key: cord-0974925-ohlciw3d authors: Lee, Jeong Hoon; Bae, Pan Kee; Kim, Hyunho; Song, Yoon Ji; Yi, So Yeon; Kwon, Jungsun; Seo, Joon-Seok; Lee, Jeong-min; Jo, Han-Sang; Park, Seon Mee; Park, Hee Sue; Shin, Kyeong Seob; Chung, Seok; Shin, Yong Beom title: A Rapid Quantitative On-site Coronavirus Disease 19 Serological Test date: 2021-06-05 journal: Biosens Bioelectron DOI: 10.1016/j.bios.2021.113406 sha: 301ebd00a7bec9ea53c14997f2ef1c83d3967e12 doc_id: 974925 cord_uid: ohlciw3d On-site severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) serological assays allow for timely in-field decisions to be made regarding patient status, also enabling population-wide screening to assist in controlling the coronavirus disease 2019 (COVID-19) pandemic. Here we propose a rapid microfluidic serological assay with two unique functions of nanointerstice filling and digitized flow control, which enable the fast/robust filling of the sample fluid as well as precise regulation of duration and volume of immune reaction. Developed microfluidic assay showed enhanced limit of detection, and 91.67% sensitivity and 100% specificity (n=152) for clinical samples of SARS CoV-2 patients. The assay enables daily monitoring of IgM/IgG titers and patterns, which could be crucial parameters for convalescence from COVID-19 and provide important insight into how the immune system responds to SARS CoV-2. The developed on-site microfluidic assay presented the mean time for IgM and IgG seroconversions, indicating that these titers plateaued days after seroconversion. The mean duration from day 0 to PCR negativity was 19.4 days (median 20 d, IQR 16–21 d), with higher IgM/IgG titres being observed when PCR positive turns into negative. Simple monitoring of these titres promotes rapid on-site detection and comprehensive understanding of the immune response of COVID-19 patients. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the aetiological agent of coronavirus disease 2019 , has spread rapidly worldwide. Different from that in other viral infections, identification and hospitalization of patients with COVID-19 may result from government-led active testing and surveillance, with potential disease severity ranging from asymptomatic to critical (Lee and 5 . Therefore, rapid and quantitative on-site serological assay investigating the dynamic 10 detection with high sensitivity and specificity (Fang et al., 2020; Lan et al., 2020) , they impose financial burdens when applied for point-of-care monitoring of populations on a large scale. Furthermore, the sensitivity of these techniques reportedly vary according to infection duration, the site and quality of specimen collection, and the viral load (Weissleder et al., 2020) For instance, RT-PCR false-negative responses in patients with . In addition to the triage/screening application, serological assays are also expected to be beneficial in determining the level of community immunity, in enabling serosurveillance at the population level to monitor COVID-19 prevalence, and for use in the field testing of therapeutic agents/vaccines (Krammer and Simon, 2020a; Shen et al., 2020) . Serological assays using whole-blood collection processes, compared to oropharyngeal (OP)/nasopharyngeal (NP) approaches, 5 significantly reduce the potential of infection, also providing better accessibility to asymptomatic or suspected-infected individuals with negative RT-PCR results (Amanat et al., 2020b; Li et al., 2005; Long et al., 2020a) . ELISAs are a popular quantitative serological assay for determining the serological kinetics of antibody responses to SARS-CoV-2. Strong correlation between ELISA results and virus neutralization 10 was reported (Amanat et al., 2020a; Okba et al., 2020 J o u r n a l P r e -p r o o f The microfluidic chip used for the COVID-19 serological assay (Fig.1A) has two unique functions of nanointerstice (NI) filling and digitized flow control. As the first key technique, the nanointerstices (NIs) were formed at the both sides of the microfluidic channel during bonding procedure of bottom and top substrates (Fig.1B) . Meniscus in the small gap of the NI (~500 nm) was formed with extremely small diameter and large pressure difference at its air-liquid interface. It enables fast and robust filling 5 of the sample liquid without any hydrophilic surface treatment, creating robust sample filling into the main