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However, measuring antigen\u2010specific and cross\u2010reactive B cells remains technically challenging due to low abundance of these cells and background binding. Here, we have assessed different antigen\u2010labeling methods to optimize the measurement of B cell specificity, cross\u2010reactivity, heterocliticity, and average affinity for antigen by flow cytometry. Antigens covalently labeled with NHS ester amine\u2010reactive dyes gave reliable results compared to other antigen labeling methods: namely conjugation of His\u2010tagged antigen on fluorescent streptavidin nanoparticles, or on soluble streptavidin tetramers, bridged via the biomolecular adapter biotin\u2010trisNTA(Ni). Blocking with unlabeled H1 or H7 clearly outcompeted H1\u2010 and\/or H7\u2010specific cells, indicating that cross\u2010reactive antibodies possess lower affinities compared to the mono\u2010reactive or the rare heteroclitic antibodies. Overall, these studies suggest that evaluating the antigen specificity and breadth of candidate vaccines, including multi\u2010season or universal vaccines, may be best accomplished using flow cytometric quantification of clonally distributed B lineage cells using antigens covalently derivatized with fluorescent dyes, which is also analogous with the standard flow cytometry approach of quantifying antigen biomarkers on cells using fluorescent monoclonal antibodies (mAbs).<\/jats:p>","DOI":"10.1002\/cytoa.70000","type":"journal-article","created":{"date-parts":[[2025,12,15]],"date-time":"2025-12-15T04:15:58Z","timestamp":1765772158000},"page":"817-836","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Detection and Validation of Influenza Virus Hemagglutinin Specific, Cross\u2010Reactive, and Heteroclitic B Cells Induced by a Combination Vaccine Adjuvant"],"prefix":"10.1002","volume":"107","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5755-6788","authenticated-orcid":false,"given":"Egest J.","family":"Pone","sequence":"first","affiliation":[{"name":"Department of Physiology &amp; Biophysics Vaccine Research &amp; Development Center, School of Medicine, University of California  Irvine California USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiin","family":"Felgner","sequence":"additional","affiliation":[{"name":"Department of Physiology &amp; Biophysics Vaccine Research &amp; Development Center, School of Medicine, University of California  Irvine California USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jenny","family":"Hernandez\u2010Davies","sequence":"additional","affiliation":[{"name":"Department of Physiology &amp; Biophysics Vaccine Research &amp; Development Center, School of Medicine, University of California  Irvine California USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Johannes S.","family":"Gach","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases School of Medicine, University of California  Irvine California USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tyler","family":"Albin","sequence":"additional","affiliation":[{"name":"Department of Chemistry University of California  Irvine California USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"D. Huw","family":"Davies","sequence":"additional","affiliation":[{"name":"Department of Physiology &amp; Biophysics Vaccine Research &amp; Development Center, School of Medicine, University of California  Irvine California USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2025,12,14]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1440\u20101711.2003.01152.x"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1111\/nyas.12012"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1159\/000234509"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.90.16.7711"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.1.264"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0161\u20105890(01)00003\u20107"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molimm.2011.07.018"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1080\/14760584.2019.1578216"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41577\u2010020\u201000479\u20107"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20101352"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1118979109"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1607931113"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.1128\/Jvi.03203\u201015"},{"key":"e_1_2_10_15_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2021.692151"},{"key":"e_1_2_10_16_1","doi-asserted-by":"publisher","DOI":"10.1034\/j.1600\u2010065X.2002.18511.x"},{"key":"e_1_2_10_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2023.106940"},{"key":"e_1_2_10_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jim.2006.09.005"},{"key":"e_1_2_10_19_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41590\u2010022\u201001367\u2010z"},{"key":"e_1_2_10_20_1","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20142284"},{"key":"e_1_2_10_21_1","first-page":"1073","article-title":"Distinct Germinal Center Selection at Local Sites Shapes Cross\u2010Protective Memory B Cell Response to Viral Escape","volume":"46","author":"Adachi Y.","year":"2016","journal-title":"European Journal of Immunology"},{"key":"e_1_2_10_22_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.01970\u201017"},{"key":"e_1_2_10_23_1","doi-asserted-by":"publisher","DOI":"10.1172\/jci.insight.129035"},{"key":"e_1_2_10_24_1","doi-asserted-by":"publisher","DOI":"10.1093\/intimm\/dxz049"},{"key":"e_1_2_10_25_1","doi-asserted-by":"publisher","DOI":"10.1172\/Jci123366"},{"key":"e_1_2_10_26_1","doi-asserted-by":"publisher","DOI":"10.1172\/jci.insight.136653"},{"key":"e_1_2_10_27_1","doi-asserted-by":"publisher","DOI":"10.1111\/imcb.12383"},{"key":"e_1_2_10_28_1","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.95.2.378"},{"key":"e_1_2_10_29_1","doi-asserted-by":"publisher","DOI":"10.1002\/jmr.2394"},{"key":"e_1_2_10_30_1","doi-asserted-by":"publisher","DOI":"10.1080\/01971520009349534"},{"key":"e_1_2_10_31_1","doi-asserted-by":"publisher","DOI":"10.1016\/s0022\u20101759(96)00227\u2010x"},{"key":"e_1_2_10_32_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.21024"},{"key":"e_1_2_10_33_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-61779-974-7_25"},{"key":"e_1_2_10_34_1","doi-asserted-by":"publisher","DOI":"10.1128\/Jvi.03422\u201013"},{"key":"e_1_2_10_35_1","first-page":"430","volume-title":"Method Enzymol","author":"Cull M. 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