key: cord-0707763-gkrwp3pd authors: Ko, Jae-Hoon; Joo, Eun-Jeong; Baek, Jin Yang; Huh, Kyungmin; Cho, Sun Young; Kang, Cheol-In; Chung, Doo Ryeon; Kim, Yae-Jean; Kang, Eun-Suk; Peck, Kyong Ran title: Evaluation of Six Anti-SARS-CoV-2 Antibody Test Kits and Practical Approaches to Optimize the Diagnostic Performance date: 2021-03-26 journal: J Microbiol Immunol Infect DOI: 10.1016/j.jmii.2021.03.008 sha: 1d94b62626e9199cb173c6d2c9966f734dc129be doc_id: 707763 cord_uid: gkrwp3pd In an investigation of six anti-SARS-CoV-2 antibody kits with different target antigen and methodology, each kit showed comparable performance. As false-positive reactions occurred independently with different kits, specificity increased to 100% when pairs of kits were used. With three-kit combination, both sensitivity (99.1%) and specificity (100%) increased. The coronavirus disease 2019 (COVID-19) pandemic due to severe acute respiratory syndrome 2 coronavirus 2 (SARS-CoV-2) infection has not been successfully controlled in most countries 3 worldwide 1 . Reverse transcriptase polymerase chain reaction (RT-PCR) test is the gold standard 4 method for the diagnosis of SARS-CoV-2 infection, while serologic antibody tests using 5 immunoassay have been utilized for sero-prevalence monitoring, risk assessment of healthcare 6 workers (HCWs), and convalescent plasma therapy 2-4 . Nevertheless, serologic tests have inherent 7 limitations as a diagnostic method: approximately seven days are required for seroconversion, a gold 8 standard method is absent, and the diagnostic performance is not as high as that of RT-PCR 2,3,5 . 9 Various formats of commercial antibody detection kits including enzyme-linked immunosorbent assay 10 (ELISA), fluorescence immunoassay (FIA), and lateral flow immunoassay (LFIA) for rapid 11 diagnostic test (RDT) have been being introduced, but the performance of these kits needs to be 12 further evaluated. Herein, we present the practical strategies to optimize the utilization of anti-SARS- 13 CoV-2 antibody test using multiple kits from different manufacturers. 16 Sera collected after more than seven days of illness from RT-PCR-confirmed asymptomatic to severe 17 COVID-19 patients were used as positive specimens 2 . In asymptomatic patients, illness days were 18 counted from the date of diagnosis. Sera from patients who had recovered from conventional 1 protein (NCP)), two FIA kits using europium particles (SD Biosensor Inc., detecting IgM and IgG 2 antibodies against NCP; and Boditech Med Inc., Chuncheon, Korea, detecting IgM and IgG 3 antibodies against NCP), and three ELISA kits (SD Biosensor Inc. detecting total antibody against 4 NCP and the receptor binding domain (RBD) of the spike protein; PCL Inc, Seoul, Korea, detecting 5 total antibody against NCP and RBD; and EUROIMMUN, Lübeck, Germany, detecting IgG antibody 6 against RBD) were used. Details of study specimens and anti-SARS-CoV-2 antibody kits are 7 presented in the Supplementary materials. This study was approved by the Institutional Review Board 8 of Samsung Medical Center. Tested serum samples 11 A total of 110 serum specimens from 74 RT-PCR-confirmed COVID-19 patients were subjected to 12 SARS-CoV-2 antibody tests. Sixty-four convalescent sera from asymptomatic and mild patients, 14 13 serial sera from three mild patients, and 32 serial sera from seven moderate to severe patients were 14 included. For negative controls, 119 serum specimens were collected including 42 convalescent sera 15 from patients infected with conventional respiratory viruses and 77 sera from HCWs. 17 Test performance of anti-SARS-CoV-2 antibody test kits, including sensitivity, specificity, positive 18 predictive value (PPV), and negative predictive value (NPV) are presented in Table 1 IgG). Although the sensitivity of the IgM band was low, the sensitivity of the IgG band was highest among the tested kits with similar specificity. The ELISA kits targeting total antibody showed high sensitivity and specificity. The ELISA kit 2 from SD Biosensor showed sensitivity/specificity of 96.3/100% and that of the PCL Inc kit was 3 98.2/100%. The ELISA kit targeting IgG (EUROIMMUN) showed sensitivity/specificity of 95.4/96.6 4 (using a cut-off value of borderline) and 93.5/100% (using a cut-off value of positive). IgG band of the SD Biosensor FIA kit. As the RDT kit and FIA kit from SD Biosensor used the same 10 product materials, false-positive reactions were shared between these kits. One specimen showed a 21 Based on the findings of independent false-positive reactions between test kits, we evaluated the 22 performance of test kits in combination (Supplementary Table 2 -3). The specimen was interpreted as 23 positive if two or more kits produced a positive result. If all the test kits yielded negative results or 24 only one kit gave a positive result, the specimen was interpreted as negative. When two different kinds of serologic kits were used in combination, sensitivity was the same or 26 J o u r n a l P r e -p r o o f tested. Second, even if the kits were manufactured by different companies, cross-reactive false 1 positivity may occur if the kits share the same recombinant virus antigen, reagent, or buffer. Third, the 2 use of multiple kits may improve performance but the cost of testing inevitably increases. Selection of 3 tests kits should be decided according to local prevalence, the purpose of testing, and the expected 4 cost. Lastly, the present study data reflects diagnostic performance of sero-converted patients, 5 evaluating serial sera of COVID-19 patients collected after seven days of illness (46 sera from 10 6 patients, median 24 days of illness) and convalescent sera after recovery (64 sera from 64 patients, 7 median 40.5 days of illness). Diagnostic performance of antibody test kits for acute SARS-CoV-2 8 infection would be considerably different. It should be further investigated whether measurement of 9 different target proteins and immunoglobulin classes would be useful in diagnosing acute SARS-CoV-10 2 infection and differentiating patients with acute, recent, past, and re-infections. 