channel even after long-term storage (Chung et al., 2009) . The NI-driven flow is then regulated by flow digitization control (Fig.1C&D) hour. Activated fluorescent beads were centrifuged at 15,000 rpm for 15 min. After removing the supernatant, the fluorescent beads were mixed with 125 µg/mL mouse anti-human IgG and mouse antihuman IgM (Thermo Fisher scientific, USA) for 2 hours. Next, 1/10 volume of 20% weight/volume skim milk (cat #232100, Gibco) was added, along with 1/10 volume of the second blocking solution (cat #ABF2BS, Absology, Korea), and incubated for 30 min at room temperature. The fluorescent beads were 5 washed three times with storage buffer (cat #IBFSB, Absology), centrifuged, and the supernatant then removed. Pellets were resuspended in MES buffer, and the concentration of dAb-conjugated fluorescent beads was determined using a UV-1800 spectrometer (Shimadzu). Fluorescent beads at a concentration of 0.2% weight/volume in 1.5 µL conjugate buffer (cat #ABCB; Absology) were loaded onto the dAb deposition zone of the bottom substrate (Fig. 1E green box) using a nanoliter dispenser (Musashi, 10 Japan) and then dried. SARS-CoV-2 nucleocapsid protein (NP), provided by the BioNano Health Guard Research Center, was used as the capture antigen. Briefly, SARS-COV-2 NP (1 mg/mL) was biotinylated using EZ-link TM 15 Sulfo-NHS-LC-Biotin (cat #21335, Thermo Scientific) for 1 hour, according to the manufacturer's protocol. Surplus biotin was removed via three 2 hour cycles of dialysis. Concentration of the biotinylated antigen (Ag) was determined using a spectrometer. Streptavidin (0.3 mg/mL; cat #SA10, Prozyme, USA) was mixed with 3 mM EDC and 3 mM NHS in 50 mM MES buffer overnight. Next, 2 µL of streptavidin solution was loaded onto the bottom substrate of the microfluidic chip (Fig. 1E red and blue box) using 20 a nanoliter dispenser. The plates were then incubated for 1 h in a humid chamber at room temperature. Streptavidin was washed with 1 M tris(hydroxymethyl)aminomethane (Tris) buffer and then dried. Biotinylated Ag (2 µL), which was diluted with 0.2% weight/volume sucrose in 1× sodium phosphate dibasic, was loaded onto the streptavidin-loaded spot of the microfluidic chip. In 1 hour of drying, the J o u r n a l P r e -p r o o f immobilized capture Ag was washed using the second washing buffer solution. Washed bottom substrate was then dried overnight and ready for bonding. The intensity profiles of test and control zones were measured using a reader (Absology, Korea) 10 min from the negative control serum samples (NC). NC samples were tested from patients before the COVID-19 pandemic emerged. 15 > 1 positive (Fig. 2B) . Titre plateau was set where signal slope 20 showed a first flattening according to the following equation. All the IgG/IgM titer curves with log2(S/COI) versus days after infections were depicted (Fig.3 B and Fig. 4 D-E) . . J o u r n a l P r e -p r o o f 8 Fresh blood samples were obtained from patients at Chungbuk National University Hospital (Cheongju, Korea) and Seoul Clinical Laboratory (SCL, Korea), in accordance with guidelines of the institutional review board (CBNUH 2020-03-025-001 at CNUH and MDCTC-20-027 at SCL). Informed consent was 5 obtained from each patient prior to the study. Collected blood samples were centrifuged at 3,000 rpm for 15 min to separate the serum. 10 µL of separated serum was mixed with 200 µL of buffer, and then applied to the inlets of the microfluidic channels. 10 RT-PCR SARS-CoV-2 tests were performed using Allplex 2019-nCOV assays (Seegene Inc, Korea), according to the manufacturer's protocol. Viral RNA was isolated from pharyngeal swabs using KingFisher Flex (ThermoFisher, USA) and reverse transcribed into complementary DNA (cDNA). Viral cDNA was then amplified, and Ct values determined, using a CFX96™ Dx System (Bio-Rad, USA). Ct values less than 40 were classified as COVID-19 positive; those with a Ct value > 40 were classified as 15 COVID-19 negative. According to PCR results, the group in which all three viral genes (E/RdRP/N) were detected was classified as positive (P); the group in which only one or two genes were detected was classified as inconclusive (I). If none of the three genes were detected, the sample was classified as negative (N). 