key: cord-0936880-3hrbea6d authors: Hu, Xiumei; An, Taixue; Situ, Bo; Hu, Yuhai; Ou, Zihao; Li, Qiang; He, Xiaojing; Zhang, Ye; Tian, Peifu; Sun, Dehua; Rui, Yongyu; Wang, Qian; Ding, Dan; Zheng, Lei title: Heat inactivation of serum interferes with the immunoanalysis of antibodies to SARS‐CoV‐2 date: 2020-06-28 journal: J Clin Lab Anal DOI: 10.1002/jcla.23411 sha: 8f88a624c68c61f379fce0d3b9b90c087b498f38 doc_id: 936880 cord_uid: 3hrbea6d BACKGROUND: The detection of serum antibodies to the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is emerging as a new tool for the coronavirus disease 2019 (COVID‐19) diagnosis. Since many coronaviruses are sensitive to heat, heating inactivation of samples at 56°C prior to testing is considered a possible method to reduce the risk of transmission, but the effect of heating on the measurement of SARS‐CoV‐2 antibodies is still unclear. METHODS: By comparing the levels of SARS‐CoV‐2 antibodies before and after heat inactivation of serum at 56°C for 30 minutes using a quantitative fluorescence immunochromatographic assay RESULTS: We showed that heat inactivation significantly interferes with the levels of antibodies to SARS‐CoV‐2. The IgM levels of all the 34 serum samples (100%) from COVID‐19 patients decreased by an average level of 53.56%. The IgG levels were decreased in 22 of 34 samples (64.71%) by an average level of 49.54%. Similar changes can also be observed in the non–COVID‐19 disease group (n = 9). Of note, 44.12% of the detected IgM levels were dropped below the cutoff value after heating, suggesting heat inactivation can lead to false‐negative results of these samples. CONCLUSION: Our results indicate that heat inactivation of serum at 56°C for 30 minutes interferes with the immunoanalysis of antibodies to SARS‐CoV‐2. Heat inactivation prior to immunoanalysis is not recommended, and the possibility of false‐negative results should be considered if the sample was pre‐inactivated by heating. The current outbreak of coronavirus disease 2019 (COVID- 19) caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is posing a serious threat to public health. [1] [2] [3] Early diagnosis of suspect cases is critical to reduce and interrupt the transmission of COVID-19 from person to person. 4 Currently, laboratory testing of viral nucleic acid by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay is the "gold standard" for COVID-19 diagnosing. 5 However, the requirement of sophisticated instruments and laboratory conditions, tedious experimental procedures, and longer detection time significantly hamper its widespread applicability. 4 Antibodies produced in the blood after COVID-19 infection are emerging as a promising class of biomarkers. 6 The antibodies to SARS-CoV-2 are specific, sensitive, and more importantly, their detection can be much faster and simpler than RT-PCR, which allows rapid screening of suspect cases to be possible. 7 All the biological specimens for COVID-19 testing should be considered to be potentially infectious. Therefore, the test must be performed by medical professionals with protective equipment in a qualified laboratory. To further reduce the risk of exposure to infectious agents, viral inactivation before sample handling is usually be recommended. 8, 9 While the sensitivity of SARS-CoV-2 to the conditions of inactivation is unknown, it is reported that many coronaviruses such as SARS are heat-sensitive and can be killed at 56°C for 30 minutes. [10] [11] [12] [13] [14] It is thus inferred that heating at 56°C could be an effective approach for SARS-CoV-2 inactivation. 15 However, the effect of heating at 56°C on COVID-19 antibody detection is unclear. The objective of this study was to compare the levels of COVID-19 antibody before and after heat inactivation. In the patients with COVID-19, the IgM signals of all the 34 serum samples (100%) decreased ( Figure 1 This study analyzed the changes in SARS-CoV-2 antibody concentration before and after heat inactivation at 56°C for 30 minutes. We found that after heat inactivation, all of the serum IgM (100%) demonstrated significantly lower levels. For IgG, 64.71% of the sample levels dropped after heat inactivation. All immunological assays are highly dependent on the recognition and binding of antigens to antibodies. The decrease in SARS-CoV-2 antibody levels may be related to their structural change in denaturation and aggregation. 16, 17 Previous studies have shown that antibodies can be denatured and lose their antigen-binding activities after heating, 18 and IgM is reported to be less thermally stable than 21 This is consistent with our results that SARS-CoV-2 IgM concentration decreased more significantly than IgG after heating. In addition, the IgG levels in 12 samples (35.29%) increased with a median of 24.22% after heating, which may be due to the increases in immunogenicity caused by the formation of IgG aggregates heating at 56°C. 16, 22, 23 It is noteworthy that after heat inactivation, 44.12% of the IgM levels from COVID-19 patients were below the cutoff value. These results suggest that heat inactivation of serum can lead to false-negative results in these samples. The signal intensity of the IgM and IgG levels of 9 serum samples from non-COVID-19 group detected by AFIA before and after heat inactivation The changes in the IgM and IgG levels of 9 serum samples from non-COVID-19 group detected by AFIA before (blue dot) and after heat inactivation (red dot) Coronavirus: covid-19 has killed more people than SARS and MERS combined, despite lower case fatality rate A novel coronavirus outbreak of global health concern The novel coronavirus originating in Wuhan, China: challenges for global health governance World Health Organization. Laboratory testing for coronavirus disease 2019 (COVID-19) in suspected human cases: interim guidance General office of the National Health Commission. 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