skip to main content


Title: Metal organic framework encapsulated tamavidin-Gluc reporter: application in COVID-19 spike antigen bioluminescent immunoassay
Enzyme linked immunosorbent assay (ELISA) is one of the most utilized serological methods to diagnose and identify etiologic agents of many infectious diseases and other physiologically important analytes. ELISA can be used either alone or adjunct to other diagnostic methods such as molecular arrays, and other serological techniques. Most ELISA assays utilize reagents that are proteinaceous in nature, which are not very stable and require cold-chain transport systems. Development of a desirable immunoassay requires stability of reagents used and its ability to be stored at room temperature without sacrificing the activity of the reagents or the protein of interest. Metal organic frameworks (MOFs) are a rapidly emerging and evolving class of porous polymeric materials used in a variety of biosensor applications. In this study, we introduce the use of MOFs to stabilize a universal reporter fusion protein, specifically, avidin-like protein (Tam-avidin2) and the small bioluminescent protein Gaussia luciferase (Gluc) forming the fusion reporter, tamavidin2-Gluc (TA2-Gluc). This fusion protein serves as a universal reporter for any assays that utilize biotin–avidin binding strategy. Using SARS-CoV2 S1 spike antigen as the model target antigen, we demonstrated that encapsulation of TA2-Gluc fusion protein using a nano-porous material, zeolitic imidazolate framework-8 (ZIF-8), allows us to store and preserve this reporter protein at room temperature for over 6 months and use it as a reporter for an ELISA assay. Our optimized assay was validated demonstrating a 0.26 μg mL −1 limit of detection, high reproducibility of assay over days, detection of spiked non-virulent SARS-COV2 pseudovirus in real sample matrix, and detection in real COVID-19 infected individuals. This result can lead to the utilization of our TA2-Gluc fusion protein reporter with other assays and potentially in diagnostic technologies in a point-of-care setting.  more » « less
Award ID(s):
1841419 2041413
NSF-PAR ID:
10390877
Author(s) / Creator(s):
; ; ; ; ;
Date Published:
Journal Name:
Sensors & Diagnostics
Volume:
1
Issue:
6
ISSN:
2635-0998
Page Range / eLocation ID:
1198 to 1208
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Dai, Tianhong ; Wu, Mei X. ; Popp, Jürgen (Ed.)
    The SARS-CoV-2 pandemic has revealed the need for rapid and inexpensive diagnostic testing to enable population-based screening for active infection. Neither standard diagnostic testing, the detection and measurement of viral RNA (via polymerase chain reaction), or serological testing (via enzyme-linked immunosorbent assay) has the capability to definitively determine active infection. The former due to a lack of ability to distinguish between replicable and inert viral RNA, and the latter due to varying immune responses (ranging from latent to a complete lack of immune response altogether). Despite many companies producing rapid point-of-care (POC) tests, none will address the global scale of testing needed and few help to combat the ever growing issue of testing resource scarcity. Here we discuss our efforts towards the development of a highly manufacturable, microfluidic device that instantly indicates active viral infection status from ~ 20 μL of nasal mucus or phlegm and requires no external power. The device features a biotin functionalized silicon nanomembrane within an acrylic body containing channels and ports for sample introduction and analysis. Virus capture and target confirmation are done using affinity-based capture and size-based occlusion respectively. Modularity of the device is proven with bead and vaccinia virus capture as we work towards testing with both pure SARS-CoV-2 virus and human samples. With success on all fronts, we could achieve an inexpensive POC diagnostic which can determine an individual’s infection status, aiding containment efforts in the current and future pandemics. In addition to direct viral detection, our method can be used as a rapid POC sample preparation tool that limits the application of PCR reagents to those samples which already display viral size and antigen-based positivity through our device. 
    more » « less
  2. Abstract Objectives

    Investigating factors that contribute to bone loss and accretion across populations in remote settings is challenging, particularly where diagnostic tools are scarce. To mitigate this challenge, we describe validation of a commercial ELISA assay to measure osteocalcin, a biomarker of bone formation, from dried blood spots (DBS).

    Methods

    We validated the Osteocalcin Human SimpleStep ELISA kit from Abcam (ab1951214) using 158 matched plasma and DBS samples. Passing‐Bablok regression analysis assessed the relationships between plasma and DBS osteocalcin concentrations. Dilutional linearity and spike and recovery experiments determined if the DBS matrix interfered with osteocalcin measurement, and intra‐ and inter‐assay coefficients of variation (CVs) were calculated. Limit of detection, analyte stability, and specific forms of osteocalcin measured by the kit were also investigated.

