{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T19:57:14Z","timestamp":1791575834239,"version":"4.3.4"},"reference-count":35,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2025,10,4]],"date-time":"2025-10-04T00:00:00Z","timestamp":1759536000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"MediDiamond Inc. through an industry-academia collaborative research project conducted at the National Health Research Institutes","award":["C12-010"],"award-info":[{"award-number":["C12-010"]}]}],"content-domain":{"domain":["www.mdpi.com"],"crossmark-restriction":true},"short-container-title":["Nanomaterials"],"abstract":"<jats:p>Nanodiamonds (NDs) are an innovative material in biomedical applications based on their excellent biocompatibility, nanoscale dimensions, and high surface area. In this study, we evaluated the potential of ND-in-oil emulsion to induce potent antibody responses in animals immunized with cobra venom. NDs demonstrated the capacity to bind complex venom proteins as stable conjugates, well dispersed in aqueous solution. Immunization of mice with cobra venom incorporated with ND-in-oil emulsion adjuvant (ND\/venom) elicited strong venom-specific antibody responses with titers comparable to those induced by venom formulation with conventional Freund\u2019s adjuvants (FA\/venom). IgG subclass analysis revealed that ND- and FA-based formulations induced a Th2-biased immune response in mice. Moreover, antibodies elicited by ND\/venom or FA\/venom immunization specifically recognized the epitopes of the lethal component of short-chain neurotoxin and conferred full protection against lethal cobra venom challenge (3LD50). Further, ND\/venom hyperimmunization was capable of inducing high levels of neutralizing antibodies in larger animals, rabbits, highlighting the potential for antivenom manufacturing. Notably, there were no obvious lesions at the injection sites of animals that received ND\/venom, in contrast to those that received FA\/venom. These findings indicated NDs as an effective and safe additive in venom formulation for antivenom production.<\/jats:p>","DOI":"10.3390\/nano15191518","type":"journal-article","created":{"date-parts":[[2025,10,6]],"date-time":"2025-10-06T08:10:51Z","timestamp":1759738251000},"page":"1518","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Evaluation of Nanodiamond-in-Oil Emulsion with Snake Venom to Enhance Potent Antibody Induction in Mice and Rabbits"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8255-8443","authenticated-orcid":false,"given":"Min-Han","family":"Lin","sequence":"first","affiliation":[{"name":"National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan 35053, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7741-2255","authenticated-orcid":false,"given":"Long-Jyun","family":"Su","sequence":"additional","affiliation":[{"name":"LuminX Biotech Co., Ltd., Taipei 11503, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8327-6835","authenticated-orcid":false,"given":"Hsin-Hung","family":"Lin","sequence":"additional","affiliation":[{"name":"LuminX Biotech Co., Ltd., Taipei 11503, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-6574-605X","authenticated-orcid":false,"given":"Liang-Yu","family":"Chen","sequence":"additional","affiliation":[{"name":"LuminX Biotech Co., Ltd., Taipei 11503, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0880-9667","authenticated-orcid":false,"given":"Asmaul","family":"Husna","sequence":"additional","affiliation":[{"name":"College of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3445-6036","authenticated-orcid":false,"given":"Wang-Chou","family":"Sung","sequence":"additional","affiliation":[{"name":"National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan 35053, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,10,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Kasturiratne, A., Wickremasinghe, A.R., de Silva, N., Gunawardena, N.K., Pathmeswaran, A., Premaratna, R., Savioli, L., Lalloo, D.G., and de Silva, H.J. (2008). The global burden of snakebite: A literature analysis and modelling based on regional estimates of envenoming and deaths. PLoS Med., 5.","DOI":"10.1371\/journal.pmed.0050218"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"17079","DOI":"10.1038\/nrdp.2017.79","article-title":"Snakebite envenoming","volume":"3","author":"Calvete","year":"2017","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1051\/jbio\/2009043","article-title":"Guidelines for the production, control and regulation of snake antivenom immunoglobulins","volume":"204","author":"Chippaux","year":"2010","journal-title":"Biol. Aujourdhui"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"747","DOI":"10.1093\/trstmh\/trv088","article-title":"Antivenom shortage is not circumstantial but structural","volume":"109","author":"Chippaux","year":"2015","journal-title":"Trans. R. Soc. Trop. Med. Hyg."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"526","DOI":"10.2471\/BLT.13.132431","article-title":"A multicomponent strategy to improve the availability of antivenom for treating snakebite envenoming","volume":"92","author":"Burnouf","year":"2014","journal-title":"Bull. World Health Organ."