POL Scientific / JBM / Volume 8 / Issue 4 / DOI: 10.14440/jbm.2022.390
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Monobiotinylated Proteins Tethered to Microspheres for Detection of Antigen-Specific Serum Antibodies

Caleb S. Whitley1 Thomas C. Mitchell1
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1 Department of Microbiology and Immunology, University of Louisville School of Medicine, 505 S. Hancock St., Louisville KY 40202
Published: 23 September 2022
(This article belongs to the Special Issue Methods to Facilitate SARS-CoV-2 and COVID-19 Research)
© 2021 by the author. Licensee POL Scientific, USA. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
Abstract

Surface modified microspheres have been leveraged as a useful way to immobilize antigen for serological studies. The use of carboxyl modified microspheres for this purpose is well-established, but commonly associated with technical challenges. Streptavidin modified microspheres require little technical expertise and thus address some of the shortcomings of carboxyl microspheres. An additional feature of streptavidin microspheres is the use of mono-biotinylated proteins, which contain a single biotinylation motif at the C-terminus. However, the relative performance of streptavidin and carboxyl microspheres is unknown. Here, we performed a head-to-head comparison of streptavidin and carboxyl microspheres. We compared antigen binding, orientation, and staining quality and found that both microspheres perform similarly based on these defined parameters. We also evaluated the utility of streptavidin microspheres bound to SARS-CoV-2 receptor binding domain (RBD), to reliably detect RBD-specific IgG1, IgG3, and IgA1 produced in individuals recently immunized with Pfizer/BioNTech mRNA COVID vaccine as ‘proof-of-concept’. We provide evidence that each of the antibody targets are detectable in serum using RBD-coated microspheres, Ig-specific ‘detector’ mAbs, and flow cytometry. We found that cross-reactivity of the detector mAbs can be minimized by antibody titration to improve differentiation between IgG1 and IgG3. We also coated streptavidin microspheres with SARS-CoV-2 delta variant RBD to determine if the streptavidin microsphere approach revealed any differences in binding of immune serum antibodies to wild-type (Wuhan) versus variant RBD (Delta). Overall, our results show that streptavidin microspheres loaded with mono-biotinylated antigen is a robust alternative to chemically cross-linking antigen to carboxyl microspheres for use in serological assays.

