Citation

  • Authors: Kainulainen MH. et al.
  • Year: 2021
  • Journal: Sci Rep 11 12330
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: Expi293F
    Description: Human embryonic kidney Fibroblast
    Known as: Expi 293-F, Expi, HEK-293 Expi

Method

-The DNA sequences for the above-described fusion proteins were cloned into an episomal vector (System Biosciences) into which a puromycin resistance cassette had been added to enable selection of pools stably expressing these proteins. -To express the proteins, human Expi293 cells, growing in Expi293 Expression medium, were transfected with FectoPRO reagent (PolyPlus Transfection). -The supernatants were clarified by slow-speed centrifugation and filtered over polyethersulfone (0.2 micron) membranes, and His-tagged proteins were purified by immobilized metal affinity chromatography using HisTrap Excel nickel columns (Cytiva). After dialysis in PBS, the proteins were flash frozen in liquid nitrogen and stored at −80 °C.

Abstract

SARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.

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