The Effect of Differentially Designed Fusion Proteins to Elicit Efficient Anti-human Thyroid Stimulating Hormone Immune Responses
-
Maysam Mard-Soltani
,
Mohamad Javad Rasaee
,
Saeed Khalili
,
Abdol-Karim Sheikhi
,
Mehdi Hedayati
,
Hossein Ghaderi-Zefrehi
,
Milad Alasvand
Abstract
The production of human thyroid stimulating hormone (hTSH) immunoassays requires specific antibodies against hTSH which is a cumbersome process. Therefore, producing specific polyclonal antibodies against engineered recombinant fusion hTSH antigens would be of great significance. The best immunogenic region of the hTSH was selected based on in silico analyses and equipped with two different fusions. Standard methods were used for protein expression, purification, verification, structural evaluation, and immunizations of the white New Zealand rabbits. Ultimately, immunized serums were used for antibody titration, purification and characterization (specificity, sensitivity and cross reactivity). The desired antigens were successfully designed, sub-cloned, expressed, confirmed and used for in vivo immunization. Structural analyses indicated that only the bigger antigen has showed changed 2 dimensional (2D) and 3D structural properties in comparison to the smaller antigen. The raised polyclonal antibodies were capable of specific and sensitive hTSH detection, while the cross reactivity with the other members of the glycoprotein hormone family was minimum and negligible. The fusion which was solely composed of the tetanus toxin epitopes led to better protein folding and was capable of immunizing the host animals resulting into high titer antibody. Therefore, the minimal fusion sequences seem to be more effective in eliciting specific antibody responses.
1. Burtis CA, Ashwood ER, Bruns DE. Tietz textbook of clinical chemistry and molecular diagnostics: Elsevier Health Sciences; 2012.St. Louis, USA: Elsevier.
2. Rabbiosi S, Vigone MC, Cortinovis F, Zamproni I, Fugazzola L, Persani L, et al. Congenital hypothyroidism with eutopic thyroid gland: analysis of clinical and biochemical features at diagnosis and after re-evaluation. J Clin Endocrinol Metab 2013; 98(4):1395-402.
3. McPherson RA, Henry JB, Pincus MR. Henry's Clinical Diagnosis and Management by Laboratory Methods: Elsevier/Saunders; 2011. Philadelphia, PA: Elsevier.
4. Spencer CA. Assay of Thyroid Hormones and Related Substances. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, et al., editors. Endotext. South Dartmouth (MA) 2000.
5. Baldo BA. Glycoprotein Hormones. Safety of Biologics Therapy: Springer 2016; 40(10):401-17.
6. Ronin C, Papandreou MJ, Sergi S, Labbe-Jullie C, Medri G, Hoffmann T, et al. Glycosylation-dependent epitope mapping of human TSH (hTSH) isoforms. Int J Rad Appl Instrum B 1990; 17(7):651-6.
7. Bidart J-M, Troalen F, Ghillani P, Puisieux A, Bohuon C, Bellet D. Immunochemical mapping of antigenic regions on the human thyrotropin beta-subunit by antipeptide antibodies. J Biol Chem 1991; 266(29):19238-44.
8. Ramakrishnan G, Pedersen K, Guenette D, Sink J, Haque R, Petri WA, et al. Utility of recombinant fragment C for assessment of anti-tetanus antibodies in plasma. Diagn Microbiol Infect Dis 2015; 82(1):11-3.
9. Schellekens H, Casadevall N. Immunogenicity of recombinant human proteins: causes and consequences. J Neurol 2004; 251(2):ii4-ii9.
10. Soria-Guerra RE, Nieto-Gomez R, Govea-Alonso DO, Rosales-Mendoza S. An overview of bioinformatics tools for epitope prediction: implications on vaccine development. J Biomed Inform 2015; 53:405-14.
11. Khalili S, Jahangiri A, Borna H, Ahmadi Zanoos K, Amani J. Computational vaccinology and epitope vaccine design by immunoinformatics. Acta Microbiol Immunol Hung 2014; 61(3):285-307.
12. Sefid F, Rasooli I, Jahangiri A, Bazmara H. Functional exposed amino acids of BauA as potential immunogen against Acinetobacter baumannii. Acta Biotheor 2015; 63(2):129-49.
13. Khalili S, Zakeri A, Hashemi ZS, Masoumikarimi M, Manesh MRR, Shariatifar N, et al. Structural analyses of the interactions between the thyme active ingredients and human serum albumin. Turkish Journal of Biochemistry 2017; 42(4):459-67
14. Khalili S, Mohammadpour H, BAROUGH MS, Kokhaei P. ILP-2 modeling and virtual screening of an FDA-approved library: a possible anticancer therapy. Turk J Med Sci 2016; 46(4):1135-43.
15. Khalili S, Jahangiri A, Hashemi ZS, Khalesi B, Mardsoltani M, Amani J. Structural pierce into molecular mechanism underlying Clostridium perfringens Epsilon toxin function. Toxicon 2017; 127:90-9.
