Enzyme-linked Immunosorbent Assay Format Research Articles

Harnessing Bioluminescent Bacteria to Develop an Enzymatic-free Enzyme-linked immunosorbent assay for the Detection of Clinically Relevant Biomarkers.

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 bacteriaAllivibrio fischeriand 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.

Development and preliminary validation of a MERS-CoV ELISA for serological testing of camels and alpacas

This study describes the development and preliminary validation of a new serological assay using MERS-CoV S1 protein in an indirect enzyme-linked immunosorbent assay (ELISA) format. This assay has the advantage of being able to test MERS-CoV serum samples in a PC2 laboratory without the need for a high-level biocontainment laboratory (PC3 or PC4), which requires highly trained and skilled staff and a high level of resources and equipment. Furthermore, this MERS-CoV S1 ELISA enables a larger number of samples to be tested quickly, with results obtained in approximately five hours. The MERS-CoV S1 ELISA demonstrated high analytical specificity, with no cross-reactivity observed in serum of animals infected with other viruses, including different coronaviruses. We tested 166 positive and 40 negative camel serum samples and have estimated the diagnostic sensitivity (DSe) to be 99.4% (95% CI: 96.7 – 100.0%) and diagnostic specificity (DSp) to be 100% (95% CI: 97.2%-100.0%) relative to the assigned serology results (ppNT and VNT) using a S/P ratio cut-off value of >0.58. The findings of this study showed that our MERS-CoV S1 ELISA was more sensitive than the commercial EUROIMMUN ELISA (Se 99.4% vs 84.9%) and comparable to the ppNT assay, and therefore could be used as a diagnostic aid in countries in the Middle East where MERS-CoV is endemic in dromedary camels. The assay reagents and protocol were easily adapted and transferred from an Australian laboratory to a laboratory in the University of Hong Kong. Thus, the results described here show that the MERS-CoV S1 ELISA represents a cheap, rapid, robust, and reliable assay to support surveillance of MERS-CoV in camels in endemic regions.

Practical use of tobravirus-based vector to produce SARS-CoV-2 antigens in plants

A plant-based heterologous expression system is an attractive option for recombinant protein production because it is based on a eukaryotic system of high feasibility, and low biological risks. Frequently, binary vector systems are used for transient gene-expression in plants. However, plant virus vector-based systems offer advantages for higher protein yields due to their self-replicating machinery. In the present study, we show an efficient protocol using a plant virus vector based on a tobravirus, pepper ringspot virus, that was employed for transient expression of severe acute respiratory syndrome coronavirus 2 partial gene fragments of the spike (named S1-N) and the nucleocapsid (named N) proteins in Nicotiana benthamiana plants. Purified proteins yield of 40–60 µg/g of fresh leaves were obtained. Both proteins, S1-N and N, showed high and specific reactivities against convalescent patients’ sera by the enzyme-linked immunosorbent assay format. The advantages and critical points in using this plant virus vector are discussed.

Off-the-shelf cryopreserved platelets for the detection of HIT and VITT antibodies

AbstractHeparin-induced thrombocytopenia (HIT) is suspected much more often than it is confirmed. Technically simple platelet factor 4 (PF4)-polyanion enzyme-linked immunosorbent assays (ELISAs) are sensitive but nonspecific. In contrast, accurate functional tests such as the serotonin release assay, heparin-induced platelet activation assay, and PF4-dependent P-selectin expression assay require fresh platelets and have complex assay end points, limiting their availability to specialized reference laboratories. To enable broad deployment of functional testing, we sought to extend platelet viability significantly by optimizing storage conditions and developed a simple functional assay end point by measuring the release of a platelet α-granule protein, thrombospondin-1 (TSP1), in an ELISA format. Platelet cryopreservation conditions were optimized by freezing platelets at controlled cooling rates that preserve activatability. Several-month-old cryopreserved platelets were treated with PF4 or heparin and were evaluated for their ability to be activated by HIT and vaccine-induced immune thrombotic thrombocytopenia (VITT) antibodies in the TSP1 release assay (TRA). HIT and spontaneous HIT patient samples induced significantly higher TSP1 release using both PF4-treated (PF4-TRA) and heparin-treated cryopreserved platelets relative to samples from patients suspected of HIT who lacked platelet-activating antibodies. This latter group included several patients that tested strongly positive in PF4-polyanion ELISA but were not platelet-activating. Four VITT patient samples tested in the TRA activated PF4-treated, but not heparin-treated, cryopreserved platelets, consistent with recent data suggesting the requirement for PF4-treated platelets for VITT antibody detection. These findings have the potential to transform the testing paradigm in HIT and VITT, making decentralized, technically simple functional testing available for rapid and accurate in-hospital diagnosis.

