Highlights
The expected 100 copies/mL LOD reported in the EUA Abbott RealTi
m
e SARS-CoV-2 assay product insert was exceeded.
All clinical samples positive for 24 non‒SARS-CoV-2 respiratory viruses were SARS-CoV-2 negative on the RealTi
m
e assay.
The assay had high sensitivity (93%) and specificity (100%) for detecting SARS-CoV-2 in clinical samples.
While diagnosis of COVID-19 relies on qualitative molecular testing for the absence or presence of SARS-CoV-2 RNA, quantitative viral load determination for SARS-CoV-2 has many potential applications in antiviral therapy and vaccine trials as well as implications for public health and quarantine guidance. To date, no quantitative SARS-CoV-2 viral load tests have been authorized for clinical use by the FDA. In this study, we modified the FDA emergency use authorized qualitative RealTi
m
e SARS-CoV-2 assay into a quantitative SARS-CoV-2 Laboratory Developed Test (LDT) using newly developed Abbott SARS-CoV-2 calibration standards. Both analytical and clinical performance of this SARS-CoV-2 quantitative LDT was evaluated using nasopharyngeal swabs (NPS). We further assessed the correlation between Ct and the ability to culture virus on Vero CCL81 cells. The SARS-CoV-2 quantitative LDT demonstrated high linearity with R
2
value of 0.992, high inter- and intra-assay reproducibility across the dynamic range (SDs ±0.08-0.14 log
10
copies/mL for inter-assay reproducibility and ±0.09 to 0.19 log
10
copies/mL for intra-assay reproducibility). Lower limit of detection was determined as 1.90 log
10
copies/mL. The highest Ct at which CPE was detected ranged between 28.21-28.49, corresponding to approximately 4.2 log
10
copies/mL. Quantitative tests, validated against viral culture capacity, may allow more accurate identification of individuals with and without infectious viral shedding from the respiratory tract.
Type II collagen binds to chondrocytes through integrins and annexin V. While the potential integrin binding sites have been identified, it is unclear which domains bind to annexin V. Proteolytic fragments of collagen are known to modulate cell signaling pathways resulting in degradation of articular cartilage; it is unknown whether annexin V binds to the fragments. The focus of our study was to determine the binding of type II collagen and its fragments to chondrocytes using flow cytometry and fluorescence microscopy. The N-telopeptide binds to annexin V, whereas the C-telopeptide and triple helical peptides do not. These data suggest that the binding of the N-telopeptide of type II collagen is through annexin V, whereas binding of the C-telopeptide and the triple helical peptide to the surface of chondrocytes are potentially facilitated through other collagen receptors, such as integrins or cell-associated matrix proteins.
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