key: cord-0733093-w965r92w authors: Nishiyama, Keine; Takahashi, Kazuki; Fukuyama, Mao; Kasuya, Motohiro; Imai, Ayuko; Usukura, Takumi; Maishi, Nako; Maeki, Masatoshi; Ishida, Akihiko; Tani, Hirofumi; Hida, Kyoko; Shigemura, Koji; Hibara, Akihide; Tokeshi, Manabu title: Facile and Rapid Detection of SARS-CoV-2 Antibody Based on a Noncompetitive Fluorescence Polarization Immunoassay in Human Serum Samples date: 2021-06-05 journal: Biosens Bioelectron DOI: 10.1016/j.bios.2021.113414 sha: 5eafb165d5ebc200ce1d4124076168d3063c7cd1 doc_id: 733093 cord_uid: w965r92w Antibody detection methods for viral infections have received broad attention due to the COVID-19 pandemic. In addition, there remains an ever-increasing need to quantitatively evaluate the immune response to develop vaccines and treatments for COVID-19. Here, we report an analytical method for the rapid and quantitative detection of SARS-CoV-2 antibody in human serum by fluorescence polarization immunoassay (FPIA). A recombinant SARS-CoV-2 receptor binding domain (RBD) protein labeled with HiLyte Fluor 647 (F-RBD) was prepared and used for FPIA. When the anti-RBD antibody in human serum binds to F-RBD, the degree of polarization (P) increases by suppressing the rotational diffusion of F-RBD. The measurement procedure required only mixing a reagent containing F-RBD with serum sample and measuring the P value with a portable fluorescence polarization analyzer after 15 min incubation. We evaluated analytical performance of the developed FPIA system using 30 samples: 20 COVID-19 positive sera and 10 negative sera. The receiver operating characteristic curve drawn with the obtained results showed that this FPIA system had high accuracy for discriminating COVID-19 positive or negative serum (AUC = 0.965). The total measurement time was about 20 min, and the serum volume required for measurement was 0.25 μL. Therefore, we successfully developed the FPIA system that enables rapid and easy quantification of SARS-CoV-2 antibody. It is believed that our FPIA system will facilitate rapid on-site identification of infected persons and deepen understanding of the immune response to COVID-19. In December 2019, an increase in patients with pneumonia of unknown cause was reported in Wuhan, China . The patients' clinical symptoms were very similar to those of SARS-CoV and MERS-CoV, and found to be due to a novel Coronaviridae family (Zhou et Here, we report a novel method for quantitatively measuring SARS-CoV-2 antibody by fluorescence polarization immunoassay (FPIA). FPIA is a well-known homogeneous immunoassay that uses a dimensionless number of the degree of fluorescence polarization (P) as a parameter (Smith and Eremin, 2008) . FPIA has the advantages of rapidity and simplicity, and it has been widely used in the food processing and medical fields (Hendrickson et al., 2020) . In order to achieve In this non-competitive FPIA-based assay, the measurement procedure consists simply of adding F-RBD into serum and measuring the P value with a portable FP analyzer (Fig. 2) . It is possible to perform antibody testing with a sample volume of 20 μL or less per measurement using the microdevice. In our developed method, the total amount of antibody that binds to RBD is Optimization and evaluation of the FPIA system J o u r n a l P r e -p r o o f FPIA is a homogeneous immunoassay and bound-free separation is not required. It is easily affected by interferences in the sample, which may cause fluctuation of the P value of blank between the samples (Nishiyama et al., 2021a) . Therefore, we investigated mitigation of the influence of interferences in serum by diluting serum with F-RBD reagent. In order to determine the appropriate dilution ratio of serum, F-RBD solution was added to healthy donor serum samples (n = 10) and the variation of P value between samples was evaluated. Fig. 3 shows the mean and standard deviation of the P value of the mixture of serum samples and F-RBD solution when diluted by F-RBD solution at each dilution ratio. The dilution ratio means the final dilution ratio of serum in the mixture, where the original serum concentration is 1. It was confirmed that the standard deviation of the P value decreased with the dilution ratio and became almost constant when the dilution ratio exceeded 80 times. Also, with 80-fold diluted serum, F-RBD of 1 μg/mL exhibited sufficient fluorescence intensity (Fig. S2) . Therefore, we decided to use a dilution factor of 80 times and 1 μg/mL F-RBD in the following experiments. Regarding the decrease in antibody concentration in sample when diluted, we discuss whether the sensitivity is sufficient for antibody detection in COVID-19-positive patients in the next section. The detection sensitivity of the FPIA system was evaluated with anti-RBD IgG antibody spiked in healthy human serum. F-RBD reagent was added into the anti-RBD antibody in serum and the P value was measured after incubation. The calibration curve for antibody in human serum is shown in Fig. 4 . The P value was confirmed to increase with the increase in antibody concentration and the prepared F-RBD was confirmed to bound with the anti-RBD antibody. The limit of detection (LOD) was calculated to be 2.9 μg / mL (final concentration in the 80 times diluted mixture: 36 ng / mL). Subsequently, we evaluated the selectivity of this FPIA system. The P value of anti-RBD antibody was compared with that of an anti-influenza A virus antibody as a negative control (Fig. S3 ). The P value of the anti-RBD antibody was found to be significantly higher than that of the negative control. From the above results, our FPIA system was demonstrated as capable of measuring anti-RBD antibody in human serum selectively with a total measurement time of about 20 min. The required serum sample volume per measurement was only 0.25 μL. The analytical performance of the developed FPIA system was evaluated with real samples. Commercially available COVID-19 positive and negative serum samples were used for the evaluation. To confirm whether the developed FPIA system has sufficient detection sensitivity to determine the antibody concentration in a real sample, the results obtained by FPIA for positive serum samples at various concentrations (dilution rates) are shown in Fig. 5 . Positive serum samples diluted with PBS were prepared, and FPIA was performed under the same conditions as the calibration curve (Fig. 4) . The P value was confirmed to decrease as the dilution rate increased (the antibody concentration decreased). The difference of the P value between the 80-fold dilution and the blank was 14.0 mP, which corresponds to ~70 μg / mL of the antibody in Fig. 4 . The antibody concentration in the positive serum can be measured even if serum diluted 80 times is used in FPIA. In order to clarify the analytical performance as a rapid diagnostic method, the LOD was compared using two commercially available LFIA kits (Kits A and B) for detecting COVID-19 antibody and the same positive serum sample ( Fig. S4 and S5 ). IgG antibody was detected only in the original serum using Kit A, and up to 16-fold dilution (corresponding to 1280-fold dilution in FPIA) using Kit B. However, in both kits, the IgG line disappeared at the following dilution ratios. Since the types of immobilized antigens in these kits are not disclosed, it is possible that antibodies that bind to epitopes different from FPIA have been detected in these kits. From the viewpoint of simple diagnosis of COVID-19, we judged that the developed FPIA system has a detection sensitivity higher than that of the commercially available LFIA kits for detecting COVID-19 antibody. We further evaluated analytical performance with 20 positive serum samples from COVID-19 infected donors and 10 negative serum samples from healthy donors. The P values obtained from each serum are shown in Fig. 6 (a) . The average P value of positive sera was higher than that of negative sera. Based on these P values, a ROC curve was prepared to evaluate discrimination ability ( Fig. 6 (b) ). The AUC value, which is an index of discrimination ability obtained from the ROC curve, was 0.965. Generally, an AUC value of 0.900 or higher is considered to indicate a very accurate analysis method (Streiner and Cairney, 2007) . Therefore, our FPIA system is highly consistent with the result of PCR or antigen testing. The candidate cut-off value was ΔmP = 0.83 or 2.17, which provided the point with the maximum sensitivity and specificity on the ROC curve. Table S1 summarizes the consistency between the conventional methods (the PCR or antigen testing) and our developed FPIA system at the above cut-off values. However, more data are needed to determine the cut-off value, increasing the measurement data will be implemented in the future. The above results demonstrated that our FPIA system has the analytical capability for COVID-19 diagnosis that is highly accurate, quick, and easy to do, and that has high throughput, We have developed a FPIA system to detect anti-SARS-CoV-2 antibodies in human serum rapidly. The total amount of anti-RBD antibody in human serum was measured by applying a fluorescence-labeled RBD to FPIA. The total analysis time was ~20 min and the required serum sample volume was 1 μL or less. Since this FPIA can be performed anywhere using a portable FP analyzer, we expect that it will be used as a fast screening method for COVID-19. In addition, based on our previously successful detection of viruses by FPIA (Nishiyama et al., 2021b), we are confident simultaneous detection of viruses and antibodies is possible. By changing the fluorescent label, applicability can be extended to other viral infections. The developed FPIA system can also be used for quantitative evaluation of COVID-19 vaccines and treatments. High-throughput measurement greatly contributes to better research efficiency. In this way, our FPIA system is a highly expandable analysis method, with many purposes in the future. A negative photoresist (SU-8 3050) was purchased from Nippon Kayaku Co., Ltd HiLyte Fluor™ 647 Labeling Kit-NH 2 were purchased from Dojindo Molecular Technologies 19 IgM/IgG rapid test LFIA kits, kit A and kit B, were purchased from GenBody Inc. (Korea) and Epigentek (USA), respectively. Preparation of Fluorescence-labeled RBD (F-RBD) CoV-2 spike RBD was labeled with HiLyte Fluor 647 using the HiLyte Fluor™ 647 Labeling Kit-NH 2 . Unreacted fluorescent molecules were separated using a modified polyethersulfone membrane (Nanosep 10 K Omega, Pall Corporation, USA). 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