Paper-based ELISA Research Articles

Comparison of protein immobilization methods with covalent bonding on paper for paper-based enzyme-linked immunosorbent assay

In this study, two methods were examined to optimize the immobilization of antibodies on paper when conducting a paper-based enzyme-linked immunosorbent assay (P-ELISA). Human IgG, as a test-capture protein, was immobilized on paper via the formation of Schiff bases. Aldehyde groups were introduced onto the surface of the paper via two methods: NaIO4 and 3-aminopropyltriethoxysilane (APTS) with glutaraldehyde (APTS-glutaraldehyde). In the assay, horseradish peroxidase-conjugated anti-human IgG (HRP-anti-IgG) binds to the immobilized human IgG, and the colorimetric reaction of 3,3′,5,5′-tetramethylbenzyzine (TMB) produces a blue color in the presence of H2O2 and HRP-anti-IgG as a model analyte. The immobilization of human IgG, the enzymatic reaction conditions, and the reduction of the chemical bond between the paper surface and immobilized human IgG all were optimized in order to improve both the analytical performance and the stability. In addition, the thickness of the paper was examined to stabilize the analytical signal. Consequently, the APTS-glutaraldehyde method was superior to the NaIO4 method in terms of sensitivity and reproducibility. Conversely, the reduction of imine to amine with NaBH4 proved to exert only minimal influence on sensitivity and stability, although it tended to degrade reproducibility. We also found that thick paper was preferential when using P-ELISA because a rigid paper substrate prevents distortion of the paper surface that is often caused by repeated washing processes.

Application of Recombinant Monoclonal Antibodies from Transgenic Chicken Bioreactors in Enzyme-Linked Immunosorbent Assay.

Transgenic chicken bioreactors can efficiently produce egg whites containing large quantities of recombinant proteins. We previously developed transgenic chickens that produce recombinant monoclonal antibodies (mAbs) against epidermal growth factor receptor 2 (HER2). However, the practical applications of mAbs derived from transgenic eggs have not yet been examined. Therefore, we aimed to evaluate whether these recombinant mAbs can be used in enzyme-linked immunosorbent assay (ELISA). Recombinant HER2 mAbs from transgenic eggs were dissolved in phosphate-buffered saline and applied directly to 96-well microplates as immobilized antibodies without purification. The performance of ELISA using the unpurified recombinant HER2 mAbs from transgenic eggs was comparable to that of ELISA using commercially available purified recombinant HER2 mAbs. Moreover, ELISA using unpurified recombinant HER2 mAbs from transgenic eggs demonstrated high antigen specificity and was successfully applied to samples from cultured cell lysates derived from HER2-positive and HER2-negative cell lines. The unpurified recombinant HER2 mAbs from transgenic eggs were also efficiently used as immobilized antibodies in paper-based ELISA. In conclusion, our findings suggest that recombinant mAbs from transgenic eggs have the potential to be used to develop economic ELISA devices. To the best of our knowledge, this study is the first to use recombinant HER2 mAbs from transgenic eggs in ELISA.

Straightforward high-temperature antibody immobilization for rapid and simple microfluidic paper-based ELISA for detection of β’-component (Onc k 5) protein, a major IgE-binding protein in salmon roe

Detection of allergenic proteins in food is required for maintaining the health of persons with food allergies. Enzyme-linked immunosorbent assay (ELISA) is a common technique for the detection of proteins. However, conventional ELISA is expensive, time-consuming, and needs technical expertise. Here, a microfluidic paper-based analytical device (µPAD) combined with a sandwich ELISA technique is developed for rapid, simple, and sensitive colorimetric detection of β’-component protein, a major allergen in salmon roe. The developed µPAD-ELISA employs a capture antibody and enzyme-labeled detection antibody. In addition to the µPAD, a technique for immobilization of the capture antibody has been developed, which completes the immobilization within 5 min by drying the antibody solution on a paper substrate in an oven at 50 °C without any chemical modification. The immobilized antibody exhibited higher binding capability to an antigen and stability against washing processes than those immobilized at room temperature. Since this technique allows simple and rapid preparation of the ELISA in the µPAD, the present µPAD-ELISA completed the analysis within only 15 min, including the immobilization step, compared to a conventional ELISA, which takes about 19 h. This µPAD-ELISA offered a good limit of detection of 1.59 ng mL−1 and reduced the amounts of reagents by a factor of about 17–67, which decreases the analysis cost. This µPAD-ELISA approach employing the present antibody immobilization is expected to be an effective strategy for the detection of allergenic proteins in food.

