Development of an Effective Neutralizing Antibody Assay for SARS-CoV-2 Diagnosis.

Introduction: Neutralizing antibodies (NAbs) are essential for preventing reinfection with SARS-CoV-2 and the recurrence of COVID-19; nonetheless, the formation of NAbs following vaccination and infection remains enigmatic due to the lack of a practical and effective NAb assay in routine laboratory settings. In this study, we developed a convenient lateral flow assay for the rapid and precise measurement of serum NAb levels within 20 minutes. Methods: Receptor-binding domain-fragment crystallizable (RBD-Fc) and angiotensin-converting enzyme 2-histidine tag (ACE2-His) were expressed by the eukaryotic expression systems of Spodoptera frugiperda clone 9 and human embryonic kidney 293T, respectively. Then, colloidal gold was synthesized and conjugated with ACE2. After optimizing various operating parameters, an NAb lateral flow assay was constructed. Subsequently, its detection limit, specificity, and stability were systematically evaluated, and clinical samples were analyzed to validate its clinical feasibility. Results: RBD-Fc and ACE2-His were obtained with 94.01% and 90.05% purity, respectively. The synthesized colloidal gold had a uniform distribution with an average diameter of 24.15 ± 2.56 nm. With a detection limit of 2 µg/mL, the proposed assay demonstrated a sensitivity of 97.80% and a specificity of 100% in 684 uninfected clinical samples. By evaluating 356 specimens from infected individuals, we observed that the overall concordance rate between the proposed assay and conventional enzyme-linked immunosorbent assay was 95.22%, and we noticed that 16.57% (59/356) of individuals still did not produce NAbs after infection (both by ELISA and the proposed assay). All the above tests by this assay can obtain results within 20 minutes by the naked eye without any additional instruments or equipment. Conclusion: The proposed assay can expediently and reliably detect anti-SARS-CoV-2 NAbs after infection, and the results provide valuable data to facilitate effective prevention and control of SARS-CoV-2. Clinical trial registration: Serum and blood samples were used under approval from the Biomedical Research Ethics Subcommittee of Henan University, and the clinical trial registration number was HUSOM-2022-052. We confirm that this study complies with the Declaration of Helsinki.

Research progress in methods for detecting neutralizing antibodies against SARS-CoV-2.

The emergence of SARS-CoV-2 has seriously affected the lives of people worldwide. Clarifying the attenuation rule of SARS-CoV-2 neutralizing antibody (NAb) in vivo is the key to prevent reinfection and recurrence of virus. Currently, the commonly used methods for detecting NAb include virus neutralization tests, pseudovirus neutralization assays, lateral flow immunochromatography and enzyme-linked immunosorbent assays. The detection of NAb not only can be used to evaluate the level of immunity after vaccination or infection but also can provide important theoretical support for virus reinfection, recurrence and vaccine iteration. In this research, the related technologies of SARS-CoV-2 NAb detection were reviewed, aiming to provide better research ideas for SARS-CoV-2 epidemic prevention and control.

Rapid Detection of Anti-SARS-CoV-2 Antibody Using a Selenium Nanoparticle-Based Lateral Flow Immunoassay.

Coronavirus disease 2019 is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is highly transmissible. Early and rapid testing is necessary to effectively prevent and control the outbreak. Detection of SARS-CoV-2 antibodies with lateral flow immunoassay can achieve this goal. In this study, SARS-CoV-2 nucleoprotein (NP) was expressed and purified. We used the selenium nanoparticle as the labeling probe coupled with the NP to prepare an antibody (IgM and IgG) detection kit. The detection limit, cross reaction, sensitivity and specificity of the kit is verified. Separate detection of IgM and IgG, such as in this assay, was performed in order to reduce mutual interference and improve the accuracy of the test results.The final purity of NP was 91.83%. Selenium nanoparticle and NP successfully combined with stable effect. The LOD of the kit was 20 ng/mL for anti-NP IgG and 60 ng/mL for anti-NP IgM, respectively. The kit does not cross reaction with RF. The sensitivity of the kit was 94.74% and the specificity was 96.23%. The assay kit does not require any special device for reading the results and the readout is a simple color change that can be evaluated with the naked eye. This kit is suitable for rapid and real-time detection of the SARS-CoV-2 antibody IgG and IgM.

Rapid detection of human heart-type fatty acid-binding protein in human plasma and blood using a colloidal gold-based lateral flow immunoassay.

