{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T01:45:31Z","timestamp":1777340731887,"version":"3.51.4"},"reference-count":100,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2021,7,21]],"date-time":"2021-07-21T00:00:00Z","timestamp":1626825600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class\/subclass usage and functional affinity. In theory, the ELISPOT\/FluoroSpot (collectively ImmunoSpot) assay platform is ideally suited for antigen-specific B-cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen-coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high-affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen-coating approach streamlines characterization of the memory B-cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune-monitoring efforts of large donor cohorts in general.<\/jats:p>","DOI":"10.3390\/cells10081843","type":"journal-article","created":{"date-parts":[[2021,7,21]],"date-time":"2021-07-21T11:53:23Z","timestamp":1626868403000},"page":"1843","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays"],"prefix":"10.3390","volume":"10","author":[{"given":"Sebastian","family":"K\u00f6ppert","sequence":"first","affiliation":[{"name":"Research & Development Department, Cellular Technology Limited, Shaker Heights, OH 44122, USA"},{"name":"Institute of Anatomy and Cell Biology, Friedrich-Alexander University Erlangen-N\u00fcrnberg, 91054 Erlangen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carla","family":"Wolf","sequence":"additional","affiliation":[{"name":"Research & Development Department, Cellular Technology Limited, Shaker Heights, OH 44122, USA"},{"name":"Institute of Anatomy and Cell Biology, Friedrich-Alexander University Erlangen-N\u00fcrnberg, 91054 Erlangen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"No\u00e9mi","family":"Becza","sequence":"additional","affiliation":[{"name":"Research & Development Department, Cellular Technology Limited, Shaker Heights, OH 44122, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0273-2235","authenticated-orcid":false,"given":"Giuseppe A.","family":"Sautto","sequence":"additional","affiliation":[{"name":"Center for Vaccines and Immunology, University of Georgia, Athens, GA 30602, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fridolin","family":"Franke","sequence":"additional","affiliation":[{"name":"Research & Development Department, Cellular Technology Limited, Shaker Heights, OH 44122, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6119-605X","authenticated-orcid":false,"given":"Stefanie","family":"Kuerten","sequence":"additional","affiliation":[{"name":"Institute of Anatomy and Cell Biology, Friedrich-Alexander University Erlangen-N\u00fcrnberg, 91054 Erlangen, Germany"},{"name":"Institute of Neuroanatomy, Medical Faculty, University of Bonn, 53115 Bonn, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1947-7469","authenticated-orcid":false,"given":"Ted M.","family":"Ross","sequence":"additional","affiliation":[{"name":"Center for Vaccines and Immunology, University of Georgia, Athens, GA 30602, USA"},{"name":"Department of Infectious Diseases, University of Georgia, Athens, GA 30602, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paul V.","family":"Lehmann","sequence":"additional","affiliation":[{"name":"Research & Development Department, Cellular Technology Limited, Shaker Heights, OH 44122, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6463-7623","authenticated-orcid":false,"given":"Greg A.","family":"Kirchenbaum","sequence":"additional","affiliation":[{"name":"Research & Development Department, Cellular Technology Limited, Shaker Heights, OH 44122, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,7,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1172\/JCI106279","article-title":"Metabolic properties of IgG subclasses in man","volume":"49","author":"Morell","year":"1970","journal-title":"J. Clin. Investig."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1002\/eji.1830180221","article-title":"The half-lives of serum immunoglobulins in adult mice","volume":"18","author":"Vieira","year":"1988","journal-title":"Eur. J. Immunol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2911","DOI":"10.1128\/JVI.06075-11","article-title":"Rapid and Massive Virus-Specific Plasmablast Responses during Acute Dengue Virus Infection in Humans","volume":"86","author":"Wrammert","year":"2012","journal-title":"J. Virol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"667","DOI":"10.1038\/nature06890","article-title":"Rapid cloning of high-affinity human monoclonal antibodies against influenza virus","volume":"453","author":"Wrammert","year":"2008","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1038\/s41591-020-0819-2","article-title":"Breadth of concomitant immune responses prior to patient recovery: A case report of non-severe COVID-19","volume":"26","author":"Thevarajan","year":"2020","journal-title":"Nat. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1146\/annurev.immunol.23.021704.115723","article-title":"Maintenance of serum antibody levels","volume":"23","author":"Manz","year":"2005","journal-title":"Annu. Rev. Immunol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"475","DOI":"10.1084\/jem.191.3.475","article-title":"bcl-2 Transgene Expression Inhibits Apoptosis in the Germinal Center and Reveals Differences in the Selection of Memory B Cells and Bone Marrow Antibody-Forming Cells","volume":"191","author":"Smith","year":"2000","journal-title":"J. Exp. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2419","DOI":"10.1084\/jem.20061254","article-title":"High affinity germinal center B cells are actively selected into the plasma cell compartment","volume":"203","author":"Phan","year":"2006","journal-title":"J. Exp. Med."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Lightman, S.M., Utley, A., and Lee, K.P. (2019). Survival of Long-Lived Plasma Cells (LLPC): Piecing Together the Puzzle. Front. Immunol., 10.","DOI":"10.3389\/fimmu.2019.00965"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1038\/nri1572","article-title":"Regulation of plasma-cell development","volume":"5","author":"Calame","year":"2005","journal-title":"Nat. Rev. Immunol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1016\/j.coi.2006.03.004","article-title":"Stromal niches, plasma cell differentiation and survival","volume":"18","author":"Moser","year":"2006","journal-title":"Curr. Opin. Immunol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1126\/science.aaz8432","article-title":"Influenza vaccine\u2013induced human bone marrow plasma cells decline within a year after vaccination","volume":"370","author":"Davis","year":"2020","journal-title":"Science"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1566","DOI":"10.1016\/j.cell.2019.04.036","article-title":"Longitudinal Analysis of the Human B Cell Response to Ebola Virus Infection","volume":"177","author":"Davis","year":"2019","journal-title":"Cell"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1016\/j.ebiom.2019.02.060","article-title":"Longitudinal analysis of acute and convalescent B cell responses in a human primary dengue serotype 2 infection model","volume":"41","author":"Nivarthi","year":"2019","journal-title":"EBioMedicine"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Turner, J.S., Kim, W., Kalaidina, E., Goss, C.W., Rauseo, A.M., Schmitz, A.J., Hansen, L., Haile, A., Klebert, M.K., and Pusic, I. (2020). SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans. Res. Sq.","DOI":"10.1038\/s41586-021-03647-4"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1128\/CVI.00674-15","article-title":"Kinetics of Epstein-Barr Virus (EBV) Neutralizing and Virus-Specific Antibodies after Primary Infection with EBV","volume":"23","author":"Bu","year":"2016","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1186\/s12985-020-01390-2","article-title":"Association of human cytomegalovirus (HCMV) neutralizing antibodies with antibodies to the HCMV glycoprotein complexes","volume":"17","author":"Shibamura","year":"2020","journal-title":"Virol. J."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"4591","DOI":"10.1128\/JVI.80.9.4591-4600.2006","article-title":"Human Cytomegalovirus Infection Elicits a Glycoprotein M (gM)\/gN-Specific Virus-Neutralizing Antibody Response","volume":"80","author":"Shimamura","year":"2006","journal-title":"J. Virol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1016\/j.coi.2008.03.016","article-title":"Plasma cell differentiation and survival","volume":"20","author":"Tarlinton","year":"2008","journal-title":"Curr. Opin. Immunol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1111","DOI":"10.1093\/cid\/ciw066","article-title":"Durability of Vaccine-Induced Immunity Against Tetanus and Diphtheria Toxins: A Cross-sectional Analysis","volume":"62","author":"Hammarlund","year":"2016","journal-title":"Clin. Infect. Dis."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Antia, A., Ahmed, H., Handel, A., Carlson, N.E., Amanna, I.J., Antia, R., and Slifka, M. (2018). Heterogeneity and longevity of antibody memory to viruses and vaccines. PLoS Biol., 16.","DOI":"10.1371\/journal.pbio.2006601"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1200","DOI":"10.1038\/s41591-020-0965-6","article-title":"Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections","volume":"26","author":"Long","year":"2020","journal-title":"Nat. Med."