Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies.

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies.

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

Effects of Race, Previous CoV2 Exposure and the Length and Type of Maintenance Immunosuppression on Protective Immunity to COVID19 Vaccine in Solid Organ Transplant Recipients

Purpose: The diverse factors affecting the vaccine induced neutralizing antibody response in solid organ transplant recipients and their immunity against CoV2 variants are needed to be well characterize to understand how we improve the vaccine efficacy. Method(s): Anti-CoV2 receptor binding domain (RBD) plasma antibody response and their neutralization potency in 29 kidney and 22 heart transplant patients was determined with recombinant RBD protein binding ELISA and by calculating the 50% virus neutralization titer of the plasma antibody with ACE2-Hu-HeLa cell based pseudo virus neutralization assay against CoV2 wild type and delta variant. Result(s): We detected strong binding and protective neutralizing plasma antibody response in SOTR who were infected with CoV-2 either prior to or after the first dose of vaccine (n=8), who showed high median IC50 value > 10,000 against both the CoV2 wild type strain and the more transmissible delta variant. In contrast to this, the CoV2 uninfected and vaccinated SOTR ( naive vaccinees, n=43) had considerably lower anti-RBD plasma antibody binding titers, and only 19% of this population possessed minimally protective neutralizing antibody titer (IC50 >50) against the wild type CoV2 strain, which further decreased to 10% against the delta variant. While IgG and IgA were dominant isotypes of anti-RBD antibody induced by the CoV2 vaccines and correlated significantly (r=0.84, p=<0.001) with the CoV2 neutralization. The COV2 uninfected SOTR vaccinees who were within 1.5 years from transplantation or African American ethnicity were less likely to have detectable vaccine induced neutralizing antibody responses than the other populations. In the naive vaccinees, administration of corticosteroids in combination with calcineurin or mTOR inhibitors and antimetabolites also negatively affected the CoV2 antibody responses, while female and younger organ transplant recipients tended towards higher IgM and IgA titers. Kidney transplant recipients showed better IgG responses vs heart transplant recipients and elevated serum creatinine levels correlated with poorer antibody response to the vaccines in both kidney and heart transplant groups. Conclusion(s): These results suggest that in the absence of immunity due to CoV2 infection, vaccination in SOTRs induces much lower protective antibody levels than in healthy controls and identifies African-American ethnicity, less than 1.5 years post transplantation as additional risk factors that further exacerbate these effects. Poor kidney function negatively affected the vaccine induced antibody response.

COVID-19 MRNA VACCINE BOOSTER in PATIENTS with SYSTEMIC AUTOIMMUNE DISEASES

Background: Patients with systemic autoimmune diseases (SADs) are often treated with drugs that interfere with the immune system and previous data showed a reduced seroconversion rate after anti-SARS-CoV2 vaccine in these subjects compared to healthy controls1. Administration of a booster dose of the vaccine could be particularly important in these patients, but data available to date are still scarce. Objectives: To evaluate the antibody response to the booster dose of mRNA SARS-CoV2 vaccine in patients with SADs and to compare it to the response after completion of the frst vaccination course. Secondly, to fnd possible correlations between a low antibody titre and patients' clinical features, with special regard to ongoing immunosuppressive therapies. Methods: Consecutive patients with an established diagnosis of SADs undergoing SARS-CoV2 vaccine were prospectively enrolled from January 2021;among them, we selected the patients who received the third vaccination dose between September and December 2021. Demographic and clinical data were collected at enrolment (sex, age, diagnosis, disease duration, ongoing therapies, previous SARS-CoV2 infection, presence of hypogammaglobulinemia);the last three elements were reassessed at each follow-up visit. Blood samples were collected 4 weeks both after the second (W4a) and the third (W4b) dose of the vaccine;a minority of patients was also tested 12 weeks after the second dose (W12). IgG antibodies to SARS-CoV2 receptor-binding domain (RBD) and neutralizing antibodies inhibiting the interaction between RBD and angiotensin converting enzyme 2 were evaluated. IgG anti-RBD were detected by solid phase assay on plates coated with recombinant RBD, while neutralising antibodies by using the kit SPIA (Spike Protein Inhibition Assay). Cut-off values were defned as the 97.5th percentile of a pre-vaccine healthy population. Statistical analysis was performed using IBM SPSS Statistics 20 and GraphPad Prism statistical packages. P values <0.05 were considered signifcant. Results: Forty-five patients (95.6% female;mean age ±SD 55.6±14.1 years;mean disease duration 12.9±10.6 years) were enrolled. Diagnosis was in most cases connective tissue disease (31/45, 68.9%), followed by infammatory arthritis (11/45, 24.4%) and systemic vasculitis (3/45, 6.7%). Two patients (4.4%) had a previous SARS-CoV2 infection and three had hypogammaglobulinemia (6.7%). At the time of the second dose, 18/45 patients were treated with glucocorticoids (GCs) [mean daily 6-methylprednisolone (6MP) dose 3.9 mg (min. 2, max. 14)], 17/45 with conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) and 12/45 with biologic DMARDs (bDMARDs). At the third dose administration, 19/45 patients were treated with GCs [mean daily 6MP dose 4.1 mg (min. 1.5, max. 10)], 18/45 with csDMARDs and 13/45 with bDMARDs. Anti-RBD IgG were positive in 42/45 patients (93.3%) at W4a, in 16/18 (88.9%) at W12 and in 42/45 (93.3%) at W4b. Neutralizing antibodies were present in 38/45 patients (84.4%) at W4a, in 14/18 (77.8%) at W12 and in 42/45 (93.3%) at W4b. Both anti-RBD IgG titers and neutralizing antibody titers signifcantly increased after the third dose if compared to W4a (p<0.0001 both) (Figure 1). Interestingly, of the 7 patients who had not developed an adequate neutralizing antibody response after the frst vaccination course, 5 mounted an adequate titer after the booster. Two non-responder patients were both on combination therapy (one with low dose of GCs plus mycophenolate mofetil, the other with methotrexate and infiximab). Conclusion: Our data suggest that in patients with SADs there is a decline in the antibody titers developed after COVID-19 vaccination, however the booster dose is effective in restoring an adequate antibody titre. These data consolidate the importance of a booster dose of COVID-19 vaccination in patients with SADs to aid in the generation of an immune response.

