spot. Results: Control spot was optimized at 0.5 µg of mouse IgG. Of three different concentrations of Pneumolysin, the spot was best visualized at 50 ng/ml Pneumolysin concentration. Conclusion: Our ICT based device could detect a commercially available recombinant Pneumolysin of S. pneumoniae. Future study is directed to validate the strip for detection of Pneumolysin using biological fluids of patients (particularly children) infected with S. pneumoniae.

Development and evaluation of sandwich ELISA for detection and quantification of staphylococcal enterotoxin‐A in food

AbstractStaphylococcus aureus is an opportunistic zoonotic pathogen which secretes 24 different types of enterotoxins (SEs) and enterotoxin‐like (SEls) proteins. Classical enterotoxins (SEA–SEE) are responsible for >95% of food poisoning outbreaks, of which SEA alone is responsible for >75% of them. This study was undertaken to develop a sandwich enzyme‐linked immunosorbent assay (ELISA) for sensitive, specific, and quantitative detection of staphylococcal enterotoxins‐A in food. Optimization of sandwich ELISA was attempted in two ways: rabbit polyclonal anti‐SEA as a capture antibody and mouse monoclonal anti‐SEA as a detector antibody, and vice versa. In the optimization of sandwich ELISA, mouse monoclonal anti‐SEA as a capture antibody and rabbit polyclonal anti‐SEA as a detector antibody yielded the highest sensitivity of 0.5–0.75 ng mL−1. The developed assay was found to be highly specific and exhibited equivalent sensitivity to a commercial kit. The developed sandwich ELISA may be utilized for the detection of staphylococcal enterotoxin‐A in food as a cheap alternative to available commercial kits. The developed sandwich ELISA may be useful for microbiological quality assurance of foods, especially in resource‐limited developing countries.

Purification of monoclonal antibodies using novel 3D printed ordered stationary phases

3D printing offers the unprecedented ability to fabricate chromatography stationary phases with bespoke 3D morphology as opposed to traditional packed beads of spherical beads. The restricted range of printable materials compatible with chromatography is considered a setback for its industrial implementation. Recently, we proposed a novel ink that exhibits favourable printing performance (printing time ∼ 100 mL/h, resolution ∼200 µm) and broadens the possibilities for a range of chromatography applications thanks to its customisable surface chemistry. In this work, this ink was used to fabricate 3D printed ordered columns with 300 µm channels for the capture and polishing of therapeutic monoclonal antibodies. The columns were initially assessed for leachables and extractables, revealing no material propensity for leaching. Columns were then functionalised with protein A and SO3 ligands to obtain affinity and strong cation exchangers, respectively. 3D printed protein A columns showed > 85% IgG recovery from harvested cell culture fluid with purities above 98%. Column reusability was evaluated over 20 cycles showing unaffected performance. Eluate samples were analysed for co-eluted protein A fragments, host cell protein and aggregates. Results demonstrate excellent HCP clearance (logarithmic reduction value of > 2.5) and protein A leakage in the range of commercial affinity resins (<100 ng/mg). SO3 functionalised columns employed for polishing achieved removal of leaked Protein A (∼ 10 ng/mg) to meet regulatory expectations of product purity. This work is the first implementation of 3D printed columns for mAb purification and provides strong evidence for their potential in industrial bioseparations.

A Novel Electrochemiluminescence (ECL) Immunoassay for the Quantitation of Monoclonal Antibody (mAb) PYX-106 in Human Serum

Background:: PYX-106 is a novel monoclonal antibody (mAb), targeting the sialic acidbinding immunoglobulin-like lectin 15 (Siglec-15) in the Tumor Microenvironment (TME). Precise measurement of PYX-106 is essential for the thorough assessment of PYX-106 pharmacokinetics in clinical investigations. Methods:: A novel Electrochemiluminescence (ECL) immunoassay for the quantitation of PYX- 106 in human serum was developed and validated. Biotinylated anti-PYX-106 antibody Bio-A1A1 was employed as the capture antibody, and ruthenylated anti-PYX-106 antibody Ru-A3G10 was utilized as the detection antibody in the ECL immunoassay on Meso Scale Discovery (MSD) platform. Results:: This assay was fully validated in terms of selectivity, accuracy, precision, hook effect, stability, etc., with a dynamic range from 50.0 to 2,500 ng/mL in human serum under the 2018 U.S. Food and Drug Administration (FDA) guidance and the 2022 U.S. FDA ICH M10 guidance. Conclusion:: PYX-106 bioanalytical assay validation was reported for the first time in a biological matrix, and this assay has been successfully applied to support a clinical trial PYX-106-101.

