anti sars cov 2 kuantitatif

Penggunaan tes Roche untuk mengukur tingkat kuantitatif dari antibodi anti-RBD SARS-CoV-2 akan membantu Moderna mendapatkan pengetahuan berharga tentang korelasi antara perlindungan dari vaksinasi dan tingkat antibodi. Hal ini dapat berperan dalam menilai jika atau kapan, seseorang membutuhkan vaksinasi ulang, atau membantu menjawab pertanyaan PemeriksaanAnti SARS-CoV-2 Kualitatif mendeteksi antibodi terhadap protein Nucleocapsid (N). Sedangkan untuk yang kuantitatif akan mendeteksi antibodi untuk protein Spike-RBD. Untuk kualitatif bisa digunakan sebagai skrining awal untuk mengetahui terinfeksi tidaknya dengan Covid 19, misalnya rapid test antibodi Therefore the results of the Anti-SARS-CoV-2 QuantiVac ELISA (IgG) can now be issued in standardised units. The standardised determination of antibody concentration plays an important role in the development of COVID-19 vaccines. The first vaccine against SARS-CoV-2 has been available since the end of last year and another one has recently COVID19 (Coronavirus Disease 19) adalah penyakit menular yang disebabkan oleh SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), yaitu jenis coronavirus yang ditemukan di Wuhan, Cina, pada Desember 2019. Jadi COVID-19 adalah nama penyakitnya dan SARS-CoV-2 adalah nama virus yang menyebabkan penyakit tersebut. RumahSakit (Rumkit) Bhayangkara Palangka Raya – Rumah Sakit (Rumkit) Bhayangkara Tk III Palangka Raya Polda Kalteng bekerjasama dengan Klinik Prodia Palangka Raya menggelar pemeriksaan antibodi Covid-19 (Laboratorium Titer Antibodi Anti Sars CoV-2 Kuantitatif) kepada Pejabat Utama Polda Kalteng di Mapolda setempat, Jumat (21/5/2021). 누누티비 우회. The development timeline of COVID-19 vaccines is unprecedented, with more than 300 vaccine developers active worldwide. Vaccine candidates developed with various technology platforms targeting different epitopes of SARS-CoV-2 are in the pipeline. Vaccine developers are using a range of immunoassays with different readouts to measure immune responses after vaccination, making comparisons of the immunogenicity of different COVID-19 vaccine candidates April, 2020, in a joint effort, the Coalition for Epidemic Preparedness Innovations CEPI, the National Institute for Biological Standards and Control NIBSC, and WHO provided vaccine developers and the entire scientific community with a research reagent for an anti-SARS-CoV-2 antibody. The availability of this material was crucial for facilitating the development of diagnostics, vaccines, and therapeutic preparations. This effort was an initial response when NIBSC, in its capacity as a WHO collaborating centre, was working on the preparation of the WHO International Standards. This work included a collaborative study that was launched in July, 2020, to test serum samples and plasma samples sourced from convalescent patients with the aim of selecting the most suitable candidate material for the WHO International Standards for anti-SARS-CoV-2 immunoglobulin. The study involved 44 laboratories from 15 countries and the use of live and pseudotype-based neutralisation assays, ELISA, rapid tests, and other methods. The outcomes of the study were submitted to WHO in November, 2020. The inter-laboratory variation was reduced more than 50 times for neutralisation and 2000 times for ELISA when assay values were reported relative to the International International Standard and International Reference Panel for anti-SARS-CoV-2 immunoglobulins were adopted by the WHO Expert Committee on Biological Standardization on Dec 10, WHO International Standard for anti-SARS-CoV-2 Scholar The International Standard allows the accurate calibration of assays to an arbitrary unit, thereby reducing inter-laboratory variation and creating a common language for reporting data. The International Standard is based on pooled human plasma from convalescent patients, which is lyophilised in ampoules, with an assigned unit of 250 international units IU per ampoule for neutralising activity. For binding assays, a unit of 1000 binding antibody units BAU per mL can be used to assist the comparison of assays detecting the same class of immunoglobulins with the same specificity eg, anti-receptor-binding domain IgG, anti-N IgM, etc The International Standard is available in the NIBSC have been launched for the harmonisation of immune response assessment across COVID-19 vaccine candidates, including the CEPI Global Centralised Laboratory for Epidemic Preparedness InnovationsCEPI establishes global network of laboratories to centralise assessment of COVID-19 vaccine Scholar CEPI centralised laboratories will achieve harmonisation of the results from different vaccine clinical trials with the use of common standard operating procedures and the same crucial reagents, including a working standard calibrated to the international basic tool for any harmonisation is the global use of an International Standard and IU to which assay data need to be calibrated with the use of a reliable method. It is therefore crucial that the International Standard is properly used by all vaccine developers, national reference laboratories, and academic groups worldwide, and that immunogenicity results are reported as an international standard unit IU/mL for neutralising antibodies and BAU/mL for binding assay formats.In this manner, the results from clinical trials expressed in IU would allow for the comparison of the immune responses after natural infection and induced by various vaccine candidates. This comparison is particularly important for the identification of correlates of protection against COVID-19; should neutralising antibodies be further supported as a component of the protective response, the expression of antibody responses in IU/mL is essential to gather a consensus from several clinical trials and other studies on the titre required for the correlate of protection against SARS-2-CoV has not yet been unequivocally defined, antibodies are likely to be at least part of the protective response. The effect of new variants on the evaluation of antibodies is obvious and unequivocal comparisons are required. Reporting the immunological responses from vaccine clinical trials against the International Standard is essential for the evaluation of clinical data submitted to national regulatory authorities as well as to WHO for emergency use listing, especially as placebo-controlled efficacy studies become operationally unfeasible. There will be a substantial effect on the use of the International Standard if regulatory authorities worldwide request data in IU/mL or BAU/mL. We also encourage journal editors and peer reviewers to ensure that the international standard is used as the benchmark in publications and that data from serology assays are reported in International Standard declare no competing TT Cramer JP Chen R Mayhew S Evolution of the COVID-19 vaccine development Rev Drug Discov. 2020; 19 WHO International Standard for anti-SARS-CoV-2 for Epidemic Preparedness InnovationsCEPI establishes global network of laboratories to centralise assessment of COVID-19 vaccine infoPublication historyPublished March 23, 2021IdentificationDOI Copyright © 2021 Published by Elsevier Ltd. All rights this article on ScienceDirectView Large ImageDownload Hi-res image Download .PPT IntroductionIt has been more than one year since the first reported case of the novel coronavirus disease 2019 COVID-19, which has already cost more than 2 million lives Fortunately, vaccines against severe acute respiratory syndrome coronavirus 2 SARS-CoV-2 have been developed with record-breaking speed and vaccine programs are ongoing worldwide to take the pandemic under During this expansion of research focus from treatment to prevention of COVID-19, the immune evasion mechanism and immunopathogenic nature of SARS-CoV-2 adds uncertainty to the efficacy of this global vaccination During natural infection, SARS-CoV-2 could avoid the innate antiviral response mediated by interferons IFNs via an array of possible strategies,4,5 which not only leads to viral replication and spreading but also could delay or impair the adaptive immune response including T cell and antibody The significant prevalence of SARS-CoV-2 RNA re-positive cases among discharged patients further raises the concern about the effectiveness and persistency of immune responses after natural Recent long-term follow-up surveys report significant decrease of SARS-CoV-2 antibody titers 5 to 8 months after infection,10,11,12 but its correlation with reduced capacity of SARS-CoV-2 neutralization and immune memory is still vaccination, equally important is the recovery and rehabilitation of COVID-19 Mild cases usually do not require hospitalization but may share similar long-lasting symptoms or discomforts with severe