Development of antibody against drug-resistant respiratory syncytial virus: Rapid detection of mutant virus using split superfolder green fluorescent protein-antibody system.

Respiratory syncytial virus (RSV) infections are associated with severe bronchiolitis or pneumonia. Although palivizumab is used to prevent RSV infections, the occurrence of palivizumab-resistant RSV strains is increasing, and these strains pose a threat to public health. Herein, we report an antibody with affinity to the S275F RSV antigen, enabling the specific detection of palivizumab-resistant RSV strains. Experimental and simulation results confirmed the affinity of the antibody to the S275F RSV antigen. Furthermore, we developed a rapid S275F RSV antigen detection method using a split superfolder green fluorescent protein (ssGFP) that can interact with the antibody. In the presence of the mutant virus antigen, ssGFP emitted fluorescence within 1 min, allowing the rapid identification of S275F RSV. We anticipate that the developed antibody would be useful for the precise diagnosis of antiviral drug-resistant RSV strains and help treat patients with RSV infections.

Development of A4 antibody for detection of neuraminidase I223R/H275Y-associated antiviral multidrug-resistant influenza virus.

The emergence and spread of antiviral drug-resistant viruses have been a worldwide challenge and a great concern for patient care. We report A4 antibody specifically recognizing and binding to the mutant I223R/H275Y neuraminidase and prove the applicability of A4 antibody for direct detection of antiviral multidrug-resistant viruses in various sensing platforms, including naked-eye detection, surface-enhanced Raman scattering-based immunoassay, and lateral flow system. The development of the A4 antibody enables fast, simple, and reliable point-of-care assays of antiviral multidrug-resistant influenza viruses. In addition to current influenza virus infection testing methods that do not provide information on the antiviral drug-resistance of the virus, diagnostic tests for antiviral multidrug-resistant viruses will improve clinical judgment in the treatment of influenza virus infections, avoid the unnecessary prescription of ineffective drugs, and improve current therapies.

Highly Sensitive in Vitro Diagnostic System of Pandemic Influenza A (H1N1) Virus Infection with Specific MicroRNA as a Biomarker.

Several microRNAs (miRNAs) have been reported to be closely related to influenza A virus infection, replication, and immune response. Therefore, the development of the infectious-disease detection system using miRNAs as biomarkers is actively underway. Herein, we identified two miRNAs (miR-181c-5p and miR-1254) as biomarkers for detection of pandemic influenza A H1N1 virus infection and proposed the catalytic hairpin assembly-based in vitro diagnostic (CIVD) system for a highly sensitive diagnosis; this system is composed of two sets of cascade hairpin probes enabling to detect miR-181c-5p and miR-1254. We demonstrated that CIVD kits could not only detect subnanomolar levels of target miRNAs but also distinguish even single-base mismatches. Moreover, this CIVD kit has shown excellent detection performance in real intracellular RNA samples and confirmed results similar to those of conventional methods (microarray and quantitative real-time polymerase chain reaction).

Fluorescence amplified sensing platforms enabling miRNA detection by self-circulation of a molecular beacon circuit.

We have proposed a novel strategy for miRNA detection through enzyme-free signal amplification by self-circulation of the hybridization between the miRNAs and molecular beacon (MB) circuits. Unlike general MB-based miRNA detection based on the one-to-one (1 : 1) hybridization between MBs and miRNA, our system consists of four species of MBs (MBs A, B, C and D) (MB circuits) and is activated by a hybridization chain reaction. MBs stably coexist as hairpin structures that hardly show fluorescence signals in the absence of target miRNA. After miRNA detection, this MB circuit is able to generate fluorescence signals and amplify the fluorescence signal, contributing to improvement in detection sensitivity under iso-thermal conditions without an enzyme. Furthermore, in vitro and in vivo studies have proven that MB circuits can detect low levels of miRNA with high sensitivity, compared to when only one MB alone is used. Therefore, the MB circuits can provide a useful platform for target miRNA detection.

Multivalent Antibody-Nanoparticle Conjugates To Enhance the Sensitivity of Surface-Enhanced Raman Scattering-Based Immunoassays.

Multivalent immunoprobes can improve the sensitivity of biosensors because increased valency can strengthen the binding affinity between the receptor and target biomolecules. Here, we report surface-enhanced Raman scattering (SERS)-based immunoassays using multivalent antibody-conjugated nanoparticles (NPs) for the first time. Multivalent antibodies were generated through the ligation of Fab fragments fused with Fc-binding peptides to immunoglobulin G. This fabrication method is easy and fast because of the elimination of heterologous protein expression, high degrees of antibody modifications, and covalent chemical ligation steps. We constructed multivalent antibody-NP conjugates (MANCs) and employed them as SERS immunoprobes. MANCs improved the sensitivity of SERS-based immunoassays by 100 times compared to standard antibody-NP conjugates. MANCs will increase the feasibility of practical SERS-based immunoassays.