Evaluation of measles IgM antibody detection assays during the 2018-2019 outbreak in New York State

Although measles was eliminated in the United States in 2000, a severe outbreak occurred between October 2018 and September 2019. New York was especially hard hit. Serology played an integral role in determining immune status (IgG) and identifying, along with molecular analyses, acute measles infections (IgM). Although an indirect immunofluorescence assay (IFA) was historically used by the New York State Department of Health for measles IgM detection, a higher throughput assay was needed to address the increased specimen numbers. Four commercial enzyme-linked immunosorbent assays (ELISAs) were evaluated for sensitivity and specificity in detecting measles IgM. Two ELISA formats were compared, indirect ELISA and IgM antibody capture. Both formats had comparable specificity as determined by cross-reactivity to non-measles specimens. Overall, the sensitivity of the capture ELISAs was greater than the indirect ELISAs and comparable to the indirect immunofluorescence assay with benefits regarding capacity, cost, and turnaround time.

Improvements to Hybridization-Ligation ELISA Methods to Overcome Bioanalytical Challenges Posed by Novel Oligonucleotide Therapeutics.

As oligonucleotides (ONs) and similar nucleic acid therapeutic modalities enter development pipelines, there is continual need to develop bioanalytical methodologies addressing unique challenges they pose. Novel ONs back bone chemistries, especially those enabling stereochemical control, and base modifications are being exploited to improve pharmacological properties, potency, and increase half-lives. These changes have strained established methods, oftentimes precluding development of assays sensitive and specific enough to meet the needs of preclinical programs. For stereopure ONs representing a single molecular species, nontrivial presence of chain-shortened metabolites in biological samples necessitate assays with high specificity. To meet these needs, this report presents a toolbox of novel techniques, easy to implement for existing hybridization-ligation enzyme-linked immunosorbent assay formats, which address this challenge and yield significant sensitivity and specificity enhancements. Ligation efficiency was improved up to 61-fold through addition of polyethylene glycol, betaine, or dimethylsulfoxide, mitigating major differences among sequence-matched ONs of varying stereopurity, enabling sensitivities below 0.100 ng/mL for quantitation. These improvements enabled further refinement of capture probe designs engendering sufficient specificity to discriminate N-1 chain-shortened metabolites at both the 5′ and 3′ end of the ONs. These generalizable methods advance the performance of mainstay bioanalytical assays, facilitating research and development of innovative ONs therapeutics.

Evaluation of poly- and monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) for their performance to detect crustacean residues in processed foods

Poly- and monoclonal antibody-based (PcAb and mAb) sandwich ELISA formats were developed to detect tropomyosin, a major crustacean allergen. Their performance was accessed by testing crustacean residue in commercial processed and model foods. Results obtained showed the constructed mAb ELISA have lower LOD (0.004 mg/kg) and LOQ (0.006 mg/kg), and higher specificity for crustacean tropomyosin than the PcAb ELISA (LOD: 0.008 mg/kg and LOQ: 0.01 mg/kg). Despite this, the PcAb ELISA system with fewer matrix interferences could discriminate against all commercial foods on the label and show higher tropomyosin recovery than the same positive samples based on a mAb ELISA test. Additionally, both ELISA systems possess adequate repeatability and precision, and can detect as low levels as 0.01% and 0.05% of incurred shrimp powder in cookies and sausages, respectively. Nevertheless, mAb ELISA system shows lower tropomyosin recovery in the same incurred foods, and fish sauce even when supplemented with 5% shrimp powder addition could not be detected by the mAb ELISA test. These findings indicated that the recovery of tropomyosin decreased with the intensity of thermal processing applied to food, while PcAb ELISA showed better matrix-tolerance and detectability in processed foods.

