The QuantuMDx Q-POC SARS-CoV-2 RT-PCR assay for rapid detection of COVID-19 at point-of-care: preliminary evaluation of a novel technology.

Accurate and rapid point-of-care (PoC) diagnostics are critical to the control of the COVID-19 pandemic. The current standard for accurate diagnosis of SARS-CoV-2 is laboratory-based reverse transcription polymerase chain reaction (RT-PCR) assays. Here, a preliminary prospective performance evaluation of the QuantuMDx Q-POC SARS-CoV-2 RT-PCR assay is reported. Between November 2020 and March 2021, 49 longitudinal combined nose/throat (NT) swabs from 29 individuals hospitalised with RT-PCR confirmed COVID-19 were obtained at St George's Hospital, London. In addition, 101 mid-nasal (MN) swabs were obtained from healthy volunteers in June 2021. These samples were used to evaluate the Q-POC SARS-CoV-2 RT-PCR assay. The primary analysis was to compare the sensitivity and specificity of the Q-POC test against a reference laboratory-based RT-PCR assay. The overall sensitivity of the Q-POC test compared with the reference test was 96.88% (83.78- 99.92% CI) for a cycle threshold (Ct) cut-off value for the reference test of 35 and 80.00% (64.35-90.95% CI) without altering the reference test's Ct cut-off value of 40. The Q-POC test is a sensitive, specific and rapid PoC test for SARS-CoV-2 at a reference Ct cut-off value of 35. The Q-POC test provides an accurate option for RT-PCR at PoC without the need for sample pre-processing and laboratory handling, enabling rapid diagnosis and clinical triage in acute care and other settings.

Dynamic interactions and intracellular fate of label-free, thin graphene oxide sheets within mammalian cells: role of lateral sheet size.

Graphene oxide (GO) holds great potential for biomedical applications, however fundamental understanding of the way it interacts with biological systems is still lacking even though it is essential for successful clinical translation. In this study, we exploit intrinsic fluorescent properties of thin GO sheets to establish the relationship between lateral dimensions of the material, its cellular uptake mechanisms and intracellular fate over time. Label-free GO with distinct lateral dimensions, small (s-GO) and ultra-small (us-GO) were thoroughly characterised both in water and in biologically relevant cell culture medium. Interactions of the material with a range of non-phagocytic mammalian cell lines (BEAS-2B, NIH/3T3, HaCaT, 293T) were studied using a combination of complementary analytical techniques (confocal microscopy, flow cytometry and TEM). The uptake mechanism was initially interrogated using a range of pharmaceutical inhibitors and validated using polystyrene beads of different diameters (0.1 and 1 µm). Subsequently, RNA-Seq was used to follow the changes in the uptake mechanism used to internalize s-GO flakes over time. Regardless of lateral dimensions, both types of GO were found to interact with the plasma membrane and to be internalized by a panel of cell lines studied. However, s-GO was internalized mainly via macropinocytosis while us-GO was mainly internalized via clathrin- and caveolae-mediated endocytosis. Importantly, we report the shift from macropinocytosis to clathrin-dependent endocytosis in the uptake of s-GO at 24 h, mediated by upregulation of mTORC1/2 pathway. Finally, we show that both s-GO and us-GO terminate in lysosomal compartments for up to 48 h. Our results offer an insight into the mechanism of interaction of GO with non-phagocytic cell lines over time that can be exploited for the design of biomedically-applicable 2D transport systems.

Rapid one-step biotinylation of biological and non-biological surfaces.

We describe a rapid one-step method to biotinylate virtually any biological or non-biological surface. Contacting a solution of biotin-spacer-lipid constructs with a surface will form a coating within seconds on non-biological surfaces or within minutes on most biological membranes including membrane viruses. The resultant biotinylated surface can then be used to interact with avidinylated conjugates, beads, vesicles, surfaces or cells.

Biofunctionalizing nanofibers with carbohydrate blood group antigens.

A rapid and simple method of biofunctionalising nylon, cellulose acetate, and polyvinyl butyral electrospun nanofibers with blood group glycans was achieved by preparing function-spacer-lipid constructs and simply contacting them to fibers with a piezo inkjet printer. A series of water dispersible amphipathic glycan-spacer constructs were synthesized representing a range ABO and related blood group antigens. After immediate contact of the amphipathic glycan-spacer constructs with nanofiber surfaces they self-assembled and were detectable by enzyme immunoassays with high sensitivity and specificity.

