key: cord-0695099-42n90433
authors: Bonelli, Fabrizio; Sarasini, Antonella; Zierold, Claudia; Calleri, Mariella; Bonetti, Alice; Vismara, Chiara; Blocki, Frank; Pallavicini, Luca; Chinali, Alberto; Campisi, Daniela; Percivalle, Elena; DiNapoli, Anna Pia; Perno, Carlo Federico; Baldanti, Fausto
title: Clinical And Analytical Performance Of An Automated Serological Test That Identifies S1/S2 Neutralizing IgG In Covid-19 Patients Semiquantitatively
date: 2020-05-20
journal: bioRxiv
DOI: 10.1101/2020.05.19.105445
sha: 0305cf8bca09e565d9522863245af670ebc7aa66
doc_id: 695099
cord_uid: 42n90433

BACKGROUND In the Covid-19 pandemic, highly selective serological testing is essential to define exposure to SARS-CoV-2 virus. Many tests have been developed, yet with variable speed to first result, and of unknown quality, particularly when considering the prediction of neutralizing capacity. OBJECTIVES/METHODS The LIAISON® SARS-CoV-2 S1/S2 IgG assay was designed to measure antibodies against the SARS-CoV-2 native S1/S2 proteins in a standardized automated chemiluminescent assay. Clinical and analytical performance of the test were validated in an observational study using residual samples (>1500) with positive or negative Covid-19 diagnosis. RESULTS The LIAISON® SARS-CoV-2 S1/S2 IgG assay proved highly selective and specific, and offers semiquantitative measures of serum or plasma levels of anti-S1/S2 IgG with neutralizing activity. The diagnostic sensitivity was 91.3% and 95.7% at >5 or ≥15 days from diagnosis respectively, and 100% when assessed against a neutralizing assay. The specificity ranged between 97% and 98.5%. The average imprecision of the assay was <5 % coefficient of variation. Assay performance at 2 different cut-offs was evaluated to optimize predictive values in settings with different % disease prevalence. CONCLUSIONS. The automated LIAISON® SARS-CoV-2 S1/S2 IgG assay brings efficient, sensitive, specific, and precise serological testing to the laboratory, with the capacity to test large amounts of samples per day: first results are available within 35 minutes with a throughput of 170 tests/hour. The test also provides a semiquantitative measure to identify samples with neutralizing antibodies, useful also for a large scale screening of convalescent plasma for safe therapeutic use. IMPORTANCE With the worldwide advance of the COVID-19 pandemic, efficient, reliable and accessible diagnostic tools are needed to support public health officials and healthcare providers in their efforts to deliver optimal medical care, and articulate sound demographic policy. DiaSorin has developed an automated serology based assay for the measurement of IgG specific to SARS CoV-2 Spike protein, and tested its clinical performance in collaboration with Italian health care professionals who provided access to large numbers of samples from infected and non-infected individuals. The assay delivers excellent sensitivity and specificity, and is able to identify samples with high levels of neutralizing antibodies. This will provide guidance in assessing the true immune status of subjects, as well as meeting the pressing need to screen donors for high titer convalescent sera for subsequent therapeutic and prophylactic use.