A simple, rapid and sensitive sandwich immunoassay based on poly(N-isopropylacrylamide) for the detection of alpha-fetoprotein

Alpha-fetoprotein (AFP), as a tumor marker, plays a vital role in the diagnosis of liver cancer. In this work, a novel sandwich immunoassay based on a thermosensitive polymer, poly(N-isopropylacrylamide) (PNIPAM), was developed for the detection of AFP. This immunoassay could realize one-step rapid reaction within 1 h, and facilitate the separation of the target molecules by incorporating PNIPAM. In this method, a conjugate of PNIPAM and capture antibody (Ab1) was successfully synthesized as a capture probe and the synthetic method of PNIPAM-Ab1 was simple, while the detection antibody (Ab2) was labeled with fluorescein isothiocyanate (FITC) to form a fluorescent detection probe. By employing a sandwich immunoassay, the method achieved quantitative determination of AFP, exhibiting a wide linear range from 5 ng/mL to 200 ng/mL and a low detection limit of 2.44 ng/mL. Furthermore, it was successfully applied to the analysis of spiked human serum samples and the screening of patients with hepatic diseases in clinical samples, indicating its potential application prospect in the diagnosis of liver cancer.

Sandwich Immuno-RCA Assay with Single Molecule Counting Readout: The Importance of Biointerface Design.

The analysis of low-abundance protein molecules in human serum is reported based on counting of the individual affinity-captured analyte on a solid sensor surface, yielding a readout format similar to digital assays. In this approach, a sandwich immunoassay with rolling circle amplification (RCA) is used for single molecule detection (SMD) through associating the target analyte with spatially distinct bright spots observed by fluorescence microscopy. The unspecific interaction of the target analyte and other immunoassay constituents with the sensor surface is of particular interest in this work, as it ultimately limits the performance of this assay. It is minimized by the design of the respective biointerface and thiol self-assembled monolayer with oligoethylene (OEG) head groups, and a poly[oligo(ethylene glycol) methacrylate] (pHOEGMA) antifouling polymer brush was used for the immobilization of the capture antibody (cAb) on the sensor surface. The assay relying on fluorescent postlabeling of long single-stranded DNA that are grafted from the detection antibody (dAb) by RCA was established with the help of combined surface plasmon resonance and surface plasmon-enhanced fluorescence monitoring of reaction kinetics. These techniques were employed for in situ measurements of conjugating of cAb to the sensor surface, tagging of short single-stranded DNA to dAb, affinity capture of the target analyte from the analyzed liquid sample, and the fluorescence readout of the RCA product. Through mitigation of adsorption of nontarget molecules on the sensor surface by tailoring of the antifouling biointerface, optimizing conjugation chemistry, and by implementing weak Coulombic repelling between dAb and the sensor surface, the limit of detection (LOD) of the assay was substantially improved. For the chosen interleukin-6 biomarker, SMD assay with LOD at a concentration of 4.3 fM was achieved for model (spiked) samples, and validation of the ability of detection of standard human serum samples is demonstrated.

Electrochemical microfluidic immunosensor with graphene-decorated gold nanoporous for T-2 mycotoxin detection