cases, which may reduce life quality after recovery from Also, cardiac magnet resonance imaging cMRI screening revealed surprisingly high prevalence of subclinical myocardial inflammation and fibrosis in recently recovered Due to the overloading of medical systems and the fear of in-hospital transmission, long-term follow-up studies of the structural and functional recovery of COVID-19-involved organs are still this prospective cohort study of recovered COVID-19 patients from Xiangyang, China, we aimed to assess long-term antibody response at 12 months after infection and comprehensively evaluate the structural and functional recovery of the lung and cardiovascular systems. We also attempted to identify potential risk factors associated with those long-term January 15 through 31 March 2020, a total of 307 patients were diagnosed with COVID-19 at Xiangyang Central Hospital, which represented of 549 cases in the downtown and of 1175 cases city-wide. During hospitalization, 12 patients succumbed to COVID-19-induced respiratory distress or lethal infection, which translated to a mortality rate of in line with the citywide mortality rate of 40/1175. All 295 survivors were invited to participate in this study and the final cohort consisted of 121 survivors including 19 recovered from severe COVID-19 Supplementary Fig. 1. Clinical procedures were performed at Xiangyang Central Hospital between 25 December 2020 and 29 January and clinical features of participantsDemographic-wise, this cohort consisted of middle-aged Chinese population with an overall comorbidity prevalence of including hypertension and diabetes as the most common preexisting conditions, which was typical for the local agricultural and industrial population with a preference of high-salt diets Table 1. The participants of this study were among the earliest confirmed COVID-19 patients with virological confirmation dates as early as January 19, 2020. Standard of care consisted of antivirals, antibiotics, immunomodulants and supplemental oxygen was given to participants following CDC guidelines Supplementary Table 1. Only 1 in this cohort received invasive ventilation Supplementary Table 1, which reflected the dismal mortality rate among critically ill patients relying on respiratory Of note, the basic characteristics of this cohort were comparable with the entire population of COVID-19 survivors treated at this hospital Supplementary Table 2.Table 1 Characteristics of participants by COVID-19 severityFull size tableAfter stratifying the cohort by severity graded according to the guideline,21 severe groups had higher ages, less females, and more comorbidities Table 1. Severe group also presented more symptoms at admission, and received more aggressive immunomodulatory therapies, supplemental oxygen, and ICU care during hospitalization Supplementary Table 1. Both severe and non-severe groups share similar lengths since symptom onset, while the severe group had shorter periods since recovery because of longer hospitalization Table 1.Long-lasting SARS-CoV-2 antibody response 1-year after infectionFirst, blood samples were screened by colloidal gold-based immunochromatographic assays GICA separately detecting IgM and IgG against At a median of 11 months post- infection, only 4% 95% CI, 2–10% participants returned positive IgM results, which included both positive and weakly positive results, while 62% 95% CI, 54–71% were IgG positive Table 1, comparing to prevalence of IgM among pre-discharge samples from the same Severe group showed higher prevalence of IgG, while the prevalence of IgM was equally low in both groups Table 1.Next, the concentration of total antibodies against the receptor-binding domain of SARS-CoV-2 spike protein RBD was quantitatively measured by chemiluminescence microparticle immunoassays CMIA.24 Although signal/cutoff S/CO ratios were lower in non-severe group, all but 1 of the results were above the positive diagnostic threshold of S/CO = when all 100 samples of unexposed individuals, which were randomly chosen from sera of in-hospital patients who had negative results from multiple PCR and serological tests for SARS-CoV-2 before and after the date of serum collection, had S/CO values participants were exposed to SARS-CoV-2 and diagnosed with COVID-19 during January to March 2020. During their COVID-19 disease courses, they have received combinations of therapies including antivirals, immunomodulatory agents, antibiotics, supplemental oxygen, and ICU outcomes of this study were immunity against SARS-CoV-2 and functional recovery of the lung and other involved organs. Immunity against SARS-CoV-2 was assessed by multiple antibody assays. The colloidal gold-based test kit gave positive, weak positive, and negative readout of anti-SARS-CoV-2 IgM and IgG separately. The quantitative chemiluminescence microparticle immunoassay for antibodies against SARS-CoV-2 RBD was performed according to manufacturer’s protocol and previous publication,24 and the results were deemed positive if the signal/cutoff S/CO ratio ≥1. For ELISA tests, results were recorded and analyzed as continuous variables and the limit of sensitivity was calculated as mean + 2 × SD of 20 serum samples negative for SARS-CoV-2 antibodies in chemiluminescence assays. Functional recovery of the lung was assessed based on 1 current CT images comparing to images taken before discharge and during earlier follow-ups, 2 pulmonary function test results, and 3 six-minute walk test results. Recovery of the heart was assessed based on ECG, echocardiogram, and cardiac MRI scans. Recovery of other potentially involved organs were assessed by laboratory tests Roche Diagnostics.Sample sizeAn initial target sample size of 108 was determined based on the assumption of a 15 ratio of severe and non-severe COVID-19 patient enrollment and α = This sample size was calculated to have 90% power to detect a 10% difference of antibody concentrations. The final sample size exceeded the target in both analysisQuantitative data were presented in violin plots with all data points shown. Patient characteristics and clinical data were summarized as incidence with prevalence or median with IQR and were assessed with Fisher’s exact test dichotomous variables or χ2 test variables with more than two categories for categorical variables and Mann–Whitney U test for continuous variables. Antibody concentrations were log-transformed before being analyzed as continuous variables. The difference of antibody concentrations between groups were assessed by the Mann–Whitney U test two groups or Kruskal–Wallis test with post hoc comparisons more than two groups. Special tests were mentioned in figure legends. Correlation was assessed by Spearman’s ρ test. Linearity between two factors was assessed by simple linear regression. Generalized linear models were used to assess factors associated with antibody titers. Analyses were performed using SPSS 26 IBM or Prism 9 GraphPad. Missing data were excluded pairwise from analyses. Significance was evaluated at α = .05 and all tests were 2-sided. *p < **p < ***p < Data availabilityReasonable requests for original dataset and clinical documents would be fulfilled by Dr. Peng Hong P. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273 2020.Article CAS PubMed PubMed Central Google Scholar Parker, E. P. K., Shrotri, M. & Kampmann, B. Keeping track of the SARS-CoV-2 vaccine pipeline. Nat. Rev. Immunol. 20, 650 2020.Article CAS PubMed Google Scholar Sette, A. & Crotty, S. Adaptive immunity to SARS-CoV-2 and COVID-19. Cell 184, 861–880 2021.Article CAS PubMed PubMed Central Google Scholar Blanco-Melo, D. et al. Imbalanced host response to SARS-CoV-2 drives development of COVID-19. Cell 181, 1036– 2020.Article CAS PubMed PubMed Central Google Scholar Lei, X. et al. Activation and evasion of type I interferon responses by SARS-CoV-2. Nat. Commun. 11, 3810 2020.Article CAS PubMed PubMed Central Google Scholar Oved, K. et al. Multi-center nationwide comparison of seven serology assays reveals a SARS-CoV-2 non-responding seronegative subpopulation. EClinicalMedicine 29, 100651 2020.Article PubMed Google Scholar Anna, F. et al. High seroprevalence but short-lived immune response to SARS-CoV-2 infection in Paris. Eur. J. Immunol. 51, 180–190 2021.Article CAS PubMed Google Scholar Lu, J. et al. Clinical, immunological and virological characterization of COVID-19 patients that test re-positive for SARS-CoV-2 by RT-PCR. EBioMedicine 59, 102960 2020.Article PubMed PubMed Central Google Scholar Yang, C. et al. Viral RNA level, serum antibody responses, and transmission risk in recovered COVID-19 patients with recurrent positive SARS-CoV-2 RNA test results a population-based observational cohort study. Emerg. Microbes Infect. 