Development and clinical evaluation of a rapid antibody lateral flow assay for the diagnosis of SARS-CoV-2 infection

Abstract

Background The novel coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has quickly spread worldwide since its outbreak in December 2019. One of the primary measures for controlling the spread of SARS-CoV-2 infection is an accurate assay for its diagnosis. SARS-CoV-2 real-time PCR kits suffer from some limitations, including false-negative results in the clinic. Therefore, there is an urgent need for the development of a rapid antibody test kit for COVID-19 diagnosis. Methods The nuclear capsid protein (N) and spike protein 1 (S1) fragments of SARS-CoV-2 were expressed in Escherichia coli, and rapid antibody-based tests for the diagnosis of SARS-CoV-2 infection were developed. To evaluate their clinical applications, the serum from COVID-19 patients, suspected COVID-19 patients, recovering COVID-19 patients, patients with general fever or pulmonary infection, doctors and nurses who worked at the fever clinic, and health professionals was analyzed by the rapid antibody test kits. The serum from patients infected with Mycoplasma pneumoniae and patients with respiratory tract infection was further analyzed to test its cross-reactivity with other respiratory pathogens. Results A 47 kDa N protein and 67 kDa S1 fragment of SARS-CoV-2 were successfully expressed, purified, and renatured. The rapid antibody test with recombinant N protein showed higher positive rate than the rapid IgM antibody test with recombinant S1 protein. Clinical evaluation showed that the rapid antibody test kit with recombinant N protein had 88.56 % analytical sensitivity and 97.42 % specificity for COVID-19 patients, 53.48 % positive rate for suspected COVID-19 patients, 57.14 % positive rate for recovering COVID-19 patients, and 0.5−0.8 % cross-reactivity with other respiratory pathogens. The analytical sensitivity of the kit did not significantly differ in COVID-19 patients with different disease courses (p < 0.01). Conclusions The rapid antibody test kit with recombinant N protein has high specificity and analytical sensitivity, and can be used for the diagnosis of SARS-CoV-2 infection combined with RT-PCR.

Influence of SARS-CoV-2 inactivation by different chemical reagents on the humoral response evaluated in a murine model

“…The viral antigens obtained after concentration and purification of SARS-CoV-2 inactivated by the different chemical reagents evaluated in this study were submitted to 10% SDS-PAGE and showed similar protein band profiles, suggesting homogeneity in the macrostructure of the viral proteins recovered after the different virus treatments, such as the suggestive molecular weight for the M protein of about 25 kda, the N-protein with about 46-58 kDa, the S2 subunit, which can be presented with 50.3-64.5 kDa, the subunit S1 around 67-85 kDa, as well as S-dimer around 180-200 kDa and S-trimer around 250 kDa ( Bosch et al, 2003 , Kuo et al, 2016 , Ke et al, 2020 , Li et al, 2021 ).…”

Update on Rapid Diagnostics for COVID-19: A Systematic Review

“…Among the 13 selected studies, 10 presented high values of sensitivity and specificity (>80%) [56][57][58]60,61,[64][65][66][67][68], and 1 reported 100% of sensitivity and specificity [56]. Lower sensitivity values were observed in the first days after infection [57,61,62,64] than the days postinfection.…”

“…Regarding immunoglobulin detection tests, we identified nine studies that assessed IgM and IgG levels [56][57][58][59][60][61][62][63][64], three evaluating only IgG [65][66][67], and one evaluating IgA, IgG, and IgM [68]. A total of 10 antibody tests were performed by lateral-flow assays (considering gold and fluorescence assays) [57][58][59][60][61][62][63][64][65]68], 1 by electrochemical enzyme immunoassay (ELISA) [56], 1 by magnetofluidic immuno-PCR platform [66], and 1 by the Split Luciferase (spLUC) antibody sensor [67].…”

“…Cross-reactions were analyzed in six studies for Mycoplasma pneumoniae [60,68], Toxoplasma gondii [60], Measles virus [60], Cytomegalovirus [60], HIV [60], Hepatitis B virus [60], Dengue virus [61,63], Typhoid fever [63], Influenza [67], patients exhibiting multiple autoantibodies [60], patients with respiratory tract infections [68], and patients with pulmonary tuberculosis and latent tuberculosis [61]. Other coronaviruses were tested only in the study by Elledge et al [67].…”

Evaluation of Simple Lateral Flow Immunoassays for Detection of SARS-CoV-2 Neutralizing Antibodies

“…However, detection of nAb is time-consuming and harbors a number of technical pitfalls [ 2 ]. Therefore, simplified and point-of-care accessible assays such as lateral flow immunoassays (LFIAs) might represent a valuable alternative [ 3 , 4 ]. The performance of these tests can vary greatly and should be examined in detail before use [ 5 , 6 ].…”