Synthesis and characterization of tracers and development of a fluorescence polarization immunoassay for amantadine with high sensitivity in chicken.

Fluorescence polarization immunoassay (FPIA) is a homogeneous and rapid analytical method that is suitable for high-throughput screening of large numbers of samples. However, FPIA typically suffers from lower sensitivity than the well-established enzyme-linked immunosorbent assay (ELISA), limiting its wide application as an analytical tool that can be run with trace levels of an analyte. Herein, a highly sensitive FPIA for detecting amantadine (AMD) in chicken is described. To achieve high sensitivity, nine chemical tracers of AMD that employ different fluoresceins, fluorescein derivatives, and haptens were synthesized and paired with four previously produced monoclonal antibodies (mAbs). The effect of the tracer structure on the sensitivity of FPIA was investigated and discussed. We found that the tracers with a linear and shorter bridge between adamantane and fluorescein generally provided higher sensitivity. After optimization, N'-(1-adamantyl) ethylenediamine (AEDA), an AMD structural analogue labeled with fluorescein isothiocyanate (FITC), achieved the lowest IC50 value (1.0ng/ml) in the FPIA, which was comparable to that of the heterologous ELISA format that used the same mAb7G2. We also investigated the possible recognition mechanism of mAbs in terms of conformational and electronic aspects. The developed FPIA was applied to chicken to detect AMD residue, demonstrating a limit of detection (LOD) of 0.9µg/kg with recoveries of 76.5-89.3% and coefficients of variation (CVs) below 14.5%. These results show that the proposed FPIA is an efficient, accurate, and convenient method for the rapid screening of AMD residues in chicken. PRACTICAL APPLICATION: The fluorescence polarization immunoassay (FPIA) was developed to determine and quantify amantadine (AMD) in chicken samples with high sensitivity. This homogeneous method avoids coating and washing steps and may provide high-throughput AMD screening in chicken in 10min with high accuracy and precision. FPIA can be used as a monitoring tool and contribute significantly to the rapid detection of AMD in chicken.

Competitive ELISA for a serologic test to detect dengue serotype-specific anti-NS1 IgGs using high-affinity UB-DNA aptamers

Serologic tests to detect specific IgGs to antigens related to viral infections are urgently needed for diagnostics and therapeutics. We present a diagnostic method for serotype-specific IgG identification of dengue infection by a competitive enzyme-linked immunosorbent assay (ELISA), using high-affinity unnatural-base-containing DNA (UB-DNA) aptamers that recognize the four categorized serotypes. Using UB-DNA aptamers specific to each serotype of dengue NS1 proteins (DEN-NS1), we developed our aptamer–antibody sandwich ELISA for dengue diagnostics. Furthermore, IgGs highly specific to DEN-NS1 inhibited the serotype-specific NS1 detection, inspiring us to develop the competitive ELISA format for dengue serotype-specific IgG detection. Blood samples from Singaporean patients with primary or secondary dengue infections confirmed the highly specific IgG detection of this format, and the IgG production initially reflected the serotype of the past infection, rather than the recent infection. Using this dengue competitive ELISA format, cross-reactivity tests of 21 plasma samples from Singaporean Zika virus-infected patients revealed two distinct patterns: 8 lacked cross-reactivity, and 13 were positive with unique dengue serotype specificities, indicating previous dengue infection. This antigen-detection ELISA and antibody-detection competitive ELISA combination using the UB-DNA aptamers identifies both past and current viral infections and will facilitate specific medical care and vaccine development for infectious diseases.

Enabling Multiplexed Electrochemical Detection of Biomarkers with High Sensitivity in Complex Biological Samples.

The ability to perform multiplexed detection of various biomarkers within complex biological fluids in a robust, rapid, sensitive, and cost-effective manner could transform clinical diagnostics and enable personalized healthcare. Electrochemical (EC) sensor technology has been explored as a way to address this challenge because it does not require optical instrumentation and it is readily compatible with both integrated circuit and microfluidic technologies; yet this approach has had little impact as a viable commercial bioanalytical tool to date. The most critical limitation hindering their clinical application is the fact that EC sensors undergo rapid biofouling when exposed to complex biological samples (e.g., blood, plasma, saliva, urine), leading to the loss of sensitivity and selectivity. Thus, to break through this barrier, we must solve this biofouling problem.In response to this challenge, our group has developed a rapid, robust, and low-cost nanocomposite-based antifouling coating for multiplexed EC sensors that enables unprecedented performance in terms of biomarker signal detection compared to reported literature. The bioinspired antifouling coating that we developed is a nanoporous composite that contains various conductive nanomaterials, including gold nanowires (AuNWs), carbon nanotubes (CNTs), or reduced graphene oxide nanoflakes (rGOx). Each study has progressively evolved this technology to provide increasing performance while simplifying process flow, reducing time, and decreasing cost. For example, after successfully developing a semipermeable nanocomposite coating containing AuNWs cross-linked to bovine serum albumin (BSA) using glutaraldehyde, we replaced the nanomaterials with reduced graphene oxide, reducing the cost by 100-fold while maintaining similar signal transduction and antifouling properties. We, subsequently, developed a localized heat-induced coating method that significantly improved the efficiency of the drop-casting coating process and occurs within the unprecedented time of <1 min (at least 3 orders of magnitude faster than state-of-the-art). Moreover, the resulting coated electrodes can be stored at room temperature for at least 5 months and still maintain full sensitivity and specificity. Importantly, this improved coating showed excellent antifouling activity against various biological fluids, including plasma, serum, whole blood, urine, and saliva.To enable affinity-based sensing of multiple biomarkers simultaneously, we have developed multiplexed EC sensors coated with the improved nanocomposite coating and then employed a sandwich enzyme-linked immunosorbent assay (ELISA) format for signal detection in which the substrate for the enzyme bound to the secondary antibody precipitates locally at the molecular binding site above the electrode surface. Using this improved EC sensor platform, we demonstrated ultrasensitive detection of a wide range of biomarkers from biological fluids, including clinical biomarkers, in both single and multiplex formats (N = 4) with assay times of 37 and 15 min when integrated with a microfluidic system. These biosensors developed demonstrate the vast potential of solving the biofouling problem, and how it can enable potential clinically important diagnostic applications. This Account reviews our antifouling surface chemistry and the multiplexed EC sensor-based biodetection method we developed and places it in context of the various innovative contributions that have been made by other researchers in this field. We are optimistic that future iterations of these systems will change the way diagnostic testing is done, and where it can be carried out, in the future.

Development of Immunoassays for Detection of Francisella tularensis Lipopolysaccharide in Tularemia Patient Samples.

Francisella tularensis is the causative agent of tularemia, a zoonotic bacterial infection that is often fatal if not diagnosed and treated promptly. Natural infection in humans is relatively rare, yet persistence in animal reservoirs, arthropod vectors, and water sources combined with a low level of clinical recognition make tularemia a serious potential threat to public health in endemic areas. F. tularensis has also garnered attention as a potential bioterror threat, as widespread dissemination could have devastating consequences on a population. A low infectious dose combined with a wide range of symptoms and a short incubation period makes timely diagnosis of tularemia difficult. Current diagnostic techniques include bacterial culture of patient samples, PCR and serological assays; however, these techniques are time consuming and require technical expertise that may not be available at the point of care. In the event of an outbreak or exposure a more efficient diagnostic platform is needed. The lipopolysaccharide (LPS) component of the bacterial outer leaflet has been identified previously by our group as a potential diagnostic target. For this study, a library of ten monoclonal antibodies specific to F. tularensis LPS were produced and confirmed to be reactive with LPS from type A and type B strains. Antibody pairs were tested in an antigen-capture enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay format to select the most sensitive pairings. The antigen-capture ELISA was then used to detect and quantify LPS in serum samples from tularemia patients for the first time to determine the viability of this molecule as a diagnostic target. In parallel, prototype lateral flow immunoassays were developed, and reactivity was assessed, demonstrating the potential utility of this assay as a rapid point-of-care test for diagnosis of tularemia.

