Polyclonal Antibody Production Against HaptenStructured KDN Molecule by Using Different  Adjuvants Alternative to Freund’s Adjuvant

Sultan  Gülçe İz; Pelin  Sağlam Metiner; lgın  Kımız; Çağlar  Kayalı; Saime İsmet  Deliloğlu Gürhan

doi:10.5152/EurJTher.2018.400

Authors

Sultan Gülçe İz Department of Bioengineering, Ege University Faculty of Engineering https://orcid.org/0000-0002-9275-6272
Pelin Sağlam Metiner Department of Bioengineering, Ege University Faculty of Engineering https://orcid.org/0000-0002-5726-1928
lgın Kımız Department of Bioengineering, Ege University Faculty of Engineering https://orcid.org/0000-0002-9639-6550
Çağlar Kayalı Department of Bioengineering, Ege University Faculty of Engineering https://orcid.org/0000-0002-3266-1757
Saime İsmet Deliloğlu Gürhan Department of Bioengineering, Ege University Faculty of Engineering https://orcid.org/0000-0003-3841-6282

DOI:

https://doi.org/10.5152/EurJTher.2018.400

Keywords:

KDN (2-keto-3-deoxy-D-glycero-D-galacto-nonionic acid), polyclonal antibody, ELISA

Abstract

Objective: KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid), a member of the sialic acid family, is a hapten-structured low-molecular-weight monosaccharide on the cell membrane, which cannot induce immune responses without a carrier protein. Since it is over-expressed on the cancerous cells’ membrane, it is thought to be a great target molecule for anti-cancer treatments. The aim of this study is to obtain high titersof the anti-KDN polyclonal antibody response without using any carrier protein against the hapten-structured KDN molecule alternative to Freund’s adjuvants.
Methods: Montanide™ ISA 61 VG, a water-in-oil adjuvant; ISA 201 VG, a water-in-oil-in-water emulsion adjuvant; and IMS 1313 VG NPR, an aqueous-dispersion-based nanoparticle (50-200 nm) microemulsion adjuvant; and Freund’s adjuvant were used as anti-KDN antibody response stimulators. FourBALB/c mice were used for each adjuvant group, and immunization was performed at eight different time points. Anti-KDN antibody levels induced after each immunization with different adjuvants were detected with indirect enzyme-linked immunosorbent assay.
Results: The adjuvant efficiency of Montanide™ ISA 61 VG water in oil adjuvant was 1.4 times higher than in Freund’s adjuvant (p<0.0001), with a maximum anti-KDN level on Day 83.
Conclusion: It’s shown that without any carrier protein conjugation molecules such as hapten-structured KDN, higher amount anti-KDN antibody titres could be obtained by using a more safe and effective Montanide™ ISA 61 VG water-in-oil adjuvant as an alternative to Freund’s adjuvants. In this regard, it may be possible to produce high-antibody titers without using any carrier molecule, especially when commercial large scale monoclonal antibodies are desired to be produced against haptens as therapeutic approaches

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References

Inoue S, Lin SL, Chang T, Wu SH, Yao CW, Chu TY, et al. Identification of free deaminated sialic acid (2-Keto-3-deoxy-Dglycero-D-galacto-nononic Acid) in human red blood cells and its elevated expression in fetal cord red blood cells and ovarian cancer cells. J BiolChem 1998; 273: 27199-204.

Gavin AL, Hoebe K, Duong B, Ota T, Martin C, Beutler B, et al. Adjuvant-enhanced antibody responses occur without Toll-like eceptor signaling. Science 2006; 314: 1936-8.

Crich D, Navuluri C. Practical synthesis of 2-Keto-3-deoxy-D-glycero-D- galactononulosonic acid (KDN). Org Lett 2011; 13: 6288-91.

Arun Raj GR, Shailaja U, Sagar K, Viswaroopan D, Kumar SN. Swarnaprashana to Swarnamritaprashana: experience inspired modification through ages. Int J Pharm Sci Res 2017; 8: 4546-50.

Inoue S, Kitajima K. KDN (DeaminatedNeuraminic Acid): Dreamful past and exciting future of the newest member of the sialic acid family. Glycoconj J 2006; 23: 277-90.

Go S, Sato C, Yin J, Kannagi R, Kitajima K. Hypoxia-enhanced expression of free deaminoneuraminic acid in human cancer cells. BiochemBiophys Res Commun 2007; 357: 537-42.

Garcia MIG, Carjaval AS, Carmona FG, Ferrer AS. Improved production of 2-keto-3-deoxy-D-glycero-galactononulosonic acid (KDN) using FastPrep-CLEAs. Process Biochem 2014; 49: 90-4.