Laser-printed paper ELISA and hydroxyapatite immobilization for colorimetric congenital anomalies screening in saliva

BackgroundAlpha-fetoprotein (AFP) is a fetal protein that can indicate congenital anomalies such as Down syndrome and spinal canal blockage when detected at abnormal levels in pregnant women. Current AFP detection methods rely on invasive blood or serum samples, which require sophisticated equipment. From the many solutions proposed, colorimetric paper-based assays excel in point-of-care settings. The concept of paper-based ELISA (p-ELISA) enhances traditional methods, aligning with the ASSURED criteria for diagnostics in resource-limited regions. Despite success in microfluidic paper-based assay devices, laser printing remains underexplored for p-ELISA. Additionally, modifying the paper surface provides an additional layer of sensitivity enhancement. ResultsIn this study, we developed a novel laser-printed paper-based ELISA (LP-pELISA) for rapid, sensitive, and noninvasive detection of AFP in saliva samples. The LP-pELISA platform was fabricated by printing hydrophobic barriers on filter paper using a laser printer, followed by depositing hydroxyapatite (HAp) as an immobilization material for the antibodies. The colorimetric detection was achieved using AuNPs functionalized with anti-AFP antibodies and silver nitrate enhancement. The LP-pELISA exhibited a linear response for AFP detection in both buffer and saliva samples over a range of 1.0–800 ng mL−1, with a limit of detection (LOD) reaching 1.0 ng mL−1. The assay also demonstrated good selectivity, repeatability, reproducibility, and stability. The LP-pELISA was further validated by testing spiked human saliva samples, showing its potential for point-of-care diagnosis of congenital disabilities. SignificanceThe LP-pELISA is a noninvasive platform showcasing simplicity, cost-effectiveness, and user-friendliness, utilizing laser printing, hydroxyapatite modification, and saliva samples to efficiently detect AFP. Beyond its application for AFP, this method's versatility extends to other biomarkers, positioning it as a catalyst for the evolution of paper-based biosensors. The LP-pELISA holds promise as a transformative tool for point-of-care diagnostics, fostering advancements in healthcare with its innovative technology.

Paper/PMMA hybrid device with a microvalve-controlled design for exosome isolation and analysis

This study proposes a paper/PMMA hybrid device designed to isolate exosomes and extract exosomal miRNA, followed by quantitative analysis. It aims to provide simplified and convenient sample preparation for potential point-of-care testing (POCT) processes. In contrast to previous work conducted by our research team, which focused on isolating exosomes and exosomal nucleic acids, this study introduces a novel approach by integrating paper and a PMMA mold with a microvalve controlled design. This innovative method enables the entire process to be performed on paper. The pressure on the paper could be adjusted by turning the screw upon the valve to change the pore size and permeability of the paper, which achieved the effect of controlling the flow rate of fluids. The paper was designed to have an immunoaffinity area for capturing exosomes and a sol-gel silica coating area for extracting miRNA. The paper-based ELISA (p-ELISA) exhibited a limit of detection and a limit of quantitation of 6 × 107 and 5.4 × 108 particles/mL, respectively, for exosome measurement. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) revealed that the Ct (threshold cycle) value for quantifying the miR-21 in the miRNAs extracted by the proposed paper/PMMA hybrid device was comparable to the Ct value of the commercial extraction kit. The developed paper/PMMA hybrid device with a microvalve-controlled design should be incorporated into the POCT system to extract exosomal miRNAs.

