{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,6]],"date-time":"2026-01-06T13:08:40Z","timestamp":1767704920358,"version":"build-2065373602"},"reference-count":51,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2018,9,29]],"date-time":"2018-09-29T00:00:00Z","timestamp":1538179200000},"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>The Bacillus collagen-like protein of anthracis (BclA), found in Bacillus anthracis spores, is an attractive target for immunoassays. Previously, using phage display we had selected llama-derived single-domain antibodies that bound to B. anthracis spore proteins including BclA. Single-domain antibodies (sdAbs), the recombinantly expressed heavy domains from the unique heavy-chain-only antibodies found in camelids, provide stable and well-expressed binding elements with excellent affinity. In addition, sdAbs offer the important advantage that they can be tailored for specific applications through protein engineering. A fusion of a BclA targeting sdAb with the enzyme Beta galactosidase (\u03b2-gal) would enable highly sensitive immunoassays with no need for a secondary reagent. First, we evaluated five anti-BclA sdAbs, including four that had been previously identified but not characterized. Each was tested to determine its binding affinity, melting temperature, producibility, and ability to function as both capture and reporter in sandwich assays for BclA. The sdAb with the best combination of properties was constructed as a fusion with \u03b2-gal and shown to enable sensitive detection. This fusion has the potential to be incorporated into highly sensitive assays for the detection of anthrax spores.<\/jats:p>","DOI":"10.3390\/antib7040036","type":"journal-article","created":{"date-parts":[[2018,10,2]],"date-time":"2018-10-02T08:23:50Z","timestamp":1538468630000},"page":"36","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Genetic Fusion of an Anti-BclA Single-Domain Antibody with Beta Galactosidase"],"prefix":"10.3390","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7545-9893","authenticated-orcid":false,"given":"George P.","family":"Anderson","sequence":"first","affiliation":[{"name":"Naval Research Laboratory, Center for Biomolecular Science and Engineering, Washington, DC 20375, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lisa C.","family":"Shriver-Lake","sequence":"additional","affiliation":[{"name":"Naval Research Laboratory, Center for Biomolecular Science and Engineering, Washington, DC 20375, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Scott A.","family":"Walper","sequence":"additional","affiliation":[{"name":"Naval Research Laboratory, Center for Biomolecular Science and Engineering, Washington, DC 20375, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lauryn","family":"Ashford","sequence":"additional","affiliation":[{"name":"The Washington Center for Internships and Academic Seminars, 1333 16th Street N.W., Washington, DC 20036, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dan","family":"Zabetakis","sequence":"additional","affiliation":[{"name":"Naval Research Laboratory, Center for Biomolecular Science and Engineering, Washington, DC 20375, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jinny L.","family":"Liu","sequence":"additional","affiliation":[{"name":"Naval Research Laboratory, Center for Biomolecular Science and Engineering, Washington, DC 20375, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joyce C.","family":"Breger","sequence":"additional","affiliation":[{"name":"Naval Research Laboratory, Center for Biomolecular Science and Engineering, Washington, DC 20375, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"P. Audrey","family":"Brozozog Lee","sequence":"additional","affiliation":[{"name":"Nova Research Inc., Alexandria, VA 22308, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ellen R.","family":"Goldman","sequence":"additional","affiliation":[{"name":"Naval Research Laboratory, Center for Biomolecular Science and Engineering, Washington, DC 20375, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2018,9,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"182","DOI":"10.1136\/jcp.56.3.182","article-title":"Bacillus anthracis","volume":"56","author":"Spencer","year":"2003","journal-title":"J. Clin. Pathol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2236","DOI":"10.1001\/jama.287.17.2236","article-title":"Anthrax as a biological weapon, 2002: Updated recommendations for management","volume":"287","author":"Inglesby","year":"2002","journal-title":"JAMA"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1263","DOI":"10.1111\/jam.12928","article-title":"Test methods and response surface models for hot, humid air decontamination of materials contaminated with dirty spores of bacillus anthracis \u2206sterne and bacillus thuringiensis al hakam","volume":"119","author":"Buhr","year":"2015","journal-title":"J. Appl. Microbiol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1074","DOI":"10.1111\/jam.13055","article-title":"Hot, humid air decontamination of a c-130 aircraft contaminated with spores of two acrystalliferous bacillus thuringiensis strains, surrogates for bacillus anthracis","volume":"120","author":"Buhr","year":"2016","journal-title":"J. Appl. Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Walper, S.A., Anderson, G.P., Brozozog Lee, P.A., Glaven, R.H., Liu, J.L., Bernstein, R.D., Zabetakis, D., Johnson, L., Czarnecki, J.M., and Goldman, E.R. (2012). Rugged single domain antibody detection elements for bacillus anthracis spores and vegetative cells. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0032801"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"152","DOI":"10.3390\/antib2010152","article-title":"Selection and characterization of single domain antibodies specific for bacillus anthracis spore proteins","volume":"2","author":"Walper","year":"2013","journal-title":"Antibodies"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"6491","DOI":"10.1128\/AEM.01282-12","article-title":"Fast and sensitive detection of bacillus anthracis spores by immunoassay","volume":"78","author":"Morel","year":"2012","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1728","DOI":"10.1128\/CVI.00235-09","article-title":"Development of antibodies against anthrose tetrasaccharide for specific detection of bacillus anthracis spores","volume":"16","author":"Kuehn","year":"2009","journal-title":"Clin. Vaccine Immunol. CVI"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1099\/mic.0.26681-0","article-title":"Identification of proteins in the exosporium of bacillus anthracis","volume":"150","author":"Redmond","year":"2004","journal-title":"Microbiology"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1903","DOI":"10.1128\/JB.185.6.1903-1910.2003","article-title":"Identification of the immunodominant protein and other proteins of the bacillus anthracis exosporium","volume":"185","author":"Steichen","year":"2003","journal-title":"J. Bacteriol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1046\/j.1365-2958.2000.03000.x","article-title":"A collagen-like surface glycoprotein is a structural component of the bacillus anthracis exosporium","volume":"45","author":"Sylvestre","year":"2002","journal-title":"Mol. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3373","DOI":"10.1128\/JB.185.11.3373-3378.2003","article-title":"Genes of bacillus cereus and bacillus anthracis encoding proteins of the exosporium","volume":"185","author":"Todd","year":"2003","journal-title":"J. Bacteriol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1038\/363446a0","article-title":"Naturally occurring antibodies devoid of light chains","volume":"363","author":"Atarhouch","year":"1993","journal-title":"Nature"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1038\/374168a0","article-title":"A new antigen receptor gene family that undergods rearrangement and extensive somatice diversificatino in sharks","volume":"374","author":"Greenberg","year":"1995","journal-title":"Nature"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"521","DOI":"10.1016\/S0014-5793(97)01062-4","article-title":"Selection and identification of single domain antibody fragments from camel heavy-chain antibodies","volume":"414","author":"Ghahroudi","year":"1997","journal-title":"FEBS Lett."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1146\/annurev-biochem-063011-092449","article-title":"Nanobodies: Natural single-domain antibodies","volume":"82","author":"Muyldermans","year":"2013","journal-title":"Annu. Rev. Biochem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1186\/1475-2859-10-44","article-title":"Biotechnological applications of recombinant single-domain antibody fragments","volume":"10","year":"2011","journal-title":"Microb. Cell Fact."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1007\/s00430-009-0116-7","article-title":"Single domain antibodies: Promising experimental and therapeutic tools in infection and immunity","volume":"198","author":"Wesolowski","year":"2009","journal-title":"Med. Microbiol. Immunol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"S213","DOI":"10.1086\/520586","article-title":"Rapid assembly of sensitive antigen-capture assays for marburg virus, using in vitro selection of llama single-domain antibodies, at biosafety level 4","volume":"196","author":"Sherwood","year":"2007","journal-title":"J. Infect. Dis."