9, 2368–2378 2020.Article CAS PubMed PubMed Central Google Scholar Choe, P. G. et al. Waning antibody responses in asymptomatic and symptomatic SARS-CoV-2 infection. Emerg. Infect. Dis. 27, 327–329 2021.Article CAS PubMed Central Google Scholar Huang, C. et al. 6-month consequences of COVID-19 in patients discharged from hospital a cohort study. Lancet 397, 220–232 2021.Article CAS PubMed PubMed Central Google Scholar Self, W. H. et al. Decline in SARS-CoV-2 antibodies after mild infection among frontline health care personnel in a multistate hospital network - 12 states, April-August 2020. Morb. Mortal. Wkly. Rep. 69, 1762–1766 2020.Article CAS Google Scholar Wajnberg, A. et al. Robust neutralizing antibodies to SARS-CoV-2 infection persist for months. Science 370, 1227–1230 2020.Article CAS PubMed PubMed Central Google Scholar Dan, J. M. et al. Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection. Science 371, eabf4063 2021.Article CAS PubMed Google Scholar Yelin, D. et al. Long-term consequences of COVID-19 research needs. Lancet Infect. Dis. 20, 1115–1117 2020.Article CAS PubMed PubMed Central Google Scholar Gandhi, R. T., Lynch, J. B. & Del Rio, C. Mild or moderate Covid-19. N. Engl. J. Med. 383, 1757–1766 2020.Article CAS PubMed Google Scholar Carfi, A., Bernabei, R. & Landi, F., Gemelli Against, Persistent symptoms in patients after acute COVID-19. JAMA 324, 603–605 2020.Article CAS PubMed PubMed Central Google Scholar Puntmann, V. O. et al. Outcomes of cardiovascular magnetic resonance imaging in patients recently recovered from coronavirus disease 2019 COVID-19. JAMA Cardiol. 5, 1265–1273 2020.Article PubMed PubMed Central Google Scholar Cortinovis, M., Perico, N. & Remuzzi, G. Long-term follow-up of recovered patients with COVID-19. Lancet 397, 173–175 2021.Article CAS PubMed PubMed Central Google Scholar Dupuis, C. et al. Association between early invasive mechanical ventilation and day-60 mortality in acute hypoxemic respiratory failure related to coronavirus disease-2019 pneumonia. Crit. Care Explor 3, e0329 2021.Article PubMed PubMed Central Google Scholar NHCPRC. National Health Commission of the People’s Republic of China. Chinese management guideline for COVID-19 version 7 [in Chinese]. 2020.Pan, Y. et al. Serological immunochromatographic approach in diagnosis with SARS-CoV-2 infected COVID-19 patients. J. Infect. 81, e28–e32 2020.Article CAS PubMed PubMed Central Google Scholar Shen, L. et al. Delayed specific IgM antibody responses observed among COVID-19 patients with severe progression. Emerg. Microbes Infect. 9, 1096–1101 2020.Article CAS PubMed PubMed Central Google Scholar Liu, W. et al. Clinical application of chemiluminescence microparticle immunoassay for SARS-CoV-2 infection diagnosis. J. Clin. Virol. 130, 104576 2020.Article CAS PubMed PubMed Central Google Scholar Atyeo, C. et al. Distinct early serological signatures track with SARS-CoV-2 survival. Immunity 53, 524– 2020.Article CAS PubMed PubMed Central Google Scholar Long, Q. X. et al. Antibody responses to SARS-CoV-2 in patients with COVID-19. Nat. Med. 26, 845–848 2020.Article CAS PubMed Google Scholar Schmidt, F. et al. Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses. J. Exp. Med. 217, 11 e20201181 2020.Article PubMed CAS Google Scholar Khoury, D. S. et al. Neutralizing antibody levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection. Nat. Med. 27, 1205–1211 2021.Article CAS PubMed Google Scholar Wang, P. et al. Antibody resistance of SARS-CoV-2 variants and Nature 593, 130–135 2021.Article CAS PubMed Google Scholar McCrohon, J. A. et al. Differentiation of heart failure related to dilated cardiomyopathy and coronary artery disease using gadolinium-enhanced cardiovascular magnetic resonance. Circulation 108, 54–59 2003.Article CAS PubMed Google Scholar Chaowu, Y. & Li, L. Histopathological basis of myocardial late gadolinium enhancement in patients with systemic hypertension. Circulation 130, 2210–2212 2014.Article PubMed Google Scholar Wadhera, R. K. et al. Variation in COVID-19 hospitalizations and deaths across New York City boroughs. JAMA 323, 2192–2195 2020.Article CAS PubMed PubMed Central Google Scholar Paremoer, L., Nandi, S., Serag, H. & Baum, F. Covid-19 pandemic and the social determinants of health. BMJ 372, n129 2021.Article PubMed PubMed Central Google Scholar Wu, Z. & McGoogan, J. M. Characteristics of and important lessons from the coronavirus disease 2019 COVID-19 outbreak in China summary of a report of 72314 cases from the Chinese center for disease control and prevention. JAMA 323, 1239–1242 2020.Article CAS PubMed Google Scholar Ji, Y., Ma, Z., Peppelenbosch, M. P. & Pan, Q. Potential association between COVID-19 mortality and health-care resource availability. Lancet Glob. Health 8, e480 2020.Article PubMed PubMed Central Google Scholar Li, Q. et al. The impact of mutations in SARS-CoV-2 spike on viral infectivity and antigenicity. Cell 182, 1284– 2020.Article CAS PubMed PubMed Central Google Scholar Trump, S. et al. Hypertension delays viral clearance and exacerbates airway hyperinflammation in patients with COVID-19. Nat. Biotechnol. 39, 705–716 2021.Article CAS PubMed Google Scholar Hu, F. et al. A compromised specific humoral immune response against the SARS-CoV-2 receptor-binding domain is related to viral persistence and periodic shedding in the gastrointestinal tract. Cell Mol. Immunol. 17, 1119–1125 2020.Article CAS PubMed Google Scholar Naidu, S. B. et al. The high mental health burden of "Long COVID" and its association with on-going physical and respiratory symptoms in all adults discharged from hospital. Eur. Respir. J. 57, 2004364 2021.Article CAS PubMed PubMed Central Google Scholar Sudre, C. H. et al. Attributes and predictors of long COVID. Nat. Med. 27, 626–631 2021.Article CAS PubMed PubMed Central Google Scholar Augustin, M. et al. Post-COVID syndrome in non-hospitalised patients with COVID-19 a longitudinal prospective cohort study. Lancet Reg. Health Eur. 6, 100122 2021.Article PubMed PubMed Central Google Scholar Peghin, M. et al. Post-COVID-19 symptoms 6 months after acute infection among hospitalized and non-hospitalized patients. Clin. Microbiol. Infect. 27, 1507–1513 2021.Article CAS PubMed PubMed Central Google Scholar Rogers, J. P. et al. Psychiatric and neuropsychiatric presentations associated with severe coronavirus infections a systematic review and meta-analysis with comparison to the COVID-19 pandemic. Lancet Psychiatry 7, 611–627 2020.Article PubMed PubMed Central Google Scholar Reichard, R. R. et al. Neuropathology of COVID-19 a spectrum of vascular and acute disseminated encephalomyelitis ADEM-like pathology. Acta Neuropathol. 140, 1–6 2020.Article CAS PubMed PubMed Central Google Scholar Varatharaj, A. et al. Neurological and neuropsychiatric complications of COVID-19 in 153 patients a UK-wide surveillance study. Lancet Psychiatry 7, 875–882 2020.Article PubMed PubMed Central Google Scholar Francone, M. et al. Chest CT score in COVID-19 patients correlation with disease severity and short-term prognosis. Eur. Radiol. 30, 6808–6817 2020.Article CAS PubMed PubMed Central Google Scholar Holland, A. E. et al. An official European Respiratory Society/American Thoracic Society technical standard field walking tests in chronic respiratory disease. Eur. Respir. J. 44, 1428–1446 2014.Article PubMed Google Scholar Hajiro, T. et al. Analysis of clinical methods used to evaluate dyspnea in patients with chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 158, 1185–1189 1998.Article CAS PubMed Google Scholar Graham, B. L. et al. Standardization of Spirometry 2019 Update. An Official American Thoracic Society and European Respiratory Society Technical Statement. Am. J. Respir. Crit. Care Med. 200, e70–e88 2019.Article PubMed PubMed Central Google Scholar Milanese, M. et al. Suggestions for lung function testing in the context of COVID-19. Respir. Med. 177, 106292 2020.Article PubMed PubMed Central Google Scholar Download referencesAcknowledgementsWe thank Chun Mao Xiangyang Central Hospital and Juan Xiao Hubei University of Arts and Science for organization and administrative support of patient recruitments and clinical examinations. We also thank Rongjie Zhao, Zhangli Li Thermo Fisher Scientific China, Shanghai, China, and GenScript Nanjing, China for technical support and protocol optimization. This work was supported by Xiangyang Science and Technology Bureau 2020YL10, 2020YL14, 2020YL17, and 2020YL39, National Natural Science Foundation of China 31501116, Shenzhen Science and Technology Innovation Commission JCYJ20190809100005672, Shenzhen Sanming Project of Medicine SZSM201911013, and US Department of Veterans Affairs 5I01BX001353.Author informationAuthor notesThese authors contributed equally Yan Zhan, Yufang Zhu, Shanshan Wang, Shijun Jia, Yunling Gao, Yingying