Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19

Abstract

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.

Long-term decay of anti-RBD IgG titers after BNT162b2 vaccination is not mirrored by loss of neutralizing bioactivity against SARS-CoV-2

“…Evidence from real-world data suggests that neutralizing antibodies (nAbs) to SARS-CoV-2 positively correlate with protection against SARS-CoV-2 infection and COVID-19 [3] , [4] , [5] , [6] by blocking the interaction of the Spike Receptor-Binding Domain (S-RBD) with the human cell receptor angiotensin converting enzyme 2 (hACE2) [7] . Thus, a number of binding immune assays and virus neutralization test (VNT) platforms have been implemented, cross-validated and eventually traced to a WHO International Standard [8] , in order to establish the usefulness of anti-SARS-CoV-2 antibodies as diagnostic tools or COP [9] , [10] , [11] , [12] , [13] . While quantitative binding assays only identify physical interactions and are mainly used for diagnostic purposes, functional neutralizing activity can only be directly investigated by means of VNTs platforms, that are grossly divided into direct VNTs, which require viable virions of SARS-CoV-2 and are considered the gold standard, and surrogate (s)VNTs, based on binding competition assays between antibodies and target receptors that mediate viral attachment and entry.…”

Clinical Application of Antibody Immunity Against SARS-CoV-2: Comprehensive Review on Immunoassay and Immunotherapy

“…Two common technologies used in antigen detection are lateral flow immunoassay (LFIA) and chemiluminescence immunoassay (CLIA) (Table 1), while antibody detection methods fall into four categories: laboratory tests for ELISA [12], CLIA [13], LFIA [14], and neutralization test [15][16][17]. The LFIA method detects antigen or antibodies by using colloidal gold test paper, which is simple in design, portable, rapid, and easy to interpret, but is limited to qualitative detection and lacks sensitivity [18].…”

“…The current gold standard method to measure nAb is the conventional virus neutralization test. In the test, virus and nAbs are mixed and incubated under appropriate conditions before testing the infection of the virus on the cell [15][16][17]. The FDA stated in one of the templates [22] that plaque reduction neutralization test (PRNT) [23] is currently considered to be the gold standard for detecting and measuring nAb titers.…”

“…Microneutralization assays and focus reduction neutralization testing are also accepted as comparable neutralization comparator methods. At the moment, the neutralization antibody assay is the gold standard for the diagnosis of antibodies and evaluation of the effectiveness of antibodies against COVID-19; however, the neutralization test requires a biosafety level 3 laboratory to manipulate the live pathogen [24] and a longer detection time; thus, it is far less popular than the methods listed above [15,17] and cannot be used for large-scale screening. Therefore, neutralization assays using pseudovirus [25] and surrogate virus [26] were developed.…”

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization

“…Eight institutions reported development or use of multiplex or protein arrays for antibody detection (Table 2). [27][28][29][30][31][32][33][34][35][36][37] Sample types include serum, plasma, DBS, saliva, and bronchoalveolar lavage (BAL) fluid. Diagnostic sensitivity and specificity for multiplex and protein array methods range from 85 -98.8 % and 95.2 -100 %, respectively.…”