In Vitro Diagnostic Assay to Detect SARS-CoV-2-Neutralizing Antibody in Patient Sera Using Engineered ACE-2 Mini-Protein.

The recent development and mass administration of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines allowed for disease control, reducing hospitalizations and mortality. Most of these vaccines target the SARS-CoV-2 Spike (S) protein antigens, culminating with the production of neutralizing antibodies (NAbs) that disrupt the attachment of the virus to ACE2 receptors on the host cells. However, several studies demonstrated that the NAbs typically rise within a few weeks after vaccination but quickly reduce months later. Thus, multiple booster administration is recommended, leading to vaccination hesitancy in many populations. Detecting serum anti-SARS-CoV-2 NAbs can instruct patients and healthcare providers on correct booster strategies. Several in vitro diagnostics kits are available; however, their high cost impairs the mass NAbs diagnostic testing. Recently, we engineered an ACE2 mimetic that interacts with the Receptor Binding Domain (RBD) of the SARS-2 S protein. Here we present the use of this engineered mini-protein (p-deface2 mut) to develop a detection assay to measure NAbs in patient sera using a competitive ELISA assay. Serum samples from twenty-one patients were tested. Nine samples (42.8%) tested positive, and twelve (57.1%) tested negative for neutralizing sera. The data correlated with the result from the standard commercial assay that uses human ACE2 protein. This confirmed that p-deface2 mut could replace human ACE2 in ELISA assays. Using bacterially expressed p-deface2 mut protein is cost-effective and may allow mass SARS-CoV-2 NAbs detection, especially in low-income countries where economical diagnostic testing is crucial. Such information will help providers decide when a booster is required, reducing risks of reinfection and preventing the administration before it is medically necessary.

Microwell plates coated with graphene oxide enable advantageous real-time immunosensing platform.

Immunoassays are fundamental analytical tools in molecular diagnostics, therapy monitoring and drug discovery. Nevertheless, they often take around 6 h and require cumbersome procedures. We introduce a breakthrough in immunosensing based on the photoluminescence quenching capabilities of graphene oxide (GO) and the versatile format offered by the famous microwell plates. Taking advantage of the highly efficient non-radiative energy transfer occurring between photoexcited fluorophores (donors) and GO (acceptor), we discovered that flurophore-labelled antibodies (Fl-Abs) are quickly and strongly quenched by the studied GO-coated microwell, whereas Fl-Abs complexed with the respective analyte are weakly quenched by the same surface due to the low affinity between the GO-coated surface and the relatively long distance between these photoluminescent complexes and the GO-coated surface. In doing so, we developed a conceptually innovative single-step immunosensing platform, avoiding blocking, separation and washing steps and exploiting a single antibody. Importantly, the biosensing response can be interrogated in real time. This leads to an advantageous immunodetection phenomenon which is observable in few minutes (e.g. 5 min). The resulting highly transformative biosensing platform operates with different photoluminescent agents and different analytes. Besides, this biosensing platform was demonstrated to operate with real samples of human urine doped with different concentrations of prostate specific antigen.