Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5′-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi

To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction–modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5′-GTAC and leave a 3′-TA overhang (5′-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90°C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure

The development of a highly sensitive and quantitative SARS-CoV-2 rapid antigen test applying newly developed monoclonal antibodies to an automated chemiluminescent flow-through membrane immunoassay device

Abstract Background Rapid and accurate diagnosis of individuals with SARS-CoV-2 infection is an effective way to prevent and control the spread of COVID-19. Although the detection of SARS‐CoV‐2 viral RNA by RT‐qPCR is the gold standard for COVID-19 testing, the use of antigen-detecting rapid diagnostic tests (Ag-RDTs) is emerging as a complementary surveillance tool as Omicron case numbers skyrocket worldwide. However, the results from Ag-RDTs are less accurate in individuals with low viral loads. Results To develop a highly sensitive and accurate Ag-RDT, 90 monoclonal antibodies were raised from guinea pigs immunized with SARS CoV-2 nucleocapsid protein (CoV-2-NP). By applying a capture antibody recognizing the structural epitope of the N-terminal domain of CoV-2-NP and a detection antibody recognizing the C-terminal tail of CoV-2-NP to an automated chemiluminescence flow-through membrane immunoassay device, we developed a novel Ag-RDT, CoV-2-POCube. The CoV-2-POCube exclusively recognizes CoV-2-NP variants but not the nucleocapsid proteins of other human coronaviruses. The CoV-2-POCube achieved a limit of detection sensitivity of 0.20 ~ 0.66 pg/mL of CoV-2-NPs, demonstrating more than 100 times greater sensitivity than commercially available SARS-CoV-2 Ag-RDTs. Conclusions CoV-2-POCube has high analytical sensitivity and can detect SARS-CoV-2 variants in 15 min without observing the high-dose hook effect, thus meeting the need for early SARS-CoV-2 diagnosis with lower viral load. CoV-2-POCube is a promising alternative to currently available diagnostic devices for faster clinical decision making in individuals with suspected COVID-19 in resource-limited settings

Evaluation of MafG interaction with Maf recognition element arrays by surface plasmon resonance imaging technique

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/71837/1/j.1356-9597.2004.00711.x.pd

Development of Serum-free Medium for Clonal Growth of Human-human Hybridomas

The effects of rheological changes in the culture medium, caused by addition of sodium carboxymethylcellulose (CMC), on the autotrophic production of poly-(n-3-hydroxybutyric acid) [P(3HB)] by Alcaligenes eutrophus was investigated in an air-lift fermentor. Addition of 0.05% CMC increased P(3HB) productivity to twice as in the control culture. The effect of CMC on the volumetric mass transfer coefficient, (K_La) and the relationship between K_La and P(3HB) productivity were investigated. The production of P(3HB) rate was not, correlated with K_La, when KL_a was measured by the sulfite oxidation method, but it was correlated with K_La, when KL_a was measured by the static method. Thus, the sulfite oxidation method was unsuitable for measurements of the KL_a of this system

Additional file 1 of The development of a highly sensitive and quantitative SARS-CoV-2 rapid antigen test applying newly developed monoclonal antibodies to an automated chemiluminescent flow-through membrane immunoassay device

Additional file 1: Fig. S1. Cell-ELISA screening of mAbs raised from immunized guinea pigs

Additional file 2 of The development of a highly sensitive and quantitative SARS-CoV-2 rapid antigen test applying newly developed monoclonal antibodies to an automated chemiluminescent flow-through membrane immunoassay device

Additional file 2: Table S1. The specific activity of forty candidate mAbs determined by ELISA and immunostaining