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1598","DOI":"10.1038\/s41564-020-00813-8","article-title":"Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans","volume":"5","author":"Seow","year":"2020","journal-title":"Nat. Microbiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"eabf4063","DOI":"10.1126\/science.abf4063","article-title":"Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection","volume":"371","author":"Dan","year":"2021","journal-title":"Science"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1038\/s41577-019-0244-2","article-title":"B cell memory: Building two walls of protection against pathogens","volume":"20","author":"Akkaya","year":"2020","journal-title":"Nat. Rev. Immunol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1111\/imr.12651","article-title":"Memory control by the B cell antigen receptor","volume":"283","author":"Engels","year":"2018","journal-title":"Immunol. Rev."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Palm, A.-K.E., and Henry, C. (2019). Remembrance of Things Past: Long-Term B Cell Memory After Infection and Vaccination. Front. Immunol., 10.","DOI":"10.3389\/fimmu.2019.01787"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Sanz, I., Wei, C., Jenks, S.A., Cashman, K.S., Tipton, C., Woodruff, M.C., Hom, J., and Lee, F.E.-H. (2019). Challenges and Opportunities for Consistent Classification of Human B Cell and Plasma Cell Populations. Front. Immunol., 10.","DOI":"10.3389\/fimmu.2019.02458"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Boonyaratanakornkit, J., and Taylor, J.J. (2019). Techniques to Study Antigen-Specific B Cell Responses. Front. Immunol., 10.","DOI":"10.3389\/fimmu.2019.01694"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"4047","DOI":"10.1128\/JVI.03422-13","article-title":"Flow Cytometry Reveals that H5N1 Vaccination Elicits Cross-Reactive Stem-Directed Antibodies from Multiple Ig Heavy-Chain Lineages","volume":"88","author":"Whittle","year":"2014","journal-title":"J. Virol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/j.jim.2008.11.012","article-title":"A method for identification of HIV gp140 binding memory B cells in human blood","volume":"343","author":"Scheid","year":"2009","journal-title":"J. Immunol. Methods"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1016\/j.cell.2020.11.029","article-title":"Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19","volume":"184","author":"Rodda","year":"2021","journal-title":"Cell"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"eabf8891","DOI":"10.1126\/sciimmunol.abf8891","article-title":"Rapid generation of durable B cell memory to SARS-CoV-2 spike and nucleocapsid proteins in COVID-19 and convalescence","volume":"5","author":"Hartley","year":"2020","journal-title":"Sci. Immunol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1636","DOI":"10.1016\/j.cell.2019.11.003","article-title":"High-Throughput Mapping of B Cell Receptor Sequences to Antigen Specificity","volume":"179","author":"Setliff","year":"2019","journal-title":"Cell"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"101428","DOI":"10.1016\/j.smim.2020.101428","article-title":"Antibody-guided structure-based vaccines","volume":"50","author":"Kwong","year":"2020","journal-title":"Semin. Immunol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1124","DOI":"10.1016\/j.str.2020.07.012","article-title":"Identification of Structurally Related Antibodies in Antibody Sequence Databases Using Rosetta-Derived Position-Specific Scoring","volume":"28","author":"Finn","year":"2020","journal-title":"Structure"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Franke, F., Kirchenbaum, G.A., Kuerten, S., and Lehmann, P.V. (2020). IL-21 in Conjunction with Anti-CD40 and IL-4 Constitutes a Potent Polyclonal B Cell Stimulator for Monitoring Antigen-Specific Memory B Cells. Cells, 9.","DOI":"10.3390\/cells9020433"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1016\/j.jim.2003.12.015","article-title":"Tracking human antigen-specific memory B cells: A sensitive and generalized ELISPOT system","volume":"286","author":"Crotty","year":"2004","journal-title":"J. Immunol. Methods"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/j.jim.2010.03.009","article-title":"An optimized assay for the enumeration of antigen-specific memory B cells in different compartments of the human body","volume":"358","author":"Cao","year":"2010","journal-title":"J. Immunol. Methods"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1260","DOI":"10.1002\/eji.200839129","article-title":"Clonal