{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,16]],"date-time":"2025-10-16T00:35:42Z","timestamp":1760574942961,"version":"build-2065373602"},"reference-count":20,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2021,11,21]],"date-time":"2021-11-21T00:00:00Z","timestamp":1637452800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":["analyticalsciencejournals.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Electrophoresis"],"published-print":{"date-parts":[[2022,2]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Rhoptry neck protein 2 (RON2) binds to the hydrophobic groove of apical membrane antigen 1 (AMA1), an interaction essential for invasion of red blood cells (RBCs) by <jats:italic>Plasmodium falciparum<\/jats:italic> (Pf) parasites. Vaccination with AMA1 alone has been shown to be immunogenic, but unprotective even against homologous challenge in human trials. However, the AMA1\u2010RON2L (L is referred to as the loop region of RON2 peptide) complex is a promising candidate, as preclinical studies with Freund's adjuvant have indicated complete protection against lethal challenge in mice and superior protection against virulent infection in <jats:italic>Aotus<\/jats:italic> monkeys. To prepare for clinical trials of the AMA1\u2010RON2L complex, identity and integrity of the candidate vaccine must be assessed, and characterization methods must be carefully designed to not dissociate the delicate complex during evaluation. In this study, we developed a native Tris\u2010glycine gel method to separate and identify the AMA1\u2010RON2L complex, which was further identified and confirmed by Western blotting using anti\u2010AMA1 monoclonal antibodies (mAbs 4G2 and 2C2) and anti\u2010RON2L polyclonal Ab coupled with mass spectrometry. The formation of complex was also confirmed by Capillary Isoelectric Focusing (cIEF). A short\u2010term (48 h and 72 h at 4\u00b0C)\u00a0stability\u00a0study of AMA1\u2010RON2L complex was also performed. The results indicate that the complex was stable for 72 h at 4\u00b0C.\u00a0Our\u00a0research demonstrates that the native Tris\u2010glycine gel separation\/Western blotting coupled with mass spectrometry and cIEF can fully characterize the identity and integrity of the AMA1\u2013RON2L complex and provide useful quality control data for the subsequent clinical trials.<\/jats:p>","DOI":"10.1002\/elps.202000365","type":"journal-article","created":{"date-parts":[[2021,10,22]],"date-time":"2021-10-22T19:24:27Z","timestamp":1634930667000},"page":"509-515","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Characterization of AMA1\u2010RON2L complex with native gel electrophoresis and capillary isoelectric focusing"],"prefix":"10.1002","volume":"43","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7397-2685","authenticated-orcid":false,"given":"Daming","family":"Zhu","sequence":"first","affiliation":[{"name":"Laboratory of Malaria Immunology and Vaccinology National Institute of Allergy and Infectious Disease National Institutes of Health  Bethesda MD USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weili","family":"Dai","sequence":"additional","affiliation":[{"name":"Laboratory of Malaria Immunology and Vaccinology National Institute of Allergy and Infectious Disease National Institutes of Health  Bethesda MD USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Prakash","family":"Srinivasan","sequence":"additional","affiliation":[{"name":"Johns Hopkins Malaria Research Institute Bloomberg School of Public Health  Baltimore MD USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Holly","family":"McClellan","sequence":"additional","affiliation":[{"name":"Laboratory of Malaria Immunology and Vaccinology National Institute of Allergy and Infectious Disease National Institutes of Health  Bethesda MD USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dennis","family":"Braden","sequence":"additional","affiliation":[{"name":"Laboratory of Malaria Immunology and Vaccinology National Institute of Allergy and Infectious Disease National Institutes of Health  Bethesda MD USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alec","family":"Allee\u2010Munoz","sequence":"additional","affiliation":[{"name":"Laboratory of Malaria Immunology and Vaccinology National Institute of Allergy and Infectious Disease National Institutes of Health  Bethesda MD USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patricia A. Gonzales","family":"Hurtado","sequence":"additional","affiliation":[{"name":"Laboratory of Malaria Immunology and Vaccinology National Institute of Allergy and Infectious Disease National Institutes of Health  Bethesda MD USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Louis H.","family":"Miller","sequence":"additional","affiliation":[{"name":"Laboratory of Malaria and Vector Research National Institute of Allergy and Infectious Disease National Institutes of Health  Bethesda MD USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patrick E.","family":"Duffy","sequence":"additional","affiliation":[{"name":"Laboratory of Malaria Immunology and Vaccinology National Institute of Allergy and Infectious Disease National Institutes of Health  Bethesda MD USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2021,11,21]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.4161\/hv.6.8.11446"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1110303108"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1002755"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms3261"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1409928111"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41541-017-0015-7"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.172.3982.440"},{"key":"e_1_2_9_9_1","first-page":"23","volume":"32","author":"Smith B. 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