T-2 is one of the most potent cytotoxic food-borne mycotoxins. In this work, we have developed and characterized an electrochemical microfluidic immunosensor for T-2 toxin quantification in wheat germ samples. T-2 toxin detection was carried out using a competitive immunoassay method based on monoclonal anti-T-2 antibodies immobilized on the poly(methyl methacrylate) (PMMA) microfluidic central channel. The platinum wire working electrode at the end of the channel was in situ modified by a single-step electrodeposition procedure with reduced graphene oxide (rGO)-nanoporous gold (NPG). T-2 toxin in the sample was allowed to compete with T-2-horseradish peroxidase (HRP) conjugated for the specific recognizing sites of immobilized anti-T-2 monoclonal antibodies. The HRP, in the presence of hydrogen peroxide (H2O2), catalyzes the oxidation of 4-tert-butylcatechol (4-TBC), whose back electrochemical reduction was detected on the nanostructured electrode at −0.15 V. Thus, at low T-2 concentrations in the sample, more enzymatically conjugated T-2 will bind to the capture antibodies, and, therefore, a higher current is expected. The detection limits found for electrochemical immunosensor, and commercial ELISA procedure were 0.10 μg kg−1 and 10 μg kg−1, and the intra- and inter-assay coefficients of variation were below 5.35% and 6.87%, respectively. Finally, our microfluidic immunosensor to T-2 toxin will significantly contribute to faster, direct, and secure in situ analysis in agricultural samples.

An rolling circle amplification-assisted CRISPR/Cas12a-based biosensor for protein detection

Glycated hemoglobin (HbA1c) has been extensively studied as a pivotal marker for monitoring and diagnosing diabetes. In this study, we introduce a highly sensitive and rapid enzyme-catalyzed rolling circle amplification (RCA)-assisted nicked-PAM CRISPR/Cas12a assay (termed “RANPC”). The RANPC system initially recognizes and captures the target using immunomagnetic beads and antibodies, forming a sandwich structure. The antibodies, upon ligating the primers, trigger the RCA reaction, resulting in the generation of single-stranded DNA with numerous repetitive sequences. The product strand of RCA then initiates the trans-cutting activity of nicked-PAM CRISPR/Cas12a, ultimately reporting the target concentration through fluorescent signaling. This study combines dual antibody capture, signal amplification through RCA, and signal output by Cas12a, resulting in high sensitivity, high specificity, and high signal-to-noise ratio. Moreover, the strategic incorporation of an incomplete PAM design in the CRISPR/Cas12a system substantially reduces background interference and enhances assay specificity relative to previous conventional Cas12a detection methods. RANPC possesses advantages such as ultra-sensitivity (with the lowest detection limit up to 0.129 pg/mL and a linear range of 12.8 pg/mL to 1 µg/mL), high specificity, rapid reaction time (approximately 50 min), and straightforward operation. Under optimized experimental conditions, we successfully detected HbA1c in eight real-world blood samples, and the results were in good agreement with the theoretical values. This suggests that this method holds promise as a new approach for the analysis of HbA1c levels in the clinical setting.

Novel assay format for total anti-adeno-associated virus antibody detection with low capsid consumption and built-in specificity control.

Aim: To develop an assay format for detection of total anti-adeno-associated virus 2 (AAV2) antibodies with low capsid material consumption. Methods: An immune complex (IC) assay format was developed. The format is based on the formation of ICs in solution and their subsequent detection using an anti-AAV2 antibody for capture and an antibody against the study species IgG for detection. Results: The feasibility of the IC assay for detection of preexisting and treatment-emergent anti-AAV2 antibodies was demonstrated in cynomolgus monkey and human serum samples, including samples from a preclinical study with AAV2-based therapies. Conclusion: The presented IC assay is an easy-to-perform total anti-AAV2 antibody assay that requires a small amount of unlabeled capsid material and provides an intrinsic specificity control.

G-quadruplex landscape and its regulation revealed by a new antibody capture method.

Our understanding of DNA G-quadruplexes (G4s) from in vitro studies has been complemented by genome-wide G4 landscapes from cultured cells. Conventionally, the formation of G4s is accepted to depend on G-repeats such that they form tetrads. However, genome-wide G4s characterized through high-throughput sequencing suggest that these structures form at a large number of regions with no such canonical G4-forming signatures. Many G4-binding proteins have been described with no evidence for any protein that binds to and stabilizes G4s. It remains unknown what fraction of G4s formed in human cells are protein-bound. The G4-chromatin immunoprecipitation (G4-ChIP) method hitherto employed to describe G4 landscapes preferentially reports G4s that get crosslinked to proteins in their proximity. Our current understanding of the G4 landscape is biased against representation of G4s which escape crosslinking as they are not stabilized by protein-binding and presumably transient. We report a protocol that captures G4s from the cells efficiently without any bias as well as eliminates the detection of G4s formed artifactually on crosslinked sheared chromatin post-fixation. We discover that G4s form sparingly at SINEs. An application of this method shows that depletion of a repeat-binding protein CGGBP1 enhances net G4 capture at CGGBP1-dependent CTCF-binding sites and regions of sharp interstrand G/C-skew transitions. Thus, we present an improved method for G4 landscape determination and by applying it we show that sequence property-specific constraints of the nuclear environment mitigate G4 formation.

