key: cord-0926515-koyvlagt authors: Li, Jinfeng; Liu, Bochao; Tang, Xi; Wu, Ze; Lu, Jinhui; Liang, Chaolan; Hou, Shuiping; Zhang, Ling; Li, Tingting; Zhao, Wei; Fu, Yongshui; Ke, Yuebin; Li, Chengyao title: Development of a smartphone based quantum-dot lateral flow immunoassay strip for ultrasensitive detection of anti-SARS-CoV-2 IgG and neutralizing antibodies date: 2022-04-26 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2022.04.042 sha: 6d348c705a64f4f46847f853a351869674f80a1f doc_id: 926515 cord_uid: koyvlagt Background : As several vaccine candidates for SARS-CoV-2 have been developed, a large proportion of people has be vaccinated worldwide so far. The rapid and accurate immunoassays are urgently demanded for detecting specific virus-neutralizing antibody (NAb), which reflect the protective effect of the vaccines among different populations. Methods : In this study, we designed a quantum-dot lateral flow immunoassay strip (QD-LFIA) on smartphone for detection of specific IgG or neutralizing antibodies to SARS-CoV-2 in human serum or whole blood samples. The recombinant receptor binding domain (RBD) of SARS-CoV-2 spike protein was used as the antigen to combine with NAb or angiotensin-converting enzyme 2 (ACE2). Results : Among 81 recovered COVID-19 patients diagnosed with NAT initially, 98.8% (80/81) were positive for IgG and 88.9% (72/81) were positive for NAb by QD-LFIA. Among 64 inactivated vaccine and 6 subunit vaccine inoculated vaccinees, 90% (63/70) were positive for IgG and 82.9% (58/70) were positive for NAb by QD-LFIA, while no cross-reaction was found from 150 healthy blood donor, 2 Flu B and 3 common cold patients. Conclusions : The established platform could achieve a rapid and accurate detection of NAb specific to SARS-CoV-2, which could be used for detecting the protective effect of the vaccines in epidemic areas of world. In December 2019, a new coronavirus pneumonia was firstly discovered in Wuhan, China and then largely reported within a few months in the rest of world, which was caused by the novel coronavirus designated as SARS-CoV-2 Zhou et al., 2020) . Some patients rapidly developed acute complications such as acute respiratory distress syndrome (ARDS) and acute respiratory failure . The main routes of SARS-CoV-2 transmission in humans were respiratory droplets and virus exposure between individuals. By the end of May 2021, more than 170 million people were diagnosed as COIVD-19 by viral nucleic acid test (NAT) and over three million deaths were announced globally, among them more than 4000 deaths were reported in China. SARS-CoV-2 is a single stranded sense RNA virus that is over 30,000 nucleotides in length and is a member of the β genus of the coronavirus family (Chan et al., 2020; Chu et al., 2020; Phelan et al., 2020) . Confirmation of the genomic viral sequence (GenBank MN908947) allowed for testing of SARS-CoV-2 RNA to determine if people were infected with the virus. Due to the samples' collection time, storage and transportation conditions, some nucleic acid tests could not detect SARS-CoV-2 from nasal or pharynx swabs (Tanner et al., 2015; Lim et al., 2018; Kim et al., 2019; Patra et al., 2019) . Many patients had to be tested repeatedly before they were finally diagnosed (Corman et al., 2020; Zhang et al., 2020) . However, testing for antibodies specific to SARS-CoV-2 could be a helpful complementary approach to confirm infection. Suspected cases, asymptomatic patients or resolvers of can be detected by the antibodies specific to SARS-CoV-2. Several vaccine candidates for SARS-CoV-2 have been developed and are currently approved for emerging use in vaccination worldwide van Doremalen et al., 2020; Yu et al., 2020) . It is generally believed that the vaccination can induce the virus-neutralizing antibody (NAb), which will reduce the likelihood of (re)infection and development of severe disease (Jackson et al., 2020; Krammer, 2020; Xia et al., 2020) . As there are individual differences after vaccination, the levels of NAb titers could be measured to predict the protective effect of the vaccines (Dagotto et al., 2020) . There were several virus neutralization assay (VNT) and pseudoviruses neutralization assay (pVNT) which could be used to evaluate the NAb titers, but required the use of live virus and cells (Crawford et al., 2020; Manenti