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.