Prospective Clinical Evaluation of the Diagnostic Accuracy of a Highly Sensitive Rapid Antigen Test Using Silver Amplification Technology for Emerging SARS-CoV-2 Variants.

The COVID-19 pandemic caused by SARS-CoV-2 remains a serious health concern worldwide due to outbreaks of SARS-CoV-2 variants that can escape vaccine-acquired immunity and infect and transmit more efficiently. Therefore, an appropriate testing method for COVID-19 is essential for effective infection control and the prevention of local outbreaks. Compared to reverse-transcription polymerase chain reaction (RT-PCR) tests, antigen tests are used for simple point-of-care testing, enabling the identification of viral infections. In this study, we tested the clinical usefulness of the FUJIFILM COVID-19 Ag test, an antigen test based on silver amplification and immunochromatographic technology. The FUJIFILM COVID-19 Ag test was shown to detect a lower viral concentration as compared to other conventional kits without significant performance loss in detecting prevalent SARS-CoV-2 variants. We tested nasopharyngeal and nasal swabs from a single patient during two different epidemic periods dominated by various SARS-CoV-2 variants. We observed that the sensitivity of the FUJIFILM COVID-19 Ag test was 95.7% and 85.7% in nasopharyngeal and nasal swabs, respectively. These results suggest that the FUJIFILM COVID-19 Ag test is highly sensitive and applicable when RT-PCR testing is unavailable. Furthermore, these results indicate that high-frequency testing using nasal swab specimens may be a valuable screening strategy.

Highly specific monoclonal antibodies and epitope identification against SARS-CoV-2 nucleocapsid protein for antigen detection tests.

The ongoing coronavirus disease 2019 (COVID-19) pandemic is a major global public health concern. Although rapid point-of-care testing for detecting viral antigen is important for management of the outbreak, the current antigen tests are less sensitive than nucleic acid testing. In our current study, we produce monoclonal antibodies (mAbs) that exclusively react with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and exhibit no cross-reactivity with other human coronaviruses, including SARS-CoV. Molecular modeling suggests that the mAbs bind to epitopes present on the exterior surface of the nucleocapsid, making them suitable for detecting SARS-CoV-2 in clinical samples. We further select the optimal pair of anti-SARS-CoV-2 nucleocapsid protein (NP) mAbs using ELISA and then use this mAb pair to develop immunochromatographic assay augmented with silver amplification technology. Our mAbs recognize the variants of concern (501Y.V1-V3) that are currently in circulation. Because of their high performance, the mAbs of this study can serve as good candidates for developing antigen detection kits for COVID-19.

Characterization and analytical validation of a new antigenic rapid diagnostic test for Ebola virus disease detection.

Hemorrhagic fever outbreaks are difficult to diagnose and control in part because of a lack of low-cost and easily accessible diagnostic structures in countries where etiologic agents are present. Furthermore, initial clinical symptoms are common and shared with other endemic diseases such as malaria or typhoid fever. Current molecular diagnostic methods such as polymerase chain reaction require trained personnel and laboratory infrastructure, hindering diagnostics at the point of need, particularly in outbreak settings. Therefore, rapid diagnostic tests such as lateral flow can be broadly deployed and are typically well-suited to rapidly diagnose hemorrhagic fever viruses, such as Ebola virus. Early detection and control of Ebola outbreaks require simple, easy-to-use assays that can detect very low amount of virus in blood. Here, we developed and characterized an immunoassay test based on immunochromatography coupled to silver amplification technology to detect the secreted glycoprotein of EBOV. The glycoprotein is among the first viral proteins to be detected in blood. This strategy aims at identifying infected patients early following onset of symptoms by detecting low amount of sGP protein in blood samples. The limit of detection achieved by this sGP-targeted kit is 2.2 x 104 genome copies/ml in plasma as assayed in a monkey analytical cohort. Clinical performance evaluation showed a specificity of 100% and a sensitivity of 85.7% when evaluated with plasma samples from healthy controls and patients infected with Zaire Ebola virus from Macenta, Guinea. This rapid and accurate diagnostic test could therefore be used in endemic countries for early detection of infected individuals in point of care settings. Moreover, it could also support efficient clinical triage in hospitals or clinical centers and thus reducing transmission rates to prevent and better manage future severe outbreaks.

Development and evaluation of silver amplification immunochromatography kit for foot-and-mouth disease virus antigen detection.