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"107229","DOI":"10.1016\/j.toxicon.2023.107229","article-title":"Physicochemical and immunological effects of adjuvant formulations with snake venom antigens for immunization of horses for antivenom production","volume":"232","author":"Fox","year":"2023","journal-title":"Toxicon"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"44","DOI":"10.3109\/1547691X.2013.772267","article-title":"Comparison of the adjuvant activity of aluminum hydroxide and calcium phosphate on the antibody response towards Bothrops asper snake venom","volume":"11","author":"Olmedo","year":"2014","journal-title":"J. Immunotoxicol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Sapsutthipas, S., Leong, P.K., Akesowan, S., Pratanaphon, R., Tan, N.H., and Ratanabanangkoon, K. (2015). Effective equine immunization protocol for production of potent poly-specific antisera against Calloselasma rhodostoma, Cryptelytrops albolabris and Daboia siamensis. PLoS Negl. Trop. Dis., 9.","DOI":"10.1371\/journal.pntd.0003609"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1016\/j.toxicon.2022.05.045","article-title":"CpG-ODN formulated with a nanostructure as adjuvant for anticrotalic serum production. Studies in mice","volume":"215","author":"Fusco","year":"2022","journal-title":"Toxicon"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"100233","DOI":"10.1016\/j.jvacx.2022.100233","article-title":"Comparison of adjuvant emulsions for their safety and ability to enhance the antibody response in horses immunized with African snake venoms","volume":"12","author":"Arguedas","year":"2022","journal-title":"Vaccine X"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/S0041-0101(96)00061-X","article-title":"Encapsulation of native crotoxin in liposomes: A safe approach for the production of antivenom and vaccination against Crotalus durissus terrificus venom","volume":"35","author":"Freitas","year":"1997","journal-title":"Toxicon"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"13046","DOI":"10.1021\/acs.analchem.3c01452","article-title":"Fluorescent nanodiamonds for tracking single polymer particles in cells and tissues","volume":"95","author":"Li","year":"2023","journal-title":"Anal. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"119933","DOI":"10.1016\/j.envres.2024.119933","article-title":"Engineering carbon-based nanomaterials for the delivery of platinum compounds: An innovative cancer disarming frontier","volume":"262","author":"Rahchamandi","year":"2024","journal-title":"Environ Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"226","DOI":"10.1007\/s00604-022-05320-7","article-title":"Nanodiamond conjugated SARS-CoV-2 spike protein: Electrochemical impedance immunosensing on a gold microelectrode","volume":"189","author":"Ramanathan","year":"2022","journal-title":"Mikrochim. Acta"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"103900","DOI":"10.1016\/j.xphs.2025.103900","article-title":"Synthesis and characterization of modified nanodiamonds for use as a potential vaccine adjuvant delivery platform for a candidate ricin toxin vaccine","volume":"114","author":"Craven","year":"2025","journal-title":"J. Pharm. Sci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1045","DOI":"10.2217\/nnm-2023-0179","article-title":"Nanodiamonds-in-oil emulsions elicit potent immune responses for effective vaccination and therapeutics","volume":"18","author":"Lin","year":"2023","journal-title":"Nanomedicine"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Ho, T.T., Pham, V.T., Nguyen, T.T., Trinh, V.T., Vi, T., Lin, H.H., Nguyen, P.M.T., Bui, H.T., Pham, N.B., and Le, T.B.T. (2021). Effects of size and surface properties of nanodiamonds on the immunogenicity of plant-based H5 protein of A\/H5N1 virus in mice. Nanomaterials, 11.","DOI":"10.3390\/nano11061597"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e20220027","DOI":"10.1590\/1678-9199-jvatitd-2022-0027","article-title":"Taiwan cobra envenoming: Serum venom concentration before and after specific treatment and relationship with debridement of necrotic wound tissue","volume":"29","author":"Wang","year":"2023","journal-title":"J. Venom. Anim. Toxins Incl. Trop. Dis."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.jprot.2015.07.015","article-title":"Cobra venom proteome and glycome determined from individual snakes of Naja atra reveal medically important dynamic range and systematic geographic variation","volume":"128","author":"Huang","year":"2015","journal-title":"J. Proteom."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1038\/s41570-022-00393-7","article-title":"The chemistry of snake venom and its medicinal potential","volume":"6","author":"Oliveira","year":"2022","journal-title":"Nat. Rev. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1252","DOI":"10.1093\/oxfordjournals.jbchem.a021889","article-title":"A novel neurotoxin, cobrotoxin b, from Naja naja atra (Taiwan cobra) venom: Purification, characterization, and gene organization","volume":"122","author":"Chang","year":"1997","journal-title":"J. Biochem."