LuAuthors and AffiliationsDepartment of Rehabilitation Medicine, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei, 441021, ChinaYan Zhan, Shanshan Wang, Peng Du, Hao Yu, Chang Liu & Peijun LiuDepartment of Laboratory Medicine, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei, 441021, ChinaYufang Zhu, Caili Zhou & Ran LiangDepartment of Radiology and Medical Imaging, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei, 441021, ChinaShijun Jia & Feng WuDepartment of Research Affairs, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei, 441021, ChinaYunling Gao & Jin ChengDepartment of Nephrology, Center of Nephrology and Urology, Sun Yat-sen University Seventh Hospital, Shenzhen, Guangdong, 518107, ChinaYingying Lu, Zhihua Zheng & Peng HongDepartment of Biomedical Science, Shenzhen Research Institute, City University of Hong Kong, Kowloon Tong, Hong Kong, ChinaYingying LuDepartment of Rehabilitation Medicine, Xiangzhou District People’s Hospital, Xiangyang, Hubei, 441000, ChinaDingwen SunDepartment of Rehabilitation Medicine, Gucheng People’s Hospital, Affiliated Gucheng Hospital of Hubei University of Arts and Science, Xiangyang, Hubei, 441700, ChinaXiaobo WangDivision of Quality Control, Xiangyang Central Blood Station, Xiangyang, Hubei, 441000, ChinaZhibing HouDepartment of Respiratory and Critical Care Medicine, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei, 441021, ChinaQiaoqiao Hu & Yulan ZhengDepartment of Pathology, Mount Sinai St. Luke’s Roosevelt Hospital Center, New York, NY, 10025, USAMiao CuiDepartment of Oncology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, 518036, ChinaGangling TongDepartment of Dermatology, Sun Yat-sen University Seventh Hospital, Shenzhen, Guangdong, 518107, ChinaYunsheng Xu & Linyu ZhuDivision of Research and Development, US Department of Veterans Affairs New York Harbor Healthcare System, Brooklyn, NY, 11209, USAPeng HongDepartment of Cell Biology, State University of New York Downstate Health Sciences University, Brooklyn, NY, 11203, USAPeng HongAuthorsYan ZhanYou can also search for this author in PubMed Google ScholarYufang ZhuYou can also search for this author in PubMed Google ScholarShanshan WangYou can also search for this author in PubMed Google ScholarShijun JiaYou can also search for this author in PubMed Google ScholarYunling GaoYou can also search for this author in PubMed Google ScholarYingying LuYou can also search for this author in PubMed Google ScholarCaili ZhouYou can also search for this author in PubMed Google ScholarRan LiangYou can also search for this author in PubMed Google ScholarDingwen SunYou can also search for this author in PubMed Google ScholarXiaobo WangYou can also search for this author in PubMed Google ScholarZhibing HouYou can also search for this author in PubMed Google ScholarQiaoqiao HuYou can also search for this author in PubMed Google ScholarPeng DuYou can also search for this author in PubMed Google ScholarHao YuYou can also search for this author in PubMed Google ScholarChang LiuYou can also search for this author in PubMed Google ScholarMiao CuiYou can also search for this author in PubMed Google ScholarGangling TongYou can also search for this author in PubMed Google ScholarZhihua ZhengYou can also search for this author in PubMed Google ScholarYunsheng XuYou can also search for this author in PubMed Google ScholarLinyu ZhuYou can also search for this author in PubMed Google ScholarJin ChengYou can also search for this author in PubMed Google ScholarFeng WuYou can also search for this author in PubMed Google ScholarYulan ZhengYou can also search for this author in PubMed Google ScholarPeijun LiuYou can also search for this author in PubMed Google ScholarPeng HongYou can also search for this author in PubMed Google ScholarContributionsY. Zhan and conceptualized the study; Y. Zhan, and recruited patients, performed physical examinations, and abstracted historic data; Y. Zhu, and performed laboratory tests and interpreted results; and conducted sonographic and radiological examinations and interpreted results; and Y. Zheng conducted PFT and interpreted results; Y. Zhan, and conducted functional tests, assessed rehabilitation status and interpreted data; and interpreted metabolic and immunological findings; Y. Zhan, and conducted data quality checks and performed statistical analyses; Y. Zhan and wrote the manuscript. All authors read and approved the final authorsCorrespondence to Feng Wu, Yulan Zheng, Peijun Liu or Peng declarations Competing interests The authors declare no competing interests. Supplementary informationRights and permissions Open Access This article is licensed under a Creative Commons Attribution International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original authors and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit Reprints and PermissionsAbout this articleCite this articleZhan, Y., Zhu, Y., Wang, S. et al. SARS-CoV-2 immunity and functional recovery of COVID-19 patients 1-year after infection. Sig Transduct Target Ther 6, 368 2021. citationReceived 06 March 2021Revised 16 September 2021Accepted 20 September 2021Published 13 October 2021DOI Loading metrics Open Access Peer-reviewed Research Article Michael Tu, Jordan Cheng, Fang Wei, Feng Li, David Chia, Omai Garner, Sukantha Chandrasekaran, Richard Bender, Charles M. Strom , David T. W. Wong Development and validation of a quantitative, non-invasive, highly sensitive and specific, electrochemical assay for anti-SARS-CoV-2 IgG antibodies in saliva Samantha H. Chiang, Michael Tu, Jordan Cheng, Fang Wei, Feng Li, David Chia, Omai Garner, Sukantha Chandrasekaran, Richard Bender, Charles M. Strom x Published July 1, 2021 Figures AbstractAmperial™ is a novel assay platform that uses immobilized antigen in a conducting polymer gel followed by detection via electrochemical measurement of oxidation-reduction reaction between H2O2/Tetrametylbenzidine and peroxidase enzyme in a completed assay complex. A highly specific and sensitive assay was developed to quantify levels of IgG antibodies to SARS-CoV-2 in saliva. After establishing linearity and limit of detection we established a reference range of 5 standard deviations above the mean. There were no false positives in 667 consecutive saliva samples obtained prior to 2019. Saliva was obtained from 34 patients who had recovered from documented COVID-19 or had documented positive serologies. All of the patients with symptoms severe enough to seek medical attention had positive antibody tests and 88% overall had positive results. We obtained blinded paired saliva and plasma samples from 14 individuals. The plasma was analyzed using an EUA-FDA cleared ELISA kit and the saliva was analyzed by our Amperial™ assay. All 5 samples with negative plasma titers were negative in saliva testing. Eight of the 9 positive plasma samples were positive in saliva and 1 had borderline results. A CLIA validation was performed as a laboratory developed test in a high complexity laboratory. A quantitative non-invasive saliva based SARS-CoV-2 antibody test was developed and validated with sufficient specificity to be useful for population-based monitoring and monitoring of individuals following vaccination. Citation Chiang SH, Tu M, Cheng J, Wei F, Li F, Chia D, et al. 2021 Development and validation of a quantitative, non-invasive, highly sensitive and specific, electrochemical assay for anti-SARS-CoV-2 IgG antibodies in saliva. PLoS ONE 167 e0251342. Chandrabose Selvaraj, Alagappa University, INDIAReceived January 14, 2021; Accepted April 25, 2021; Published July 1, 2021Copyright © 2021 Chiang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are Availability Data is available on figshare DW is supported by U54HL119893, UCLA Keck Foundation Research Award Program. SC is supported by F30DE027615. This study was partially funded by Liquid Diagnostics, LLC LD. The funder provided reimbursement to MT as a paid consultant. This author contributed to this study by performing some experiments and in manuscript preparation. He did not contribute to the decision to publish, data collection or interests CS in an unpaid CEO of LD. CS, MT, RB, and DW are equity holders in LD. LD is the exclusive license holder for the Amperial™ technology from the University of California and hopes to commercialize products based on this technology. This does not alter our adherence to PLOS ONE policies on sharing data an materials. IntroductionA novel corona virus, severe acute respiratory syndrome coronavirus 2 SARS-CoV-2, has caused a global pandemic causing major disruptions world-wide [1]. Multiple high-throughput PCR based tests have been developed that are reasonably sensitive and specific, however the same cannot be said for antibody testing, prompting The Center for Disease Control