Immunochemical method for penthiopyrad detection through thermodynamic and kinetic characterization of monoclonal antibodies

Immunoassays are nowadays being employed for rapid contaminant analysis in clinical, environmental, and agrochemical samples. A thorough characterization of the antibody‒antigen interaction can bring light to the immunoreagent selection process in order to develop sensitive and robust tests. Thus, determination of equilibrium and reaction rate constants is usually recommendable. However, this can be quite tricky for low molecular weight compounds, and competitive strategies are commonly followed to estimate apparent affinity values. In the present study, a collection of monoclonal antibodies to penthiopyrad was raised for the first time, and apparent equilibrium constants were assessed by the Langmuir model using three different competitive enzyme-linked immunosorbent assay formats. The obtained KD values from antibody-coated assays were quite close to the corresponding KD values calculated from surface plasmon resonance (SPR) evaluation. These studies were employed to select a pair of immunoreagents for immunoassay development. The KD value for penthiopyrad of the selected antibody obtained by SPR was 0.28 nM. The optimized direct assay showed an IC50 value for penthiopyrad of 0.42 nM (0.15 ng mL−1) in buffer. The limit of quantification for grape, must, and wine samples was 10 ng mL−1. An excellent correlation was found when immunochemical results were compared with those from LC-MS/MS. As an application case, it was determined that 58% of penthiopyrad was still found in wine after fermentation.

Production of trimeric SARS-CoV-2 spike protein by CHO cells for serological COVID-19 testing.

We describe scalable and cost-efficient production of full length, His-tagged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein trimer by Chinese hamster ovary (CHO) cells that can be used to detect SARS-CoV-2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both human embryonic kidney (HEK) and CHO cells mediated by polyethyleneiminewas increased significantly (up to 10.9-fold) by a reduction in culture temperature to 32°C to permit extended duration cultures. Based on these data GS-CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9-fold to 53 mg/L. Purification of recombinant spike by Ni-chelate affinity chromatography initially yielded a variety of co-eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in enzyme-linked immunosorbent assay format to detect immunoglobulin Gantibodies against SARS-CoV-2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (COVID-19) symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS-CoV-2 trimer which can be used as antigen for mass serological testing.

Simplified Digital Enzyme-Linked Immunosorbent Assay Using Tyramide Signal Amplification and Fibrin Hydrogels.

Many protein biomarkers occur at very low concentrations in biofluids like blood and saliva, and ultrasensitive detection methods are required in order to measure them. Approaches such as digital enzyme-linked immunosorbent assays (ELISA) and single molecule arrays (Simoa) have been developed to accurately quantitate protein concentrations as low as attomolar levels. Although these techniques are being implemented in research and clinical laboratories to develop ultrasensitive clinical diagnostic assays, the size and cost of the instruments required to run these digital assays have precluded them from being implemented into point-of-care diagnostic formats. Here, we report the development of a simplified digital ELISA format that is more amenable to point-of-care technologies, referred to as catalyzed reporter deposition digital ELISA (CARD-dELISA). On-bead signal generation using the CARD tyramide signal amplification technique is combined with bead immobilization in fibrin hydrogels for single molecule counting in a simplified workflow format. CARD-dELISA allows for ultrasensitive protein detection (IL-6: ∼1 fM) with a dynamic range similar to the conventional Simoa assay. We use CARD-dELISA to measure IL-6 in saliva samples and show good agreement with conventional Simoa.

Development of a Highly Sensitive Enzyme-Linked Immunosorbent Assay for Mouse Soluble Epoxide Hydrolase Detection by Combining a Polyclonal Capture Antibody with a Nanobody Tracer.

