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These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into <jats:italic>Chlamydomonas reinhardtii<\/jats:italic> chloroplasts and their products purified from algae lysates and assayed for <jats:italic>in vitro<\/jats:italic> biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (Bo<jats:styled-content style=\"fixed-case\">NT<\/jats:styled-content>\/A) with similar affinities as camelid antibodies produced in <jats:italic>Escherichia coli<\/jats:italic>, and they are similarly able to protect primary rat neurons from intoxication by Bo<jats:styled-content style=\"fixed-case\">NT<\/jats:styled-content>\/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in <jats:italic>E.\u00a0coli<\/jats:italic>. These camelid antibody domains are potent antigen\u2010binding proteins and the heterodimer fusion protein containing two <jats:styled-content style=\"fixed-case\">V<jats:sub>H<\/jats:sub>H<\/jats:styled-content> domains was capable of neutralizing Bo<jats:styled-content style=\"fixed-case\">NT<\/jats:styled-content>\/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (<jats:styled-content style=\"fixed-case\">GI<\/jats:styled-content>) tract of mice treated orally with antitoxin\u2010producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism.<\/jats:p>","DOI":"10.1111\/pbi.12244","type":"journal-article","created":{"date-parts":[[2014,9,17]],"date-time":"2014-09-17T07:13:38Z","timestamp":1410938018000},"page":"117-124","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":51,"title":["Algal chloroplast produced camelid <scp>V<sub>H<\/sub>H<\/scp> antitoxins are capable of neutralizing botulinum neurotoxin"],"prefix":"10.1111","volume":"13","author":[{"given":"Daniel J.","family":"Barrera","sequence":"first","affiliation":[{"name":"Division of Biological Sciences The California Center for Algae Biotechnology UC\u2013San Diego La Jolla CA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julian N.","family":"Rosenberg","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering Johns Hopkins University Baltimore MD USA"},{"name":"Synaptic Research LLC Baltimore MD USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joanna G.","family":"Chiu","sequence":"additional","affiliation":[{"name":"Division of Biological Sciences The California Center for Algae Biotechnology UC\u2013San Diego La Jolla CA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yung\u2010Nien","family":"Chang","sequence":"additional","affiliation":[{"name":"Synaptic Research LLC Baltimore MD USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle","family":"Debatis","sequence":"additional","affiliation":[{"name":"Department of Infectious Disease and Global Health Tufts Cummings School of Veterinary Medicine North Grafton MA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Soo\u2010Mun","family":"Ngoi","sequence":"additional","affiliation":[{"name":"Department of Medicine UC\u2013San Diego La Jolla CA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John T.","family":"Chang","sequence":"additional","affiliation":[{"name":"Department of Medicine UC\u2013San Diego La Jolla CA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Charles B.","family":"Shoemaker","sequence":"additional","affiliation":[{"name":"Department of Infectious Disease and Global Health Tufts Cummings School of Veterinary Medicine North Grafton MA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"George A.","family":"Oyler","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering Johns Hopkins University Baltimore MD USA"},{"name":"Synaptic Research LLC Baltimore MD USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephen P.","family":"Mayfield","sequence":"additional","affiliation":[{"name":"Division of Biological Sciences The California Center for Algae Biotechnology UC\u2013San Diego La Jolla CA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2014,9,17]]},"reference":[{"key":"e_1_2_6_2_1","doi-asserted-by":"publisher","DOI":"10.1001\/jama.285.8.1059"},{"key":"e_1_2_6_3_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa051926"},{"key":"e_1_2_6_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/9781118567166.ch27"},{"key":"e_1_2_6_5_1","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.00405-09"},{"key":"e_1_2_6_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.plantsci.2009.12.005"},{"key":"e_1_2_6_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/prot.20378"},{"key":"e_1_2_6_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt1028"},{"key":"e_1_2_6_9_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0037179"},{"key":"e_1_2_6_10_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.00714-13"},{"key":"e_1_2_6_11_1","doi-asserted-by":"publisher","DOI":"10.1111\/tpj.12316"},{"key":"e_1_2_6_12_1","doi-asserted-by":"publisher","DOI":"10.1111\/pbi.12008"},{"key":"e_1_2_6_13_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00425-012-1642-x"},{"key":"e_1_2_6_14_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1467-7652.2007.00249.x"},{"key":"e_1_2_6_15_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0029941"},{"key":"e_1_2_6_16_1","first-page":"301","article-title":"A simple method for chloroplast transformation in Chlamydomonas reinhardtii","volume":"274","author":"Ramesh V.M.","year":"2004","journal-title":"Methods Mol. 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