CDC to issue guidelines entitled “Interim Guidelines for COVID-19 Antibody Testing” [2]. This publication describes the variability of in-home antibody tests and the lack of specificity required to make home-based antibody testing a valuable tool for epidemiologic surveillance. Having a reliable self-collection antibody test may be of enormous help in epidemiologic studies of background immunity, testing symptomatic individuals without RNA based testing during their acute illness, and screening health care providers and first responders to establish prior COVID-19 infection. Such a test may also be valuable in following vaccinated patients to assess the kinetics of anti-SARS-CoV-2 antibody production following inoculation. Multiple serological tests based on serum or plasma have been developed and marketed, with ELISA and lateral flow methods predominating. However, many methods suffer from low sensitivities and specificities [2–6]. Antibodies begin appearing in the first week following the development of symptoms. IgG, IgM, and IgA are detectable with IgA appearing somewhat earlier than IgG and IgM. Most patients seroconvert by 2 weeks following symptoms. Unlike IgA and IgM, IgG persists for several months following infection [7–9]. In a published study of 1,797 Icelandic individuals recovered from qPCR documented COVID-19 disease, 91% were IgG seropositive and antibody levels remained stable for 4 months after initial symptoms [10]. Notably of individuals quarantined due to exposure but untested for virus, with negative qPCR results, tested positive for IgG antibodies. Of 18,609 patients who were both unexposed and asymptomatic, the seropositivity rate was [11]. Since health care systems are burdened with care for COVID-19 patients, having a test that does not require phlebotomy would be extremely beneficial. To that end, investigations have been carried out using home finger prick blood sampling and even some home blood spot testing lateral flow strips [5–7]. However, home finger stick is invasive and not acceptable to some individuals, and requires a health care professional to administer the test to vulnerable individuals such as the elderly and children. In addition, home blood collection tests are less accurate than phlebotomy, with specificities less than 98%. In a low prevalence disease, the positive predictive value for a test with 98% specificity is less than 50% [7, 11]. Saliva is an oral fluid that is obtained easily and non-invasively. Proteomic studies show that the immunoglobulin profile in saliva is nearly identical to that of plasma [12]. Therefore, saliva is an excellent medium for COVID-19 antibody measurement. There are several commercially available collection devices to facilitate saliva collection, stabilization of IgG, and transport. A recently published study demonstrated excellent correlation between levels of COVID-19 antibodies in serum and saliva [13]. In order to be useful in population-based screening and to determine individual immunity in exposed populations, a SARS-CoV-2 antibody test must be highly specific because of the low seroprevalence rate in the population [2, 14]. In addition, the ability to quantify antibody levels is important for vaccine development and in monitoring for waning immunity [2, 14]. The only published saliva based assay for SARS-CoV-2 antibodies had only 89% sensitivity with 98% specificity [13], leading to a positive predictive value of only 49% in a population with a 2% prevalence of COVID-19 exposure. Our goal was to develop a non-invasive saliva based quantitative test for COVID-19 antibodies with exquisite sensitivity. We reviewed existing literature to find the SARS-CoV-2 antigen domain with the highest specificity and the ability to distinguish between the COVID-19 virus and other related Coronaviruses. The S1 domain is the most specific in terms of cross reactivity with other Corona and other respiratory viruses. As recombinant S1 antigen is readily available from at least 2 vendors, we chose the S1 antigen for our assay development. Levels of IgM and IgA deteriorate rapidly following recovery from COVID-19 infection; IgG levels remain detectable for several weeks to months [10]. Since the intended use of our assay is for population-based screening and vaccine efficacy monitoring, we chose to assay IgG only. The Amperial™ technology, formerly known as Electric Field Induced Release and Measurement EFIRM™, is a novel platform capable of performing quantitation of target molecules in both blood and saliva [15]. The device works by immobilizing capture moieties on the surface of an electrode structure for capturing target analytes and then quantifying the target analyte through electrochemically measuring oxidation-reduction between a hydrogen peroxide and tetramethylbenzidine substrate and peroxidase enzyme in a completed assay sandwich. The assay takes place on electrodes packaged in the format of a traditional 96-well microtiter plate, making the assay technique highly compatible and scalable with existing lab liquid handling instruments. We developed quantitative Amperial™ assays for IgG, IgM, and IgA antibodies to the S1 spike protein antigen of SARS-CoV-2. This test is highly sensitive >88% and specific > for patients with COVID-19 infections and correlates well with plasma ELISA analysis. The unique assay described in this article is completely non-invasive, allows home-collection, is quantitative, and has shown no false positives in 667 unexposed individuals, leading to a specificity of at least The assay has strong utility for clinical laboratories as it does not require purification/extraction of the saliva specimen, but the sample can simply be pipetted out of the collection device, diluted, and pipetted to the assay plate. The turnaround time of the assay is also fast, requiring less than 1 hour for a complete assay to be run. The widespread use of this test may be of great value in identifying individuals with prior exposure to SARS-CoV-2, to follow patients longitudinally to determine the kinetics of diminishing antibody concentration, and may be of special value in the longitudinal monitoring of vaccinated individuals to assess continued serologic immunity. Materials and methodsThe schematic of the Amperial™ SARS-CoV-2 IgG antibody is shown in Fig 1. The principle of the Amperial™ platform is that a biomolecule in this case SARS-CoV-2 Spike protein S1 antigen is added to a liquid pyrrole solution that is then pipetted into the bottom of microtiter wells containing a gold electrode at the bottom of each well. After the solution is added to each well, the plate is placed into the Amperial™ Reader and subjected to an electric current leading to polymerization. This procedure results in each well becoming coated with a conducting polymer gel containing the S1 antigen. Following the polymerization, diluted saliva, plasma, or serum is added to the well. Specific anti-S1 antibodies bind to the S1 antigen in the polymer. After rigorous washing procedures, the bound antibody is detected by using biotinylated anti-human IgG and then the signal is amplified by a standard streptavidin / horseradish peroxidase reaction that produces an electric current measured by the Amperial™ Reader in the nanoampere nA scale. The instrument is capable of accurately measuring current in the picoampere pA range, so the measurement is well within the ability of the instrument [13, 14, 16, 17]. The measurement of current rather than optical absorbance, as is done in the typical ELISA, has two important advantages over standard ELISA. Firstly, it allows precise quantitation of the amount of bound antibody and secondly, the measurement of current rather than optical absorbance allows increased sensitivity. Since antibody levels in saliva are lower than in plasma [13, 16], this increased sensitivity is crucial. The precise details of the assay are described in the next paragraph. COVID-19 Spike-1 Antigen Sanyou-Bio, Shanghai, China was diluted to a concentration of μg / mL, added to each well of the microtiter plate, and co-polymerized with pyrrole Sigma-Aldrich, St. Louis, MO onto the bare gold electrodes by applying a cyclic square wave electric field at 350 mV for 1 second and 1100 mV for 1 second. In total, polymerization proceeded for 4 cycles of 2 seconds each. Following this electro-polymerization procedure, 6 wash cycles were performed using 1x PBS with Tween-20 PBS-T using a 96-channel Biotek 405LS plate washer programmed to aspirate and dispense 400 μL of solution per cycle. Following the application of the polymer layer, 30 μL of saliva diluted at a 110 ratio in Casein/PBS Thermo-Fisher, Waltham, MA was pipetted into each well and incubated for 10 minutes at room temperature. Unbound components were removed by performing 6 wash