Ultra-Fast Glyco-Coating of Non-Biological Surfaces.

The ability to glycosylate surfaces has medical and diagnostic applications, but there is no technology currently recognized as being able to coat any surface without the need for prior chemical modification of the surface. Recently, a family of constructs called function-spacer-lipids (FSL) has been used to glycosylate cells. Because it is known that lipid-based material can adsorb onto surfaces, we explored the potential and performance of cell-labelling FSL constructs to "glycosylate" non-biological surfaces. Using blood group A antigen as an indicator, the performance of a several variations of FSL constructs to modify a large variety of non-biological surfaces was evaluated. It was found the FSL constructs when optimised could in a few seconds glycosylate almost any non-biological surface including metals, glass, plastics, rubbers and other polymers. Although the FSL glycan coating was non-covalent, and therefore temporary, it was sufficiently robust with appropriate selection of spacer and surface that it could capture anti-glycan antibodies, immobilize cells (via antibody), and withstand incubation in serum and extensive buffer washing, making it suitable for diagnostic and research applications.

Monoclonal anti-A activity against the FORS1 (Forssman) antigen.

BACKGROUND: The FORS blood group system (originally recognized as the Apae phenotype) was discovered by sporadic activity against polyclonal anti-A reagents and activity against the lectin Helix pomatia. The extent of monoclonal anti-A reagent activity against the FORS1 antigen is serologically and immunochemically incomplete. STUDY DESIGN AND METHODS: In the absence of natural FORS1-positive red blood cells (RBCs), kodecytes were created with synthetic disaccharide and pentasaccharide Forssman function-spacer-lipid (FSL) constructs, Fsdi -kodecytes, and FORS1-kodecytes, respectively. FSL constructs were also applied to solid surfaces and used in solid-phase enzyme immunoassays. A range of characterized monoclonal anti-A and anti-B reagents were then serologically and immunochemically characterized against these Forssman antigens. Polyclonal human anti-A, anti-B, the lectin H. pomatia serologic reagents; and canine RBCs were used as serologic controls. RESULTS: None of 19 different monoclonal anti-A reagents were able to detect the pentasaccharide Forssman on FORS1-kodecytes, while three reagents were able to detect disaccharide Forssman on Fsdi -kodecytes. Most anti-A reagents were immunochemically reactive with both the di- and the pentasaccharide Forssman antigens in the solid-phase assays. Historic polyclonal human anti-A and the lectin H. pomatia reacted strongly with the FORS1-kodecytes, correlating with the discovery of the Apae phenotype and supporting the use of FORS1-kodecytes as FORS1 surrogates. CONCLUSIONS: Monoclonal anti-A reagents, despite showing reactivity against the FORS1 antigen in solid-phase assays are unlikely to cause the agglutination of FORS1 antigen-positive RBCs.

Mapping the fine specificity of ABO monoclonal reagents with A and B type-specific function-spacer-lipid constructs in kodecytes and inkjet printed on paper.

BACKGROUND: Monoclonal (MoAb) reagents are routinely used and are usually very reliable for the serologic determination of ABO blood types. However, the fine specificity and cross-reactivity of these reagents are often unknown, particularly against synthetic antigens used in some diagnostic assays. If nonserologic assays or very sensitive techniques other than those specifically prescribed by the manufacturer are used, then there is a risk of incorrect interpretation of results. STUDY DESIGN AND METHODS: Forty-seven MoAbs and two polyclonal ABO reagents were tested against red blood cell (RBC) kodecytes prepared with A trisaccharide, A Type 1, A Type 2, A Type 3, A Type 4, B trisaccharide, B Type 1, B Type 2, acquired B trisaccharide, and Le(a) trisaccharide function-spacer-lipid (FSL) constructs. Natural RBCs were tested in parallel. In addition these FSL constructs were printed onto paper with a desktop inkjet printer and used in a novel immunoassay that identifies reactivity through the appearance of alphanumeric characters. RESULTS: Mapping of MoAbs with kodecytes and printed FSL constructs revealed a series of broad recognition patterns. All ABO MoAbs tested were reactive with the RBC dominant Type 2 ABO antigens. Unexpectedly some anti-A reagents were reactive against the B Type 1 antigen, while others were poorly reactive with trisaccharide antigens. CONCLUSIONS: All ABO MoAbs detect the RBC dominant Type 2 ABO antigens; however, some reagents may show minor reactivity with inappropriate blood group antigens, which needs to be considered when using these reagents in alternative or highly sensitive analytic systems.