Tokyo Institute of Technology and Tokyo Medical and Dental University succeed in rapid quantification of coronavirus proteins using fluorescent antibodies

Assistant Professor Bo Zhu and Professor Hiroshi Ueda of the Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Junior Associate Professor Nobuyuki Nosaka, Professor Kenji Wakabayashi and Professor Ryuji Koike of the Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, and their colleagues announced on December 15 last year, that together they have succeeded in detecting the presence and quantity of coronavirus in a short space of time using antibody fragments known as Quenchbodies (Q-bodies) that fluoresce when they bind to an antigen. When detecting SARS-CoV-2 (COVID-19) using clinical samples, they were able to rapidly detect SARS-CoV-2 N-proteins of less than 0.3 nM with the same sensitivity as the immunochromatography method in around five minutes. It is hoped that this outcome will lead to the development of new methods of diagnosing COVID-19 infections. The researchers' results were selected for HOT Articles 2022 in Analyst, an international science journal.

Antigen-antibody reactions are used for immunoassays that detect various substances. A research group consisting of Ueda and his colleagues previously developed a Q-body that detects antigens with high sensitivity. It achieved this thanks to the chemical modification of a fluorescent label onto parts of antibody fragments with antigen binding sites, which increases the fluorescence during antigen binding. They succeeded in detecting tumor-suppressor protein p53 and the influenza virus HA protein, among other molecules.

In this research, the group aimed to detect SARS-CoV-2 proteins using a Q-body quickly, and in an easy-to-do fashion. First, they obtained human recombinant antibody A7, which binds strongly to the S1 (spike) protein that exists on the virus surface and plays a key role in infection, from a human antibody-displayed phage library, and succeeded in creating Fab antibody fragments that bind to almost all variants of the virus. They then created a Q-body by using the fluorescent label ATTO 520 to modify the N-terminal in two places.

When the group explored infection neutralization capacity using a pseudovirus, they were able to confirm that the Q-body detected the pseudovirus at 10⁵ copies/mL in five minutes. However, there were issues, as the S1 protein was likely to mutate and the detection sensitivity decreased in variants.

Thus, the group investigated the development of a Q-body that targets the N (RNA-binding) protein, which is less likely to mutate. They succeeded in doing so using the structure of the nCoV396 antibody, the high compatibility of which had already been reported. However, the Q-body for N-protein detection needed time for the bonding reaction, requiring more than 10 minutes for the maximum response.

The group therefore explored adding multiple types of macromolecules thought to be effective in accelerating antigen-antibody reactions. They confirmed that adding 5% polyethylene glycol significantly reduced the reaction time and substantially improved the detection limit.

When the group used clinical samples to verify the functions of the Q-body for N-protein detection they had created, they ascertained that it could discern positive and negative results. It was able to detect N-proteins of less than 0.3 nM in five minutes. This functionality is around the same as the current immunochromatography method, but it is simple and quick to use (the immunochromatography method takes around 15 minutes), so it is hoped that this could be used as a new alternative.

Q-bodies can be used for the detection of various antigens, and in the future will be developed in response to emerging infectious diseases. Ueda and his colleagues will consider putting this into use for COVID-19 after discussions with HikariQ Health, a venture company that originated from the Tokyo Institute of Technology.