Plant-produced recombinant SARS-CoV-2 receptor-binding domain; an economical, scalable biomaterial source for COVID-19 diagnosis

doi:10.3877/cma.j.issn.2096-112X.2021.01.006

Biomaterials Translational ›› 2021, Vol. 2 ›› Issue (1): 43-49.doi: 10.3877/cma.j.issn.2096-112X.2021.01.006

• RESEARCH ARTICLE • Previous Articles Next Articles

Plant-produced recombinant SARS-CoV-2 receptor-binding domain; an economical, scalable biomaterial source for COVID-19 diagnosis

Kaewta Rattanapisit¹, Gorawit Yusakul², Balamurugan Shanmugaraj¹, Kittinop Kittirotruji¹, Phassorn Suwatsrisakul¹, Eakachai Prompetchara³^,⁴, Suthira Taychakhoonavud⁵^,^*(), Waranyoo Phoolcharoen⁶^,⁷^,^*()

1 Baiya Phytopharm Co., Ltd., Bangkok, Thailand
2 School of Pharmacy, Walailak University, Nakhon Si Thammarat, Thailand
3 Centre of Excellence in Vaccine Research and Development (Chula Vaccine Research Centre, Chula VRC), Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
4 Department of Laboratory Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
5 Department of Social and Administrative Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
6 Research Unit for Plant-produced Pharmaceuticals, Chulalongkorn University, Bangkok, Thailand
7 Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand

Received:2020-11-19 Revised:2021-03-12 Accepted:2021-03-17 Online:2021-03-31 Published:2021-03-28
Contact: Suthira Taychakhoonavud,Waranyoo Phoolcharoen E-mail:suthira.t@chula.ac.th;waranyoo.p@chula.ac.th

Abstract

Abstract:

The outbreak of the novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread rapidly causing a severe global health burden. The standard COVID-19 diagnosis relies heavily on molecular tests to detect viral RNA in patient samples; however, this method is costly, requires highly-equipped laboratories, multiple reagents, skilled laboratory technicians, and takes 3-6 hours to complete. To overcome these limitations, we developed a plant-based production platform for the SARS-CoV-2 receptor-binding domain as an economical source of detection reagents for a lateral-flow immunoassay strip (LFIA) which is suitable for detection of IgM/IgG antibodies in human samples. Further, we validated the plant-produced SARS-CoV-2 receptor-binding domain-based LFIA as a useful diagnostic tool for COVID-19. A total of 51 confirmed COVID-19 serum samples were tested using the LFIA, and the obtained results were consistent with those from polymerase chain reaction assays, while providing sensitivity and specificity of 94.1% and 98%, respectively. The developed LFIA is rapid, scalable, user-friendly, and relatively inexpensive with a simple test procedure, making it useful for the routine monitoring of COVID-19 in clinical settings. This study was approved on March 19, 2020 by the Ethics Committee of the Faculty of Medicine, Chulalongkorn University (COA No. 354/2020 and IRB No. 236/63).

Key words: COVID-19, lateral flow immunoassay, Nicotiana benthamiana, point-of-care testing, recombinant protein, SARS-CoV-2

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Figures/Tables 5

Figure 1. Schematic representation for the transient expression of SARS-CoV-2 RBD protein in plants. RBD: receptor-binding domain; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.

Figure 2. Schematic diagram of the T-DNA region of the geminiviral vector used in this study. pBY2e: geminiviral vector; RBD: receptor-binding domain; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; UTR: untranslated region.

Figure 3. Schematic illustration of the developed Baiya’ Rapid COVID-19’ IgM/IgG test kit. Different test results are shown. AuNPs: gold nanoparticles; C: control line; G: test line for human IgG; M: test line for human IgM; RBD: receptor-binding domain; S: Sample pad; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.

Figure 4. Western blot analysis of purified SARS-CoV-2 RBD protein from Nicotiana benthamiana agroinfiltrated with pBY2e-SARS-CoV-2-RBD. (A, B) The purified SARS-CoV-2 RBD protein was loaded at 4 μg/lane under reducing conditions and visualized with InstantBlue? (A) The purified SARS-CoV-2 RBD protein was loaded at 200 ng/lane under reducing conditions and detected with a horseradish peroxidase-conjugated rabbit anti-His antibody (B). M represents the protein molecular weight ladder, and lane 1 shows the purified SARS-CoV-2 RBD. pBY2e: geminiviral vector; RBD: receptor-binding domain; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.

Figure 5. Representative test strips showing the results of serum samples collected from different patients in Thailand. The test sample is added to the sample well of the test cassette, where AuNP-RBD-IgY conjugates react with human anti-SARS-CoV-2 IgG and IgM in the sample. The sample then migrates through capillary action through the test and control lines. Sample #2 was found to be negative for both IgM and IgG, #86 was positive for IgM and IgG, while #32 was positive for IgM only. AuNPs: gold nanoparticles; C: control line; G: test line for human IgG; M: test line for human IgM; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.

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Plant-produced recombinant SARS-CoV-2 receptor-binding domain; an economical, scalable biomaterial source for COVID-19 diagnosis

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