dissection of the human memory B-cell repertoire following infection and vaccination","volume":"39","author":"Pinna","year":"2009","journal-title":"Eur. J. Immunol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.jim.2013.05.007","article-title":"Optimization and qualification of a memory B-cell ELISpot for the detection of vaccine-induced memory responses in HIV vaccine trials","volume":"394","author":"Walsh","year":"2013","journal-title":"J. Immunol. Methods"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.jim.2013.02.009","article-title":"Optimization of a human IgG B-cell ELISpot assay for the analysis of vaccine-induced B-cell responses","volume":"391","author":"Jahnmatz","year":"2013","journal-title":"J. Immunol. Methods"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"97","DOI":"10.12688\/wellcomeopenres.11386.1","article-title":"Optimisation of ex vivo memory B cell expansion\/differentiation for interrogation of rare peripheral memory B cell subset responses","volume":"2","author":"Muir","year":"2017","journal-title":"Wellcome Open Res."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/0022-1759(83)90308-3","article-title":"A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells","volume":"65","author":"Czerkinsky","year":"1983","journal-title":"J. Immunol. Methods"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"21","DOI":"10.3390\/cells4010021","article-title":"High Reproducibility of ELISPOT Counts from Nine Different Laboratories","volume":"4","author":"Sundararaman","year":"2015","journal-title":"Cells"},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Carter, M.J., Mitchell, R.M., Meyer Sauteur, P.M., Kelly, D.F., and Tr\u00fcck, J. (2017). The Antibody-Secreting Cell Response to Infection: Kinetics and Clinical Applications. Front. Immunol., 8.","DOI":"10.3389\/fimmu.2017.00630"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1084\/jem.20101352","article-title":"Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection","volume":"208","author":"Wrammert","year":"2011","journal-title":"J. Exp. Med."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"13133","DOI":"10.1073\/pnas.1414070111","article-title":"Induction of broadly cross-reactive antibody responses to the influenza HA stem region following H5N1 vaccination in humans","volume":"111","author":"Ellebedy","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Bhaumik, S.K., Priyamvada, L., Kauffman, R.C., Lai, L., Natrajan, M.S., Cho, A., Rouphael, N., Suthar, M.S., Mulligan, M.J., and Wrammert, J. (2019). Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient. Viruses, 11.","DOI":"10.3390\/v11010019"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1016\/0022-1759(90)90014-M","article-title":"Measurement of the humoral immune response against streptococcus pneumoniae type 14-derived antigens by an ELISA and ELISPOT assay based on biotin-avidin technology","volume":"126","author":"Verheul","year":"1990","journal-title":"J. Immunol. Methods"},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Gaultier, G.N., McCready, W., and Ulanova, M. (2019). The effect of pneumococcal immunization on total and antigen-specific B cells in patients with severe chronic kidney disease. BMC Immunol., 20.","DOI":"10.1186\/s12865-019-0325-9"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41426-018-0197-8","article-title":"Use of ELISpot assay to study HBs-specific B cell responses in vaccinated and HBV infected humans","volume":"7","author":"Tian","year":"2018","journal-title":"Emerg. Microbes Infect."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.clim.2014.02.014","article-title":"Identification of a B cell-dependent subpopulation of multiple sclerosis by measurements of brain-reactive B cells in the blood","volume":"152","author":"Kuerten","year":"2014","journal-title":"Clin. Immunol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1186\/s13075-015-0551-3","article-title":"Long-lived plasma cells are early and constantly generated in New Zealand Black\/New Zealand White F1 mice and their therapeutic depletion requires a combined targeting of autoreactive plasma cells and their precursors","volume":"17","author":"Taddeo","year":"2015","journal-title":"Arthritis Res. Ther."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"3011","DOI":"10.4049\/jimmunol.1302003","article-title":"Long-Term B Cell Depletion in Murine Lupus Eliminates Autoantibody-Secreting Cells and Is Associated with Alterations in the Kidney Plasma Cell Niche","volume":"192","author":"Wang","year":"2014","journal-title":"J. Immunol."