Continuous multi-column capture of monoclonal antibodies with convective diffusive membrane adsorbers.

Downstream processing is the bottleneck in the continuous manufacturing of monoclonal antibodies (mAbs). To overcome throughput limitations, two different continuous processes with a novel convective diffusive protein A membrane adsorber (MA) were investigated: the rapid cycling parallel multi-column chromatography (RC-PMCC) process and the rapid cycling simulated moving bed (RC-BioSMB) process. First, breakthrough curve experiments were performed to investigate the influence of the flow rate on the mAb dynamic binding capacity and to calculate the duration of the loading steps. In addition, customized control software was developed for an automated MA exchange in case of pressure increase due to membrane fouling to enable robust, uninterrupted, and continuous processing. Both processes were performed for 4 days with 0.61 g L-1 mAb-containing filtrate and process performance, product purity, productivity, and buffer consumption were compared. The mAb was recovered with a yield of approximately 90% and productivities of 1010 g L-1  d-1 (RC-PMCC) and 574 g L-1  d-1 (RC-BioSMB). At the same time, high removal of process-related impurities was achieved with both processes, whereas the buffer consumption was lower for the RC-BioSMB process. Finally, the attainable productivity for perfusion bioreactors of different sizes with suitable MA sizes was calculated to demonstrate the potential to operate both processes on a manufacturing scale with bioreactor volumes of up to 2000 L.

Electrochemical biosensor using direct electron transfer and an antibody–aptamer hybrid sandwich for target detection in complex biological samples

Direct electron transfer (DET) between an electrode and redox labels is feasible in electrochemical biosensors using small aptamer–aptamer sandwiches; however, its application is limited in biosensors that rely on larger antibody–antibody sandwiches. The development of sandwich-type biosensors utilizing DET is challenged by the scarcity of aptamer–aptamer sandwich pairs with high affinity in complex biological samples. Here, we introduce an electrochemical biosensor using an antibody–aptamer hybrid sandwich for detecting thrombin in human serum. The biosensor enables rapid DET through an antibody–aptamer hybrid configuration comprising (i) an antibody capture probe that provides high and specific affinity to the target in human serum, (ii) the target thrombin, and (iii) an aptamer detection probe that facilitates convenient terminal conjugation with long flexible spacer DNA and polylinker peptide containing multiple amine-reactive phenazine ethosulfate (arPES) redox labels, allowing the conjugated labels to easily approach the electrode. Rapid repeated DET using arPES-catalyzed NADH oxidation strongly enhanced the electrochemical signals. Properly sized spacer and polylinker provided low nonspecific adsorption of the aptamer probe conjugated with multiple arPESs and low interference with the binding of the aptamer probe. Methods for immobilizing thiol-terminated antibodies on Au electrodes were compared and optimized. The developed biosensor using the antibody–aptamer hybrid sandwich exhibited high sensitivity and selectivity in detecting thrombin, surpassing the limitations of an aptamer–aptamer sandwich owing to the low affinity of thrombin aptamers in human serum. The calculated detection limit of the biosensor was ∼1.5 pM in buffer and ∼2.7 nM in human serum.

Quantification of Hepatitis E Virus ORF2 Protein by a Novel Sandwich ELISA.