20 J o u r n a l P r e -p r o o f 9 The developed on-site quantitative point-of-care (POC) microfluidic assay for SARS-CoV-2 requires 50 µL aliquot of serum (10 µL) of a patient mixed with buffer (200 µL) loaded into the inlet of each microfluidic channel (Fig. 1A) . Each microfluidic channel has two NIs at both sidewalls as presented (Fig. 1B) (Kim et al., 2015; Yoon et al., 2017) . The microfludic chip was designed to have two microchannels; one for IgM and the other for IgG (See Fig.1E). Test and control zones (Fig.1E red 5 Fig.1E) 10 We observed small standard deviations, representing good reproducibility on the mean values: 0.25(std 0.14), 1.8(std 0.1) and 18.01(std 0.97) for IgM and 0.26(std 0.14), 2.0(std 0.12), and 16(std 0.96) for IgG ( Fig.2A) . Fluorescence intensity cut-off values for IgM and IgG in the developed microfluidic serological platform were determined using negative control serum samples (n = 244) collected prior to Oct 30, 2019, 15 which preceded emergence of the COVID-19 pandemic (Fig. 2B) . A total of 152 samples, including 60 from patients with COVID-19 and 92 from healthy volunteers, were used to verify the sensitivity and specificity of the microfluidic platform. All serum samples were confirmed using RT-PCR and ELISA IgG/IgM analyses. Evaluation of the microfluidic platform revealed a sensitivity and specificity of 91.67% and 100%, respectively (Fig. 2C) . Accuracy (overall agreement) was 96.7 %. The results are shown in Eleven patients (6 female and 5 male), aged 38 to 90 years, at Chungbuk National University Hospital were randomly selected and monitored during their hospitalization following clinical confirmation of COVID-19 using RT-PCR. Day of onset (day 0) was defined as the first day of a positive RT-PCR result. IgM/IgG titres were monitored during hospitalization until the time of discharge, which was based on 5 confirmation with a final negative RT-PCR result. The IgM/IgG evaluation revealed the kinetics of seroconversion and the plateauing of titre levels, which were then compared with clinical severity, categorized as either severe, moderate, mild, or asymptomatic, as defined by WHO (World Health Organization, n.d.), chest X-ray/CT radiologic findings, body temperature, and RT-PCR results from the analysis of upper/lower respiratory specimens collected using NP/OP and sputum (Fig. 3A) . Three Seven patients (P1-P7, Group A) presented typical patterns of virus-specific IgM/IgG antibody titres, with their levels increasing and then plateauing (Fig. 3B) . The body temperature of three Group A patients with fever (P1-P3) returned to normal prior to seroconversion. Graphs of IgM/IgG levels in six patients (P1-P5 and P7) in Group A diagnosed with typical COVID-19-associated pneumonia, based on consistent with those previously reported (Long et al., 2020a) . Graphs of IgM/IgG levels of an asymptomatic patient in Group A (P6) revealed high IgM/IgG titres, indicating that virus clearance occurred after antibody levels plateaued (World Health Organization, 2020). As previously described, seroconversion is the time period after the development of a specific antibody when that antibody becomes detectable in the blood (Cooper et al., 1985; Long et al., 2020a) . Even with recent studies on 5 COVID-19 and seroconversion, the duration and nature of immune responses against SARS-CoV-2 infection remain unclear (Bentivegna et al., 2020; Long et al., 2020a; Yongchen et al., 2020) . (Conklin et al., 2020) . However, there were differences in the range of time between our current findings and those previously reported, with a variation of 2.2 to 3 days. These differences may have been caused by differences in the reference dates (day 0). The referred group was counted from 15 the onset of symptoms, while our group was counted from the time of infection being confirmed by RT-PCR; the onset of symptoms is typically earlier than the RT-PCR confirmation date (Yong et al., 2020) . As shown in Fig. 4A , IgM and IgG titres plateaued on days 9.4 (median 9 d, IQR 6-12 d) and 10 (median 9 d, IQR 7-12 d), respectively. When plateaued values of both IgM and IgG were kept higher than the cut-off value we found that patients were confirmed to be RT-PCR negative on average 19.4 days 20 (median 20 d, IQR 16-21 d) after the initial confirmation of infection. This was consistent with a previous study reporting that serum IgM and IgG plateaued 7 and 8.2 days, respectively, after infection, based on the average time between infection and RT-PCR confirmation (Conklin et al., 2020) . Two of the patients in our current study, P6 and P7, did not have IgM/IgG levels measured during the first week of infection, only having four time points in which IgM and IgG were measured due to delayed hospitalization. 