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"3109","DOI":"10.1172\/JCI57834","article-title":"Limited efficacy of inactivated influenza vaccine in elderly individuals is associated with decreased production of vaccine-specific antibodies","volume":"121","author":"Sasaki","year":"2011","journal-title":"J. Clin. Investig."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1089\/vim.2013.0099","article-title":"Detection of Influenza A\/H1N1-Specific Human IgG-Secreting B Cells in Older Adults by ELISPOT Assay","volume":"27","author":"Painter","year":"2014","journal-title":"Viral Immunol."},{"key":"ref_58","unstructured":"Kalyuzhny, A.E. (2018). Detecting all Immunoglobulin Classes and Subclasses in a Multiplex 7 Color ImmunoSpot\u00ae Assay. Handbook of ELISPOT: Methods and Protocols, Springer."},{"key":"ref_59","unstructured":"Kalyuzhny, A.E. (2018). Multi-Color FLUOROSPOT Counting Using ImmunoSpot\u00ae Fluoro-X\u2122 Suite. Handbook of ELISPOT: Methods and Protocols, Springer."},{"key":"ref_60","unstructured":"Kalyuzhny, A.E. (2012). How ELISPOT Morphology Reflects on the Productivity and Kinetics of Cells\u2019 Secretory Activity. Handbook of ELISPOT: Methods and Protocols, Humana Press."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"5574","DOI":"10.1128\/JVI.03203-15","article-title":"B Cell Responses during Secondary Dengue Virus Infection Are Dominated by Highly Cross-Reactive, Memory-Derived Plasmablasts","volume":"90","author":"Priyamvada","year":"2016","journal-title":"J. Virol."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"3798","DOI":"10.4049\/jimmunol.1701174","article-title":"Infection of Ferrets with Influenza Virus Elicits a Light Chain\u2013Biased Antibody Response against Hemagglutinin","volume":"199","author":"Kirchenbaum","year":"2017","journal-title":"J. Immunol."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.jim.2009.05.005","article-title":"The third dimension of ELISPOTs: Quantifying antibody secretion from individual plasma cells","volume":"346","author":"Bromage","year":"2009","journal-title":"J. Immunol. Methods"},{"key":"ref_64","unstructured":"Kurien, B.T., and Scofield, R.H. (2015). Membrane Microplates for One- and Two-Color ELISPOT and FLUOROSPOT Assays. Western Blotting: Methods and Protocols, Springer."},{"key":"ref_65","unstructured":"Kalyuzhny, A.E. (2012). Overview of Membranes and Membrane Plates Used in Research and Diagnostic ELISPOT Assays. Handbook of ELISPOT: Methods and Protocols, Humana Press."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"313","DOI":"10.3390\/cells1030313","article-title":"Optimal Thawing of Cryopreserved Peripheral Blood Mononuclear Cells for Use in High-Throughput Human Immune Monitoring Studies","volume":"1","author":"Ramachandran","year":"2012","journal-title":"Cells"},{"key":"ref_67","doi-asserted-by":"crossref","unstructured":"Fecher, P., Caspell, R., Naeem, V., Karulin, A.Y., Kuerten, S., and Lehmann, P.V. (2018). B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody. Cells, 7.","DOI":"10.3390\/cells7060050"},{"key":"ref_68","doi-asserted-by":"crossref","unstructured":"Ecker, J.W., Kirchenbaum, G.A., Pierce, S.R., Skarlupka, A.L., Abreu, R.B., Cooper, R.E., Taylor-Mulneix, D., Ross, T.M., and Sautto, G.A. (2020). High-Yield Expression and Purification of Recombinant Influenza Virus Proteins from Stably-Transfected Mammalian Cell Lines. Vaccines, 8.","DOI":"10.3390\/vaccines8030462"},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"1501","DOI":"10.1126\/science.abd0826","article-title":"Structure-based design of prefusion-stabilized SARS-CoV-2 spikes","volume":"369","author":"Hsieh","year":"2020","journal-title":"Science"},{"key":"ref_70","unstructured":"(2021, May 30). The Following Reagent was Produced under HHSN272201400008C and Obtained through BEI Resources, N., NIH: Vector pCAGGS Containing the SARS-Related Coronavirus 2, Wuhan-Hu-1 Spike Glycoprotein Receptor Binding Domain (RBD), NR-52309. Available online: https:\/\/www.beiresources.org\/Catalog\/BEIPlasmidVectors\/NR-52309.aspx."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"375","DOI":"10.4049\/jimmunol.1900379","article-title":"A Computationally Optimized Broadly Reactive Antigen Subtype\u2013Specific Influenza Vaccine Strategy Elicits Unique Potent Broadly Neutralizing Antibodies against Hemagglutinin","volume":"204","author":"Sautto","year":"2020","journal-title":"J. Immunol."