Evaluation of an E. coli-expressed spike protein-based in-house ELISA system for assessment of antibody responses after COVID-19 infection and vaccination

Sitti Nurisyah; Mitsuhiro Iyori; Ammar A. Hasyim; Khaeriah Amru; Kei Itani; Kurumi Nakamura; Kartika H. Zainal; Handayani Halik; Irawaty Djaharuddin; Agussalim Bukhari; Puji BS. Asih; Din Syafruddin; Shigeto Yoshida; Irfan Idris; Yenni Yusuf

doi:10.52225/narra.v5i1.1250

Authors

Sitti Nurisyah Department of Pulmonology and Respiratory Medicine, Universitas Hasanuddin, Makassar, Indonesia; Dr. Tadjuddin Chalid Hospital, Makassar, Indonesia https://orcid.org/0000-0003-2285-7286
Mitsuhiro Iyori Research Institute of Pharmaceutical Science, Musashino University, Nishitokyo, Japan https://orcid.org/0000-0001-9940-7342
Ammar A. Hasyim Laboratory of Vaccinology and Applied Immunology, Kanazawa University, Ishikawa, Japan https://orcid.org/0000-0003-2799-1652
Khaeriah Amru Dr. Tadjuddin Chalid Hospital, Makassar, Indonesia; Department of Medical Education, Universitas Hasanuddin, Makassar, Indonesia https://orcid.org/0009-0008-3017-5828
Kei Itani Laboratory of Vaccinology and Applied Immunology, Kanazawa University, Ishikawa, Japan
Kurumi Nakamura Laboratory of Vaccinology and Applied Immunology, Kanazawa University, Ishikawa, Japan https://orcid.org/0009-0007-7205-6440
Kartika H. Zainal Laboratory of Vaccinology and Applied Immunology, Kanazawa University, Ishikawa, Japan
Handayani Halik Universitas Megarezky, Makassar, Indonesia
Irawaty Djaharuddin Department of Pulmonology and Respiratory Medicine, Universitas Hasanuddin, Makassar, Indonesia; Dr. Wahidin Soedirohusodo Hospital, Makassar, Indonesia https://orcid.org/0000-0002-5240-4950
Agussalim Bukhari Department of Clinical Nutrition, Universitas Hasanuddin, Makassar, Indonesia
Puji BS. Asih National Research and Innovation Agency, Jakarta, Indonesia https://orcid.org/0000-0002-4582-9133
Din Syafruddin Department of Parasitology, Universitas Hasanuddin, Makassar, Indonesia https://orcid.org/0000-0001-7141-2545
Shigeto Yoshida Laboratory of Vaccinology and Applied Immunology, Kanazawa University, Ishikawa, Japan
Irfan Idris Department of Physiology, Universitas Hasanuddin, Makassar, Indonesia https://orcid.org/0000-0002-7350-8687
Yenni Yusuf Department of Parasitology, Universitas Hasanuddin, Makassar, Indonesia https://orcid.org/0000-0001-7310-6381

DOI:

https://doi.org/10.52225/narra.v5i1.1250

Keywords:

Capture antigen, COVID-19, ELISA, E. coli expression system, antibody detection

Abstract

Evaluating long-term immunity after COVID-19 infection and vaccination is critical for managing potential outbreaks. The aim of this study was to develop a cost-effective in-house enzyme-linked immunosorbent assay (ELISA) based on Escherichia coli-expressed SARS-CoV-2 spike protein (E-S1) for antibody detection and to evaluate its performance. The system was validated by comparing the in-house ELISA results with those obtained using a commercial ELISA with HEK293-expressed spike protein (H-S1). Recombinant SARS-CoV-2 spike protein was produced in E. coli, purified, and validated for antigenicity via ELISA. Indirect ELISAs with both E-S1 and H-S1 antigens were performed on 386 serum samples from COVID-19 survivors, vaccinated individuals, and pre-pandemic controls collected at different time points. The E-S1 ELISA showed a statistically significant but weak correlation with H-S1 ELISA across all samples (r=0.205; p=0.0001). Stronger correlations were observed among vaccinated individuals with prior infection on day 90 (r=0.6017; p<0.001) and in naïve vaccine recipients on day 30 (r=0.5361; p=0.0003). Pre-pandemic sera from a rural population in Sumba Island exhibited high background reactivity in E-S1 ELISA, likely due to anti-E. coli antibodies, while urban pre-pandemic sera from Jakarta showed a stronger correlation with H-S1 ELISA. This suggests potential regional or immune background differences influencing assay performance. Although E-S1 retained antigenic properties, its diagnostic utility is limited by non-specific reactivity and reduced sensitivity compared to H-S1. In conclusion, E. coli expression systems may not be ideal for producing spike protein-based ELISA antigens specific to SARS-CoV-2. Alternative expression systems, such as human or baculovirus, could enhance diagnostic accuracy and specificity for COVID-19 antibody detection.