Deep learning-assisted ultra-accurate smartphone testing of paper-based colorimetric ELISA assays

Smartphone has long been considered as one excellent platform for disease screening and diagnosis, especially when combined with microfluidic paper-based analytical devices (μPADs) that feature low cost, ease of use, and pump-free operations. In this paper, we report a deep learning-assisted smartphone platform for ultra-accurate testing of paper-based microfluidic colorimetric enzyme-linked immunosorbent assay (c-ELISA). Different from existing smartphone-based μPAD platforms, whose sensing reliability is suffered from uncontrolled ambient lighting conditions, our platform is able to eliminate those random lighting influences for enhanced sensing accuracy. We first constructed a dataset that contains c-ELISA results (n=2048) of rabbit IgG as the model target on μPADs under eight controlled lighting conditions. Those images are then used to train four different mainstream deep learning algorithms. By training with these images, the deep learning algorithms can well eliminate the influences of lighting conditions. Among them, the GoogLeNet algorithm gives the highest accuracy (>97%) in quantitative rabbit IgG concentration classification/prediction, which also provides 4% higher area under curve (AUC) value than that of the traditional curve fitting results analysis method. In addition, we fully automate the whole sensing process and achieve the "image in, answer out" to maximize the convenience of the smartphone. A simple and user-friendly smartphone application has been developed that controls the whole process. This newly developed platform further enhances the sensing performance of μPADs for use by laypersons in low-resource areas and can be facilely adapted to the real disease protein biomarkers detection by c-ELISA on μPADs.

Paper-Based Detection Device for Microenvironment Examination: Measuring Neurotransmitters and Cytokines in the Mice Acupoint.

(1) Background: The medical practice of acupuncture involves the insertion of a specialized stainless needle into a specific body point, often called an acupoint, to initiate a perceived phenomenon of de-qi sensation. Therefore, the term “de-qi” describes bodily sensations experienced by the recipient during acupuncture, which may include feelings of soreness, heaviness, fullness, numbness, and migration. However, while acupuncture treatments reportedly result in acupoint activation and an increased release of neurotransmitters or cytokines, detecting these substances released into the acupoint microenvironment is often missed or delayed in clinical and basic practice. (2) Methods: To address this situation, we employed a paper-based enzyme-linked immunosorbent assay method to examine acupoint environmental changes using minute volumes of easily accessible acupoint fluids. (3) Results: Our results indicated that while levels of adenosine triphosphate (ATP), interleukin-1β, interleukin-6, glutamate, substance P, and histamine were all increased in the experimental group following electroacupuncture (EA) treatment, contrary results were observed in the sham EA and transient receptor potential vanilloid 1 (Trpv1−/−) groups. Subsequently, TRPV1 and its associated molecules were augmented in mouse dorsal root ganglion, spinal cord, thalamus, and the somatosensory cortex, then examined by Western blotting and immunofluorescence techniques. Investigations revealed that these elevations were still unobserved in the sham EA or EA in the Trpv1−/− groups. Furthermore, results showed that while administering ATP could mimic EA function, it could be reversed by the ATP P2 receptor antagonist, suramin. (4) Conclusions: Our data provide novel information, indicating that changes in neurotransmitter and cytokine levels can offer insight into acupuncture mechanisms and clinical targeting.

Paper-based ELISA for fast CA 15–3 detection in point-of-care

The paper-based Enzyme-Linked Immunosorbent Assay (P-ELISA) is a promising tool for diagnostic applications because the paper matrix is characterised by low cost, short analysis time, portability, and a high surface-to-volume ratio that allows the use of a small sample and reagent volume of a few microliters. In addition, colorimetric assays are suitable for low-resource areas due to their simplicity and naked-eye detectability. Although several works have been reported using paper-based colorimetric sensors for cancer biomarkers, P-ELISA for cancer antigen 15–3 (CA 15–3) has never been reported.Thus, this work reports the development of a rapid, simple and relatively inexpensive paper-based colorimetric assay for the detection of CA 15–3 as cancer biomarker. The assay was developed on a filter paper that was previously washed and chemically oxidized with periodate to generate aldehyde functional groups on the cellulose surface. After covalent binding of the first antibody, detection was performed by a colorimetric reaction based on the oxidation of the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by a peroxidase enzyme in the presence of H2O2. The colorimetric measurement was based on digital image acquisition analysed using ImageJ software. The linear range of the assay in buffer ranged 2 to 1100 U/mL. The performance of the assay was also successfully tested in human serum from Cormay®, offering a linear range from 2 to 200 U/mL. Thus, this P-ELISA sensor is suitable for the analysis of serum samples, since the physiological value in cancer patients is 30 U/mL. We believe that this proof-of-concept has the potential to be extended to be applied to other protein biomarkers and to be a suitable tool for cancer screening in developing countries.

