Preparation of the luciferase-labeled antibody for improving the detection sensitivity of viral antigen

Abstract

Background Viral antigen detection test is the most common method used to detect viruses in the field rapidly. However, due to the low sensitivity, it can only be used as an auxiliary diagnosis method for virus infection. Improving sensitivity is crucial for developing more accurate viral antigen tests. Nano luciferase (Nluc) is a sensitive reporter that has not been used in virus detection. Results In this study, we produced an intracellularly Nluc labeled detection antibody (Nluc-ch2C5) and evaluated its ability to improve the detection sensitivity of respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens. Compared with the traditional horse-radish peroxidase (HRP) labeled antibody (HRP-ch2C5), Nluc-ch2C5 was 41 times more sensitive for inactivated SARS-CoV-2 virus by sandwich chemiluminescence ELISA. Then we applied Nluc-ch2C5 to establish an automatic magnet chemiluminescence immune assay (AMCA) for the SARS-CoV-2 viral spike protein, the limit of detection was 68 pfu/reaction. The clinical sensitivity and specificity reached 75% (24/32) and 100% (48/48) using 32 PCR-positive and 48 PCR-negative swab samples for clinical evaluation, which is more sensitive than the commercial ELSA kit and colloid gold strip kit. Conclusions Here, monoclonal antibody ch2C5 served as a model antibody and the SARS-CoV-2 served as a model pathogen. The Nluc labeled detecting antibody (Nluc-ch2C5) significantly improved the detection sensitivity of SARS-CoV-2 antigen. This labeling principle applies to other viral infections, so this labeling and test format could be expected to play an important role in detecting other virus antigens.

A Slow but Steady NanoLuc: R162A mutation results in a decreased, but stable, NanoLuc activity

“…Another area of research where NLuc has been utilized extensively is in the development of protein fragment complementation assay, typically utilized for monitoring protein-protein interaction in living cells. 24-26 Additional applications of NLuc include nanoparticle conjugates utilized for in vivo imaging 27 , sensitive viral infection detection 28 , to evaluate one of metabolic intermediate in fatty acid metabolism 29 or to test antibiotic susceptibility 30 . Further, NLuc has been successfully applied in the development of various Bioluminescence Resonance Energy Transfer (BRET)-based biomolecular assays 4-7, 31-33 such as to study molecular/organelle interactions 34, 35 , for optogenetics purposes 36 , for drug descovery 37, 38 , as genetic encoded light source for a photodynamic anticancer therapy 39 , biomolecule detection 40 , monitoring molecular tension 41 , and assays to monitor SARS-CoV-2 protease activity 42, 43 .…”

Advances in phage display based nano immunosensors for cholera toxin

“…A diverse range of antibodies targeting various cholera toxins, such as monoclonal antibodies ( 29 ), polyclonal antibodies ( 30 ), and recombinant antibodies ( 31 ), have been prepared. Currently, antibodies remain pivotal immune reagents and their preparation continues to evolve ( 32 , 33 ). However, traditional antibody production can be intricate, time-consuming, and costly, hindering broad application ( 34 ).…”