key: cord-0810014-c9b0q3g9
authors: DomBourian, Melkon G.; Annen, Kyle; Huey, Leah; Andersen, Gillian; Merkel, Patricia A.; Jung, Sarah; Dominguez, Samuel R.; Knight, Vijaya
title: Analysis of COVID-19 convalescent plasma for SARS-CoV-2 IgG using two commercial immunoassays
date: 2020-08-20
journal: J Immunol Methods
DOI: 10.1016/j.jim.2020.112837
sha: df61a98ca9b706e8725d0e9b3a736db20685925b
doc_id: 810014
cord_uid: c9b0q3g9

Coronavirus Disease 2019 (COVID-19) convalescent plasma (CCP) was approved by the FDA for use in severe cases of COVID-19 under an emergency Investigational New Drug (IND) protocol. Eligibility criteria for CCP donors includes documentation of evidence of COVID-19 either by viral RNA detection at the time of illness or positive SARS-CoV-2 IgG after recovery if diagnostic testing for COVID-19 was not performed at the time of illness. In addition to analysis of CCP, analysis of SARS-CoV-2 IgG provides information for possible past exposure and may support diagnosis when SARS-CoV-2 PCR is negative and clinical suspicion for COVID-19 is high. Furthermore, assays with high sensitivity and specificity for SARS-CoV-2 IgG are critical for understanding community exposure rates to SARS-CoV-2. Currently, there are several assays that test for antibodies to SARS-CoV-2 using a variety of methods, including point-of-care lateral flow-based devices, high throughput immunoassay analyzers, and manual methods such as ELISA. These assays target a number of SARS-CoV-2 antigens, including the nucleocapsid protein (N), full length spike protein (S), S1 subunit, or receptor binding domain (RBD) of the S protein. Given the heterogeneity among methods for, and antigenic targets used in SARS-CoV-2 antibody assays, it is necessary for careful evaluation of these assays prior to implementation for clinical use. We compared two assays that had received the CE mark of regulatory approval and that used either the N antigen or S1-RBD antigen as the target for analysis of a large set of CCP samples. Our data indicates that sensitivity and specificity vary between these assays and that more than one antigenic target may be required to improve the sensitivity and specificity of IgG detection to SARS-CoV-2.

that sensitivity and specificity vary between these assays and that more than one antigenic target may be required to improve the sensitivity and specificity of IgG detection to SARS-CoV-2.

Coronavirus disease 2019 , caused by the RNA virus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has rapidly spread worldwide since its discovery in December 2019 1 

World Health Organization (WHO) declared COVID-19 a pandemic in March 2020, and currently there are over 6 million cases and greater than 350,000 deaths globally 2 . Early in the pandemic, our facility established a collection program for the experimental COVID-19 intravenous therapy known as COVID-19 Convalescent Plasma (CCP), which is derived from individuals who have recovered from COVID-19.

Plasma antibodies from a recovered COVID-19 patient might shorten illness duration, reduce morbidity, and potentially prevent death when administered to a COVID-19 patient with active infection 3 . In addition, convalescent plasma has been used in previous viral epidemics such as those caused by SARS-CoV-1, Ebola, and Influenza A virus type H1N1 4-7 .

The United States Food and Drug Administration(FDA) authorized CCP for compassionate use on March 27, 2020 and provided guidance for CCP collection. The fundamental eligibility requirement is a confirmed positive SARS-CoV-2 PCR or serology test, with the most recent guidance mandating at least 14-days of COVID-19 recovery prior to donation 8 . Aside from assisting in eligibility determination, serological testing informs CCP donor management decisions related to continual eligibility as detectable antibody in donors would be of benefit to potential recipients. In addition to the analysis of CCP, SARS-CoV-2 antibody testing provides information related to past exposure that would be useful in epidemiological studies to understand disease seroprevalence as well as confirm vaccine response during clinical trials. In the patient care setting, as with many antibody tests for infectious disease, SARS-J o u r n a l P r e -p r o o f

CoV-2 antibody testing is not used for primary diagnosis. However, it may serve to support diagnosis in SARS-CoV-2 PCR-negative cases if the patients' viral loads are below the limit of detection in PCR testing, but there is a high clinical suspicion for COVID-19.