cycles of PBS-T using the plate washer. Biotinylated anti-human IgG secondary antibody Thermofisher, Waltham, MA at a stock concentration of mg / mL was diluted 1500 in Casein/PBS and 30 μL pipetted to the surface of each well and incubated for 10 minutes at room temperature followed by 6 wash cycles using PBS-T. Subsequently, 30 μL of Poly-HRP80 Fitzgerald Industries, Acton, MA at a stock concentration of 2 μg / mL was diluted 125 in Casein/PBS, added to the wells, and incubated at 10 minutes at room temperature. Following a final wash using 6 cycles of PBS-T, current generation is accomplished by pipetting 60 μL of 1-Step Ultra TMB Thermofisher, Waltham, MA to the surface of the electrode and placing the plate into the Amperial™ reader where current is measured at -200 mV for 60 seconds. The current in nA is measured 3 times for each well. The process for reading the entire 96 well plate requires approximately 3 minutes. Plasma quantitative Amperial™ assay for SARS-CoV-2 IgG The protocol is similar to the Amperial™ SARS-CoV-2 IgG antibody for saliva samples. Following the application of the polymer layer, 30 μL of plasma diluted at a 1100 ratio in Casein/PBS Thermo-Fisher, Waltham, MA was pipetted into each well and incubated for 10 minutes at room temperature. The standard curve for plasma contains the following points 300 ng / ml, 150 ng / ml, 75 ng / ml, ng / ml, ng / ml, and 0 ng / ml. Plasma SARS-CoV-2 ELISA assay We purchased FDA EUA ELISA kits EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgG antibodies EUROIMMUN US, Mountain Lakes, NJ, Product ID EI 2606–9601 G, Lot E2001513BK. We processed samples exactly as described in the package insert. Human subjects Volunteers, with prior positive qPCR tests for COVID-19 infection or positive antibody tests using currently available FDA EUA-cleared antibody tests were consented via a written consent. Subjects enrolled were all over the age of 18. Subject participants responded to a questionnaire regarding severity of symptoms, onset of symptoms, and method of diagnosis UCLA IRB 06-05-042. Severity of symptoms were self-graded on the following 7-point scale 0 Asymptomatic 1 Mild Barely noticed, perhaps slight fever and cough 2 Moderate felt moderately ill but did not need to seek medical care 3 Sought medical Care but was not admitted to hospital 4 Hospitalized 5 Admitted to ICU 6 Placed on Ventilator A set of 13 paired saliva and plasma samples were provided by the Orasure™ Company. Saliva collection All COVID-19 samples were obtained using the Orasure™ FDA-cleared saliva collection device and used according to manufacturer instructions. The Orasure™ collection device consists of an absorbent pad on the end of a scored plastic wand. The individual places the pad between cheek and gum for a period of 2–5 minutes. Subsequently the wand and pad are placed into a tube containing transport medium, the top of the stick is broken off, and the tube is sealed for transport. The sealed tube is placed into a zip-lock bag and shipped by any standard method. According to the package insert, samples are stable at ambient temperature for 21 days see results below and Orasure™ website. An alternate sample collection method involves the individual swabbing the pad 4 times in the gingival tooth junction prior to placing the pad between the cheek and gum. This method has been shown to improve IgG yield in some patients with low antibody levels personal communication with Orasure Technologies, Inc.. Participant recruitment method Positive samples determined either through a positive SARS-CoV-2 viral test or antibody test were acquired beginning May 2020 to July 2020 via the described Orasure™ Oral Fluid Collection Device Kit previous described. Subjects were recruited into the study via electronic correspondence during the early stages of the COVID-19 pandemic in regions affected by COVID-19 California, Illinois, New York, New Jersey. Subjects are all over the age of 18. Subjects are not representative of the general population. Samples collected pre-2012 were used as controls. Saliva was collected from healthy individual volunteers at meetings of the American Dental Association between 2006 and 2011. Consent was obtained under IRB approval UCLA IRB 06-05-042. Both male and females, mostly non-smokers, 18–80 years of age, and differing ethnicities were included. All subjects were consented prior to collection. Each subject expectorated ~ 5 mL of whole saliva in a 50cc conical tube set on ice. The saliva was processed within 1/2 hour of collection. Samples were spun in a refrigerated centrifuge at 2600 X g for 15 minutes at 4°C. The supernatant cell-free saliva was then pipetted into two-2 mL cryotubes and μL Superase-In Ambion, Austin, TX was added as a preservative. Each tube was inverted to mix. The samples were frozen in dry ice and later stored in -80°C. Sample size and statistical methods Due to the nature of the pandemic and the evolving nature of EUA diagnostics during the early phases of the pandemic, no power calculations were performed for study size but instead the FDA/EUA recommendation of 30 subjects was followed. For components of work that required comparisons between groups, student’s T-test was conducted. p value, corresponds to a 95% confidence or p value, corresponds to 99% confidence. Data analysis performed was using GraphPad Prism Results Linearity Fig 2 demonstrates the dynamic range and linearity of the assay. In these experiments varying amounts of monoclonal human anti-S1 IgG was added to a saliva sample from a healthy volunteer and subjected to the assay. Fig 2 shows a range of to 6 ng/ml. The Y-axis shows nano-amperage measured nA. The X-axis represents spike-in concentrations of IgG. The assay begins to become saturated at about 3 ng / ml. Fig 3 shows dilutions down to ng / ml to ng / ml and shows linearity in that range. This allows us to create a standard curve containing the following points 3 ng / ml, ng / ml, ng / ml, ng / ml, ng / ml, and 0 ng / ml. Fig 2. Dynamic range and linear range of Amperial™ anti-Spike S1 IgG Amount of spike in anti-SARS-CoV-2 IgG in ng / ml. Y-axis Normalized current in nA. Panel A 0–5 ng / ml Panel B ng / ml. Inhibition assay In order to demonstrate the specificity for the assay on actual clinical samples, we used the saliva from 3 recovered patients who had high levels of SARS-CoV-2 antibodies and added exogenous S1 antigen in varying amounts prior to analysis on the Amperial™ assay. The exogenous S1 antigen should compete for binding sites and therefore extinguish the nA signal. Fig 3 shows the results of this experiment. The red, purple, and green represent 3 different patients. The X-axis demonstrates increasing concentration of exogenous S1 added to the saliva before subjecting it to the assay. As shown, saliva pre-incubated with S1 antigen extinguishes the detectable IgG signal proportionately, therefore demonstrating the specificity of the assay to S1 antigen in clinical samples. Matrix effects Since we are be comparing samples collected by various methods, it is vital to determine if any significant matrix effects could interfere with data interpretation. We examined the 3 different collection methods used in this study Expectoration/centrifugation, Orasure™ without swabbing and Orasure™ with swabbing. Two methods of collection using the Orasure™ Oral Fluid Collection Device were tested. The first method non-swabbing collects saliva by placing an absorbent pad into the lower gum area for 2–5 minutes and then placing the saturated collection pad into a preservative collection tube. The second method swabbing adds the step of first gently rubbing the collection pad along gum line, between the gum and cheek, 5 times, before placing the device in the lower gum area for 2–5 minutes, and then immersing the saturated collection pad into the collection tube. Healthy donors n = 5 collected their saliva using these two different methods. The control pre-2012 samples were collected with an expectoration protocol for whole saliva collection falcon tubes, processing centrifuge, stabilization, and storage. Five samples collected by each of the 3 methods and were analyzed in duplicate. The results are shown in Fig 4 under the heading “No spike in.” There are no differences among 3 sample types. We then added monoclonal human anti-S1 IgG to each sample and again ran them in duplicate Fig 4 above caption Spike-in ng / ml IgG. A non-parametric Student t-test was performed with no significant differences between any of the collection methods. Stability The Orasure™ collector is an FDA-cleared device for the analysis of anti-HIV IgG. The package insert describes a 21-day stability at ambient temperature. We wished to establish the stability of anti-COVID-19 IgG using this collector. Passive