key: cord-0255290-yljwir4u authors: Kung, Y.-A.; Huang, C.-G.; Huang, S.-Y.; Liu, K.-T.; Huang, P.-N.; Yu, K.-Y.; Yang, S.-L.; Chen, C.-P.; Cheng, C.-Y.; Lin, Y.-T.; Liu, Y.-C.; Chen, G.-W.; Shih, S.-R. title: Antibody titers measured by commercial assays are correlated with neutralizing antibody titers calibrated by international standards date: 2021-07-22 journal: nan DOI: 10.1101/2021.07.16.21260618 sha: 523171d3777304b04dd7bb874980309671d4bf79 doc_id: 255290 cord_uid: yljwir4u The World Health Organization (WHO) has highlighted the importance of an international standard (IS) for SARS-CoV-2 neutralizing antibody titer detection, with the aim of calibrating different diagnostic techniques. In this study, IS was applied to calibrate neutralizing antibody titers (IU/mL) and binding antibody titers (BAU/mL) in response to SARS-CoV-2 vaccines. Serum samples were collected from participants receiving the Moderna (n = 20) and Pfizer (n = 20) vaccines at three time points: pre-vaccination, after one dose, and after two doses. We obtained geometric mean titers of 1404.16 and 928.75 IU/mL for neutralizing antibodies after two doses of the Moderna and Pfizer vaccines, respectively. These values provide an important baseline for vaccine development and the implementation of non-inferiority trials. We also compared three commercially available kits from Roche, Abbott, and MeDiPro for the detection of COVID-19 antibodies based on binding affinity to S1 and/or RBD. Our results demonstrated that antibody titers measured by commercial assays are highly correlated with neutralizing antibody titers calibrated by IS. replicates. Geometric mean titers (GMTs) were calculated with 95% confidence 130 intervals (CIs) using GraphPad Prism version 8 (GraphPad Software, Inc., CA, USA). 131 132 ELISA 133 For indirect ELISA, a 96-well plate was coated with 2 μg/mL S1, RBD, and N protein 134 (Sino Biological, Beijing, China) diluted in phosphate-buffered saline and incubated 135 overnight at 4°C. Each well was blocked with 300 μL of StartingBlock™ T20 blocking 136 buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at 37°C. Serum from 137 vaccinated donors or NIBSC 20/136 standard was diluted in blocking buffer, and 100 138 μL of the sample was added to a 96-well plate, followed by incubation for 1 h at 37°C. 139 After washing, horseradish peroxidase (HRP)-tagged anti-human antibodies (Abcam, 140 Cambridge, UK), diluted 1:10,000 in blocking buffer, were added to the wells (100 141 μL/well), and the plate was incubated for 1 h at 37°C. Samples with N antibodies were 142 incubated for 30 min at 37°C. The chromogenic reagent 3,3,5,5-tetramethylbenzidine 143 (TMB) was mixed with an equal volume of Color A and B (R&D Systems, Minneapolis, 144 MN, USA). The TMB reaction time for S1 and RBD ELISA was 5 min and for N 145 protein ELISA was 10 min. After the reaction, stop solution (R&D Systems) was added 146 to the wells, and the absorbance was measured immediately at 450 nm using a Synergy 147 2 Microplate Reader (Bio-Tek, Winooski, VT, USA). 148 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Each serum sample was analyzed by the MeDiPro SARS-CoV-2 antibody ELISA, 150 Roche Elecsys ® Anti-SARS-CoV-2 S assay, and Abbott AdviseDx SARS-CoV-2 IgG 151 II assay, according to the manufacturers' instructions. The MeDiPro SARS-CoV-2 152 antibody ELISA detected antibodies against S1 and RBD, and values <34.47 IU/mL 153 were considered negative. The electrochemiluminescence immunoassay (ECLIA) (i.e., 154 Roche Elecsys Anti-SARS-CoV-2 S assay) was used for the detection of antibodies 155 against the RBD of S protein; <0.80 U/mL was considered negative and ≥0.80 U/mL 156 was considered positive for anti-SARS-CoV-2 S protein. The Abbott AdviseDx SARS-157 CoV-2 IgG II assay is a chemiluminescent microparticle immunoassay (CMIA) for the 158 detection of IgG antibodies to the RBD of S protein; the cut-off value was 50.0 AU/mL. 159 WHO IS sera (20/130, 20/136, and 20/268) (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Statistical analyses were performed using GraphPad Prism version 8 (GraphPad 169 Software, Inc., CA, USA). Pearson's correlation coefficients (r) were used to determine 170 the correlation between the titers obtained by the different serological assays and the 171 live SARS-CoV-2 NT assay. Statistical significance was set at P < 0.05. 172 173 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.16.21260618 doi: medRxiv preprint The neutralizing antibody titer is important for evaluating protection against viral 175 infection after vaccination. Dynamic neutralizing antibody titers were observed in 176 individuals who had been fully vaccinated. In detail, we obtained serum samples from reference for non-inferiority tests of candidate vaccines. 192 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10. 1101 /2021 Although the live virus neutralization assay is the gold standard for determining 193 neutralizing antibody titers, the operation is time-consuming and requires a BSL-3 194 laboratory. We developed binding assays to detect anti-S1, anti-RBD, and anti-N 195 antibodies, followed by NIBSC 20/136 (WHO IS) for conversion to BAU/mL ( Figure 196 2). Increases in anti-S1 and anti-RBD antibodies were observed after the first and 197 second doses (Figure 2A and B) . Interestingly, anti-N antibodies were detected in a few 198 individuals pre-and post-vaccination, suggesting that the individuals had COVID-19 199 before or after vaccination. 200 To evaluate whether our binding assay based on anti-S1 or anti-RBD in BAU/mL 201 reflects neutralizing antibody titers in IU/mL, the correlation between the two values 202 was determined, as shown in Figure 2C . Both the S1 and RBD antibody titers were 203 highly correlated with the NT titers (r = 0.9040 and 0.9298, respectively), suggesting 204 that the binding assays based on anti-S1 or RBD can be used as surrogates to measure 205 neutralizing antibodies. 