(95% CI 94.1% -99.1%). The relationship between the LIAISON ® SARS-CoV-2 S1/S2 151 IgG assay and NT assay-negative or NT assay-positive samples portrays a nearly 152 complete separation between the 2 groups with medians of 2.4 AU/mL (95% CI 2.2 to 153 2.6 AU/mL), and 61.8 AU/mL (95% CI 50.3 to 70.7 AU/mL), respectively ( Figure 4 ). In 154 Figure 5A , the LIAISON ® SARS-CoV-2 S1/S2 IgG assay's measurements were 155 separated into 3 semi-quantitative groups (<40 AU/mL, 40-80 AU/mL, and >80 AU/mL) 156 and related to NT assay titers ≥1:160, which is the threshold recommended by the FDA 157 guidelines for use in convalescent blood transfusion. 39% (17/43), 56% (24/43), and 158 87% (33/38) of the samples, respectively, had NT assay titers ≥1:160 (5). Furthermore, 159 since the FDA guidelines also admit NT assay titers of ≥1:80 as acceptable, additional 160 leeway is granted towards use of the LIAISON ® SARS-CoV-2 S1/S2 IgG assay to pre-161 screen or assess blood donor samples for potential convalescent plasma/serum 162 therapy: 92% (35/38) and 79% (34/43) of the > 80 AU/mL and 40-80 AU/mL groups, 163 respectively, had NT assay titers ≥1:80 ( Figure 5B ). 164 Analytical Performance 165 The LIAISON ® SARS-CoV-2 S1/S2 assay was evaluated for intra-assay 166 imprecision using 6 samples with moderate, low, or negative S1/S2 IgG levels. The 167 average intra-assay imprecision was 2.8 %CV (range 2.0-3.4%CV), and total-assay 168 imprecision averaged 3.2%CV (range 2.7-3.9%CV) ( Table 5) . 169 Cross-reactivity with other coronaviruses was tested against 10 patient samples 170 positive by their respective RT-PCR tests to other coronaviruses that maintained 171 negative NT assay results by SARS-Cov-2. Their LIAISON values ranged from 1.81 to and 15 AU/mL indicating the absence of cross-reactivity with the other coronaviruses 174 tested (Table 6 ). Additionally, cross-reactivity was assessed in samples from patients 175 with conditions caused by other viruses, other organisms, or with atypical immune 176 system activity. As shown in Table 7 , 3 out of 160 assessed specimens (1.9%) resulted 177 positive with the LIAISON ® SARS-CoV-2 S1/S2 IgG assay. Potentially interfering 178 substances such as triglycerides (3000 mg/dL), cholesterol (400 mg/dL), hemoglobin 179 (1000 mg/dL) conjugated and unconjugated bilirubin (40 mg/dL), acetaminophen (500 180 mg/mL and ibuprofen (500 mg/mL) showed no interference at the indicated 181 concentrations. The LIAISON ® SARS-CoV-2 S1/S2 IgG assay demonstrated a negative 182 bias up to a 16% in S1/S2 IgG-positive specimens with biotin concentrations above 183 3500 ng/mL, a concentration 15-fold higher than that induced following ingestion of a 20 184 mg/day biotin supplement (6). Envelope, Nucleocapsid and Spike that are referred to as M, E, N and S, respectively. 194 While N protein elicits cell mediated immunity attributable to two predominant CD8 T 195 cell epitopes (9), of the remaining three structural proteins, S protein is widely recognized as that most specific with regard to generating protective, neutralizing 197 antibodies (10, 11).

The specificities reported from in vitro diagnostic immunoassays (12-14) are 199 impacted greatly by the fidelity of preservation of both linear and conformational 200 epitopes of the given analyte being measured for presentation to specific 201 immunoglobulins within a patient's serum sample (here SARS-Cov-2 S1/S2). Specificity plates. This procedure induces significant structural deformation and denaturation, with 206 the consequent loss of native conformation, as well as occlusion of access to both 207 conformational and linear epitopes beneath the protein stuck to the plastic titer plate's 208 surface. As explained in Materials and Methods, our system allows for optimal 209 maintenance of Spike protein conformation. Consequently, the LIAISON ® SARS-CoV-2 210 S1/S2 IgG assay rendered no false positive results from NT assay-negative, RT-PCR-211 positive samples for related coronavirus members, and its performance is sensitive, 212 specific, and precise as evaluated in >1500 samples.