whole saliva was collected from four healthy individuals using 50 mL falcon tubes and spiked with anti-Spike S1 IgG to reach a final concentration of 300 ng / ml. Aliquots of mL of saliva were placed into 50 mL tubes and then the sponge of the Orasure™ collector was submerged into the saliva for five minutes and processed as described in Methods. The collected saliva was then aliquoted into PCR tubes and left at ambient temperature 21°C for 0, 1, 3, 7, and 14 days before storage at -80°C. After 14 days, samples were thawed and assayed using the anti-Spike S1 IgG Amperial™ assay to assess stability. At 14 days, 95% of the original signal remained, demonstrating the 14-day stability of anti-SARS-CoV-2 antibodies collected in Orasure™ containers see Fig 5. Fig 5. Stability study performed on spike-in of SARS-CoV-2 IgG into healthy saliva specimen using two different methods a research SOP which involves expectoration into a falcon tube and the Orasure™ Oral Fluid collection device.The collect saliva was aliquoted and left at ambient temp for 0, 1, 3, 7, 14 days. Results were normalized relative to the measured assay signal of a sample at day 0. Results show that the sample is stable with no significant degradation for up to 14 days. Specificity and reference range Once we established no significant differences between the tube collection method and the Orasure™ collector method, we analyzed a series of 667 samples collected between 2006 and 2009 at the annual meeting of the American Dental Association. Scatter plots of these data for both nA and ng / ml are shown in Fig 6A and 6B. We established the mean and standard deviation for both raw nA values and concentration in ng / ml. In order to maximize specificity, we selected a reference range > 5 SD above the mean. A 5 sigma level would lead to a specificity of In fact, we have never seen a healthy sample above the 5 sigma level. As will be seen, the sensitivity of the assay remains greater than 88% even with this rigorous specificity. Fig 6. Healthy reference range of Amperial™ saliva anti-SARS-CoV-2 IgG assay of 667 unexposed subjects in A normalized current ΔnA with mean = and cutoff = and B concentration ng / ml with mean = and cutoff = Recovered COVID-19 patients Fig 7 displays the scatter plot for 667 healthy controls and 34 volunteer patients who recovered from COVID-19 infection. All patients were a minimum of 14 days post onset of symptoms and some patients were as many as 15 weeks post symptoms. The 5 sigma cutoff is shown by the green dotted line. A more detailed discussion of the recovered patients appears in the following section. The data show that all healthy patients are negative and 30 of the 34 recovered patients are positive. These data demonstrate a sensitivity of 88% and a specificity of > It is important to note that not all recovered patients have detectable antibody [10] so the 4 patients with undetectable antibody may be biologically negative and not the result of lack of sensitivity of the assay. Fig 8 demonstrates the relationship of anti-S1 IgG levels to severity of symptoms. Table 1 is a tabular summary of these data. All patients who had severity indexes ≥3 sought medical attention, admitted to hospital, admitted to ICU, on ventilator had positive antibody levels. Although 4 patients with mild symptoms had antibody levels in the normal range, both asymptomatic patients had appreciable antibody levels. These patients were close contacts of more severely affected patients. The highest antibody level recorded is severity index level 2 patient moderate symptoms, did not seek medical care. It is important to note that both asymptomatic patients had easily detectable antibody levels in saliva, suggesting this test may be useful in general population screening. Paired saliva and plasma samples We obtained 14 paired, blinded plasma and saliva samples. The plasma was analyzed by an FDA EUA-cleared ELISA test purchased from EUROIMMUN see Methods. The saliva samples, collected in Orasure™ buffer, were analyzed by the Amperial™ assay described in Methods. After unblinding, we discovered 8 recovered COVID patients and 5 healthy patients in this series. All 5 healthy patients were negative in both the saliva and plasma assays. In 7 of the 8 recovered patients, both plasma and saliva tests were positive. There was one sample with a discrepancy between saliva and plasma, with the plasma positive and the saliva in the indeterminate range. The EUROIMMUNE ELISA assay is a semi-quantitative assay and yields an absorbance ratio rather than a quantity. Fig 9 demonstrates the relationship between the saliva quantitative results and plasma absorbance ratio for the paired plasma and saliva samples. There is a clear relationship between the 2 levels, with the higher plasma absorbance ratios associated with higher saliva quantitation. Fig 9. COVID-19 antibody level in paired saliva and plasma of COVID-19 n = 8 subjects in a blinded randomized antibodies level are measured by EUROIMMUN ELISA reported in ratio proportion of OD of calibrator to OD of sample and saliva antibodies are measured by Amperial™ in pg / ml. Green dashed line indicates 5 SD reference range cutoff of Amperial™ test and red dashed line is reference range for EUROIMMUN ELISA. developed a research quality assay to quantify anti-SARS-CoV-2 IgG levels in plasma see Methods. We analyzed the 13 plasma samples using this assay. The results of this experiment are shown in Fig 10. Panel A shows a log / log plot of plasma versus saliva levels showing a clustering with high plasma levels associated with high saliva levels. Panel B shows the box plot of these values, demonstrating that plasma levels are approximately 50X those of saliva. This observation explains the necessity for an extremely sensitive assay such as the Amperial™ assay in order to detect antibodies in saliva. Of note, the publication regarding saliva SARS-CoV-2 IgG detection reports levels of 25–60 mcg / ml, 1000 times less sensitive than our assay. Fig 10. Relationship of plasma anti-SARS-CoV-2 IgG levels to saliva levels measured by Amperial™ assays.A Panel A shows a log / log plot of plasma versus saliva levels showing a clustering of the positive values with high plasma levels associated with high saliva levels on the Amperial™ platform. B Box plot of COVID-19 n = 8 and healthy n = 5 subjects demonstrating that the normalized plasma levels are approximately 50X those of saliva. Longitudinal tracking of antibody levels Three of our volunteers supplied samples at weekly intervals so we could determine the stability of their antibody levels. Results appear in Fig 11. The 5 standard deviation cutoff is again shown with the dashed green line. All 3 patients continued to have detectable levels for more than 12 weeks, with the longest interval of 15 weeks. All tests were positive in all patients and antibody levels in all 3 patients remained clearly positive during the time interval studied. Patients C1 and C3 seem to have a rise in antibody level between 11 and 12 weeks post initial symptoms followed by a return to baseline level. Patient C2 might also have had a spike in antibody levels at 10 weeks. This may be result of the amnestic B-cell population becoming established. There is insufficient data at this time to determine if this is a generalized pattern. CLIA evaluation We performed a full CLIA laboratory developed test evaluation for the Amperial™ COVID-19 IgG Antibody test. The validation assayed 72 unaffected patients and 30 recovered patients and demonstrated 100% sensitivity and specificity. The intra-assay and inter-assay variability were and respectively. DiscussionWe have developed an exquisitely specific, sensitive, non-invasive saliva based quantitative assay for anti-SARS-CoV-2 IgG antibodies. Our goal was to create a quantitative assay with sufficient positive predictive value to be useful to inform individuals regarding previous infection with COVID-19. By establishing a reference range of 5 sigma above than the mean we have a theoretical analytical specificity of We plan to repeat the analysis of all positive samples to further increase analytical specificity. Since our test is non-invasive with home-collection we can also offer repeat testing on a second sample to further increase specificity. These procedures will minimize the false positives due to purely technical issues. There is still the possibility of biological false positives, however, due to cross reactivity with other infectious or environmental agents. The S1 antigen appears to be specific for SARS-CoV-2 [2, 3, 10] and in our series of 667 samples collected prior to 2019 we observed no false positive results. We cannot predict the eventual clinical specificity of this assay. At a minimum, the specificity is 667 / 668 or assuming