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Design and Use of Synthetic Peptides for Detection of Antibodies in Serum or Plasma of Coronavirus Disease 2019 (COVID-19) Patients

Ruby Anne N. King 1, Janice C. Caoili 2, Romulo J. de Castro 3, Salvador Eugenio C. Caoili 1, Fresthel Monica M. Climacosa 1,4

1 Biomedical Innovations Research for Translational Health Science Laboratory, Department of Biochemistry and Molecular Biology, College of Medicine, University of the Philippines Manila

2 Section of Infectious Diseases, Department of Medicine, Makati Medical Center

3 Center for Informatics, University of San Agustin

4 Department of Medical Microbiology, College of Public Health, University of the Philippines Manila

Using publicly available severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic sequences of Philippine origin from the GISAID EpiFlu Database, we selected core peptide sequences from the four viral structural proteins and modified them by incorporating cysteine residues at their N- and C-terminal ends, in order to facilitate end-to-end peptide polymerization via disulfide-bond formation and thereby produce synthetic antigens suitable for antibody detection. Here, we report the preliminary results for our peptide M1 (CADSNGTITVEELKKLLEQC), an artificial sequence consisting of an 18-residue wild-type core sequence derived from the SARS-CoV-2 membrane glycoprotein and flanked by cysteine residues. We dissolved crude M1 in dimethyl sulfoxide and diluted the resulting solution with aqueous urea to facilitate disulfide-bond formation between cysteine residues. We then analyzed the reaction products using polyacrylamide gel electrophoresis, which revealed Coomassie blue-stained material of lower average electrophoretic mobility under non-reducing versus reducing conditions that was consistent with polymerization of M1 via intermolecular disulfide-bond formation. We found that polymerized M1 captured antibodies from convalescent COVID-19 patient plasma (CCPP) more efficiently than monomeric M1 in an indirect enzyme-linked immunosorbent assay (ELISA) and that the binding thus observed was blocked by preincubation of the antibodies with M1. We also observed significantly higher average ELISA signal intensity among CCPP samples compared to archived human plasma samples that were collected prior to the onset of human-to-human SARS-CoV-2 transmission. Our results suggest the potential utility of M1 for antibody detection in contexts of immunodiagnosis and immunosurveillance, particularly in settings where vaccination against COVID-19 is expected to induce production of antibodies against the SARS-CoV-2 spike protein rather than the membrane glycoprotein.