Enzyme-linked immunosorbent assays (ELISA) for the detection of soluble epoxide hydrolase (sEH), a key enzyme in the metabolism of fatty acids and a biomarker, may increasingly represent an important diagnostic tool. However, there is a lack of ELISAs for mouse sEH quantification, thus resulting in a bottleneck in understanding the pathogenesis of many diseases related to sEH based on mouse models. In this work, nanobodies recognizing mouse sEH were obtained through rebiopanning against mouse sEH in the previous phage display library of human sEH. Later, we developed four ELISAs involving a combination of anti-mouse sEH polyclonal antibodies (pAbs) and nanobodies. It was found that the double antibodies worked as dual filters and had a huge impact on both the sensitivity and selectivity of sandwich immunoassays. The switch from anti-human sEH pAbs to anti-mouse sEH pAbs led to over a 100-fold increase in the sensitivity and a dramatic decrease of the limit of detection to a picogram per milliliter range in format B (pAb/biotin-VHH/streptavidin-poly-horseradish peroxidase). Moreover, we found that the four sandwich ELISAs might demonstrate excellent selectivities to mouse sEH, despite the antibodies alone showing significant cross-reactivity to the matrix, indicating the enhanced selectivity of double antibodies as dual filters. Eventually, for the first time, the ELISA (format B) was successfully used to measure the mouse sEH level in cancer cells with ultralow abundances. The ELISAs proposed here represent a sensitive tool for tracking sEH in various biological processes and also provide deep insights into developing sandwich immunoassays against various targets in terms of both the sensitivity and selectivity.

Detection of C-Reactive Protein Using Histag-HRP Functionalized Nanoconjugate with Signal Amplified Immunoassay.

Ultrasensitive detection of biomarkers is highly significant for disease prognosis and public health treatment. Despite wide acceptance in routine laboratory tests, the conventional enzyme-linked immunosorbent assay (ELISA) has been of limited use for early biomarker detection due to insufficient sensitivity and multiple long incubation time. Several nanoprobes have been introduced to circumvent the limitation, however, rapid, simple, and chemical-free nanoprobe synthesis and sensitive detection methods, particularly for ELISA, are still lacking. In this study, we have synthesized a gold nanoprobe, conjugated with multiple 6X-histidine (6X-his) peptide and nickel-horseradish peroxidase (Ni2+-HRP), for enhancing the colorimetric signal in ELISA. The developed nanoprobe has been tested for the detection of immunologically significant C-reactive protein (CRP) in ELISA format. The performance of designed probe is validated by testing standard and serum samples, and the detection limit of 32.0 pg/mL with R2 = 0.98 is confirmed. Furthermore, a comparative analysis of the developed nanoprobe was performed with ELISA developed on conventional guidelines, the proposed immunoassay showed an increase of 12-fold sensitivity for detecting CRP due to the high loading of 6Xhis peptide and binding of multiple Ni2+-HRP on a gold nanoparticle. Additionally, the proposed assay provides a simple, fast, and cost-efficient (not requiring multiple antibodies) detection of CRP with easy nanoprobe synthesis. Moreover, the developed Histag-HRP functionalized nanoconjugate immunoassay is flexible and can be applied to other biomarkers efficiently by using disease specific antibody.

Development and Validation of a S1 Protein-Based ELISA for the Specific Detection of Antibodies against Equine Coronavirus.

Equine coronavirus (ECoV) is considered to be involved in enteric diseases in foals. Recently, several outbreaks of ECoV infection have also been reported in adult horses from the USA, France and Japan. Epidemiological studies of ECoV infection are still limited, and the seroprevalence of ECoV infection in Europe is unknown. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) method utilizing ECoV spike S1 protein was developed in two formats, and further validated by analyzing 27 paired serum samples (acute and convalescent sera) from horses involved in an ECoV outbreak and 1084 sera of horses with unknown ECoV exposure. Both formats showed high diagnostic accuracy compared to virus neutralization (VN) assay. Receiver-operating characteristic (ROC) analyses were performed to determine the best cut-off values for both ELISA formats, assuming a test specificity of 99%. Employing the developed ELISA method, we detected seroconversion in 70.4% of horses from an ECoV outbreak. Among the 1084 horse sera, seropositivity varied from 25.9% (young horses) to 82.8% (adult horses) in Dutch horse populations. Further, sera of Icelandic horses were included in this study and a significant number of sera (62%) were found to be positive. Overall, the results demonstrated that the ECoV S1-based ELISA has reliable diagnostic performance compared to the VN assay and is a useful assay to support seroconversion in horses involved with ECoV outbreaks and to estimate ECoV seroprevalence in populations of horses.