the next control sample tested would be a false positive, but the specificity is likely to be higher. Our current sensitivity is 100% for patients with symptoms severe enough to seek medical care. For all patients, including mildly asymptomatic patients, our clinical sensitivity is 88%. Since the Amperial™ assay only requires 6 μL of collection fluid, several assays can be performed from the same sample. This allows all positives to be repeated to confirm the positive results and further increase the specificity of the assay. We will offer testing of a second, independent sample for all patients testing positive. Since saliva collection is easily be performed at home, obtaining a second sample is not difficult. For any laboratory test, the PPV is proportional to the prevalence of positivity in the population. A recent study demonstrated a prevalence of between to 6% in Britain [17]. Using the minimum specificity of and a prevalence of 6% the Amperial™ saliva assay would have a minimum PPV of 96%. In contrast, a published saliva antibody detection assay reported a specificity of 98% with a similar sensitivity 89%. This specificity leads to PPV of only 69% making it an ineffective tool for population screening. Our data demonstrate that the Imperial™ assay is appropriate for longitudinal screening of antibody levels, a particular utility in vaccine trials and in population monitoring following mass immunization. Since this assay is quantitative and levels appear to be stable with time, patients may be monitored from home at frequent intervals. If antibodies raised in response to vaccination do not include IgG antibodies to S1 antigen, it is easy to rapidly develop Amperial™ antibody tests to any antigen. This requires adding the new antigen to the pyrrole solution and does not require significant alteration of assay conditions. A particular advantage of this assay is convenience. The Orasure™ collector is simple and easy to use and does not require professional monitoring for adequate collection. Home collection relieves the burden to an already stressed health care system. Vulnerable populations such as children and the elderly can be guided through the collection process by parents or other adults. It is possible to obtain repeat samples to confirm positives and to perform longitudinal testing since the only requirement for testing is shipping the collecting kit. The Amperial™ IgG test is plate-based and high-throughput. An entire plate is easily processed in 2 hours, leading to rapid turnaround time once the sample enters the laboratory. There is no pre-processing of the sample required; samples are taken directly from the collection vial and placed into the assay. With standard liquid handlers, the assay may be easily automated allowing for extremely high-throughput since the Amperial™ reader is only required for the polymerization step of less than a minute at the beginning of the assay and 3 minutes for the measurement phase at the end of the assay. Published data [13] and our own demonstrate a correlation between blood results and saliva results indicating that the IgG present in saliva is most likely derived from the plasma through filtration. Our data shows that saliva IgG levels are approximately 50-fold less than those in plasma necessitating a highly sensitive assay in order to detect the IgG levels in saliva. There is some discussion in the literature of the role antibody testing may have in managing the COVID-19 epidemic. Alter and Seder published an editorial in the New England Journal of Medicine arguing, “Contrary to recent reports suggesting that SARS-CoV-2 RNA testing alone, in the absence of antibodies, will be sufficient to track and contain the pandemic, the cost, complexity, and transient nature of RNA testing for pathogen detection render it an incomplete metric of viral spread at the population level. Instead, the accurate assessment of antibodies during a pandemic can provide important population-based data on pathogen exposure, facilitate an understanding of the role of antibodies in protective immunity, and guide vaccine development [14]”. ConclusionIn this article, we describe the development of a non-invasive, home collection based, exquisitely specific, and acceptably sensitive test for the presence of anti-SARS-CoV-2 antibodies in saliva. This may be an important tool in controlling the pandemic and facilitating and understanding of the role of antibody production in COVID-19 immunity. Longitudinal monitoring of anti-SARS-CoV-2 IgG levels could also play a valuable role in vaccine development and deployment by allowing longitudinal quantitative assessment of antibody levels. If the presence of detectable anti-COVID-19 IgG is shown to be an indicator of immunity to reinfection, measurement of these antibodies could allow individuals to safely return to work, school and community. The Amperial™ SARS-CoV-2 assay fulfills the requirements for all of these applications. References1. Guan W, Ni Z, Hu Y, Liang W, Ou C, He J, et al. Clinical Characteristics of Coronavirus Disease 2019 in China. New Eng J Med. 2020 Apr;382181708–20. pmid32109013 View Article PubMed/NCBI Google Scholar 2. Interim Guidelines for COVID-19 Antibody Testing. Center for Disease Control and Prevention. 2020 Aug 1. [Cited 2020 Nov 5] 3. Hoffman T, Nissen K, Krambach J, Ronnberg B, Akaberi D, Esmaeilzadeh , et al. Evaluation of a COVID-19 IgM and IgG rapid test; an efficient tool for assessment of past exposure to SARS-CoV-2. Infection Ecology and Epidemiology. 2020 Jan 1;1011754538. pmid32363011 View Article PubMed/NCBI Google Scholar 4. Montesinos I, Gruson D, Kabamba B, Dahma H, Wijngaert S, Reza S, et al. Evaluation of two automated and three rapid lateral flow immunoassays for the detection of SARS CoV-2 antibodies. Journal of Clinical Virology. 2020 Jul;128104413. pmid32403010 View Article PubMed/NCBI Google Scholar 5. Döhla M, Boesecke C, Schulte B, Diegmann C, Sib E, Richter E, et al. Rapid point-of-care testing for SARS-CoV-2 in a community screening setting shows low sensitivity. Public Health. 2020 May;182170–172. pmid32334183 View Article PubMed/NCBI Google Scholar 6. Rashid Z, Othman S, Samat M, Ali U, Wong K. Diagnostic performance of COVID-19 serology assays, Malaysian J Pathol, 2020 Apr;42113–21. View Article Google Scholar 7. Thevis M, Knoop A, Schafer M, Dufaux B, Schrader Y, Thomas A, et al. Can dried blood spots DBS contribute to conducting comprehensive SARS-CoV-2 antibody tests? Drug Test Analy. 2020 Jul;127994–7. pmid32386354 View Article PubMed/NCBI Google Scholar 8. Sun B, Feng Y, Mo X, Zheng P, Wang Q, Li P, et al. Kinetics of SARS-CoV-2 specific IgM and IgG responses in COVID-19 patients. Emerging Microbes and Infections. 2020 Jan 1;91940–948. pmid32357808 View Article PubMed/NCBI Google Scholar 9. Shi Y, Wang Y, Shao C, Huang J, Gan J, Huang X, et al. COVID-19 infection the perspectives on immune response. Cell Death and Differentiation. 2020 May;275 1451–1454. pmid32205856 View Article PubMed/NCBI Google Scholar 10. Gudbjartsson D, Norddahl G, Melsted P, Gunnarsdottir K, Holm H, Eythorsson E, et al. Humeral immune response to SARS-CoV-2 in Iceland. N Engl J Med. 2020 Oct 29;383181724–1734. pmid32871063 View Article PubMed/NCBI Google Scholar 11. Okba N, Muller M, Li W, Wang C, GeurtsvanKessel C, Corman V, et al. Severe Acute Respiratory Syndrome Coronavirus 2 –Specific Antibody Responses in Coronavirus Disease Patients. Emerg Infect Dis. 2020 Jul;26 71478–88. pmid32267220 View Article PubMed/NCBI Google Scholar 12. Hettegger P, Huber J, Pabecker K, Soldo R, Kegler U, Nöhammer C, et al. High similarity of IgG antibody profiles in blood and saliva opens opportunities for saliva based serology. PLoS ONE. 2019 Jun 20;146e0218456. pmid31220138 View Article PubMed/NCBI Google Scholar 13. Isho B, Abe KT, Zuo M, Jamal A, Rathod B, Wang J, et al. Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients. Sci Immunol. 2020 Oct 8;552eabe5511. pmid33033173 View Article PubMed/NCBI Google Scholar 14. Alter G, Seder R. The power of Antibody-Based Surveillance. N Engl J Med. 2020 Oct 29;383181782–1784. pmid32871061 View Article PubMed/NCBI Google Scholar 15. Wei F, Patel P, Liao W, Chaudhry K, Zhang L, Arellano-Garcia M, et al. Electrochemical Sensor for Multiplex Biomarkers Detection. Clinical Cancer Research. 2009;15 4446–4452. pmid19509137 View Article PubMed/NCBI Google Scholar 16. Ceron J, Lamy E, Martinez-Subiela S, Lopez-Jornet P, Capela-Silva F, Eckersall P, et al. Use of Saliva for Diagnosis and Monitoring of SARS-CoV-2 A General Perspective. J Clin Med. 2020 May 15;951491. pmid32429101 View Article PubMed/NCBI Google Scholar 17. Imperial College London COVID-19 Virus Home Antibody Testing. 2020 June to September. [Cited 2020 November 5]

anti sars cov 2 kuantitatif