LLAMA (Lama glama) ANTISERUM AGAINST SARS-CoV-2

The receptor-binding domain (RBD) of SARS-CoV-2 Spike protein constitutes the key access for the virus inside the host cell. A positive correlation between titers of anti-RBD immunoglobulin G and antibodies (Ab) capable of neutralizing the virus has been demonstrated. In this context, passive immunotherapies such as convalescent plasma and hyperimmune equine serum have gained relevance as therapies against COVID-19. Another promising alternative is the use of polyclonal Ab from llamas (Lama glama), because of their unique features. For this reason, we aimed to obtain recombinant RBD as an immunogen to generate anti-RBD Ab in llamas. To achieve this, HEK 293 cells were transfected and transduced with the RDB encoding sequence, resulting in higher yields with this last method. The RBD was purified by affinity chromatography. An immunization schedule was designed and evaluated on two male animals, which were initially inoculated with RBD, followed by periodical boosters. Exploratory bleedings were performed in order to evaluate the reached titers, and larger bleedings in order to obtain enriched plasma with anti-RBD Ac. Ac quantification was accomplished by an in-house ELISA. Results showed that the immunization scheme was successful, achieving a maximum titer of 168000 at 28 days post-immunization. The results lay the foundations for the production of polyclonal anti-RBD Ab.

Recombinant Decoy Exhibits Broad Protection against Omicron and Resistance Potential to Future Variants.

The Omicron variant has swept through most countries and become a dominant circulating strain, replacing the Delta variant. The evolutionary history of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suggests that the onset of another variant (possibly another variant of concern (VOC) is inevitable. Therefore, the development of therapeutics that enable treatments for all Omicron-included VOCs/variants of interest (VOIs) and future variants is desired. Recently, the recombinant receptor decoy therapeutic angiotensin-converting enzyme 2 (ACE2)-Fc has exhibited good safety in a phase 1 clinical trial; therefore, its variant-resistant profile needs to be understood. Here, we conducted a comprehensive evaluation of its neutralization breadth against the Omicron variant and other VOCs/VOIs. Furthermore, to evaluate its resistance to future variants, we investigated its ability to neutralize various single-residue mutated variants. Next, we demonstrated its resistance to evasion via an experiment that rapidly and effectively stimulates virus evolution with a replication-competent virus model. In addition, we evaluated its efficacy for cocktail therapy. The combination of ACE2-Fc and neutralizing antibodies showed both efficacy and breadth in the simulation experiment. The underlying mechanism was revealed to be a synergistic effect in the cocktails. Collectively, this study deepens the understanding of the resistance profile of recombinant receptor decoy therapeutics and highlights the potential value of ACE2-Fc and neutralizing antibody cocktails in the subsequent anti-SARS-CoV-2 campaign. Furthermore, we also provide an effective method to study the resistance profile of antiviral agents and rapidly screen for potential cocktails to combat future variants.