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.jim.2013.04.001","article-title":"Single domain antibody alkaline phosphatase fusion proteins for antigen detection\u2014Analysis of affinity and thermal stability of single domain antibody","volume":"393","author":"Liu","year":"2013","journal-title":"J. Immunol. Methods"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1016\/j.ab.2013.10.031","article-title":"Thermostable single domain antibody\u2013maltose binding protein fusion for Bacillus anthracis spore protein bcla detection","volume":"447","author":"Walper","year":"2014","journal-title":"Anal. Biochem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/j.jim.2014.06.004","article-title":"Bioconjugates of rhizavidin with single domain antibodies as bifunctional immunoreagents","volume":"411","author":"Liu","year":"2014","journal-title":"J. Immunol. Methods"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"664","DOI":"10.1021\/bc049964w","article-title":"An s-layer heavy chain camel antibody fusion protein for generation of a nanopatterned sensing layer to detect the prostate-specific antigen by surface plasmon resonance technology","volume":"15","author":"Pleschberger","year":"2004","journal-title":"Bioconjugate Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"807","DOI":"10.1038\/srep00807","article-title":"Hapten mediated display and pairing of recombinant antibodies accelerates assay assembly for biothreat countermeasures","volume":"2","author":"Sherwood","year":"2012","journal-title":"Sci. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1387","DOI":"10.1021\/ac504305z","article-title":"Development of a nanobody\u2013alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin a in cereal","volume":"87","author":"Liu","year":"2015","journal-title":"Anal. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Sherwood, L.J., and Hayhurst, A. (2013). Ebolavirus nucleoprotein C-termini potently attract single domain antibodies enabling monoclonal affinity reagent sandwich assay (MARSA) formulation. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0061232"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"P153","DOI":"10.1016\/S0022-1759(99)00216-1","article-title":"Cell-elisa using \u03b2-galactosidase conjugated antibodies","volume":"234","author":"Liu","year":"2000","journal-title":"J. Immunol. Methods"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"6570","DOI":"10.1021\/acs.analchem.5b00322","article-title":"Ultrasensitive detection of ricin toxin in multiple sample matrixes using single-domain antibodies","volume":"87","author":"Gaylord","year":"2015","journal-title":"Anal. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/S0014-5793(02)03775-4","article-title":"Engineering of Escherichia coli \u03b2-galactosidase for solvent display of a functional scfv antibody fragment","volume":"533","author":"Villaverde","year":"2003","journal-title":"FEBS Lett."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"953","DOI":"10.1128\/jb.177.4.953-963.1995","article-title":"Beta-galactosidase is inactivated by intermolecular disulfide bonds and is toxic when secreted to the periplasm of Escherichia coli","volume":"177","author":"Snyder","year":"1995","journal-title":"J. Bacteriol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3343","DOI":"10.1128\/JB.01843-14","article-title":"Folding lacz in the periplasm of Escherichia coli","volume":"196","author":"Dwyer","year":"2014","journal-title":"J. Bacteriol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"10881","DOI":"10.1093\/nar\/16.22.10881","article-title":"Multiple sequence alignment with hierarchical-clustering","volume":"16","author":"Corpet","year":"1988","journal-title":"Nucleic Acids Res."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Walper, S.A., Liu, J.L., Zabetakis, D., Anderson, G.P., and Goldman, E.R. (2014). Development and evaluation of single domain antibodies for vaccinia and the L1 antigen. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0106263"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1016\/j.toxicon.2017.06.002","article-title":"Evaluation of anti-botulinum neurotoxin single domain antibodies with additional optimization for improved production and stability","volume":"135","author":"Zabetakis","year":"2017","journal-title":"Toxicon"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1016\/j.molimm.2012.07.010","article-title":"Selection and evaluation of single domain antibodies toward ms2 phage and coat protein","volume":"53","author":"Liu","year":"2013","journal-title":"Mol. Immunol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"93089","DOI":"10.1039\/C5RA21187E","article-title":"Kinetic enhancement of the diffusion-limited enzyme beta-galactosidase when displayed with quantum dots","volume":"5","author":"Oh","year":"2015","journal-title":"RSC Adv."