Lemus R, Karol MH. Conjugation of Haptens. Methods Mol Med 2008; 138: 167-82.

Torres OB, Jalah R, Rice KC, Li F, Antoline JFG, Iyer MR, et al. Characterization and optimization of heroin hapten-BSA conjugates: method development for the synthesis of reproducible hapten-based vaccines. Anal BioalChem 2014; 406: 5927-37.

Rajesh K, Rana KV, Suri CR. Characterization of hapten-protein conjugates: antibody generation and immunoassay development for pesticides monitoring. Bionanoscience 2013; 3: 137-44.

Bröker M, Berti F, Schneider J, Vojtek I. Polysaccharide conjugate vaccine protein carriers as a ‘’neglected valency” - Potential and limitations. Vaccine 2017; 35: 3286-94.

Rajput ZI, Hu SH, Xiao CW, Arijo AG. Adjuvant effects of saponins on animal immune responses. J Zhejiang UnivSci B 2007; 8: 153-61.

Kandil OM, Nassar SA, Nasr SM, Shalaby HAM, Hendawy S, El Moghazy FM. Synergetic effect of Egyptian propolis in immunization of BALB/c mice against bovine cysticercosis. Asian Pac J Trop Biomed 2015; 5: 324-30.

Thompson FM, Porter DW, Okitsu LS, Westerfeld N, Vogel D, Todryk S, et al. Evidence of blood stage efficacy with a virosomal Malaria Vaccine in a phase IIa clinical trial. PLoS One 2008; 3:1493.

Huijbers EJM, Femel J, Andersson K, Björkelund H, Hellman L, Olsson AK. The non-toxic and biodegradable adjuvant Montanide ISA 720/CpG can replace Freund’s in a cancer vaccine targeting ED-B-a prerequisite for clinical development. Vaccine 2012; 30: 225-30.

Cargnelutti DE, Salomon MC, Celedon V, Gracia Bustos MF, Morea G, Cuello-Carrion FD, et al. İmmunization with antigenic extracts of Leishmania associated with Montanide ISA 763 adjuvant induces partial protection in BALB/c mice against Leishmania (Leishmania) amazonensis infection. J Microbiolİmmünol Infect 2016; 49: 24-32.

Lone NA, Spackman E, Kapczynski D. İmmunologic evaluation of 10 different adjuvants for use in vaccines for chickens against highly pathogenic avian influenza virüs. Vaccine 2017; 35: 3401-8.

Wang F, Xie B, Wang B, Troy FA. LC-MS/MS glycomic analyses of free and conjugated forms of the sialic acids, Neu5Ac, Neu5Gc and KDN in human throat cancers. Glycobiology 2015; 25: 1362-74.

Elgert KD. Antibody Structure and Function. In: Elgert KD, editor. Immunology: Understanding the Immune System. New Jersey, USA: John Wiley & Sons Inc; 1998. p.58-78.

Huisman H, Wynveen P, Setter PW. Studies on the immune responce and preparation of antibodies against a large panel of conjugated neurotransmitters and biogenic amines: specific polyclonal antibody responce and tolerance. J Neurochem 2010; 112: 829-41.

Karahan M, Tuğlu S, Akdeste ZM. Polyelectrolytes and its biological properties. Sigma J EngNat Sci 2013; 31: 85-106.

Büyüktanır Ö. Current Biotechnological Bacterial Vaccines. Atatürk Üniversitesi Vet Bi. Derg 2010; 5: 97-105.

El-Bagoury GF, Sharawi SSA, Salama LS, Hammad MY. Comparative evaluation of different inactivated Rift valley fever virus vaccine adjuvanted with montanide oil ISA 61 VG, montanide oil ISA 201 VG, and aluminum hydroxide gel. BVMJ 2015; 29: 224-8.

İbrahim Eel-S, Gamal WM, Hassan AI, MahdySel-D, Hegazy AZ, Abdel-Atty MM. Comparative study on the immunopotentiator effect of ISA 201, ISA 61, ISA 50, ISA 206 used in trivalent foot and mouth disease vaccine. Vet World 2015; 8: 1189-98.

Jang SI, Lillehoj HS, Lee SH, Lee KW, Lillehoj EP, Bertrand F, et al. MontanideTM IMS 1313 N VG PR nanoparticle adjuvant enhances antigen-specific immune responses to profilin following mucosal vaccination against Eimeriaacervulina. Vet Parasitol 2011; 182: 163-70.

Campanero-Rhodes MA, Solis D, Carrera E, de la Cruz MJ, Diaz-Maurino T. Rat liver contains age-regulated cytosolic 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid (Kdn). Glycobiology 1999; 9: 527-32