SARS-CoV-2 antibody analysis is evolving at a rapid pace with several assays coming to market, some having received CE-mark (European Economic Area) approval, and a few, more recently, having received FDA Emergency Use Authorization (EUA) 9, 10 . However, assay analytical evaluation and robust comparative data among assays is lacking. Currently, there are a number of methodologies including ELISA, automated immunoassay, and lateral flow-based point of care devices. These assays generally target one of the virus's four main structural proteins including the spike (S), small envelope (E), membrane (M) and nucleocapsid (N) glycoproteins 11 . The S protein has additional antigenic targets in the S1 domain and receptor binding domain (RBD) 12 . Here, we evaluated the analytical performance of two commercially available ELISA-based SARS-CoV-2 serological assays targeting different viral antigens utilizing a relatively large CCP donor sample set. (Table 1) ; and (c) de-identified samples that were collected prior to November 2019 (pre-pandemic samples).

Two commercial ELISAs, Epitope Diagnostics Inc. (EDI) (San Diego, CA) that is CE-marked and Euroimmun ELISA (Lubeck, Germany), that is both CE-marked and FDA EUA approved were compared in this study. The manufacturer's claims for sensitivity and specificity of these assays are shown in Table 2 . For this study, the assays were used per the manufacturers' specifications.

The Epitope Diagnostics Inc. (EDI) ELISA (San Diego, CA) utilizes the SARS-CoV-2 recombinant nucleocapsid antigen. Positive and negative assay controls, and samples diluted 1:100 with the kitspecific COVID-19 IgG sample diluent were added to the wells. Following a 30-minute incubation at room temperature, the plates were washed 5 times using the kit-specific wash buffer and anti-human IgG horseradish peroxidase (HRP)-conjugated detection antibody was added. The plate was incubated for 30 minutes at room temperature, followed by 5 washes, and addition of the substrate tetramethylbenzidine (TMB). The reaction was stopped with 0.5 M sulfuric acid after 20 minutes, and the plate was read at 450 nm within 10 minutes of halting the reaction.

Journal Pre-proof 2.3.2. The Euroimmun ELISA (Lubeck, Germany) assay utilizes the S1 domain, including the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. A kit-specific calibrator, positive and negative controls, and samples diluted 1:101 with the kit-specific dilution buffer were added to pre-coated wells.

Following a 1-hour incubation at 37°C, the plates were washed 3 times with wash buffer. Anti-human IgG-HRP conjugated detection antibody was added and the plates incubated for another 30 minutes at 37°C. Plates were then washed 3 times, and substrate TMB was added. Color development was halted after 30 minutes at room temperature with 0.5 M sulfuric acid, and the plate was read at 450 nm within 10 minutes of halting the reaction. J o u r n a l P r e -p r o o f 

A Novel Coronavirus from Patients with Pneumonia in China

Effectiveness of convalescent plasma therapy in severe COVID-19 patients

Use of convalescent plasma therapy in SARS patients in Hong Kong

Treatment with Convalescent Plasma for Influenza A (H5N1)

Use of Convalescent Whole Blood or Plasma collected from Patients recovered from Ebola Virus Disease for Transfusion, as an Empirical Treatment during Outbreaks. Interim Guidance for National Health Authorities and Blood Transfusion Services

jsessionid=C5 A4BB870D449DC36A9E2E2778725FB4?sequence=1 (accessed 05

Convalescent Plasma Treatment Reduced Mortality in Patients With Severe Pandemic Influenza A (H1N1) 2009 Virus Infection. device-exemption-ide-process-cber/recommendations-investigational-covid-19-convalescent-plasma

New York State Department of Health "Health Advisory: COVID-19 Serology Testing

Infectious Disease Society of America, 2020. IDSA COVID-19 Antibody Testing Primer

Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor