Fusing Peptide Epitopes for Advanced Multiplex Serological Testing for SARS-CoV-2 Antibody Detection

Ali H Aldoukhi; Panayiotis Bilalis; Dana M Alhattab; Alexander U Valle-Pérez; Hepi H Susapto; Rosario Pérez-Pedroza; Emiliano Backhoff-García; Sarah M Alsawaf; Salwa Alshehri; Hattan Boshah; Abdulelah A Alrashoudi; Waleed A Aljabr; Manal Alaamery; May Alrashed; Rana M Hasanato; Raed A Farzan; Roua A Alsubki; Manola Moretti; Malak S Abedalthagafi; Charlotte A E Hauser

doi:10.1021/acsbiomedchemau.3c00010

Fusing Peptide Epitopes for Advanced Multiplex Serological Testing for SARS-CoV-2 Antibody Detection

ACS Bio Med Chem Au. 2023 Aug 30;4(1):37-52. doi: 10.1021/acsbiomedchemau.3c00010. eCollection 2024 Feb 21.

Authors

Ali H Aldoukhi^{1

2}, Panayiotis Bilalis^{1

2}, Dana M Alhattab^{1

2}, Alexander U Valle-Pérez^{1

2}, Hepi H Susapto^{1

2}, Rosario Pérez-Pedroza^{1

2}, Emiliano Backhoff-García¹, Sarah M Alsawaf^{1

2}, Salwa Alshehri^{1

2}, Hattan Boshah^{1

2}, Abdulelah A Alrashoudi^{1

2}, Waleed A Aljabr³, Manal Alaamery^{4

5

6}, May Alrashed^{7

8}, Rana M Hasanato⁹, Raed A Farzan^{7

8}, Roua A Alsubki^{7

8}, Manola Moretti^{1

2}, Malak S Abedalthagafi¹⁰, Charlotte A E Hauser^{1

2

11}

Affiliations

¹ Laboratory for Nanomedicine, Division of Biological and Environmental Science and Engineering (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
² Computational Bioscience Research Center (CBRC), King Abdullah University of Science and Technology, Thuwal 23955-69900, Saudi Arabia.
³ Research Centre, King Fahad Medical City, Riyadh 12231, Saudi Arabia.
⁴ Developmental Medicine Department, King Abdullah International Medical Research Center, King Abdulaziz Medical City, Ministry of National Guard-Health Affairs, King Saud Bin Abdulaziz University for Health Sciences, Riyadh 11426, Saudi Arabia.
⁵ KACST-BWH Centre of Excellence for Biomedicine, Joint Centers of Excellence Program, King Abdulaziz City for Science and Technology (KACST), Riyadh 12371, Saudi Arabia.
⁶ Saudi Human Genome Project (SHGP), Satellite Lab at King Abdulaziz Medical City (KAMC), Ministry of National Guard Health Affairs (MNG-HA), King Abdulaziz City for Science and Technology (KACST), Riyadh 11426, Saudi Arabia.
⁷ Department of Clinical Laboratory Science, College of Applied Medical Sciences, King Saud University, Riyadh 11433, Saudi Arabia.
⁸ Chair of Medical and Molecular Genetics Research, King Saud University, Riyadh 11433, Saudi Arabia.
⁹ Department of Pathology and Laboratory Medicine, King Saud University, Riyadh 11433, Saudi Arabia.
¹⁰ Pathology and Laboratory Medicine, Emory School of Medicine, Atlanta, Georgia 30329, United States.
¹¹ Red Sea Research Center, Division of Biological and Environmental Science and Engineering (BESE), King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia.

Abstract

The tragic COVID-19 pandemic, which has seen a total of 655 million cases worldwide and a death toll of over 6.6 million seems finally tailing off. Even so, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise, the severity of which cannot be predicted in advance. This is concerning for the maintenance and stability of public health, since immune evasion and increased transmissibility may arise. Therefore, it is crucial to continue monitoring antibody responses to SARS-CoV-2 in the general population. As a complement to polymerase chain reaction tests, multiplex immunoassays are elegant tools that use individual protein or peptide antigens simultaneously to provide a high level of sensitivity and specificity. To further improve these aspects of SARS-CoV-2 antibody detection, as well as accuracy, we have developed an advanced serological peptide-based multiplex assay using antigen-fused peptide epitopes derived from both the spike and the nucleocapsid proteins. The significance of the epitopes selected for antibody detection has been verified by in silico molecular docking simulations between the peptide epitopes and reported SARS-CoV-2 antibodies. Peptides can be more easily and quickly modified and synthesized than full length proteins and can, therefore, be used in a more cost-effective manner. Three different fusion-epitope peptides (FEPs) were synthesized and tested by enzyme-linked immunosorbent assay (ELISA). A total of 145 blood serum samples were used, compromising 110 COVID-19 serum samples from COVID-19 patients and 35 negative control serum samples taken from COVID-19-free individuals before the outbreak. Interestingly, our data demonstrate that the sensitivity, specificity, and accuracy of the results for the FEP antigens are higher than for single peptide epitopes or mixtures of single peptide epitopes. Our FEP concept can be applied to different multiplex immunoassays testing not only for SARS-CoV-2 but also for various other pathogens. A significantly improved peptide-based serological assay may support the development of commercial point-of-care tests, such as lateral-flow-assays.