ELISA Path: SARS‑CoV‑2 (RBD) Total Antibody

ELISA for detection of antibodies to the SARS-CoV-2 Receptor Binding Domain (RBD)
This ELISA assay employs a bridging technology for detection of antibodies (immunoglobulins IgG, IgA, IgM) to the SARS-CoV-2 Receptor Binding Domain (RBD) in solutions, e.g., serum/plasma. The kit contains all reagents and buffers needed for the assay. It also contains one uncoated ELISA strip-plate. The ELISA has been validated with human serum/plasma samples from COVID-19 convalescent individuals and uninfected control individuals. The ELISA is species independent. Analysis of serum/plasma samples from uninfected mouse, cynomolgus macaque, rhesus macaque, rat, cat, cow, dog, rabbit, and pig, all resulted in OD values below cut off.

Assay principle
The ELISA plate is coated (overnight) with Strep-Tactin®XT. Sample and the RBD-bridge mix are added after blocking. The RBD-bridge mix contains two components, recombinant RBD expressed with a Twin-Strep-tag®, and recombinant RBD expressed with WASP peptide tag. Antibodies with specificity to RBD will bind RBD-Twin-Strep-tag® with one arm and RBD-WASP tag with the other, creating a bridge. The Twin-Strep-tag will bind to the coated Strep-Tactin®XT, allowing the whole bridge complex to be captured. The captured complex is detected by addition of anti-WASP-HRP. Addition of TMB substrate will result in a colored substrate product. The reaction is stopped with sulfuric acid and optical density determined using an ELISA plate reader.