The use of convalescent serum to treat subjects with acute SARS-Cov-2 has 214 growing appeal for meeting the immediate challenges being imposed upon increasingly CoV-2 S1/S2 IgG assay was designed to detect IgG with neutralizing potential, and is 218 shown here to have very good sensitivity and specificity in identifying samples with positive neutralization titers. Furthermore, if used in a semi-quantitative manner, higher 220 LIAISON units are indicative of higher NT assay titers, and provide a pre-screen tool to 221 assess large numbers of samples. While neutralization tests provide the recognized 222 benchmark, they are not practical for implementation on a large scale screening basis, 223 due to requirements for high biosecurity containment laboratories, and the need for 224 highly trained personnel to execute labor-intensive protocols. With our system, clear 225 separation of NT assay-negative samples from NT assay-positive samples was 226 achieved. In fact, with 40-80 AU/mL levels measured by the LIAISON ® SARS-CoV-2 227 S1/S2 IgG assay, the probability to have neutralization titers ≥1:80 and >1:160 was 79% 228 and 56%, while with >80 AU/ml the probability of having neutralization titers >1:80 and 229 >1:160 was 92%, and 87%, respectively. This may be useful for the efficient screening 230 of convalescent plasma for safe therapeutic use.

The LIAISON ® SARS-CoV-2 S1/S2 IgG assay's sensitivity increases significantly 232 as the immune response matures, as one would expect for an IgG-based serology 233 assay's assessment of a host response to viral infection (Table 1 & 2, and Figure 3 ).

Here, sensitivities of 33.3% at <5 days but >91% at ≥5 days post admission on samples 235 from 104 Italian patients whose RT-PCR tests were positive at the time of diagnosis are 236 reported. Serology tests are now being utilized to gain an initial assessment of infection 237 prevalence with reported numbers of ~20% and ~3% from New York and California, 238 respectively (19). In California, a negative test with the LIAISON assay would have an 239 accompanying NPV of >99.5%, and in NYC a NPV of >97.5%, regardless of cut-off, 240 indicating that staying at home and avoiding exposures would be the best PPV of 80% derived from the higher cut-off, though overall less sensitive, would provide 243 a positive test result, affording more confidence of the subject's true positivity, while the 244 lower cut-off would present some ambiguity as regards any subject's real level of 245 protection (PPV of 62%). In New York, however, regardless of the cut-off, the PPV of 246 89-95% affords a much greater degree of confidence that an individual would have 247 protective levels of antibody. When testing in a hospital setting, the lower cut-off may be 248 preferable to ensure a higher NPV, even though the overall specificity may be 249 decreased.

In conclusion the automated LIAISON ® SARS-CoV-2 S1/S2 IgG assay brings 251 efficient, sensitive, specific, and precise serological testing to the laboratory. Further, 252 the assay is amenable for semi-quantitative efficient pre-screening of samples for 253 neutralizing antibody content, to be used in convalescent plasma therapy. 

Assay format. The LIAISON ® SARS-CoV-2 S1/S2 IgG chemiluminescent assay 257 is a recently developed assay from DiaSorin designed to detect IgG antibodies in the 258 serum or plasma of subjects and patients exposed to the SARS-CoV-2 virus. The assay 259 consists of paramagnetic microparticles (PMPs) coated with S1 and S2 fragments of the susceptible to significant denaturation consequent to passive adsorption to these 266 hydrophobic surfaces (23, 24). Distally biotinylated-S1 and biotinylated-S2 proteins 267 were tethered to the surface of paramagnetic particles coated with streptavidin to 268 assure optimal presentation of both S1 and S2 for access and recognition by specific 269 immunoglobulin within pathologic serum samples.

The automated assay format consists of a first incubation step (10 minutes ibuprofen and biotin were assessed with the LIAISON ® SARS-CoV-2 S1/S2 IgG assay. RT-PCR admitted to the hospital or ICU were tested with the LIAISON ® SARS-CoV-2 509 S1/S2 IgG assay. A value of 9 AU/mL was used as the cut-off for positivity. Thresholds for the LIAISON ® SARS-CoV-2 S1/S2 IgG Assay at Cut-offs of 9 and 15 517 AU/mL 518 LIAISON ® SARS-CoV-2 S1/S2 IgG Assay

Cut-off PPV 95% CI NPV 95% CI HCoV-HKU1 1 0

HCoV-229E 1 0

HCoV-untyped strain 4 0

Total 10 0 IgG: immunoglobulin G

Using 9 and 15 AU/mL as Cut-offs from Samples with Known Neutralizing Titers 521 Defined as Negative (< 1:40) or Positive for Neutralizing Antibodies

Specificity 98.3% 95