206 All 120 serum samples were tested by commercial serological assays, including 207 the MeDiPro SARS-CoV-2 antibody ELISA, Roche Elecsys ® Anti-SARS-CoV-2 S, 208 and Abbott AdviseDx SARS-CoV-2 IgG II assay. Roche and Abbott serological assays 209 are widely used in clinical laboratories to detect SARS-CoV-2 antibodies worldwide. 210 They detect the antibody against the RBD of the S antigen, yielding qualitative and 211 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.16.21260618 doi: medRxiv preprint semi-quantitative results. MeDiPro is a Taiwan FDA-approved kit for quantifying S1-212 and RBD-binding antibodies; it assumes that data for S1 and RBD fusion proteins can 213 accurately predict the NT titer. We used real NT titers (IU/mL) from BSL-3 as a 214 standard to assess whether these serological assays reflect the neutralization titers based 215 on the detection of antibodies against S1 and/or RBD. The highest correlation was 216 observed between the titers obtained by the MeDiPro and the live SARS-CoV-2 NT 217 assays (r = 0.9111) ( Figure 3A ). Roche and Abbott RBD antibody titers also had good 218 correlation coefficients of 0.7294 and 0.8466, respectively ( Figure 3B and C). With 219 respect to the live virus microneutralization assays, the Roche and Abbott serological 220 assays can correctly detect RBD antibodies in all 77 NT-positive samples (Table 1) , 221 whereas MeDiPro produced six negative results. Among these six negative results, the 222 NT titer of four samples is equal to 34.47 IU/mL, which is the limit of the detection in 223 MeDiPro assay. Therefore, both the Roche and Abbot serological assays have 100% 224 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 CoV-2 neutralization antibody assay is an effective option for detecting SARS-Cov-2 231 neutralizing antibodies without requiring a live virus neutralization assay at a BSL-3 232 laboratory. 233 234 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.16.21260618 doi: medRxiv preprint Antibodies increase gradually within a few weeks after vaccination, and the timespan 236 may vary among individuals (13, 14) . Therefore, it is necessary to test neutralizing 237 antibodies to determine whether protective antibodies will be elevated after vaccination. 238 Vaccinated individuals may still have to take measures to avoid infection. Accordingly, 239 such assays are very important for protecting vaccinated individuals as well as for the 240 control and prevention of epidemics (15). 241 Anti-N antibodies may reveal whether vaccinated individuals were infected by the 242 virus before or after the vaccination dose (16). For example, as shown in Figure 2B , 243 one individual who received the Pfizer vaccine showed a dramatic increase in N to 556 244 BAU/mL. S1 for the same individual, after the first and second doses, were 2,094 and 245 1,240 BAU/mL, respectively. In the same individual, after the first and second doses, 246 the RBD levels were 1,618 and 927 BAU/mL, respectively. It has been reported that 247 prior infection with SARS-CoV-2 may boost B cells and significantly elevate antibody 248 production (17, 18). 249 Roche and Abbott Covid-19 diagnostic kits are being used extensively in clinical 250 laboratories (19-21). They are semi-quantitative and were originally designed to 251 confirm an infected case. In this study, we utilized standard sera to develop an approach 252 that utilizes these two kits to quantitate antibody titers after vaccination. The Pearson's 253 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 correlation coefficients (r) between these antibody titers and neutralization antibody 254 titers were 0.7294 and 0.8466, respectively. Titers obtained by MeDiPro, designed to 255 detect neutralizing antibody titers, were highly correlated with titers obtained by live 256 SARS-CoV-2 NT assays (r = 0.9111). (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 Distinct antibody and memory B cell responses in 352 SARS-CoV-2 naive and recovered individuals following mRNA vaccination A guide to vaccinology: from basic principles to 355 new developments Efficacy Needed for a COVID-19 Coronavirus Vaccine to Prevent or Stop an 359 Epidemic as the Sole Intervention Accuracy of a nucleocapsid protein antigen rapid test in the 362 diagnosis of SARS-CoV-2 infection TP+FN) 92.2% (84.0%-96.4%) 100% (95 100% (95 NPV=TN/(TN+FN) 87.0% (74.3%-93.9%) 100% All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.16.21260618 doi: medRxiv preprint All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 22, 2021. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 22, 2021. The responses of antibodies against S1, RBD, and N protein were detected in 120 serum 402 samples from 20 individuals receiving Moderna mRNA-1273 (A) and 20 individuals 403 All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101/2021.07.16.21260618 doi: medRxiv preprint 26 receiving Pfizer BNT162b2 vaccines (B). The geometric mean titers (GMTs) with 95% 404 CI are shown pre-vaccination, after the first dose, and after the second dose. (C) 405Correlation between the live virus neutralization titer (IU/mL) and antibody binding 406 unit (BAU/mL) in 120 serum samples. Vertical dashed lines indicate the limit of 407 detection (NT = 34.47). The Pearson's correlation coefficients (r) are provided for S1 408 or RBD antibody responses to live virus neutralization titer (IU/mL). 409 410 411 All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 22, 2021. ; https://doi.org/10.1101 https://doi.org/10. /2021