et al., 2020) . A surrogate virus neutralization assay (sVNT) mimicking the virus-host interaction in an ELISA plate well, was established so far, but still need to be completed in several hours (Tan et al., 2020) . Though there are several neutralizing antibody methods, more rapid and simple tests are still required strongly by the vaccinated individuals. In this study, we designed a smartphone based quantum-dot lateral flow immunoassay strip (QD-LFIA) for detection of specific IgG and NAb to SARS-CoV-2 in human serum or whole blood samples. The testing can be carried out within 30 min and this novel assay is ultrasensitive, cost-effective, rapid and simple, which can be used for on-site detection of specific IgG and NAb to SARS-CoV-2 infection in humans. Bovine serum albumin (BSA) and goat anti-mouse polyclonal immunoglobulin G (G- An aliquot of QDs were conjugated with a specific amount of anti-His mAb via covalent attachment. Briefly, 1.5 μL of EDC (1 mg/mL) and 1.5 μL of sulfo-NHS (2 mg/mL) solutions were mixed and initially added into 25 μL of QDs suspended in 475 μL activating buffer (25 mM MES, pH 6.1). The mixture was incubated for 30 min at room temperature with gentle stirring. After the NHS-ester was formed, the QDs was washed two times with binding buffer (10mM phosphate buffer, pH7.0) by centrifugation at 10000×g for 15 min to remove unreacted sulfo-NHS and EDC and was resuspended in 475 μL binding buffer. 20 μg of anti-His antibody was added to the activated QDs and the mixture was incubated for 2 h with gentle rotation. The washing process was repeated two times to remove unbound chemicals or proteins and were suspended in blocking buffer and incubated for another 30 min with gentle mixing to cover any remaining unbound hydrophobic sites on the surface of the QDs. Finally, the QD@anti-His mAb were rinsed two times using washing buffer and suspended in 250 μL storing buffer. The G-mIgG (1 mg/mL), and M-hIgG (1 mg/mL), ACE2 (0.2 mg/mL) were spotted onto the NC membrane with dispensing parameter of 1 μL/cm (from the absorbent pad to the sample pad) to generate the C, T1, and T2 lines, respectively ( Figure 1A ). The coated NC was dried at 37C for 30 min. The sample pad was pretreated with an optimized treatment buffer and then dried at 37C overnight. Fluorescence signals on the T1/T2 and C lines of the strip were captured using a selfproduced portable fluorescence strip reader and were uploaded to a smartphone via Wi-Fi for data processing. The ELISA tests for 81 convalescent serum samples from COVID-19 patients and 70 serum samples from vaccinees were performed following the instruction of Wantai SARS-CoV-2 IgG ELISA kit (Wantai Biological Pharmacy, Beijing, China). All samples were tested in serial dilutions beginning at 1:50 and endpoint titers were defined as the highest plasma dilution that yielded an absorbance > cut-off value. The cut-off value was calculated as the mean of three negative controls plus 3 SD (mean+3SD). The SARS-CoV-2 pseudoviruses expressing a luciferase reporter gene were generated as described previously (Yang et al., 2004) . Briefly, the packaging construct psPAX2 and luciferase reporter plasmid pLenti-CMV Puro-Luc (Miaoling Biotechnology Co., Ltd), and spike protein expressing pcDNA3. This system included a QD-LFIA strip and a portable fluorescence strip reader connecting with smartphone platform. The QD-LFIA strip contained sample pad, NC membrane with C and T1/T2 lines, and absorbent pad ( Figure 1A ). The working principle was shown in Figure 1B and 1C when a sample was added to the sample pad. In order to report the testing results conveniently, a portable fluorescence strip reader connecting with smartphone platform was invented for QD-LFIA strip ( Figure 2 ). As shown in Figure 1B , if the sample did not contain the IgG or NAb specific to SARS-CoV-2, the RBD-His would react with QD@anti-His mAb, migrate and bind to ACE2 on the T2 line of NC membrane. The excessive complexes would continue to move along the membrane until captured on the C line by secondary goat antimouse antibodies. The C line was always visible, which indicated that the test strip and procedure worked well. As shown in Figure 1C , if the sample contained the IgG or NAb specific to SARS-CoV-2, the IgG or NAb would combine with RBD-His, then reacted with