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"7202","DOI":"10.1021\/ac100961x","article-title":"Binding kinetics of antiricin single domain antibodies and improved detection using a b chain specific binder","volume":"82","author":"Anderson","year":"2010","journal-title":"Anal. Chem."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1016\/j.pep.2014.01.003","article-title":"Negative tail fusions can improve ruggedness of single domain antibodies","volume":"95","author":"Goldman","year":"2014","journal-title":"Protein Expr. Purif."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"3794","DOI":"10.1021\/ja8049063","article-title":"High-throughput thermal scanning: A general, rapid dye-binding thermal shift screen for protein engineering","volume":"131","author":"Lavinder","year":"2009","journal-title":"J. Am. Chem. Soc."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1093\/protein\/gzs090","article-title":"An integrated approach to extreme thermostabilization and affinity maturation of an antibody","volume":"26","author":"McConnell","year":"2013","journal-title":"Protein Eng. Des. Sel."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"36489","DOI":"10.1074\/jbc.M707078200","article-title":"Stabilization of an immunoglobulin fold domain by an engineered disulfide bond at the buried hydrophobic region","volume":"282","author":"Hagihara","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"478","DOI":"10.1016\/j.jmb.2008.01.022","article-title":"Disulfide bond introduction for general stabilization of immunoglobulin heavy-chain variable domains","volume":"377","author":"Saerens","year":"2008","journal-title":"J. Mol. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"3101","DOI":"10.1002\/prot.24671","article-title":"Structural and mutational analysis of a monomeric and dimeric form of a single domain antibody with implications for protein misfolding","volume":"82","author":"George","year":"2014","journal-title":"Proteins Struct. Funct. Bioinform."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1016\/j.jmb.2005.07.038","article-title":"Identification of a universal vhh framework to graft non-canonical antigen-binding loops of camel single-domain antibodies","volume":"352","author":"Saerens","year":"2005","journal-title":"J. Mol. Biol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1016\/j.pep.2011.03.005","article-title":"Improved quantitative and qualitative production of single-domain intrabodies mediated by the co-expression of erv1p sulfhydryl oxidase","volume":"79","author":"Veggiani","year":"2011","journal-title":"Protein Expr. Purif."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1186\/1475-2859-12-97","article-title":"High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli","volume":"12","author":"Zarschler","year":"2013","journal-title":"Microb. Cell Fact."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"140","DOI":"10.1186\/s12934-014-0140-1","article-title":"Bacterial cytoplasm as an effective cell compartment for producing functional vhh-based affinity reagents and camelidae igg-like recombinant antibodies","volume":"13","author":"Djender","year":"2014","journal-title":"Microb. Cell Fact."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"12622","DOI":"10.1021\/ja8053018","article-title":"Detection of single-molecule DNA hybridization using enzymatic amplification in an array of femtoliter-sized reaction vessels","volume":"130","author":"Li","year":"2008","journal-title":"J. Am. Chem. Soc."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1258","DOI":"10.1021\/ac3027178","article-title":"Optical methods for single molecule detection and analysis","volume":"85","author":"Walt","year":"2013","journal-title":"Anal. Chem."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"3195","DOI":"10.1039\/C4LC00277F","article-title":"Protein measurements in microwells","volume":"14","author":"Walt","year":"2014","journal-title":"Lab Chip"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1007\/s11419-016-0336-7","article-title":"Rapid and ultrasensitive detection of botulinum neurotoxin serotype A1 in human serum and urine using single-molecule array method","volume":"35","author":"Dinh","year":"2017","journal-title":"Forensic Toxicol."}],"container-title":["Antibodies"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4468\/7\/4\/36\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T15:23:07Z","timestamp":1760196187000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4468\/7\/4\/36"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,9,29]]},"references-count":51,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2018,12]]}},"alternative-id":["antib7040036"],"URL":"https:\/\/doi.org\/10.3390\/antib7040036","relation":{},"ISSN":["2073-4468"],"issn-type":[{"type":"electronic","value":"2073-4468"}],"subject":[],"published":{"date-parts":[[2018,9,29]]}}}