A silver amplification immunochromatography (SAI) kit for the detection of all seven serotypes of foot-and-mouth disease virus (FMDV)-FMDV-Ag SAI-was developed using the monoclonal antibody 1H5 recognizing the highly conserved N terminus region of VP2. The FMDV-Ag SAI can be used under conditions of high biosecurity containment as it does not require any apparatus. The FMDV-Ag SAI exhibited 10-100 times higher sensitivity against the five serotypes (O, A, Asia1, C, and SAT1) and similar sensitivity against SAT2 and SAT3, compared with the Svanodip® FMDV-Ag kit immunochromatography kit. The Svanodip kit showed inhibitory results with several saliva samples but not with the FMDV-Ag SAI kit. In a validation study using clinical samples (n = 132; vesicular epithelium = 92, vesicular lesion swabs = 20, saliva = 20) in Mongolia, the sensitivity of FMDV-Ag SAI in comparison with real-time reverse transcription-polymerase chain reaction revealed the following data: vesicular epithelium, 85.4% (76/89); vesicular lesion swab, 46.7% (7/17); and saliva, 36.8% (7/19). No cross-reactivity with the non-FMDV vesicular-forming viruses and taxonomically related viruses of the Picornaviridae family occurred. The FMDV-Ag SAI is a highly sensitive diagnostic tool that enables pen-side diagnosis without requiring the use of any equipment.

Clinical evaluation of highly sensitive silver amplification immunochromatography systems for rapid diagnosis of influenza.

In this study, the clinical usefulness of silver amplification immunochromatography (SAI) influenza virus detection kits, which employed a photographic development technology to increase the sensitivity of the conventional immunochromatographic assay was evaluated. Influenza A and B virus strains of nasopharyngeal aspirates obtained from influenza patients were tested at different dilutions on the SAI system and conventional immunochromatographic assay kit (ESPLINE Influenza A & B-N), and detection limits were calculated for comparison. The detection ability of the SAI system was 8 times higher for Influenza A viruses and 32-64 times higher for Influenza B viruses. Then 1118 respiratory specimens were obtained from patients who presented with influenza-like symptoms between 2009 and 2012. The sensitivities of the SAI system were 91.2% for type A and 94.4% for type B viruses and higher than those of the conventional kit. The SAI system also showed excellent specificities, 95.8% for type A and 98.0% for type B viruses, and was able to detect influenza viruses even within 6h after the disease onset with 90% sensitivity. In conclusion, the SAI system is useful for diagnosis of influenza from early stages of the illness.

Development of a highly sensitive immunochromatographic detection kit for H5 influenza virus hemagglutinin using silver amplification.

H5N1, a highly pathogenic avian influenza virus (HPAIV), has become a serious epizootic threat to the poultry population in Asia. In addition, significant numbers of human cases of HPAIV infection have been reported to date. To prevent the spread of HPAIV among humans and to allow for timely medical intervention, a rapid and high sensitive method is needed to detect and subtype the causative HPAIVs. In the present study, a silver amplification technique used in photographic development was combined with immunochromatography technologies and a highly sensitive and rapid diagnostic test to detect the hemagglutinin of H5 influenza viruses was developed. The sensitivity of the test kit was increased 500 times by silver amplification. The sensitivity of the method was more than 10 times higher than those of conventional rapid influenza diagnostic tests, which detect viral nucleoproteins. The diagnostic system developed in the present study can therefore provide rapid and highly sensitive results and will be useful for diagnosis of H5 HPAIV infection in humans and animals.

MCM-enzyme-supramolecular hydrogel hybrid as a fluorescence sensing material for polyanions of biological significance.

Polyanions are important sensing targets because of their wide variety of biological activities. We report a novel polyanion-selective fluorescence sensing system composed of a hybrid material of supramolecular hydrogel, enzymes, and aminoethyl-modified MCM41-type mesoporous silica particles (NH(2)-MCM41) encapsulating anionic fluorescent dyes. The rational combination of the polyanion-exchange ability of NH(2)-MCM41 and semi-wet supramolecular hydrogel matrix successfully produced three distinct domains; namely, cationic nanopores, hydrophobic nano/microfibers, and aqueous bulk gel phase, which are orthogonal to each other. The coupling of anion-selective probe release from NH(2)-MCM41 with translocation of the probe facilitated by enzymatic reaction enabled fluorescence resonance energy transfer-type sensing in the hybrid materials for polyanions such as heparin, chondroitin sulfate, sucrose octasulfate, and so forth. The enzymatic dephosphorylation catalyzed by phosphatase (alkaline phosphatase or acid phosphatase) that is embedded in gel matrix with retention of activity also contributed to improving the sensing selectivity toward polysulfates relative to polyphosphates. It is clear that the orthogonal domain formation of these materials and maintaining the mobility of the fluorescent dyes between the three domains are crucial for the rapid and convenient sensing provided by this system.

Photo-responsive gel droplet as a nano- or pico-litre container comprising a supramolecular hydrogel.

Photo-responsive gel droplets having nano- or pico-L volume that showed photo-induced gel-sol transition were successfully developed, for which the inter-droplet mass transport and the subsequent enzymatic reactions in the interior of the gel droplets were photo-triggered.