QD@anti-His mAb. When the complexes migrated on the NC membrane, the NAb saturated RBD complexes could not bind to the immobilized ACE2 on the T2 line, while IgG saturated RBD complexes would bind to the mouse anti-human IgG (T1). The more NAb molecules or titers were presented in the sample, the lower density of T2 line was appeared. Finally, the strip was inserted into the self-produced fluorescence reader and fluorescent images of the T2/T1/C lines were captured and transferred to a smartphone through Wi-Fi (Figure 2 ). The fluorescence lines were then selected and the fluorescence intensity of the T1, T2, or C lines was converted to peak areas. The value of the T1/C or T2/C was then calculated, and the sample was determined to be negative or positive after comparing the result with the cut-off value. To enhance the performance of this assay, the amount of anti-His mAb labeled quantum-dots (QDs) To reduce nonspecific interactions, we optimized the sample buffer and treatment buffer used for the QD-LFIA ( Supplementary Figure 7 and 8 ). An optimized sample buffer (PBS + 1% BSA + 0.1% Triton X-100, pH7.4) was chosen for this assay and a mixed treatment buffer (PBST with 1% BSA and 0.1% gelatin, pH7.4) was optimized for blocking the sample pad. Fifty healthy blood donor plasma samples (provided by Guangzhou Blood Center) were tested negative for specific IgG or NAb to SARS-CoV-2 by ELISA or pVNT, respectively. By using these negative control samples, the appropriate cut-off values of specific IgG or NAb to SARS-CoV-2 in QD-FLIA were defined as 0.021(T1/C, mean plus 3 SD) or 2.783 (T2/C, mean minus 3 SD), respectively. The IgG titer of a convalescent COVID-19 patients (No.1 patient in Supplementary Table 2 ) was 6400 by ELISA while the NAb IC 50 was 666 by pVNT, which was highest among all the samples. Thus, the serum sample of this patient was used as reference standard to distinguish positive from negative samples. To establish the standard curve, we used different diluted serum samples of reference standard from 1:1 to 1:10000 and obtained the light intensity for each dilution using the selfproduced device. The LOD for detecting the standard was 19.2 (IgG titer) and the linear range was 64 to 6400 ( Figure 3A ). For NAb testing, the reference standard with 1:30 dilution was tested positive by QD-LFIA of which IC50 was 21 by pVNT ( Figure 3B, C) . The different diluted reference standard from 1:1 to 1:10000 was listed in Figure 3D . were positive for NAb by QD-LFIA, which were also consistent with the results of ELISA or pVNT (Table 1 and Supplementary Table 3 (Table 1 and Supplementary Table 3) . The titers of IgG tested by QD-LFIA had quantitative correlation to the results by ELISA ( Figure 4A , C) and NAb IC 50 tested by pVNT ( Figure To examine the specificity of QD-LFIA, 150 plasma samples of healthy blood donors, 2 Flu B and 3 common cold patients' serum samples were tested. All samples were detected negative, which suggested that the specificity of QD-LFIA was 100% for testing of IgG or NAb (Table 1) . A blood donor sample (BD5) infected with SARS 17 years ago was additionally detected positive by ELISA (Xu et al., 2021) , but negative by QD-LFIA (Supplementary Figure 9) . To determine the stability of QD-LFIA, we measured the activity and performance of this assay at different time points and temperatures (Supplementary Figure 10) . The results demonstrated that QD-LFIA had constant stability for testing period up to 30 days at room temperature or at temperatures between 10 ºC and 50 ºC. The accuracy of QD-LFIA was evaluated by examining the recovery rate and relative standard deviation (RSD). Three different dilutions of standard sample were analyzed, of which the recovery rates and RSD were around 95% and 5%, respectively (Supplementary Table 4 ). This finding showed that the QD-LFIA performed well in both accuracy and precision. In order to verify if this method could be used for on-site detection in grassroot hospitals or self-check at home, we tried whole blood samples on QD-LFIA. The representative results were presented in Supplementary Figure 11 , suggesting that this assay could be applied to detect whole blood samples. A brand new coronavirus from a patient's pharyngeal swab sample was discovered by the Chinese Center for Disease Control and Prevention (CDC) on