A novel polymer-based nitrocellulose platform for implementing a multiplexed microfluidic paper-based enzyme-linked immunosorbent assay

Nitrocellulose (NC) membranes, as porous paper-like substrates with high protein-binding capabilities, are very popular in the field of point-of-care immunoassays. However, generating robust hydrophobic structures in NC membranes to fabricate microfluidic paper-based analytical devices (μPADs) remains a great challenge. At present, the main method relies on an expensive wax printer. In addition, NC membranes very easy to adhere during the printing process due to electrostatic adsorption. Herein, we developed a facile, fast and low-cost strategy to fabricate μPADs in NC membranes by screen-printing polyurethane acrylate (PUA) as a barrier material for defining flow channels and reaction zones. Moreover, hydrophobic barriers based on UV-curable PUA can resist various surfactant solutions and organic solvents that are generally used in immunoassays and biochemical reactions. To validate the feasibility of this PUA-based NC membrane for immunoassays in point-of-care testing (POCT), we further designed and assembled a rotational paper-based analytical device for implementing a multiplexed enzyme-linked immunosorbent assay (ELISA) in a simple manner. Using the proposed device under the optimal conditions, alpha fetoprotein (AFP) and carcinoembryonic antigen (CEA) could be identified, with limits of detection of 136 pg/mL and 174 pg/mL, respectively, which are below the threshold values of these two cancer biomarkers for clinical diagnosis. We believe that this reliable device provides a promising platform for the diagnosis of disease based on ELISA or other related bioassays in limited settings or remote regions.

Improving the Sensitivity of Nanofibrous Membrane-Based ELISA for On-Site Antibiotics Detection

An ultrasensitive and portable colorimetric enzyme-linked immunosorbent assay (ELISA) sensor for antibiotics was fabricated by immobilizing antibodies inside the largely porous and highly hydrophilic nanofibrous membranes. Different from regular electrospun nanofibrous membranes where antibodies may frequently be blocked by the heterogeneous porous structure and sterically crowded loaded on the surface, the controlled microporous structure and increased hydrophilicity of nanofibrous membranes could improve the diffusion properties of antibodies, reduce the sterically crowding effect, and dramatically improve the sensitivity of the membrane-based ELISA. The limitation of detection (LOD) for chloramphenicol (CAP) reached 0.005 ng/mL, around 200 times lower than the conventional paper-based ELISA, making quantitative analysis and portable on-site detection achievable via the use of smartphones. The successful design and fabrication of the nanofibrous membrane-based ELISA with novel features overcome the structural drawbacks of regular electrospun nanofibrous membranes and provide new paths to develop highly sensitive on-site detection of hazardous chemical agents.

Deep Learning-Assisted Ultra-Accurate Smartphone Testing of Paper-Based Colorimetric ELISA Assays

Deep Learning-Assisted Ultra-Accurate Smartphone Testing of Paper-Based Colorimetric ELISA Assays

A paper-based ELISA for rapid sensitive determination of anaphylaxis-related MRGPRX2 in human peripheral blood

Mas-related G-protein-coupled receptor X2 (MRGPRX2) has recently been reported to be associated with anaphylaxis. Detection of MRGPRX2 levels in human peripheral blood might serve as a powerful tool for predicting the predisposition of patients to anaphylactic reactions. For rapid measurement of MRGPRX2, we established a paper-based double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody and horseradish peroxidase (HRP)-labelled rabbit polyclonal antibody as capture antibody and detection antibody, respectively. We avoided chemical functionalization of the cellulose paper by introducing bovine serum albumin (BSA) to provide COOH and NH2 groups for covalent immobilization of the capture antibody. Through amide condensation, a two-layer immobilization strategy was applied with BSA-BSA and BSA-capture antibody networks as the first and second layers, respectively. This strategy improved the quantity, activity and stability of the immobilized antibody. We then established a paper-based ELISA to detect MRGPRX2 in human peripheral blood. Our method is less laborious, easier to implement, and more cost-effective than conventional ELISA, while offering similar sensitivity, specificity, and accuracy. Therefore, it could serve as an innovative clinical point-of-care diagnostic tool, especially in areas that lack advanced clinical equipment.