Highly Sensitive ELISA Using Membrane-Based Microwave-Mediated Electrochemical Immunoassay for Thyroid-Stimulating Hormone Detection

Thyroid stimulating hormone (TSH) plays a critical role in regulating physiological symptoms in human, thus, it is important to detect TSH with high sensitivity and specificity is urgently needed in the human serum sample. In this paper, we proposed a strategy to detect TSH antigen using a membrane-based microwave-mediated analytical device developed for highly responsive electrochemical immunoassay with sandwich enzyme-linked immunosorbent assay (ELISA) format. Experimental assay was done with different TSH antigen concentrations. It was found that the immunoassay was able to detect TSH antigen as low as 0.2 mIU/L. The total detection time from preparation of samples to measurement was within 60 min. The specificity of this immunoassay was proven that against unspecific proteins as the almost low signal was obtained for all tested solutions. Finally, exploratory experiments using standard human serum samples with TSH antigens were carried out prior to a successful trial using clinical human serum samples. Our results indicate that this membrane-based microwave-mediated analytical electrochemical immunoassay device could be used as a new platform for low cost, sensitive, specific, swift, and point-of-care diagnosis of TSH. Moreover, the advance system could define a powerful point-of-care tool in the next generation of technology for detecting and signaling biomarkers at early stages by using electrochemical immunosensor.

Development and Characterization of Monoclonal Antibodies for the Mycotoxin Citreoviridin.

Citreoviridin (CTV) in an inhibitor of mitochondrial ATPase that has been isolated from molded yellow rice and linked to the human disease Shoshin-kakke (acute cardiac beriberi). The disease results from a deficiency of thiamine, however, purified CTV can reproduce the symptoms in experimental animals. The link between CTV and Shoshin-kakke has been difficult to resolve, in part because cases of the disease are rare. In addition to rice, CTV has been found in maize, pecan nuts, and wheat products. A method to screen for CTV and its geometric isomer, iso-CTV, in commodities was developed, based upon the isolation of two novel monoclonal antibodies (mAb). In an antigen-immobilized competitive enzyme-linked immunosorbent assay format (CI-ELISA), the observed IC50s for CTV were 11 ng/mL and 18 ng/mL (mAbs 2-2 and 2-4, respectively). The assays were relatively tolerant to methanol and acetonitrile, which allowed their application to the detection of CTV in spiked polished white rice. For quantification, a standard mixture of CTV and iso-CTV was used, along with matrix matched calibration. The dynamic range of the ELISA using mAb 2-4 was equivalent to 0.23 to 2.22 mg/kg in rice. Recoveries over the range of 0.36 to 7.23 mg/kg averaged 97 ± 10%. The results suggest that the mAb 2-4-based immunoassay can be applied to the screening of white rice for CTV. Both mAbs were also observed to significantly enhance the fluorescence of the toxin.

Enhanced processing of von Willebrand factor reflects disease severity and discriminates severe portal hypertension in cirrhosis.

Portal hypertension in cirrhosis is associated with endothelial dysfunction, impaired wound healing, and decreased platelet count. Increased von Willebrand factor (VWF) formation has been suggested as a compensatory mechanism, but the role of VWF processing has not been directly assessed. The aim was to measure the processing of activated VWF (VWF-A) in addition to VWF release (VWF-N) to investigate the association of primary hemostasis with disease activity and portal hypertension in liver cirrhosis. Plasma samples from 105 participants undergoing liver vein catheterization and with liver cirrhosis of varying severity were included in the study together with 20 controls without liver disease. Competitive enzyme-linked immunosorbent assay format was used to estimate biomarkers of VWF turnover using neo-epitope-specific monoclonal antibodies. VWF-N levels and VWF-A levels were significantly elevated in cirrhotic patients compared with controls (P<0.0001), and both markers could discriminate mild from severe cirrhosis (VWF-N, P<0.0001; VWF-A, P<0.05). Both markers correlated well with increasing portal hypertension and could identify patients with clinically significant portal hypertension (VWF-N, area under the curve: 0.78; VWF-A, area under the curve: 0.67). Only VWF-A significantly separated compensated from decompensated patients (P<0.05). The data indicate that both VWF release and processing of active VWF are increased in cirrhosis, reflecting ongoing wound healing initiation. VWF-N and VWF-A may specifically contain information to assess the presence and severity of PHT as an early indicator of cirrhosis, and for acute damage in decompensated cirrhosis. Whether the increased wound healing affects long-term outcome needs to be addressed in future studies.