key: cord-0824017-uj0i2anr 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: 2020-03-16 journal: nan DOI: 10.1101/2020.03.12.20034231 sha: 9afddf25a02706489b4e5f73d96d7b8e64cb68f4 doc_id: 824017 cord_uid: uj0i2anr 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 ℃ 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. By comparing the levels of SARS-CoV-2 antibodies before and after heat inactivation of serum at 56 ℃ for 30 minutes using a quantitative fluorescence immunochromatographic assay, we shown 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 diseases group (n=9). Of note, 44.12% of the detected IgM levels were dropped below the cut-off value after heating, suggesting heat inactivation can lead to false-negative results of these samples. Our results indicate that heat inactivation of serum at 56 ℃ 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, 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 are usually be recommended. 8, 9 While the sensitivity of SARS-CoV-2 to the conditions of inactivation is unknown, it is reported that many coronavirus such as SARS are heat-sensitive and can be killed at 56 ℃ for 30 minutes. [10] [11] [12] [13] [14] It is thus inferred that heating at 56 ℃ could be an effective approach for SARS-CoV-2 inactivation. 15 However, the effect of heating at 56 ℃ on COVID-19 antibody detection is unclear. The objective of this study was to compare the levels of . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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 March 16, 2020. . https://doi.org/10.1101/2020.03.12.20034231 doi: medRxiv preprint ([95% CI,21.37%-66.63%]). None of the measured antibodies became higher than the cut-off value after heating. . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 16, 2020. This study analyzes the changes in SARS-CoV-2 antibody concentration before and after heat inactivation at 56 ℃ 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 of 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 IgG 19,20 due to their different compositions and structures of heavy chains. 21 This is consistent with our results that SARS-CoV-2 IgM concentration decreased more significantly than IgG after heating. In . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 16, 2020. . https://doi.org/10.1101/2020.03.12.20034231 doi: medRxiv preprint 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 immunogenicity caused by the formation of IgG aggregates heating at 56 . 16, 22, 23 It is noteworthy that after heat inactivation, 44.12% of the IgM levels form COVID-19 patients were below the cut-off value. These results suggests that heat inactivation of serum can lead to false-negative results in these samples. Heat inactivation of serum at 56 [7] Li Z, Yi Y, Luo X, Xiong N, Liu Y, Li S, Sun R, Wang Y, Hu B, Chen W, Zhang Y, Wang J, Huang B, Lin . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 16, 2020. . https://doi.org/10.1101/2020.03.12.20034231 doi: medRxiv preprint 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 Genernal office of the National Health Commission, Guide of diagnosis and treatment of novel coronavirus (nCoV) pneumonia, 6th Version Development and clinical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis World Health Organization, Infection prevention and control during health care when novel coronavirus (nCoV) infection is suspected: interim guidance Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV Heat inactivation of the Middle East respiratory syndrome coronavirus, Influenza and Other Respiratory Viruses Heat sensitivity of a SARS-associated coronavirus introduced into plasma products Inactivation of SARS coronavirus by means of povidone-iodine, physical conditions and chemical reagents Stability of SARS coronavirus in human specimens and environment and its sensitivity to heating and UV irradiation Coronavirus disease 2019: coronaviruses and blood safety Antibody structure, instability, and formulation Effect of heat treatment on the antigen-binding activity of anti-peroxidase immunoglobulins in bovine colostrum IgM and lactoferrin contents of human milk during early lactation and the effect of processing and storage Protection, 1983, 46(1): 4-7.[19] Li S Q, Bomser J A, Zhang Q H, Effects of pulsed electric fields and heat treatment on stability and secondary structure of bovine immunoglobulin G, Journal of Agricultural and Food Chemistry, 2005, . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a 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 March 16, 2020. . https://doi.org/10.1101/2020.03.12.20034231 doi: medRxiv preprint