A Multi-Epitope Fusion Protein-Based p-ELISA Method for Diagnosing Bovine and Goat Brucellosis.

In recent years, the incidence of brucellosis has increased annually, causing tremendous economic losses to animal husbandry in a lot of countries. Therefore, developing rapid, sensitive, and specific diagnostic techniques is critical to control the spread of brucellosis. In this study, bioinformatics technology was used to predict the B cell epitopes of the main outer membrane proteins of Brucella, and the diagnostic efficacy of each epitope was verified by an indirect enzyme-linked immunosorbent assay (iELISA). Then, a fusion protein containing 22 verified epitopes was prokaryotically expressed and used as an antigen in paper-based ELISA (p-ELISA) for serodiagnosis of brucellosis. The multi-epitope-based p-ELISA was evaluated using a collection of brucellosis-positive and -negative sera collected from bovine and goat, respectively. Receiver operating characteristic (ROC) curve analysis showed that the sensitivity and specificity of detection-ELISA in diagnosing goat brucellosis were 98.85 and 98.51%. The positive and the negative predictive values were 99.29 and 98.15%, respectively. In diagnosing bovine brucellosis, the sensitivity and specificity of this method were 97.85 and 96.61%, with the positive and negative predictive values being identified as 98.28 and 97.33%, respectively. This study demonstrated that the B cell epitopes contained in major antigenic proteins of Brucella can be a very useful antigen source in developing a highly sensitive and specific method for serodiagnosis of brucellosis.

Pulling-Force Spinning Top for Serum Separation Combined with Paper-Based Microfluidic Devices in COVID-19 ELISA Diagnosis.

The spread of Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), resulting in a global pandemic with around four million deaths. Although there are a variety of nucleic acid-based tests for detecting SARS-CoV-2, these methods have a relatively high cost and require expensive supporting equipment. To overcome these limitations and improve the efficiency of SARS-CoV-2 diagnosis, we developed a microfluidic platform that collected serum by a pulling-force spinning top and paper-based microfluidic enzyme-linked immunosorbent assay (ELISA) for quantitative IgA/IgM/IgG measurements in an instrument-free way. We further validated the paper-based microfluidic ELISA analysis of SARS-CoV-2 receptor-binding domain (RBD)-specific IgA/IgM/IgG antibodies from human blood samples as a good measurement with higher sensitivity compared with traditional IgM/IgG detection (99.7% vs 95.6%) for early illness onset patients. In conclusion, we provide an alternative solution for the diagnosis of SARS-CoV-2 in a portable manner by this smart integration of pulling-force spinning top and paper-based microfluidic immunoassay.

Turntable Paper-Based Device to Detect Escherichia coli.

Escherichia coli has been known to cause a variety of infectious diseases. The conventional enzyme-linked immunosorbent assay (ELISA) is a well-known method widely used to diagnose a variety of infectious diseases. This method is expensive and requires considerable time and effort to conduct and complete multiple integral steps. We previously proposed the use of paper-based ELISA to rapidly detect the presence of E. coli. This approach has demonstrated utility for point-of-care (POC) urinary tract infection diagnoses. Paper-based ELISA, while advantageous, still requires the execution of several procedural steps. Here, we discuss the design and experimental implementation of a turntable paper-based device to simplify the paper-based ELISA protocols for the detection of E. coli. In this process, antibodies or reagents are preloaded onto zones of a paper-based device and allowed to dry before use. We successfully used this device to detect E. coli with a detection limit of 105 colony-forming units (colony-forming unit [CFU])/mL.

A manual and portable centrifuge combined with a paper-based immunoassay for myocardial infarction diagnosis

Myocardial infarction (MI) causes hundreds of thousands of deaths annually. Early diagnosis of MI by detecting the concentration of troponin I in plasma can help limit mortality rates. However, plasma separation and early diagnosis are particularly difficult to achieve in resource-limited settings. In this study, we demonstrate a manual and portable centrifuge along with a paper analytical device to measure troponin I for early MI diagnosis. With this so-called “Handfuge,” pure plasma can be achieved with a yield of up to 47.1% and purity as high as 99.9% in less than 3 min without electricity. The resulting plasma can then be directly used for detecting troponin I using a paper-based enzyme-linked immunosorbent assay (ELISA), with a detection limit as low as 0.03 ng/mL. The whole detection process consumes just 10 µL of blood, which is similar to the volume obtained by a clinical finger-prick. The proposed centrifuge together with the paper analytical device possesses the potential to replace existing techniques in troponin I detection for early MI diagnoses in resource-limited regions of the world.