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Ratanabanangkoon, K., Tan, K.Y., Eursakun, S., Tan, C.H., Simsiriwong, P., Pamornsakda, T., Wiriyarat, W., Klinpayom, C., and Tan, N.H. (2016). A simple and novel strategy for the production of a pan-specific antiserum against elapid snakes of Asia. PLoS Negl. Trop. Dis., 10.","DOI":"10.1371\/journal.pntd.0004565"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Liu, B.S., Jiang, B.R., Hu, K.C., Liu, C.H., Hsieh, W.C., Lin, M.H., and Sung, W.C. (2021). Development of a broad-spectrum antiserum against cobra venoms using recombinant three-finger toxins. Toxins, 13.","DOI":"10.3390\/toxins13080556"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"100204","DOI":"10.1016\/j.toxcx.2024.100204","article-title":"Plug and play virus-like particles for the generation of anti-toxin antibodies","volume":"23","author":"Edge","year":"2024","journal-title":"Toxicon X"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Ramos, H.R., Junqueira-de-Azevedo Ide, L., Novo, J.B., Castro, K., Duarte, C.G., Machado-de-\u00c1vila, R.A., Chavez-Olortegui, C., and Ho, P.L. (2016). A heterologous multiepitope DNA prime\/recombinant protein boost immunisation strategy for the development of an antiserum against micrurus corallinus (Coral snake) venom. PLoS Negl. Trop. Dis., 10.","DOI":"10.1371\/journal.pntd.0004484"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Clement, H., Corrales-Garc\u00eda, L.L., Bola\u00f1os, D., Corzo, G., and Villegas, E. (2019). Immunogenic properties of recombinant enzymes from Bothrops ammodytoides towards the generation of neutralizing antibodies against Its own venom. Toxins, 11.","DOI":"10.3390\/toxins11120702"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Liu, B.S., Wu, W.G., Lin, M.H., Li, C.H., Jiang, B.R., Wu, S.C., Leng, C.H., and Sung, W.C. (2017). Identification of immunoreactive peptides of toxins to simultaneously assess the neutralization potency of antivenoms against neurotoxicity and cytotoxicity of Naja atra venom. Toxins, 10.","DOI":"10.3390\/toxins10010010"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"981","DOI":"10.1128\/CVI.00156-06","article-title":"Distinct contributions of vaccine-induced immunoglobulin G1 (IgG1) and IgG2a antibodies to protective immunity against influenza","volume":"13","author":"Huber","year":"2006","journal-title":"Clin. Vaccine Immunol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"787","DOI":"10.1038\/nri2868","article-title":"Vaccine delivery: A matter of size, geometry, kinetics and molecular patterns","volume":"10","author":"Bachmann","year":"2010","journal-title":"Nat. Rev. Immunol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e004709","DOI":"10.1136\/jitc-2022-004709","article-title":"Peptide emulsions in incomplete Freund\u2019s adjuvant create effective nurseries promoting egress of systemic CD4(+) and CD8(+) T cells for immunotherapy of cancer","volume":"10","author":"Melssen","year":"2022","journal-title":"J. Immunother. Cancer"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"915","DOI":"10.1016\/j.addr.2007.05.017","article-title":"Nanotechnology in vaccine delivery","volume":"60","author":"Peek","year":"2008","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"5397","DOI":"10.1039\/D0CS01127D","article-title":"Understanding nanoparticle endocytosis to improve targeting strategies in nanomedicine","volume":"50","author":"Susnik","year":"2021","journal-title":"Chem. Soc. Rev."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e24662","DOI":"10.1016\/j.heliyon.2024.e24662","article-title":"Research progress on emulsion vaccine adjuvants","volume":"10","author":"Huang","year":"2024","journal-title":"Heliyon"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Bekbossynova, A., Zharylgap, A., and Filchakova, O. (2021). Venom-derived neurotoxins targeting nicotinic acetylcholine receptors. Molecules, 26.","DOI":"10.3390\/molecules26113373"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1016\/j.actatropica.2015.05.020","article-title":"Immunological cross-reactivity and neutralization of the principal toxins of Naja sumatrana and related cobra venoms by a Thai polyvalent antivenom (Neuro Polyvalent Snake Antivenom)","volume":"149","author":"Leong","year":"2015","journal-title":"Acta Trop."}],"container-title":["Nanomaterials"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-4991\/15\/19\/1518\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,6]],"date-time":"2025-10-06T08:42:53Z","timestamp":1759740173000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-4991\/15\/19\/1518"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,4]]},"references-count":35,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2025,10]]}},"alternative-id":["nano15191518"],"URL":"https:\/\/doi.org\/10.3390\/nano15191518","relation":{},"ISSN":["2079-4991"],"issn-type":[{"value":"2079-4991","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,4]]}}}