January 7, 2020, which was temporarily named 2019-nCoV, and subsequently designated as SARS-CoV-2 by the World Health Organization (WHO) (Cheng and Shan, 2020) . SARS-CoV-2 has caused a global pandemic with novel coronavirus pneumonia disease . More than 170 million of people were diagnosed with COVID-19 and over three millions of deaths were reported so far. This pandemic has made many communities or cities closed, which caused huge economic losses worldwide. Currently, the re-opening or re-starting of social activities are initiating in Asian and European countries where the SARS-CoV-2 infection has been contained. Along with nucleic acid testing of SARS-CoV-2, the antibody tests are largely needed as well. The development of rapid and accurate antibody detection methods is highly welcomed. Detection of specific antibody to SARS-CoV-2 can reflect the status of virus infection among different populations, and also can provide serological evidence for clinical diagnosis of COVID-19 patients. Several COVID-19 vaccines have been developed for emerging use of vaccination (Keech et al., 2020; Logunov et al., 2020; Poland et al., 2020; Zhu et al., 2020) , including mRNA-1273, NVX-CoV2373, BNT162b2, ChAdOx1 nCoV-19, CoronaVac and two Chinese inactivated virus vaccines. Billions of people have already been vaccinated or will be vaccinated in the near future. Immunity to SARS-CoV-2 induced by these vaccines could protect against reinfection and reduce the risk of becoming critically ill. A critical challenge at present is to identify the vaccine efficacy based on immune protection from SARS-CoV-2 infection and thereby assist in the further development of vaccines. Although antiviral cellular immunity certainly contribute to the protection from SARS-CoV-2 infection, the main protective effect of vaccines was based on neutralizing antibodies (Khoury et al., 2021) . There are conventional live virus neutralization test (VNT) or pVNT assays for measuring neutralizing antibody titers, which require virus cell cultures for few days in P2 or P3 laboratory. The surrogate VNT based on ELISA can measure NAb effect to block the virus-host interaction in microplate, which still needs 2 hours to finish and a microplate reader to read the results. In this study, we developed a quantum-dot based LFIA (QD-LFIA) and combined with a self-produced portable fluorescence real-time camera reader, which could detect specific IgG or NAb to SARS-CoV-2 infection in human serum or whole blood samples. The testing can be carried out within 30 min. The results could be read by a self-produced portable fluorescence reader which was cost-effective compared with the traditional microplate reader. Finally, the results could be sent to a smartphone through Wi-Fi transmission for reporting. As some of anti-SARS-CoV could neutralize or recognize SARS-CoV-2 (Gavor et al., 2020; Yuan et al., 2020) , a serum sample from a blood donor infected with SARS-CoV 17 years ago (BD5, Xu, et al. 2020 ) was used to test if there was false positive result by the cross-reaction between SARS-CoV and SARS-CoV-2. As this serum sample was tested negative by QD-LFIA, it suggested that this assay might not be cross-reactive with anti-SARS-CoV. As no new SARS case has been reported over seventeen years, it was difficult to collect more anti-SARS-CoV positive samples to make this conclusion. In addition, 150 healthy blood donors' plasma samples, 2 Flu B and 3 common cold patients' serum samples were tested for the specificity of QD-LFIA, and all samples were detected negative. These results suggested that this assay was not cross-reactive with anti-SARS-CoV or antibodies to other coronavirus. the results of NAb titers tested by pVNT or QD-LFIA. In conclusion, this assay will be useful for grassroot hospitals and institutions where are in urgent need for cost-effective, rapid and simple methods for on-site detection of specific IgG or NAb to SARS-CoV-2 infection in humans. Zhifei vaccinees 6 6(100%) 6(100%) 6(100%) 6(100%) 6(100%) 6(100%) The working principle of QD-LFIA strip. Correlation between QD-LFIA and ELISA, or QD-LFIA and pVNT for testing of 49 vaccinees' samples. 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The authors thank the Guangzhou and Shenzhen blood centers for providing the healthy blood donor samples used in this study.