A Sensitive and Simple Enzyme-Linked Immunosorbent Assay Using Polymer as Carrier.

In this study, a new and sensitive enzyme-linked immunosorbent assay (ELISA) was developed by introducing a polymer as a reaction carrier. The results suggest that the newly developed ELISA method is more convenient than the existing paper-based ELISA method and applicable to a wider range of environments. In addition, the sensitivity of the new method is much higher than that of the existing paper-based ELISA method and even higher than that of the traditional ELISA method.

Preliminary Assessment of Burn Depth by Paper-Based ELISA for the Detection of Angiogenin in Burn Blister Fluid-A Proof of Concept.

Rapid assessment of burn depth is important for burn wound management. Superficial partial-thickness burn (SPTB) wounds heal without scars, but deep partial-thickness burn (DPTB) wounds require a longer healing time and have a higher risk of scar formation. We previously found that DPTB blister fluid displayed a higher angiogenin level than SPTB blister fluid by conventional ELISA. In this study, we developed a paper-based ELISA (P-ELISA) technique for rapid assessment of angiogenin concentration in burn blister fluid. We collected six samples of SPTB blister fluid, six samples of DPTB blister fluid, and seven normal healthy serum samples for analysis. We again chose ELISA to measure and compare angiogenin levels across all of our samples, but we developed a P-ELISA tool and compared sample results from that tool to the results from conventional ELISA. As with conventional ELISA, DPTB blister fluid displayed higher angiogenin levels than SPTB in P-ELISA. Furthermore, our P-ELISA results showed a moderate correlation with conventional ELISA results. This new diagnostic technique facilitates rapid and convenient assessment of burn depth by evaluating a key molecule in burn blister fluid. It presents a novel and easy-to-learn approach that may be suitable for clinically determining burn depth with diagnostic precision.

Enhanced paper-based ELISA for simultaneous EVs/exosome isolation and detection using streptavidin agarose-based immobilization.

EVs/exosomes are considered as the next generation of biomarkers, including for liquid biopsies. Consequently, the quantification of EVs/exosomes is crucial for facilitating EV/exosome research and applications. Paper-based enzyme-linked immunosorbent assay (p-ELISA) is a portable diagnostic system with low cost that is simple and easy to use; however, it shows low sensitivity and linearity. In this study, we develop p-ELISA for targeting EVs/exosomes by using streptavidin agarose resin-based immobilization (SARBI). This method reduces assay preparation times, provides strong binding, and retains good sensitivity and linearity. The time required for the total assay, including preparation steps and surface immobilization, was shortened to ∼2 h. We evaluated SARBI p-ELISA systems with/without CD63 capture Ab and then with fetal bovine serum (FBS) and EVs/exosome-depleted fetal bovine serum (dFBS). The results provide evidence supporting the selective capture ability of SARBI p-ELISA. We obtain semiquantitative p-ELISA results using an exosome standard (ES) and human serum (HS), with R2 values of 0.95 and 0.92, respectively.

Detection of the SARS-CoV-2 humanized antibody with paper-based ELISA.

This work reports the development of a rapid, simple and inexpensive colorimetric paper-based assay for the detection of the severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper device was prepared with lamination for easy sample handling and coated with the recombinant SARS-CoV-2 nucleocapsid antigen. This assay employed a colorimetric reaction, which is followed by horseradish peroxidase (HRP) conjugated detecting antibody in the presence of the 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The colorimetric readout was evaluated and quantified for specificity and sensitivity. The characterization of this assay includes determining the linear regression curve, the limit of detection (LOD), the repeatability, and testing complex biological samples. We found that the LOD of the assay was 9.00 ng μL-1 (0.112 IU mL-1). The relative standard deviation was approximately 10% for a sample number of n = 3. We believe that our proof-of-concept assay has the potential to be developed for clinical screening of the SARS-CoV-2 humanized antibody as a tool to confirm infected active cases or to confirm SARS-CoV-2 immune cases during the process of vaccine development.