},{"key":"ref_72","unstructured":"(2021, May 30). The Following Reagent was Deposited by the Centers for Disease Control and Prevention and Obtained through BEI Resources, N., NIH: SARS-Related Coronavirus 2, Isolate USA-WA1\/2020, Heat Inactivated, NR-52286. Available online: https:\/\/www.beiresources.org\/Catalog\/antigen\/NR-52286.aspx."},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"5874572","DOI":"10.1155\/2017\/5874572","article-title":"Generation of Monoclonal Antibodies against Immunoglobulin Proteins of the Domestic Ferret (Mustela putorius furo)","volume":"2017","author":"Kirchenbaum","year":"2017","journal-title":"J. Immunol. Res."},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"906","DOI":"10.1007\/s10875-021-00997-6","article-title":"Serologic Cross-Reactivity of SARS-CoV-2 with Endemic and Seasonal Betacoronaviruses","volume":"41","author":"Hicks","year":"2021","journal-title":"J. Clin. Immunol."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"100040","DOI":"10.1016\/j.xcrm.2020.100040","article-title":"Rapid Generation of Neutralizing Antibody Responses in COVID-19 Patients","volume":"1","author":"Suthar","year":"2020","journal-title":"Cell Rep. Med."},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"2605","DOI":"10.4049\/jimmunol.2001420","article-title":"Evaluation of Cellular and Serological Responses to Acute SARS-CoV-2 Infection Demonstrates the Functional Importance of the Receptor-Binding Domain","volume":"206","author":"Mantus","year":"2021","journal-title":"J. Immunol."},{"key":"ref_77","doi-asserted-by":"crossref","unstructured":"M\u00fcnz, C. (2015). Infectious Mononucleosis. Epstein Barr Virus Volume 1: One Herpes Virus: Many Diseases, Springer International Publishing.","DOI":"10.1007\/978-3-319-22822-8"},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1016\/j.coviro.2015.09.007","article-title":"Epstein-Barr virus latency: Current and future perspectives","volume":"14","author":"Kempkes","year":"2015","journal-title":"Curr. Opin. Virol."},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"2975","DOI":"10.4049\/jimmunol.165.6.2975","article-title":"EBV Persistence Involves Strict Selection of Latently Infected B Cells","volume":"165","author":"Joseph","year":"2000","journal-title":"J. Immunol."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"438204","DOI":"10.6064\/2012\/438204","article-title":"The Epstein-Barr Virus EBNA1 Protein","volume":"2012","author":"Frappier","year":"2012","journal-title":"Scientifica"},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"570","DOI":"10.1073\/pnas.84.2.570","article-title":"Antibody responses to Epstein-Barr virus-determined nuclear antigen (EBNA)-1 and EBNA-2 in acute and chronic Epstein-Barr virus infection","volume":"84","author":"Henle","year":"1987","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"e02033-16","DOI":"10.1128\/JVI.02033-16","article-title":"Neutralization of Diverse Human Cytomegalovirus Strains Conferred by Antibodies Targeting Viral gH\/gL\/pUL128-131 Pentameric Complex","volume":"91","author":"Ha","year":"2017","journal-title":"J. Virol."},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"649","DOI":"10.1038\/331649c0","article-title":"AIDS envelope protein patent","volume":"331","author":"Ezzell","year":"1988","journal-title":"Nature"},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"e01991-20","DOI":"10.1128\/mBio.01991-20","article-title":"S Protein-Reactive IgG and Memory B Cell Production after Human SARS-CoV-2 Infection Includes Broad Reactivity to the S2 Subunit","volume":"11","author":"Embong","year":"2020","journal-title":"mBio"},{"key":"ref_85","doi-asserted-by":"crossref","first-page":"e1245","DOI":"10.1002\/cti2.1245","article-title":"Pattern of circulating SARS-CoV-2-specific antibody-secreting and memory B-cell generation in patients with acute COVID-19","volume":"10","author":"Byazrova","year":"2021","journal-title":"Clin. Transl. Immunol."},{"key":"ref_86","unstructured":"Yao, L., Wang, G.-L., Shen, Y., Wang, Z.-Y., Zhan, B.-D., Duan, L.-J., Lu, B., Shi, C., Gao, Y.-M., and Peng, H.-H. (2021). Persistence of Antibody and Cellular Immune Responses in COVID-19 patients over Nine Months after Infection. J. Infect. Dis."},{"key":"ref_87","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1016\/j.medj.2021.02.001","article-title":"Persistence of SARS-CoV-2-specific B and T cell responses in convalescent COVID-19 patients 6\u20138 months after the infection","volume":"2","author":"Sherina","year":"2021","journal-title":"Med"},{"key":"ref_88","doi-asserted-by":"crossref","unstructured":"Ansari, A., Arya, R., Sachan, S., Jha, S.N., Kalia, A., Lall, A., Sette, A., Grifoni, A., Weiskopf, D., and Coshic, P. (2021). Immune Memory in Mild COVID-19 Patients and Unexposed Donors Reveals Persistent T Cell Responses After SARS-CoV-2 Infection. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.636768"},{"key":"ref_89","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1016\/S0076-6879(00)26058-8","article-title":"Purification of proteins using polyhistidine affinity tags","volume":"326","author":"Bornhorst","year":"2000","journal-title":"Methods Enzymol."},{"key":"ref_90","doi-asserted-by":"crossref","first-page":"581093","DOI":"10.1155\/2013\/581093","article-title":"Several affinity tags commonly used in chromatographic purification","volume":"2013","author":"Zhao","year":"2013","journal-title":"J. Anal. Methods Chem."},{"key":"ref_91","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1016\/S0165-022X(01)00213-5","article-title":"The FLAG\u2122 peptide, a versatile fusion tag for the purification of recombinant proteins","volume":"49","author":"Einhauer","year":"2001","journal-title":"J. Biochem. Biophys. Methods"},{"key":"ref_92","doi-asserted-by":"crossref","first-page":"1841","DOI":"10.1007\/s10529-012-0966-8","article-title":"A new idea for simple and rapid monitoring of gene expression: Requirement of nucleotide sequences encoding an N-terminal HA tag in the T7 promoter-driven expression in E. coli","volume":"34","author":"Moon","year":"2012","journal-title":"Biotechnol. Lett."},{"key":"ref_93","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1006\/prep.2001.1514","article-title":"A Comprehensive System for Protein Purification and Biochemical Analysis Based on Antibodies to c-myc Peptide","volume":"23","author":"Hillman","year":"2001","journal-title":"Protein Expr. Purif."},{"key":"ref_94","doi-asserted-by":"crossref","first-page":"753","DOI":"10.1006\/jmbi.1996.0061","article-title":"Molecular Interaction Between the Strep-tag Affinity Peptide and its Cognate Target, Streptavidin","volume":"255","author":"Schmidt","year":"1996","journal-title":"J. Mol. Biol."},{"key":"ref_95","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s00604-015-1623-4","article-title":"Oriented immobilization of proteins on solid supports for use in biosensors and biochips: A review","volume":"183","author":"Liu","year":"2016","journal-title":"Microchim. Acta"},{"key":"ref_96","doi-asserted-by":"crossref","unstructured":"Nu\u00f1ez, I.A., Carlock, M.A., Allen, J.D., Owino, S.O., Moehling, K.K., Nowalk, P., Susick, M., Diagle, K., Sweeney, K., and Mundle, S. (2017). Impact of age and pre-existing influenza immune responses in humans receiving split inactivated influenza vaccine on the induction of the breadth of antibodies to influenza A strains. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0185666"},{"key":"ref_97","doi-asserted-by":"crossref","first-page":"26745","DOI":"10.1073\/pnas.1915620116","article-title":"Affinity maturation in a human humoral response to influenza hemagglutinin","volume":"116","author":"McCarthy","year":"2019","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_98","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1016\/j.jim.2008.09.025","article-title":"Frequencies of human influenza-specific antibody secreting cells or plasmablasts post vaccination from fresh and frozen peripheral blood mononuclear cells","volume":"340","author":"Kyu","year":"2009","journal-title":"J. Immunol. Methods"},{"key":"ref_99","doi-asserted-by":"crossref","first-page":"2437","DOI":"10.4049\/jimmunol.2000717","article-title":"Expansion of SARS-CoV-2\u2013Specific Antibody-Secreting Cells and Generation of Neutralizing Antibodies in Hospitalized COVID-19 Patients","volume":"205","author":"Glans","year":"2020","journal-title":"J. Immunol."},{"key":"ref_100","doi-asserted-by":"crossref","unstructured":"Demonbreun, A.R., McDade, T.W., Pesce, L., Vaught, L.A., Reiser, N.L., Bogdanovic, E., Velez, M.P., Hsieh, R.R., Simons, L.M., and Saber, R. (2021). Patterns and persistence of SARS-CoV-2 IgG antibodies in Chicago to monitor COVID-19 exposure. JCI Insight, 6.","DOI":"10.1172\/jci.insight.146148"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/10\/8\/1843\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:32:46Z","timestamp":1760164366000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/10\/8\/1843"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,7,21]]},"references-count":100,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2021,8]]}},"alternative-id":["cells10081843"],"URL":"https:\/\/doi.org\/10.3390\/cells10081843","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2021.06.02.21258073","asserted-by":"object"}]},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,7,21]]}}}