Non-RBD binding antibody neutralize SARSCoV- 2

Since November 2019, the COVID-19 pandemic has been going on around the world, according to the WHO, more 5.5 million people have died. The main strategy for developing therapeutic antibodies is to obtain human viral neutralizing antibodies directed to the receptor-binding domains (RBD) of the SARS-CoV-2 S-protein. However, it is known that the immune response of humans and mice to different antigens is different, therefore, studies of B-cell epitopes of SARS-CoV-2 S-protein with mouse monoclonal antibodies may allow us to find new virus neutralizing epitopes. Eighteen monoclonal antibodies (mAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were obtained using hybridoma technology from mice immunized with inactivated SARS-CoV-2. ELISA demonstrated that selected 16 mAbs bound recombinant spike (S) protein and 2 mAbs bound recombinant nucleocapsid (N) protein. The equilibrium dissociation constants of the obtained mAbs against S protein ranged from 0.08 to 10 nM. Three mAbs bound recombinant RBD of S protein, the equilibrium dissociation constants of the mAbs against RBD ranged from 0.2 to 3 nM. Anti-RBD mAbs did not neutralize SARS-CoV-2 in the plaque reduction neutralization test. mAbs RS2 demonstrated a dose-dependent inhibition of plaque formation after infection with SARS-CoV-2. The kD and IC50 values for this antibody were 0.2 nM and 400 mcg/ml, respectively. To determine the S protein region responsible for binding to mAb RS2 S1, S2 and RBD subunit of S protein SARS-CoV-2 were expressed in CHO cells. Unfortunately, the localization of the epitope recognized by neutralizing mAb RS2 was not identified using ELISA or western blot analysis. Moreover, mAb RS2 do not recognized full sized recombinant S-protein in western blot analysis. The obtained results demonstrated that the epitope recognized by neutralizing mAb RS2 were discontinuous and have quaternary structure.

Anakinra in COVID-19 and pericarditis

Background: Anakinra, an interleukin 1β recombinant receptor antagonist, could be used to reduce corticosteroid-dosage in colchicine-resistant pericarditis. Recently anakinra has been approved to treat patients presenting covid-19 related pneumonia requiring supplementary oxygen at risk of developing ARDS (identified by the finding of sieric suPAR at least 6 ng/mL). Case Report: A 88-years-old woman with history of hypertensive heart disease, diabetes mellitus and hyperthyroidism. Echocardiography showed pericardial effusion with signs of early-stage cardiac tamponade requiring pericardiocentesis. Post-procedural AF occurred. Considering the potential high bleeding risk caused by an high dose of NSAIDs, has been set up medical treatment with colchicine and corticosteroid. During hospitalization patient developed COVID-19. At the same time pericardial fluid cultures were negative to infectious and non-infectious etiologies. Despite the persisting chest pain (sharp and pleuritic, improved by sitting up and leaning forward), elevated serum CRP and a pericardiocentesis residual right ventricle pericardial effusion, there has been a fast improvement of the clinical picture and of the laboratory findings after the treatment with Anakinra. Conclusions: given that Interleukin-1 (IL-1) is a pro-inflammatory cytokine that has been associated with severe COVID-19, in this case treatment with IL-1 inhibitors (Anakinra) has been exploited to reduce COVID-19-associated mortality and to treat patients with symptoms refractory to first lines of therapy.

Correlation between the atellica SARS-COV-2 IGG assay results and the presence of SARS-COV-2 neutralizing antibodies

Background-aim: Antibodies against SARS-CoV-2 detected by routine immunoassays ensure the existence of antibodies binding the virus, but not necessarily the elimination of the infection. Instead, neutralizing antibodies protect against SARS-CoV-2. Virus neutralization test remains the gold standard, but other neutralization assays have been developed to estimate the neutralizing potential against SARS-CoV-2, among them the FDA approved cPass SARS-CoV-2 Neutralization Antibody Detection (cPass) assay. We studied the correlation between the results obtained with SARS-CoV-2 IgG (Siemens Healthineers) and cPass (GenScript) assays in 218 patients. Methods: SARS-CoV-2 IgG is a 2-step sandwich immunoassay automated on Atellica analyzer. It is an assay based on indirect chemiluminescent technology used for the qualitative and quantitative detection of IgG antibodies (sCOVG) against SARS-CoV-2. cPass assay is a blocking ELISA detection assay using the HRP-conjugated recombinant receptor binding domain (RBD) from the viral spike protein and the human Angiotensin-Converting Enzyme 2 (ACE2). The interaction between HRP-RBD and ACE2 will be blocked if neutralizing antibodies against SARS-CoV-2 RBD are present in the sample. Results higher than 30% indicate the presence of neutralizing antibodies. Result: We observed that the presence of SARS-CoV-2 neutralizing antibodies correlated with sCOVG results, observing that neutralizing antibodies using the manufacturer cutoff (>30%) were present in 5% of samples with sCOVG <1, 83% of samples with sCOVG between 1 and 2, 96% of samples with sCOVG between 2,01 and 10, and 100% of samples with sCOVG >10. On the other hand we studied the agreement between the titers obtained for both tests using Passing Bablok regression analysis, obtaining a regression coefficient of 0.881. Finally, we compared the accuracy of the results of the sCOVG and the cPass tests. The area under the curve obtained in the comparison between both tests was 0,969. Furthermore, we observed a concordance between both tests considering the respective cut-off points (≥1 for sCOVG and ≥ 30 for cPass assay) in 96% of patients included in our study. Conclusions: We concluded that the Atellica SARS-CoV-2 IgG assay correlates well with the detection of neutralizing antibodies.

