Exploration of low serum volume tests for SARS-CoV-2 antibodies


In a recent study published on medRxiv* preprint server, researchers have developed several non-commercial (in-house) low-volume serum assays to detect and characterize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies.

Study: Development and evaluation of low-volume assays to detect and characterize anti-SARS-CoV-2 antibodies. Image Credit: Kateryna Kon/Shutterstock

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.

About the study

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 results of the tests were compared with those of the neutralization tests based on the neutralizing titer of the antibody (ND50) values.


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.

For each antigen, Pan ELISAs performed better than IgG-specific tests in distinguishing COVID-19 cases from pre-pandemic controls. SARS-CoV-2 positive cases demonstrated greater area under the curve (AUC) after receptor operator characteristics (ROC) analyses.

In IgA and IgG isotype titers, improved discrimination between pre-pandemic and COVID-19 samples was observed using Nluc-labeled RBD and unlabeled RBD whereas those observed in IgM titers were not not optimal. As observed in Pan ELISAs, the S-RBD bridging assay showed better post-ROC AUC analysis to distinguish COVID-19 samples from controls. Despite the same antigen usage, RBD LIPS assays demonstrated better AUCs than RBD-specific ELISAs for all Ig isotypes.

In the threshold analysis, all tests accurately distinguished COVID-19 cases from pre-pandemic samples (AUC between 0.95 and 0.997). The S-RBD bridging LIPS assay demonstrated optimal and near-perfect performance (AUC = 0.997). All test thresholds showed >96% specificity. The S Pan ELISA demonstrated the highest specificity of 97.3%. In addition, the screening trials showed less intra-assay and inter-assay variation.

In sensitivity analysis, the S Pan ELISA and S-RBD bridging LIPS assays showed the highest sensitivities for detecting COVID-19 (between 92.4 and 95.7% for >21 days post-infection ). Compared to the commercial Roche test, the S-RBD Bridging LIPS test and the S Pan ELISA showed optimal sensitivity for the detection of COVID-19, > 93% sensitivity compared to 85.5% for the Roche test. The ELISA N Pan and RBD Pan tests showed a sensitivity of 76.97 and 76.3%, respectively. The strongest agreements were observed between the S and RBD Pan ELISA assays and between the IgG and total antibody assays (Bridging or Pan). Binding antibody unit (BAU)/ml values ​​in internal testing were comparable to the World Health Organization (WHO)/National Institute for Biological Standards and Control (NIBSC) reference.

In neutralization assays, the tests reasonably neutralized COVID-19 samples (ND50 >125) with results in the high positive range for S- and RBD-specific assays (e.g., >0.72 normalized OD on the S Pan assay and >20 units on the S-RBD Bridging assay). ELISA S IgA and IgG assays showed the strongest correlation with half-maximal neutralization titers.

Overall, the study results showed that low-volume assays are an economically viable option with high diagnostic accuracy for the detection and characterization of SARS-CoV-2 antibodies. Such tests would be of great benefit to low- and middle-income countries where commercial tests requiring 100 times higher serum volumes are not always feasible.

*Important Notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.


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