Elecsys anti sars cov 2 n

Serum SARS‐CoV‐2‐RBD‐total antibodies and nucleocapsid (N)‐reactive IgG antibodies were measured using the Roche Elecsys® Anti‐SARS‐CoV‐2 S and Elecsys® Anti‐SARS‐CoV‐2 N assays (Roche Diagnostics), respectively. Neutralizing antibodies (NtAb) targeting the S protein were measured using a GFP‐expressing vesicular stomatitis virus pseudotyped with the Wuhan‐Hu‐1 and Omicron BA.1 and BA.2 variants, as previously described¹⁰ (Supplementary Material). SARS‐CoV‐2‐S specific‐IFNγ‐producing CD4⁺ and CD8⁺ T cells were enumerated by whole‐blood flow cytometry for intracellular cytokine staining ICS (BD Fastimmune, Becton Dickinson and Company Biosciences, San Jose, CA), as previously reported¹⁰, ¹¹ (Supplementary Material).

Anti-N seropositive was investigated using the electrochemiluminescence immunoassay (ECLIA) (Elecsys® Anti-SARS-CoV-2 N, Roche Diagnostics GmbH, Mannheim, Germany) to detect anti-N total Ig against the original Wuhan strain on the Roche Cobas e411 platform. The results of anti-N total Ig antibodies were evaluated using the manufacturer’s initial cut-off index (COI) of 1.0. The number of infections was estimated based on the number of individuals who tested positive for total anti-N Ig and/or had a history of COVID-19 infection.

SARS-CoV-2 neutralization titers, expressed as the ID₅₀, were assessed using pseudotyped lentiviruses presenting SARS-CoV-2 spike mutations for the D614G (Wuhan-1 containing a single D614G spike mutation), B1.617.2 (Delta), B.1.351 (Beta) and B.1.1.529 (Omicron BA.1) variants, as described previously^{22 (link),41 (link)}. A random subset of samples (25 per selected vaccine arm, distributed equally between age strata and sites) was analyzed for neutralization titers to the Omicron BA.4/BA.5, BA.2.12.1, BA.2.75.2, BA.2.75, BA.4.6, BF.7 and BQ.1.1 subvariants in a separate laboratory^{42 (link)}. Electrochemiluminescence immunoassays were used for the detection of anti-N (Elecsys Anti-SARS-CoV-2 N; Roche) at baseline^{41 (link)}.

The antibodies anti-S are produced as a consequence of both natural infection and vaccination by the BNT162b2 vaccine that induces the production of the antigen S. In order to discriminate these conditions, the presence of antibodies anti-N was analyzed since it is directly linked to the SARS-CoV-2 infection but not to the vaccination. In this study, the Elecsys^® Anti-SARS-CoV-2 N (Roche Diagnostics GmbH, Mannheim, Germany), which uses a recombinant protein representing the N antigen in a double-antigen sandwich assay format, was used to identify subjects with a history of previous SARS-CoV-2 infection and to exclude immunization due to infection rather than vaccination. This test showed 97.2% sensitivity and 99.8% specificity for detecting preceding SARS-CoV-2 infection [20 (link)].

Electro-chemiluminescence immunoassay (ECLIA) was used to quantify Ig anti-N (Elecsys® Anti-SARS-CoV-2 N, Roche) and anti-RBD (Elecsys® Anti-SARS-CoV-2 S, Roche), ran (on cobas e602) and analysed as per the manufacturer instructions, with a threshold defining positivity at index value = 0.8 U/ml. Where indicated, samples with a value > or =2500 in the standard anti-RBD ECLIA assay were measured again after a 50x dilution. Direct ELISA was used to quantify IgG, IgM and IgA anti-full-length spike. The assay was adapted from Amanat et al.^{30 (link)}. and semi-automized to measure IgM, IgG and IgA in 384-well format, according to a protocol to be detailed elsewhere. Assay performance was determined by testing 1000 pre-pandemic sera and 40 COVID-19 patients diagnosed at least 10 days prior to sera collection. ROC curve analysis determined a specificity of 99.3%, 99.2%, 99.2%, and a sensitivity of 95.9%, 61.2%, and 73.7% for IgG, IgM, and IgA, respectively. The threshold defining positivity correspond to normalized OD (ODnorm) = 1. Serial titration of 67 COVID-19 patients established the assay has a dynamic range of 3 logs titre.

Anti-spike (S) and anti-nucleocapsid (N) antibodies were measured using the Roche Elecsys® Anti-SARS-CoV-2 S and Roche Elecsys® Anti-SARS-CoV-2 N assays at the Public Health England (now the United Kingdom Health Security Agency) Laboratories at Porton Down, UK. The Roche S assay is reported in units per milliliter (U/ml), which are standardized 1:1 to the WHO binding antibody units/ml (BAU/ml). Seroconversion is defined for S as a response equal to or greater than 0.8 U/ml, and for N as a response equal to or greater than 1.0 COI.