Exploring low serum volume assays for SARS-CoV-2 antibodies

In a recent study posted to the medRxiv* preprint server, researchers developed several non-commercial (in-house) low serum volume assays for detecting and characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies.

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

Antibody testing is important to assess SARS-CoV-2 seroprevalence, especially to identify asymptomatic cases among individuals residing in remote locations and low- and middle-income nations. Current coronavirus disease 2019 (COVID-19) diagnostic assays are expensive and technique-sensitive, warranting the need for cheap and simple tests for anti-SARS-CoV-2 antibodies.

About the study

In the present study, researchers developed low-serum volume (<10 µl) assays for COVID-19 diagnosis and optimizing discrimination between pre-pandemic controls and COVID-19 cases.

Serum samples were obtained from 984 pre-pandemic (known negative) individuals and from 269 suspected or polymerase chain reaction (PCR)-confirmed COVID-19 (known positive) patients.

The assays included total antibody and isotype-specific, standardized, low serum volume assays such as enzyme-linked immunosorbent assays (ELISA) for detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid (N) protein or spike (S) protein and its receptor-binding domain (RBD). They also developed anti-RBD isotype-specific luciferase immunoprecipitation system (LIPS) assays and a novel S-RBD bridging LIPS total-antibody assay for COVID-19 diagnosis.

For every antigen, ELISAs were performed to assess the total antibody or Pan Immunoglobulin G (IgG) titers using commercial anti-human IgG secondary antiserum assays that detect all the isotypes (Pan antibodies), or IgA, IgM, and IgG isotypes using class-specific antibodies. In addition, LIPS assays measuring RBD-specific antibodies were developed. To measure IgA and IgG isotypes, an unlabelled RBD competing with Nluc-labelled RBD was used. For the evaluation of total antibody titers with high affinity for RBD, the novel S-RBD bridging assay was used.

The assay 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 Index for achieving balanced specificity and sensitivity. The assay results were compared with that of neutralization assays based on the neutralizing titer of antibody (ND50) values.


In the study, the team developed 12 ELISAs that detected total antibodies or antibody isotypes to SARS-CoV-2 N, or S RBD. In the blind analysis, the S-RBD bridging LIPS and the S pan ELISA demonstrated >92% and >97% sensitivity and specificity, respectively among SARs-CoV-2-positive samples after 21 days of symptom onset or polymerase chain reaction (PCR) results.  

For every antigen, the Pan ELISAs performed better than the IgG-specific assays to distinguish COVID-19 cases from the pre-pandemic controls. SARS-CoV-2-positive cases demonstrated a greater area under the curve (AUCs) after the receiver operator characteristic (ROC) analyses.

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

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

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

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

Overall, the study findings 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 assays would be highly beneficial for low and middle-income nations where commercial assays requiring 100-times higher serum volumes may not always be feasible.

*Important notice

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

Journal reference:
Pooja Toshniwal Paharia

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Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.


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