Correlation of COVID-19 Severity and Immunoglobulin Presence Against Spike and Nucleocapsid Proteins in SARS-CoV-2.

Data on the human immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins have been applied to vaccine development and diagnosing coronavirus disease 2019 (COVID-19), but little research has been done on the relationship between the human immune response and COVID-19 severity. We herein sought to determine whether there is a correlation between the immunoglobulin level and COVID-19 severity. Clinical samples were collected from 102 patients with COVID-19. Of these, 65 and 37 patients had mild and severe symptoms, respectively. An enzyme-linked immunosorbent assay using the recombinant SARS-CoV-2 nucleocapsid (N) protein, spike (S) protein, and synthetic peptides covering N and S as antigens was performed to measure the IgM and IgG levels. The correlation between the immunoglobulin level and COVID-19 severity was then analyzed. A significant difference in the level of IgG antibodies against N and of IgM antibodies against the receptor binding domain of the S protein was observed between patients with nonsevere and severe COVID-19 symptoms, and the level of IgG antibodies against N was found to be higher in patients with severe symptoms whereas the level of IgM antibodies against the S peptides was higher in patients with nonsevere symptoms. The level of specific antibodies against SARS-CoV-2 structural proteins might correlate with COVID-19 severity. If so, this fact may be useful for predicting the prognosis of the disease and in determining the appropriate treatment with greater precision.

DEVELOPMENT of COVID-19 MONOCLONAL ANTIBODIES and RECOMBINANT PROTEINS AS REAGENTS for BIOMEDICAL RESEARCH and DIAGNOSTIC TESTS

Since SARS-COV-2 virus spread worldwide and COVID-19 turned rapidly into a pandemic illness, the necessity for vaccines and diagnostic tests became crucial. The viral surface is decorated with Spike, the major antigenic determinant and main target for vaccine development. Within Spike, the receptor binding domain (RBD), constitutes the main target of highly neutralizing antibodies found in COVID-19 convalescent plasma. Besides vaccination, another important aspect of Spike (and RBD) is their use as immunogen for the development of poli- and monoclonal antibodies (mAbs) for therapeutic and diagnostic purposes. Here we report the development and preliminary biochemical characterization of a set of monoclonal antibodies against the Spike RBD domain along with the recombinant expression of two mayor COVID-19 protein reagents: the viral Spike RBD domain and the extracellular domain of the human receptor ACE2. RBD and the extracellular domain of ACE2 (aa 1-740) were obtained through transient gene transfection (TGE) in two different mammalian cell culture systems: HEK293T adherent monolayers and Expi293F™ suspension cultures. Due to its low cost and ease scale-up, all transfections were carried with polyethyleneimine (PEI). Expressed proteins were purified from culture supernatants by immobilized metal affinity chromatography. Anti-RBD mAbs were developed from two different immunization schemes: one aimed to elicit antibodies with viral neutralizing potential, and the other with the ability to recognize denatured RBD for routine lab immunoassays. To achieve this, the first group of mice was immunized with RBD in aluminum salts (RBD/Al) and the other with RBD emulsified in Freunds adjuvant (RBD/FA). Polyclonal and monoclonal antibody reactivities against native or denatured RBD forms were then assessed by ELISA. Complete RBD denaturation was followed by intrinsic fluorescence spectral changes upon different physicochemical stress treatments. As expected, RBD/Al immunized mice developed an antibody response shifted to native RBD while those immunized with RBD/FA showed a high response against both forms of the protein. In accordance with the observed polyclonal response, RBD/FA derived mAbs recognize both, native and denatured RBD. On the contrary, hybridomas generated from the RBD/Al protocol mostly recognize RBD in its native state. Further ELISA binding assays revealed that all RBD/FA derived mAbs can form a trimeric complex with ACE2 and RBD, denoting they would not have viral neutralizing activity. ELISA competition assays with the RBD/ACE2 complex aimed to determine the neutralization potential of the RBD/Al derived mAbs are under way. Overall, the anti-Spike RBD mAbs and the recombinant RBD and ACE2 proteins presented here constitute valuable tools for diverse COVID-19 academic research projects and local immunity surveillance testing.