The incidence of acute myocardial infarction (AMI) is currently increasing. Early detection is important for the treatment and prognosis of patients with AMI. Heart-type fatty acid-binding protein (H-FABP) may be used as an early marker of AMI due to its high sensitivity, specificity and prognostic value. Therefore, in the present study, H-FABP was used as a biomarker in a double-antibody sandwich method and colloidal gold-based lateral flow immunoassay to develop a rapid detection kit for H-FABP with a processing time of only 5 min. The sensitivity of the kit in plasma and whole blood was 1 ng/ml and this method had good specificity, exhibiting no cross-reaction with cardiac troponin I, myoglobin or creatine kinase-Mb. The kits had good shelf life and stability, as they were able to be stored at 40˚C for 30 days. A total of 12 clinical samples were collected for detection and the coincidence rate with the ELISA method was up to 91.67%. Therefore, the present study provided a simple, rapid and economical early-detection in-home testing kit.

Rapid Detection of Ractopamine and Salbutamol in Swine Urine by Immunochromatography Based on Selenium Nanoparticles.

PURPOSE: The purpose of this study was to establish a lateral flow immunoassay using selenium nanoparticles (Se-NPs) as a probe to detect ractopamine (RAC) and salbutamol (SAL) in swine urine. METHODS: SDS and PEG were used as templates to prepare Se-NPs; anti-RAC monoclonal antibodies or anti-SAL monoclonal antibodies were labelled with Se-NPs; and rapid detection kits were prepared. The sensitivity, specificity, and stability were measured, and actual samples were analysed. RESULTS: The Se-NPs were spherical with a diameter of 40.63 ± 5.91 nm, and were conjugated successfully with an anti-RAC antibody to give a total diameter of 82.33 ± 17.91 nm. The detection limit of a RAC kit in swine urine was 1 ng/mL, and that of a SAL kit was 3 ng/mL. Both procedures could be completed within 5 minutes. No cross-reaction occurred with clenbuterol, bambuterol and phenylethanolamine A. Samples were tested consistently across different batches of kits for swine urine. The results of the kits were identical to those of actual clinical samples analysed by ELISA, and the coincidence rate was 100%. CONCLUSION: The assay kit does not require any special device for reading the results, and the readout is a simple colour change that can be evaluated with the naked eye. It is easy to operate, sensitive, specific, and stable This kit is suitable for the rapid and real-time detection of RAC and SAL residues in swine urine samples. CLINICAL TRIAL REGISTRATION: Swine urines samples were used under approval from the Experimental Animal Ethics committee of the Joint National Laboratory for Antibody Drug Engineering, Henan University.

A point-of-care selenium nanoparticle-based test for the combined detection of anti-SARS-CoV-2 IgM and IgG in human serum and blood.

COVID-19 is a widespread and highly contagious disease in the human population. COVID-19 is caused by SARS-CoV-2 infection. There is still a great demand for point-of-care tests for detection, epidemic prevention and epidemiological investigation, both now and after the epidemic. We present a lateral flow immunoassay kit based on a selenium nanoparticle-modified SARS-CoV-2 nucleoprotein, which detects anti-SARS-CoV-2 IgM and anti-SARS-CoV-2 IgG in human serum, and the results can be read by the naked eye in 10 minutes. We expressed and purified the SARS-CoV-2 nucleoprotein in HEK293 cells, with a purity of 98.14% and a concentration of 5 mg mL-1. Selenium nanoparticles were synthesized by l-ascorbic acid reduction of seleninic acid at room temperature. After conjugation with the nucleoprotein, a lateral flow kit was successfully prepared. The IgM and IgG detection limits of the lateral flow kit reached 20 ng mL-1 and 5 ng mL-1, respectively, in human serum. A clinical study sample comprising 90 COVID-19-diagnosed patients and 263 non-infected controls was used to demonstrate a sensitivity and specificity of 93.33% and 97.34%, respectively, based on RT-PCR and clinical results. No cross-reactions with rheumatoid factor and positive serum for anti-nuclear antibodies, influenza A, and influenza B were observed. Moreover, the lateral flow kit remained stable after storage for 30 days at 37 °C. Our results demonstrate that the selenium nanoparticle lateral flow kit can conveniently, rapidly, and sensitively detect anti-SARS-CoV-2 IgM and IgG in human serum and blood; it can also be suitable for the epidemiological investigation of COVID-19.