    Results

    Mean plasma osteocalcin value was 218.2 ng/mL (range 64.6‐618.1 ng/mL). Linear relationships existed between plasma and DBS concentrations of osteocalcin, with no apparent bias in plasma vs DBS concentrations. There was no apparent interference of the DBS matrix with measurement of osteocalcin in DBS. Intra‐assay CV for DBS was ~8%, while average inter‐assay CV was 14.8%. Limit of detection was 0.34 ng/mL. Osteocalcin concentrations were stable in DBS stored at −28°C and room temperature, but not those stored at 37°C. This ELISA kit detects total osteocalcin.

    Conclusions

    Osteocalcin, a bone formation biomarker, can be measured from DBS. Combined with a previously validated DBS assay for TRACP‐5b, a bone resorption biomarker, these assays have the potential to help researchers disentangle the many factors contributing to bone strength.

     
    more » « less
  3. Abstract

    Antibody detection assays are essential for evaluating immunity of individuals against a given virus, and this has been particularly relevant during the COVID‐19 pandemic. Current serology assays either require a laboratory setting and take >1 hr (i.e., enzyme‐linked immunosorbent assay [ELISA]) or are rapid but only qualitative in nature and cannot accurately track antibody levels over time (i.e., lateral flow assay [LFA]). Therefore, there is a need for development of a rapid and simple but also quantitative assay that can evaluate antibody levels in patients accurately over time. We have developed an assay that uses a split nanoluciferase fused to the spike or nucleocapsid proteins of the SARS‐CoV‐2 virus to enable luminescent‐based detection of spike‐ or nucleocapsid‐binding antibodies in serum, plasma, and whole blood samples. The resulting approach is simple, rapid, and quantitative and is highly amenable to low‐/medium‐throughput scale using plate‐based assays, high‐throughput scale using robotics, and point‐of‐care applications. In this article, we describe how to perform the assay in a laboratory setting using a plate reader or liquid‐handling robotics and in a point‐of‐care setting using a handheld, battery‐powered luminometer. Together, these assays allow antibody detection to be easily performed in multiple settings by simplifying and reducing assay time in a laboratory or clinical environment and by allowing for antibody detection in point‐of‐care, nonlaboratory settings. © 2022 Wiley Periodicals LLC.

    Basic Protocol: SARS‐CoV‐2 antibody detection using the split‐luciferase assay on a medium‐throughput scale with a laboratory luminometer

    Alternate Protocol 1: High‐throughput‐based protocol for SARS‐CoV‐2 antibody detection using a robotic platform

    Alternate Protocol 2: Point‐of‐care‐based protocol for SARS‐CoV‐2 antibody detection using a handheld luminometer

    Support Protocol: Determining positive/negative cutoffs for test samples and standardizing the assay between days

     
    more » « less
  4. We report on the novel observation about the gain in nanomechanical stability of the SARS-CoV-2 (CoV2) spike (S) protein in comparison with SARS-CoV from 2002 (CoV1). Our findings have several biological implications in the subfamily of coronaviruses, as they suggest that the receptor binding domain (RBD) (∼200 amino acids) plays a fundamental role as a damping element of the massive viral particle's motion prior to cell-recognition, while also facilitating viral attachment, fusion and entry. The mechanical stability via pulling of the RBD is 250 pN and 200 pN for CoV2 and CoV1 respectively, and the additional stability observed for CoV2 (∼50 pN) might play a role in the increasing spread of COVID-19. 
    more » « less
  5. In the context of continued spread of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 and the emergence of new variants, the demand for rapid, accurate, and frequent detection is increasing. Moreover, the new predominant strain, Omicron variant, manifests more similar clinical features to those of other common respiratory infections. The concurrent detection of multiple potential pathogens helps distinguish SARS-CoV-2 infection from other diseases with overlapping symptoms, which is significant for providing tailored treatment to patients and containing the outbreak. Here, we report a lab-on-a-chip biosensing platform for SARS-CoV-2 detection based on the subwavelength grating micro-ring resonator. The sensing surface is functionalized by specific antibody against SARS-CoV-2 spike protein, which could produce redshifts of resonant peaks by antigen–antibody combination, thus achieving quantitative detection. Additionally, the sensor chip is integrated with a microfluidic chip featuring an anti-backflow Y-shaped structure that enables the concurrent detection of two analytes. In this study, we realized the detection and differentiation of COVID-19 and influenza A H1N1. Experimental results indicate that the limit of detection of our device reaches 100 fg/ml (1.31 fM) within 15 min detecting time, and cross-reactivity tests manifest the specificity of the optical diagnostic assay. Furthermore, the integrated packaging and streamlined workflow facilitate its use for clinical applications. Thus, the biosensing platform presents a promising approach for attaining highly sensitive, selective, multiplexed, and quantitative point-of-care diagnosis and distinction between COVID-19 and influenza.

     
    more » « less