Exploring low serum volume assays for SARS-CoV-2 antibodies
Antibody testing is important to assess the seroprevalence of SARS-CoV-2, especially to identify asymptomatic cases among people residing in remote areas and low- and middle-income countries. Current diagnostic tests for coronavirus disease 2019 (COVID-19) are expensive and technically sensitive, justifying the need for simple and inexpensive tests for antibodies to SARS-CoV-2.
In the present study, researchers developed low serum volume assays (
Serum samples were obtained from 984 pre-pandemic individuals (known negatives) and 269 suspected or confirmed COVID-19 patients by polymerase chain reaction (PCR) (known positives).
Tests included standardized, low serum volume, total and isotype-specific antibody tests, such as enzyme-linked immunosorbent assays (ELISA) to detect total or antibody isotypes against nucleocapsid protein ( N) of SARS-CoV-2 or the spike (S) protein and its receptor binding domain (RBD). They have also developed anti-RBD isotype-specific luciferase immunoprecipitation system (LIPS) assays and a novel S-RBD bridging LIPS total antibody assay for the diagnosis of COVID-19.
For each antigen, ELISA tests were performed to assess total antibody or Pan IgG (IgG) titers using commercial anti-human IgG secondary antiserum assays that detect all isotypes (Pan antibodies), or IgA, IgM, and IgG isotypes using class-specific antibodies. Additionally, LIPS assays measuring RBD-specific antibodies have been developed. To measure IgA and IgG isotypes, an unlabeled RBD in competition with an Nluc-labeled RBD was used. For the evaluation of total antibody titers with high affinity for RBD, the new S-RBD bridging assay was used.
Dosage thresholds were determined based on three criteria: 1) 99th percentile of pre-pandemic controls; 2) 98th percentile of pre-pandemic controls and 3) highest Youden's index to achieve balanced specificity and sensitivity. The test results were compared to those of neutralization tests based on neutralizing titer antibody values (ND50).
In the study, the team developed 12 ELISAs that detected total antibodies or antibody isotypes against SARS-CoV-2 N or S RBD. In blinded analysis, S-RBD bridging LIPS and S pan ELISA demonstrated >92% and >97% sensitivity and specificity, respectively among SARs-CoV-2 positive samples after 21 days of onset symptoms or polymerase chain reaction (PCR) results.