Rapid and simple detection of Tamiflu-resistant influenza virus: Development of oseltamivir derivative-based lateral flow biosensor for point-of-care (POC) diagnostics.

We have developed a novel oseltamivir derivative (oseltamivir hexylthiol; OHT) that exhibits a higher binding affinity for Tamiflu-resistant virus (Tamiflu resistance) than for the wild-type virus (Tamiflu-susceptible virus; WT) as an antibody. First, OHT-modified gold nanoparticles (OHT-GNPs) are used in a simple colorimetric assay as nanoprobes for the Tamiflu-resistant virus. In the presence of Tamiflu-resistant virus, they show a colorimetric change from deep red to purple because of the OHT-GNP aggregation driven by strong interactions between OHT and neuraminidase (NA) on the surface of the Tamiflu-resistance. Moreover, the color gradually turns purple as the concentration of the Tamiflu-resistant virus increases, allowing the determination of the presence of the virus with the naked eye. Furthermore, an OHT-based lateral flow assay (LFA) has been developed as a rapid and easy detection device for Tamiflu resistance. It shows detection specificity for various virus concentrations of Tamiflu-resistant virus even for the mixture of WT and Tamiflu-resistant viruses, where the limit of detection (LOD) is 5 × 102 ~ 103 PFU per test (=1 × 104 PFU/mL). It has been confirmed that this platform can provide accurate information on whether a virus exhibits Tamiflu resistance, thus supporting the selection of appropriate treatments using point-of-care (POC) diagnostics.

Attomolar detection of extracellular microRNAs released from living prostate cancer cells by a plasmonic nanowire interstice sensor.

Prostate cancer (PC) is the second leading cause of cancer death for men worldwide. The serum prostate-specific antigen level test has been widely used to screen for PC. This method, however, exhibits a high false-positive rate, leading to over-diagnosis and over-treatment of PC patients. Extracellular microRNAs (miRNAs) recently provided valuable information including the site and the status of the cancers and thus emerged as new biomarkers for several cancers. Among them, miR141 and miR375 are the most pronounced biomarkers for the diagnosis of high-risk PC. Herein, we report an attomolar detection of miR141 and miR375 released from living PC cells by using a plasmonic nanowire interstice (PNI) sensor. This sensor showed a very low detection limit of 100 aM as well as a wide dynamic range from 100 aM to 100 pM for all target miRNAs. In addition, the PNI sensor could discriminate perfectly the diverse single-base mismatches in the miRNAs. More importantly, the PNI sensor successfully detected the extracellular miR141 and miR375 released from living PC cell lines (LNCaP and PC-3), proving the diagnostic ability of the sensor for PC. We anticipate that the present PNI sensor can hold great promise for the precise diagnosis and prognosis of various cancer patients as well as PC patients.

A facile, rapid and sensitive detection of MRSA using a CRISPR-mediated DNA FISH method, antibody-like dCas9/sgRNA complex.

Rapid and reliable diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is crucial for guiding effective patient treatment and preventing the spread of MRSA infections. Nonetheless, further simplification of MRSA detection procedures to shorten detection time and reduce labor relative to that of conventional methods remains a challenge. Here, we have demonstrated a Clustered regularly interspaced palindromic repeats (CRISPR)-mediated DNA-FISH method for the simple, rapid and highly sensitive detection of MRSA; this method uses CRISPR associated protein 9/single-guide RNA (dCas9/sgRNA) complex as a targeting material and SYBR Green I (SG I) as a fluorescent probe. A dCas9/sgRNA-SG I based detection approach has advantages over monoclonal antibody in conventional immunoassay systems due to its ability to interact with the target gene in a sequence-specific manner. The detection limit of MRSA was as low as 10 cfu/ml and was found to be sufficient to effectively detect MRSA. Unlike conventional gene diagnosis methods in which PCR must be accompanied or genes are isolated and analyzed, the target gene can be detected within 30min with high sensitivity without performing a gene separation step by using cell lysates. We showed that the fluorescence signal of the MRSA cell lysate was more than 10-fold higher than that of methicillin-susceptible S. aureus (MSSA). Importantly, the present approach can be applied to any target other than MRSA by simply changing the single-guide RNA (sgRNA) sequence. Because dCas9/sgRNA-SG I based detection approach has proved to be easy, fast, sensitive, and cost-efficient, it can be applied directly at the point of care to detect various pathogens as well as MRSA in this study.