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. 2022 Oct 6;2(10):e521. doi: 10.1002/cpz1.521

Figure 1.

Figure 1

Overview of the split‐luciferase assay for antibody detection. This assay detects patient antibodies by using protein‐based biosensors that are fusions of split nanoluciferase (SmBiT/LgBiT) and a viral protein produced by SARS‐CoV‐2. Sensors have been developed to both the spike receptor‐binding domain (S‐RBD) protein and nucleocapsid N‐terminal domain (N‐NTD) protein of the virus. Fifty percent of SARS‐CoV‐2 specific antibodies bind one SmBiT and one LgBiT fusion protein, and thus the split nanoluciferase enzyme is reconstituted as an active enzyme. Twenty‐five percent of antibodies bind two SmBiT fusions, and 25% bind two LgBiT fusions. The Basic Protocol describes how this assay can be performed in a medium‐throughput manner in the laboratory with multichannel pipettes and a tabletop luminometer. Alternate Protocol 1 describes how this protocol can be performed in high throughput with a robotics platform. Alternate Protocol 2 describes how this assay can be further applied for use in a point‐of‐care or resource‐limited setting using a handheld luminometer.