11 In conclusion, in the present analysis of anti-SARS-CoV-2 antibody test kits, false-positive 12 reactions occurred independently between test kits. Using a combination of two different kits 13 increased specificity to 100% and combination of more than two kits further increased performance. We would like to express our sincerest condolences to the patients and families who suffered from the 3 COVID-19 outbreak. We greatly appreciate the patients and HCWs who voluntarily participated in 4 this study. Finally, we would like to thank SD Biosensor Inc., Boditech Med Inc., and PCL Inc. for the 5 donation of their antibody test kits. A total of 110 RT-PCR confirmed positive specimens and 119 negative control specimens were used, otherwise indicated. * 109 positive specimens were used. † 108 positive specimens and 118 negative control specimens were used. ‡ 108 positive specimens and 117 negative control specimens were used. standard method is absent, and the diagnostic performance is not as high as that of RT-PCR 2,3,5 . Various formats of commercial antibody detection kits including enzyme-linked immunosorbent assay 10 (ELISA), fluorescence immunoassay (FIA), and lateral flow immunoassay (LFIA) for rapid 11 diagnostic test (RDT) have been being introduced, but the performance of these kits needs to be 12 further evaluated. Herein, we present the practical strategies to optimize the utilization of anti-SARS- CoV-2 antibody test using multiple kits from different manufacturers. 16 Sera collected after more than seven days of illness from RT-PCR-confirmed asymptomatic to severe 17 COVID-19 patients were used as positive specimens 2 . In asymptomatic patients, illness days were (using a cut-off value of borderline) and 93.5/100% (using a cut-off value of positive). only one kit gave a positive result, the specimen was interpreted as negative. decreased, but specificity increased to 100%. When three different kinds of kits were used, both 1 sensitivity and specificity increased (99.1% and 100%, respectively). Serologic testing for an emerging viral disease has a wide range of clinical implications 2-9 . However, 4 immunoassay methods inevitably have false-positive and false-negative results, and there is no gold 5 standard method to detect the presence of virus-specific antibodies 3,5 . Although the neutralization test 6 is highly specific and reflects functional activity, low levels of binding antibodies may not be detected 7 by neutralizing tests 2 . Several anti-SARS-CoV-2 antibody test kits using immunoassay methods 8 exhibited high sensitivity, but sub-optimal specificity remains a concern. In the present investigation, 9 false-positive reactions occurred independently between kits. Cross-reactive false-positive results were observed only between RDT and FIA kits from the same manufacturer, which shared raw 11 materials. False-positive reactions on anti-SARS-CoV-2 antibody test kits are likely to occur 12 specifically to individual materials, such as recombinant SARS-CoV-2 protein, reagents, or buffers. These findings suggest that false-positivity could be decreased if we use multiple test kits. 14 As false-positive reactions occurred independently between test kits, the specificity of the kits 15 increased to 100% when two different kits were used together, but the sensitivity inevitably decreased. When three kits were used together, both sensitivity and specificity increased to 99.1% and 100%, 17 respectively. To optimize the performance of serologic testing, whether to use two or more kits in 18 combination should be decided according to the purpose of the test. For the case of sero-prevalence 19 studies in low COVID-19 prevalence areas, specificity and cost are important due to large sample 20 sizes and low PPV. Two-step confirmation using two different kits would be cost-effective in such 21 situation. Meanwhile, for the diagnosis of multisystem inflammatory syndrome in children, both 22 sensitivity and specificity are important and more than two kits could be used to optimize 23 performance 3,9 . The present analysis has several limitations. First, the number of negative control specimens was In conclusion, in the present analysis of anti-SARS-CoV-2 antibody test kits, false-positive 12 reactions occurred independently between test kits. Using a combination of two different kits 13 increased specificity to 100% and combination of more than two kits further increased performance. 14 The number and type of kits used should be decided based on the outbreak situation and the purpose 15 of testing. Prevention (#2020-ER5328-00). WHO. WHO Coronavirus Disease (COVID-19) Dashboard Neutralizing Antibody Production in 8 Asymptomatic and Mild COVID-19 Patients WHO. WHO Coronavirus Disease (COVID-19) Dashboard Asymptomatic and Mild COVID-19 Patients Serologic Evaluation of Healthcare 13 Workers Caring for COVID-19 Patients in the Republic of Korea Antibody 16 tests for identification of current and past infection with SARS-CoV-2 Clinical application of rapid diagnostic 19 test kit for SARS-CoV-2 antibodies into the field of patient care Challenges of convalescent plasma 22 infusion therapy in Middle East respiratory coronavirus infection: a single centre 23 experience Serologic Evaluation of MERS 25 Screening Strategy for Healthcare Personnel During a Hospital-Associated Outbreak Syndrome in Children Related to COVID-19: the First Case in Korea