Keywords
COVID
microspheres
serology
vaccine
flow cytometry
References

1. Fong CH, Cai JP, Dissanayake TK, Chen LL, Choi CY, Wong LH, et al. Improved Detection of Antibodies against SARS-CoV-2 by Microsphere-Based Antibody Assay. Int J Mol Sci. 2020 Sep;21(18):E6595. https://doi.org/10.3390/ijms21186595 PMID:32916926
2. Dobaño C, Vidal M, Santano R, Jiménez A, Chi J, Barrios D, et al. Highly Sensitive and Specific Multiplex Antibody Assays To Quantify Immunoglobulins M, A, and G against SARS-CoV-2 Antigens. J Clin Microbiol. 2021 Jan;59(2):e01731-20. https://doi.org/10.1128/JCM.01731-20 PMID:33127841
3. Rogier EW, Moss DM, Mace KE, Chang M, Jean SE, Bullard SM, et al. Use of Bead-Based Serologic Assay to Evaluate Chikungunya Virus Epidemic, Haiti. Emerg Infect Dis. 2018 Jun;24(6):995–1001. https://doi.org/10.3201/eid2406.171447 PMID:29774861
4. Fraley E, LeMaster C, Geanes E, Banerjee D, Khanal S, Grundberg E, et al. Humoral immune responses during SARS-CoV-2 mRNA vaccine administration in seropositive and seronegative individuals. BMC Med. 2021 Jul;19(1):169. https://doi.org/10.1186/s12916-021-02055-9 PMID:34304742
5. Tcherniaeva I, den Hartog G, Berbers G, van der Klis F. The development of a bead-based multiplex immunoassay for the detection of IgG antibodies to CMV and EBV. J Immunol Methods. 2018 Nov;462:1–8. https://doi.org/10.1016/j.jim.2018.07.003 PMID:30056034
6. Bugiel M, Fantana H, Bormuth V, Trushko A, Schiemann F, Howard J, et al. Versatile microsphere attachment of GFP-labeled motors and other tagged proteins with preserved functionality. J Biol Methods. 2015;2(4):e30. https://doi.org/10.14440/jbm.2015.79
7. O’callaghan CA, Byford MF, Wyer JR, Willcox BE, Jakobsen BK, McMichael AJ, et al. BirA enzyme: production and application in the study of membrane receptor-ligand interactions by site-specific biotinylation. Anal Biochem. 1999 Jan;266(1):9–15. https://doi.org/10.1006/abio.1998.2930 PMID:9887208
8. Ioannou M, Papageorgiou DN, Ogryzko V, Strouboulis J. Mammalian expression vectors for metabolic biotinylation tandem affinity tagging by co-expression in cis of a mammalian codon-optimized BirA biotin ligase. BMC Res Notes. 2018 Jun;11(1):390. https://doi.org/10.1186/s13104-018-3500-9 PMID:29898783
9. Cantarero LA, Butler JE, Osborne JW. The adsorptive characteristics of proteins for polystyrene and their significance in solid-phase imunoassays. Anal Biochem. 1980 Jul;105(2):375–82. https://doi.org/10.1016/0003-2697(80)90473-X PMID:7006446
10. FlowJo For Antibody Titrations- Separation Index and Concatenation. Flow Cytometry Core, Carbone Cancer Center, University of Wisconsin. Cited on May 5, 2016. Available from: http://www.uwhealth.org/flowlab.
11. Morell A, Skvaril F, Noseda G, Barandun S. Metabolic properties of human IgA subclasses. Clin Exp Immunol. 1973 Apr;13(4):521–8. PMID:4717094
12. Davis SK, Selva KJ, Kent SJ, Chung AW. Serum IgA Fc effector functions in infectious disease and cancer. Immunol Cell Biol. 2020 Apr;98(4):276–86. https://doi.org/10.1111/imcb.12306 PMID:31785006
13. Christensen PA, Olsen RJ, Long SW, Subedi S, Davis JJ, Hodjat P, et al. Delta Variants of SARS-CoV-2 Cause Significantly Increased Vaccine Breakthrough COVID-19 Cases in Houston, Texas. Am J Pathol. 2022 Feb;192(2):320–31. https://doi.org/10.1016/j.ajpath.2021.10.019 PMID:34774517
14. Davis C, Logan N, Tyson G, Orton R, Harvey WT, Perkins JS, et al.; COVID-19 Genomics UK (COG-UK) Consortium; COVID-19 DeplOyed VaccinE (DOVE) Cohort Study investigators. Reduced neutralisation of the Delta (B.1.617.2) SARS-CoV-2 variant of concern following vaccination. PLoS Pathog. 2021 Dec;17(12):e1010022. https://doi.org/10.1371/journal.ppat.1010022 PMID:34855916
15. Edara VV, Pinsky BA, Suthar MS, Lai L, Davis-Gardner ME, Floyd K, et al. Infection and Vaccine-Induced Neutralizing-Antibody Responses to the SARS-CoV-2 B.1.617 Variants. N Engl J Med. 2021 Aug;385(7):664–6. https://doi.org/10.1056/NEJMc2107799 PMID:34233096
16. Kumar S, Thambiraja TS, Karuppanan K, Subramaniam G. Omicron and Delta variant of SARS-CoV-2: A comparative computational study of spike protein. J Med Virol. 2022 Apr;94(4):1641–9. https://doi.org/10.1002/jmv.27526 PMID:34914115
17. Amanat F, Thapa M, Lei T, Ahmed SM, Adelsberg DC, Carreño JM, et al.; Personalized Virology Initiative. SARS-CoV-2 mRNA vaccination induces functionally diverse antibodies to NTD, RBD, and S2. Cell. 2021 Jul;184(15):3936–3948.e10. https://doi.org/10.1016/j.cell.2021.06.005 PMID:34192529
18. Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A, Lockhart S, et al.; C4591001 Clinical Trial Group. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med. 2020 Dec;383(27):2603–15. https://doi.org/10.1056/NEJMoa2034577 PMID:33301246
19. Levin EG, Lustig Y, Cohen C, Fluss R, Indenbaum V, Amit S, et al. Waning Immune Humoral Response to BNT162b2 Covid-19 Vaccine over 6 Months. N Engl J Med. 2021 Dec;385(24):e84. https://doi.org/10.1056/NEJMoa2114583 PMID:34614326
20. Tartof SY, Slezak JM, Fischer H, Hong V, Ackerson BK, Ranasinghe ON, et al. Effectiveness of mRNA BNT162b2 COVID-19 vaccine up to 6 months in a large integrated health system in the USA: a retrospective cohort study. Lancet. 2021 Oct;398(10309):1407–16. https://doi.org/10.1016/S0140-6736(21)02183-8 PMID:34619098
21. Jantarabenjakul W, Sodsai P, Chantasrisawad N, Jitsatja A, Ninwattana S, Thippamom N, et al. Dynamics of Neutralizing Antibody and T-Cell Responses to SARS-CoV-2 and Variants of Concern after Primary Immunization with CoronaVac and Booster with BNT162b2 or ChAdOx1 in Health Care Workers. Vaccines (Basel). 2022 Apr;10(5):639. https://doi.org/10.3390/vaccines10050639 PMID:35632395

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Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TBA, Published by POL Scientific