Hepatitis E virus (HEV) causes acute hepatitis in humans, which can progress to chronicity in immunosuppressed individuals. Almost all reported HEV infections are caused by Paslahepevirus balayani genotypes 1-4. The structural ORF2 protein is the major antigen detected in the blood of HEV-infected individuals. ELISA assays to detect IgM antibodies to HEV are the first-line diagnostic tests; however, they showed variable performance with frequently discordant results. A qualitative HEV antigen (ORF2) ELISA is currently available for research use. Here, we report a novel quantitative sandwich ELISA to measure HEV ORF2 protein in 3 matrix types. An optimal pair of capture and detection antibodies was selected among 12 unique combinations tested. A sandwich ELISA protocol was developed using these mAbs and biotin-streptavidin technology. The protocol was further optimized to quantify ORF2 antigen in different matrices by interpolating from a standard curve with a linear range of 3.17 to 50.8 femtomoles/mL. Using this method, ORF2 protein was detected in the cell culture medium of Huh7 cells as early as 2-3 days after transfection with HEV genome RNA and in a medium of human hepatocytes infected with HEV. ORF2 antigen was readily detected in the first 2 weeks post-HEV infection in gerbil sera. In immunosuppressed gerbils, ORF2 was detected up to 6 weeks, and the levels were significantly higher between 3 and 6 weeks post-infection. HEV ORF2 antigen levels showed a strong positive correlation with HEV RNA levels in both cell culture medium and gerbil sera. Our novel sandwich ELISA detected at least 7.3 femtomoles/mL ORF2 protein in human plasma spiked with cell culture propagated HEV and detected ORF2 protein in human plasma samples that tested positive for HEV RNA but negative for anti-HEV antibodies. Further, the assay was nonreactive, with negative human plasma, and HBV or HCV-positive human plasma demonstrating specificity. Overall, our ORF2 antigen ELISA will be useful for quantifying ORF2 antigen in cell culture medium, gerbil serum, and human plasma. Further studies are warranted to evaluate its utility in HEV clinical diagnosis.

Rapid and Convenient Single-Chain Variable Fragment-Employed Electrochemical C-Reactive Protein Detection System.

Although IgG-free immunosensors are in high demand owing to ethical concerns, the development of convenient immunosensors that alternatively integrate recombinantly produced antibody fragments, such as single-chain variable fragments (scFvs), remains challenging. The low affinity of antibody fragments, unlike IgG, caused by monovalent binding to targets often leads to decreased sensitivity. We improved the affinity owing to the bivalent effect by fabricating a bivalent antibody-enzyme complex (AEC) composed of two scFvs and a single glucose dehydrogenase, and developed a rapid and convenient scFv-employed electrochemical detection system for the C-reactive protein (CRP), which is a homopentameric protein biomarker of systemic inflammation. The development of a point-of-care testing (POCT) system is highly desirable; however, no scFv-based CRP-POCT immunosensors have been developed. As expected, the bivalent AEC showed higher affinity than the single scFv and contributed to the high sensitivity of CRP detection. The electrochemical CRP detection using scFv-immobilized magnetic beads and the bivalent AEC as capture and detection antibodies, respectively, was achieved in 20 min without washing steps in human serum and the linear range was 1-10 nM with the limit of detection of 2.9 nM, which has potential to meet the criteria required for POCT application in rapidity, convenience, and hand-held detection devices without employing IgGs.

Establishment of enzyme-linked immunosorbent assay for beef and lamb contents in cooked meat

In this study, an enzyme-linked immunosorbent assay (ELISA) was established to detect beef and lamb components, and its performance was tested. Double-antibody sandwich ELISA was adopted and determined a coating concentration of capture antibody 3G5 of 1:4 000, a working concentration of enzyme-labeled antibody 2E7-horseradish peroxidase (HRP) of 1:1 000, a sample incubation time of 60 min and a detection antibody reaction time of 60 min. The specificity, sensitivity, repeatability and stability of this assay were determined. The limit of detection for beef and lamb skeletal muscle troponin I was 45 mg/kg, the inter-assay and intra-assay recovery rates ranged from 80.4% to 115.7%, the coefficients of variation were below 13.6%, and the cross reaction rates of the tissue components of chicken, duck and fish were below 13.4%. The sandwich ELISA method established in this study is stable and has high accuracy. The test results were consistent with the polymerase chain reaction (PCR) method at 50 and 100 g/kg. Therefore, this ELISA method can be used to quantitatively detect beef and lamb components in meat products.