25 However, data from these patients supported the inference for the final confirmation of a negative RT-PCR result. We also observed that IgM/IgG antibody responses of three patients with fever (P1-P3) and four patients without fever (P4-P7) in Group A were similar (Fig. 4B and 4C) . In the fever group, the body temperature of the patients returned to less than 37.5℃ when IgG levels plateaued. IgG titres in the non-fever group decreased, which was consistent with a previous report stating that 93.3% of 5 asymptomatic individuals had a reduction in IgG levels (Z. Liu et al., 2020) . 10 (Fig. 4E) . Similar infer could be applied to the 15 patient 6 (Fig.4B) 20 seven days of symptom onset (Long et al., 2020a) . The serological assay could confirm response to 25 The microfluidic assay provides a predictive tool for the effective surveillance of patient status and 15 (Amanat et al., 2020b; Duan et al., 2020) . Recently, immunity 20 hospitali ation duration (red line , date of negative CR results ased on tests for 2 consecutive days 5 relative to days after initial confirmation of the infection y CR ( lue ox . All patients were temporally aligned to the day of IgG levels plateauing ( lue circle . IgM seroconversion, IgG seroconversion, and IgM plateau were are indicated y the light lue ar, red ar, and red circle, respectively. B) Daily timeline graphs. atient age, sex, and COVID-9 testing results are provided, as well as, CR test results ( ositive: red ox, Inconclusive: gray ox, Negative: lue ox , and the serological assay including the during hospitali ation. neumonia was confirmed during hospitali ation y chest CT or X-ray and are indicated with lack arrow. J o u r n a l P r e -p r o o f Correlation of Chest CT and RT-PCR Testing for Coronavirus Disease A serological assay to detect SARS-CoV-2 seroconversion in humans New IgM 15 seroconversion and positive RT-PCR test after exposure to the virus in recovered COVID-19 patient Nanointerstice-driven microflow Evaluation of Serological SARS-CoV-2 Lateral Flow Assays for Rapid Point of Care Testing ACUTE AIDS RETROVIRUS INFECTION: Definition of a Clinical Illness Associated with Seroconversion Effectiveness of convalescent plasma therapy in severe COVID-19 patients Sensitivity of Chest CT for COVID-19: 10 Comparison to RT-PCR Authorized Serology Test Performance [WWW Document An evaluation of COVID-19 serological assays informs future diagnostics and exposure assessment Nanointerstice-driven microflow patterns in physical interrupts Microfluidic immunoassay for point-of-care testing using simple fluid vent control Serology assays to manage COVID-19. Science (80-. ) Serology assays to manage COVID-19 Comparison of four new commercial serologic assays for determination of SARS-CoV-2 IgG Positive RT-PCR Test Results in Patients Recovered From COVID-19 Testing on the move: South Korea's rapid response to the COVID-19 pandemic Structure of SARS Coronavirus Spike Receptor-Binding Domain Complexed with Receptor. Science (80-. ) Disappearance of antibodies to SARS-CoV-2 in a -COVID-19 patient after recovery Lymphocyte subset (CD4+, CD8+) counts reflect the severity of infection and predict the clinical outcomes in patients with COVID-19 20 immunological assessment of asymptomatic SARS-CoV-2 infections Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease Patients Prevalence of Asymptomatic SARS-CoV-2 Infection Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma Ethical Implementation of Immunity Passports During the COVID-19 COVID-19 diagnostics in context orld ealth Organi ation, 2020. opulation-ased age-stratified seroepidemiological investigation protocol for Clinical management of severe acute respiratory infection (SARI) when COVID-19 20 disease is suspected Serological tests facilitate identification of asymptomatic SARS-CoV-2 infection in Wuhan Connecting clusters of COVID-19: an epidemiological and serological investigation Different longitudinal patterns of nucleic acid and serology testing results based on disease severity of COVID-19 patients Generation of digitized microfluidic filling flow by vent control