FAST SARS COV-2 DETEC v1
Authors: Huang not provided , not provided linyang , E Voss James 
Published: September 2020

Our SARS-CoV-2 detection method is a proximity-based assay that uses NanoLuc® Binary Technology (NanoBiT) developed by Promega. It relies on the well-defined monoclonal antibodies (mAbs) targeting epitopes on the SARS-CoV-2 spike protein. Several non-competing epitopes for the receptor binding domain and three non-competing epitopes for spike (S-A, S-B, and S-C) were identified by isolated mAbs in our and other papers (Rogers et al., 2020 Science and Wec et al., 2020 Science). Briefly, these mAbs are either labeled with Large BiT (LgBiT; 18kDa) or Small BiT (SmBiT; 11 amino acid peptide). Upon binding of the labeled antibodies to the spike protein on the surface of SARS-CoV-2 virion, these two subunits come into close proximity if the epitopes recognized by mAbs are close to each other and with appropriate angles, reassemble into a functional luciferase enzyme and generate luminescent light in the presence of substrates (Fig. A). So, unlike the PCR assay, this assay does not require special training to isolate nucleic acids, to reverse transcribe the isolated RNAs, and to set up PCR reactions. Our method is simplified into only two steps, addition of labeled mAbs for 30 mins and addition of substrates for signal detection. Another advantage is that the assay is run in 96-well plates and can be adapted to 384-well plates, allowing us to easily scale up to meet the unmet needs for SARS-CoV-2 detection without heavy investment. One person can handle about 30 to 50 of 96-well plates after simple training process. And the only costs of the spike protein-based SARS-CoV-2 detection are the plates, labeled mAbs in addition to need for a luminometer. We have developed a rapid, inexpensive SARS-CoV-2 test capable of detecting infections using >200 ml. Virus-positive samples will emit luminescence within 20 minutes of performing this test, which can be detected by widely available laboratory equipment (luminometers), and in principle, smartphone cameras. Our method uses the NanoLuc® Binary Technology (NanoBiT) developed by Promega, and recently discovered, high affinity, strain specific, monoclonal antibodies (mAbs) targeting adjacent epitopes on the viral Spike (S) protein. Briefly, one of the mABs is labeled with ‘Large BiT’ (LgBiT), an 18 kDa Luciferase apoenzyme, and the other is labelled with ‘Small BiT’ (SmBiT), an 11 amino acid peptide that binds to LgBit only when in close proximity. When bound, the complex forms a functional luciferase holoenzyme capable of emitting light in the presence of substrate. Only when the two antibodies bind their respective epitopes on the viral S protein do NanoBiT subunits bind and generate a detectable luminescence signal (Figure 1). We are able to detect 4 x 103 SARS-CoV-2 virions/ml of saliva using this system that is almost equivalent in sensitivity to RT-PCR. The normal range of virus concentrations in the saliva of infected individuals is 104-108/ml. The assay is simplified into only two steps: 1) addition of labelled mAbs, and 2) addition of substrates for signal detection. Samples can be read in 96-well and 384-well plates using Luminometers which are widely available in labs globally. Inexpensive detection using smartphone cameras should also be possible, which would enable widespread global adoption of the technology.