ABSTRACT
Regular checkups for thyroid-stimulating hormone (TSH) levels are essential for the diagnosis of thyroid disease. The enzyme-linked immunosorbent assay (ELISA) technique is a standard method for detecting TSH in the serum or plasma of hospitalized patients. A recently developed next-generation ELISA, the digital immunoassay (d-IA), has facilitated detection of molecules with ultra-high-sensitivity. In this study, we developed a TSH assay system using the d-IA platform. By utilizing the ultrasensitivity of d-IA, we were able to use a sample volume of as little as 5 µL for each assay (the dead volume was 5 µL). The limits of blank, detection, and quantification (i.e., functional sensitivity), were 0.000346, 0.001953, and 0.002280 µIU/mL, respectively, and the precision of the total coefficient of variation did not exceed 10%. The correlation between serum and plasma levels indicated good agreement. Thus, our system successfully measured TSH using d-IA with a small sample volume and equal functional sensitivity to the current third generation like ARCHITECT TSH assay, which has a functional sensitivity of 0.0038 µIU/mL.
ABSTRACT
BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has had a significant impact on public health and the global economy. Several diagnostic tools are available for the detection of infectious diseases, with reverse transcription-polymerase chain reaction (RT-PCR) testing specifically recommended for viral RNA detection. However, this diagnostic method is costly, complex, and time-consuming. Although it does not have sufficient sensitivity, antigen detection by an immunoassay is an inexpensive and simpler alternative to RT-PCR. Here, we developed an ultrahigh sensitivity digital immunoassay (d-IA) for detecting SARS-CoV-2 nucleocapsid (N) protein as antigens using a fully automated desktop analyzer based on a digital enzyme-linked immunosorbent assay. METHODS: We developed a fully automated d-IA desktop analyzer and measured the viral N protein as an antigen in nasopharyngeal (NP) swabs from patients with coronavirus disease. We studied nasopharyngeal swabs of 159 and 88 patients who were RT-PCR-negative and RT-PCR-positive, respectively. RESULTS: The limit of detection of SARS-CoV-2 d-IA was 0.0043 pg/mL of N protein. The cutoff value was 0.029 pg/mL, with a negative RT-PCR distribution. The sensitivity of RT-PCR-positive specimens was estimated to be 94.3% (83/88). The assay time was 28 min. CONCLUSIONS: Our d-IA system, which includes a novel fully automated desktop analyzer, enabled detection of the SARS-CoV-2 N-protein with a comparable sensitivity to RT-PCR within 30 min. Thus, d-IA shows potential for SARS-CoV-2 detection across multiple diagnostic centers including small clinics, hospitals, airport quarantines, and clinical laboratories.
ABSTRACT
A peptide containing a single disulfide bond was sequenced using high-energy collision-induced dissociation (HE-CID) in conjunction with a high mass resolution time-of-flight tandem mass spectrometer equipped with a matrix-assisted laser desorption/ionization source. This mass spectrometer, which has spiral ion trajectory, allowed both high mass resolution and high precursor ion selectivity. It is difficult to obtain sufficient product ions from peptides containing disulfide bonds using HE-CID due to the single collision in the gas phase. To compensate for insufficient dissociation, the disulfide bond was cleaved via an in-source reduction process using 1,5-diaminonaphthalene, a reducing matrix. After applying the reduction in the ionization, subsequent sequencing using HE-CID provided the detailed structural information of the peptide containing the single disulfide bond.
