Citation
- Authors: Zhao H. et al.
- Year: 2022
- Journal: N Biotechnol 72 139-148
- Applications: in vitro / DNA / FectoPRO
- Cell type: Expi293F
Description: Human embryonic kidney Fibroblast
Known as: Expi 293-F, Expi, HEK-293 Expi
Method
Recombinant S1-RBD was produced in 293 F cells (Catalog number: R79007, Thermo Scientific,Waltham, MA, USA) cultured in Freestyle 293 medium and transfected at 2 × 106 cells/ml, using FectoPro transfection reagent (Polyplus transfection, Illkirch, France) in a volume of 200 ml in a 1000 ml E-flask at 130 rpm in a Multitron 4 incubator (Infors, Velp, the Netherlands). Transfected cells were kept at 37ºC and the supernatant was harvested after 72 h and filtered using Polydisc AS 0.45 µm (Whatman; Cytiva, Marlborough, MA, USA) and loaded onto a 5 ml HisTrap Excel column (Cytiva).
Abstract
A homogeneous PCR-based assay for sensitive and specific detection of antibodies in serum or dried blood spots (DBS) is presented and the method is used to monitor individuals infected with or vaccinated against SARS-CoV-2. Detection probes were prepared by conjugating the recombinant spike protein subunit 1 (S1), containing the receptor binding domain (RBD) of SARS-CoV-2, to each of a pair of specific oligonucleotides. The same was done for the nucleocapsid protein (NP). Upon incubation with serum or DBS samples, the bi- or multivalency of the antibodies (IgG, IgA or IgM) brings pairs of viral proteins with their conjugated oligonucleotides in proximity, allowing the antibodies to be detected by a modified proximity extension assay (PEA). Anti-S1 and anti-NP antibodies could be detected simultaneously from one incubation reaction. This Antibody PEA (AbPEA) test uses only 1 µl of neat or up to 100,000-fold diluted serum or one ø1.2 mm disc cut from a DBS. All 100 investigated sera and 21 DBS collected prior to the COVID-19 outbreak were negative, demonstrating a 100% specificity. The area under the curve, as evaluated by Receiver Operating Characteristic (ROC) analysis reached 0.998 (95%CI: 0.993-1) for samples taken from 11 days after symptoms onset. The kinetics of antibody responses were monitored after a first and second vaccination using serially collected DBS from 14 individuals. AbPEA offers highly specific and sensitive solution-phase antibody detection without requirement for secondary antibodies, no elution step when using DBS sample in a simple procedure that lends itself to multiplex survey of antibody responses.