{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,27]],"date-time":"2026-04-27T18:45:42Z","timestamp":1777315542904,"version":"3.51.4"},"reference-count":38,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2024,8,1]],"date-time":"2024-08-01T00:00:00Z","timestamp":1722470400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibodies"],"abstract":"<jats:p>Critical blood shortages plague healthcare systems, particularly in lower-income and middle-income countries. This affects patients requiring regular transfusions and creates challenges during emergencies where universal blood is vital. To address these shortages and support blood banks during emergencies, this study reports a method for increasing the compatibility of blood group A red blood cells (RBCs) by blocking surface antigen-A using anti-A single chain fragment variable (scFv). To enhance stability, the scFv was first modified with the addition of interdomain disulfide bonds. The most effective location for this modification was found to be H44-L232 of mutant-1a scFv. ScFv was then produced from E.coli BL21(DE3) and purified using a three-step process. Purified scFvs were then used to block maximum number of antigens-A on RBCs, and it was found that only monomers were functional, while dimers formed through incorrect domain-swapping were non-functional. These antigen-blocked RBCs displayed no clumping in hemagglutination testing with incompatible blood plasma. The dissociation constant KD was found to be 0.724 \u03bcM. Antigen-blocked RBCs have the potential to be given to other blood groups during emergencies. This innovative approach could significantly increase the pool of usable blood, potentially saving countless lives.<\/jats:p>","DOI":"10.3390\/antib13030064","type":"journal-article","created":{"date-parts":[[2024,8,1]],"date-time":"2024-08-01T11:34:20Z","timestamp":1722512060000},"page":"64","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Prevention of Blood Incompatibility Related Hemagglutination: Blocking of Antigen A on Red Blood Cells Using In Silico Designed Recombinant Anti-A scFv"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5721-1480","authenticated-orcid":false,"given":"Saleha","family":"Hafeez","sequence":"first","affiliation":[{"name":"Atta-Ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, H-12 Sector, Islamabad 44000, Pakistan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Najam","family":"Zaidi","sequence":"additional","affiliation":[{"name":"Atta-Ur-Rahman School of Applied Biosciences, National University of Sciences and Technology, H-12 Sector, Islamabad 44000, Pakistan"},{"name":"Pak-Austria Fachhochschule Institute of Applied Sciences and Technology, Haripur 22600, Pakistan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,8,1]]},"reference":[{"key":"ref_1","unstructured":"(2023, March 13). Global Status Report on Blood Safety and Availability. Available online: https:\/\/apps.who.int\/iris\/handle\/10665\/254987."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e055017","DOI":"10.1136\/bmjopen-2021-055017","article-title":"Status of hospital-based blood transfusion services in low-income and middle-income countries: A cross-sectional international survey","volume":"12","author":"Barnes","year":"2022","journal-title":"BMJ Open"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1016\/j.tracli.2020.10.006","article-title":"The impact of COVID-19 on transfusion-dependent thalassemia patients of Karachi, Pakistan: A single-center experience","volume":"28","author":"Azim","year":"2021","journal-title":"Transfus. Clin. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"336","DOI":"10.1111\/trf.16798","article-title":"COVID-19 and the impact on blood availability and transfusion practices in low- and middle-income countries","volume":"62","author":"Barnes","year":"2022","journal-title":"Transfusion"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3027","DOI":"10.1111\/trf.16640","article-title":"The transfusion management of beta thalassemia in the United States","volume":"61","author":"Lal","year":"2021","journal-title":"Transfusion"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.aan.2007.07.005","article-title":"Advances in transfusion medicine","volume":"25","author":"Nordmeyer","year":"2007","journal-title":"Adv. Anesth."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"7182","DOI":"10.1093\/bja\/aet376","article-title":"Update on massive transfusion","volume":"111","author":"Pham","year":"2013","journal-title":"Br. J. Anaesth."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1074\/jbc.REV119.008164","article-title":"Toward universal donor blood: Enzymatic conversion of A and B to O type","volume":"295","author":"Rahfeld","year":"2020","journal-title":"J. Biol. Chem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1016\/j.tracli.2023.06.007","article-title":"O-negative blood shortage management in a University Hospital: Impact of transfusing RHD-positive red blood cells to RHD-negative patients","volume":"30","author":"Luyten","year":"2023","journal-title":"Transfus. Clin. Biol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Hafeez, S., and Zaidi, N.S.S. (2023). Red blood cell substitutes: Liposome encapsulated hemoglobin and magnetite nanoparticle conjugates as oxygen carriers. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24021618"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/j.tracli.2003.12.002","article-title":"Universal red blood cells enzymatic conversion of blood group A and B antigens","volume":"11","author":"Olsson","year":"2004","journal-title":"Transfus. Clin. Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1","DOI":"10.5779\/hypothesis.v14i1.515","article-title":"Production of scFvs as anti-ABO antibodies antagonist for prevention of hemolytic transfusion reactions resulting ABO blood groups incompatibility between donor and recipient","volume":"14","author":"Mohammadi","year":"2016","journal-title":"Hypothesis"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2059","DOI":"10.1074\/jbc.M104364200","article-title":"Structure of an anti-blood group A FV and improvement of its binding affinity without loss of specificity","volume":"277","author":"Thomas","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3390\/ijms12010001","article-title":"Stabilization of the single-chain fragment variable by an interdomain disulfide bond and its effect on antibody affinity","volume":"12","author":"Zhao","year":"2010","journal-title":"Int. J. Mol. Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1038\/nmeth.3213","article-title":"The I-TASSER suite: Protein structure and function prediction","volume":"12","author":"Yang","year":"2015","journal-title":"Nat. Methods"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1511","DOI":"10.1021\/acs.jcim.7b00190","article-title":"Playmolecule ProteinPrepare: A web application for protein preparation for molecular dynamics simulations","volume":"57","author":"Giorgino","year":"2017","journal-title":"J. Chem. Inf. Model."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.softx.2015.06.001","article-title":"Gromacs: High performance molecular simulations through multi-level parallelism from laptops to supercomputers","volume":"1","author":"Abraham","year":"2015","journal-title":"SoftwareX"},{"key":"ref_18","unstructured":"Woods Group (2023, February 21). GLYCAM Web. Complex Carbohydrate Research Center, University of Georgia, Athens, GA. 2005\u20132023. Available online: http:\/\/glycam.org."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2785","DOI":"10.1002\/jcc.21256","article-title":"Autodock4 and AutoDockTools4: Automated docking with selective receptor flexiblity","volume":"16","author":"Morris","year":"2009","journal-title":"J. Comput. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1668","DOI":"10.1002\/jcc.20290","article-title":"The Amber Biomolecular Simulation Programs","volume":"26","author":"Case","year":"2005","journal-title":"J. Comput. Chem."},{"key":"ref_21","unstructured":"(2021). BIOVIA, Version 21.1.0, Dassault Syst\u00e8mes. Available online: https:\/\/www.3ds.com\/."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"536","DOI":"10.1093\/nar\/24.3.536","article-title":"High efficiency 5 min transformation of Escherichia coli","volume":"24","author":"Pope","year":"1996","journal-title":"Nucleic Acids Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of structural proteins during the Assembly of the head of bacteriophage T4","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature"},{"key":"ref_24","first-page":"e53575","article-title":"An ELISA based binding and competition method to rapidly determine ligand-receptor interactions","volume":"109","author":"Syedbasha","year":"2016","journal-title":"JoVE"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1080\/15476910601161691","article-title":"Use of whole sheep red blood cells in Elisa to assess immunosuppressionin vivo","volume":"4","author":"Koganei","year":"2007","journal-title":"J. Immunotoxicol."},{"key":"ref_26","unstructured":"(2023). Nonlinear Regression Curve Fitting was Performed Using GraphPad Prism Version 9.5.1, GraphPad Software. Available online: https:\/\/www.graphpad.com\/."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1186\/1475-2859-8-26","article-title":"Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli","volume":"8","year":"2009","journal-title":"Microb. Cell Factories"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1910","DOI":"10.1021\/ac7018624","article-title":"Engineering peptide linkers for scFv immunosensors","volume":"80","author":"Shen","year":"2008","journal-title":"Anal. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"5449","DOI":"10.1038\/s41598-022-09324-4","article-title":"Effect of non-repetitive linker on in vitro and in vivo properties of an anti-VEGF scfv","volume":"12","author":"Arslan","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_30","unstructured":"National Institutes of Health (2022, July 20). Mesh Browser. U.S. National Library of Medicine, Available online: https:\/\/meshb.nlm.nih.gov\/record\/ui?name=Blocking+Antibodies."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"321","DOI":"10.1093\/protein\/gzs021","article-title":"Towards a universal disulphide stabilised single chain FV format: Importance of interchain disulphide bond location and VL-VH orientation","volume":"25","author":"Weatherill","year":"2012","journal-title":"PEDS"},{"key":"ref_32","unstructured":"Dean, L. (2023, October 13). Blood Groups and Red Cell Antigens, Available online: https:\/\/www.ncbi.nlm.nih.gov\/books\/NBK2261\/."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"12918","DOI":"10.1021\/bi9810407","article-title":"Factors influencing the dimer to monomer transition of an antibody single-chain FV fragment","volume":"37","author":"Arndt","year":"1998","journal-title":"Biochemistry"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1093\/protein\/13.8.565","article-title":"ScFv multimers of the anti-neuraminidase antibody NC10: Shortening of the linker in single-chain FV fragment assembled in VL to VH orientation drives the formation of dimers, trimers, tetramers and higher molecular mass multimers","volume":"13","author":"Dolezal","year":"2000","journal-title":"Protein Eng. Des. Sel."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1016\/S0022-2836(03)00526-6","article-title":"Effect of domain order on the activity of bacterially produced bispecific single-chain FV antibodies","volume":"330","author":"Kipriyanov","year":"2003","journal-title":"J. Mol. Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"20120835","DOI":"10.1098\/rsif.2012.0835","article-title":"On the binding affinity of macromolecular interactions: Daring to ask why proteins interact","volume":"10","author":"Kastritis","year":"2013","journal-title":"J. R. Soc. Interface"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"13579","DOI":"10.1016\/S0021-9258(19)76467-9","article-title":"Nucleotide and translated amino acid sequences of cDNA coding for the variable regions of the light and heavy chains of mouse hybridoma antibodies to blood group A and B substances","volume":"262","author":"Chen","year":"1987","journal-title":"J. Biol. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Muthana, S.M., Xia, L., Campbell, C.T., Zhang, Y., and Gildersleeve, J.C. (2015). Competition between serum IgG, IgM, and IgA Anti-Glycan antibodies. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0119298"}],"container-title":["Antibodies"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4468\/13\/3\/64\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T15:28:01Z","timestamp":1760110081000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4468\/13\/3\/64"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,8,1]]},"references-count":38,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2024,9]]}},"alternative-id":["antib13030064"],"URL":"https:\/\/doi.org\/10.3390\/antib13030064","relation":{},"ISSN":["2073-4468"],"issn-type":[{"value":"2073-4468","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,8,1]]}}}