16. Khalili S, Rasaee M, Bamdad T. 3D structure of DKK1 indicates its involvement in both canonical and non-canonical Wnt pathways. Mol Biol (Mosk) 2017; 51(1):180-92.
17. Khalili S, Rahbar MR, Dezfulian MH, Jahangiri A. In silico analyses of Wilms׳ tumor protein to designing a novel multi-epitope DNA vaccine against cancer. J Theor Biol 2015; 379:66-78.
18. Jahangiri A, Rasooli I, Owlia P, Fooladi AAI, Salimian J. In silico design of an immunogen against Acinetobacter baumannii based on a novel model for native structure of outer membrane protein A. Microb Pathog 2017; 105:201-10.
19. Mohammadpour H, Pourfathollah AA, Zarif MN, Khalili S. Key role of Dkk3 protein in inhibition of cancer cell proliferation: an in silico identification. J Theor Biol 2016; 393:98-104.
20. Mohammadpour H, Khalili S, Hashemi ZS. Kremen is beyond a subsidiary co-receptor of Wnt signaling: an in silico validation. Turkish Journal of Biology 2015; 39(3):501-10.
21. Houen G. Peptide Antibodies: Past, Present, and Future. Peptide Antibodies: Methods Mol Biol 2015; 1348:1-6.
22. Lim S-L, Ichinose H, Shinoda T, Ueda H. Noncompetitive detection of low molecular weight peptides by open sandwich immunoassay. Anal Chem 2007; 79(16):6193-200.
23. Brown-Augsburger P, Masnyk M, Santa P, Gingerich RL, Bowsher RR. An immunoadsorption strategy to produce specific antisera against analogs of human proteins: development of sensitive and specific radioimmunoassays for two analogs of human leptin. J Pharm Biomed Anal 2000; 23(4):687-96.
24. Structural Genomics C, China Structural Genomics C, Northeast Structural Genomics C, Graslund S, Nordlund P, Weigelt J, et al. Protein production and purification. Nat Methods 2008; 5(2):135-46.
25. Jia B, Jeon CO. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives. Open Biol 2016; 6(8).
26. Liu JK. The history of monoclonal antibody development - Progress, remaining challenges and future innovations. Ann Med Surg (Lond) 2014; 3(4):113-6.
27. Arur S, Schedl T. Generation and purification of highly specific antibodies for detecting post-translationally modified proteins in vivo. Nat Protoc 2014; 9(2):375-95.
28. Fryauff DJ, Mouzin E, Church LW, Ratiwayanto S, Hadiputranto H, Sutamihardja MA, et al. Lymphocyte response to tetanus toxin T-cell epitopes: effects of tetanus vaccination and concurrent malaria prophylaxis. Vaccine 1999; 17(1):59-63.
29. Chambers RS, Johnston SA. High-level generation of polyclonal antibodies by genetic immunization. Nat Biotechnol 2003; 21(9):1088-92.
30. Fodey TL, Greer NM, Crooks SR. Antibody production: Low dose immunogen vs. low incorporation hapten using salmeterol as a model. Anal Chim Acta 2009; 637(1-2):328-32.
31. Clementi ME, Marini S, Condo SG, Giardina B. Antibodies against small molecules. Ann Ist Super Sanita 1991; 27(1):139-43.
32. Mardsoltani M, Rasaee MJ, Sheikhi A, Hedayati M. Eliciting an antibody response against a recombinant TSH containing fusion protein. J Immunoassay Immunochem 2017; 38(3):257-70.
33. Graciani FS, Ximenes VF. Investigation of human albumin-induced circular dichroism in dansylglycine. PLoS One 2013; 8(10):e76849.
34. Matsuo K, Hiramatsu H, Gekko K, Namatame H, Taniguchi M, Woody RW. Characterization of intermolecular structure of beta(2)-microglobulin core fragments in amyloid fibrils by vacuum-ultraviolet circular dichroism spectroscopy and circular dichroism theory. J Phys Chem B 2014; 118(11):2785-95.
35. Micsonai A, Wien F, Kernya L, Lee YH, Goto Y, Refregiers M, et al. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc Natl Acad Sci USA 2015; 112(24):E3095-103.
36. Nagatomo S, Nagai M, Ogura T, Kitagawa T. Near-UV circular dichroism and UV resonance Raman spectra of tryptophan residues as a structural marker of proteins. J Phys Chem B 2013; 117(32):9343-53.
37. Panina-Bordignon P, Tan A, Termijtelen A, Demotz S, Corradin G, Lanzavecchia A. Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur J Immunol 1989; 19(12):2237-42.
38. Dintzis HM, Dintzis RZ, Vogelstein B. Molecular determinants of immunogenicity: the immunon model of immune response. Proc Natl Acad Sci USA 1976; 73(10):3671-5.
39. Bachmann MF, Rohrer UH, Kündig TM, Bürki K, Hengartner H, Zinkernagel RM. The influence of antigen organization on B cell responsiveness. Science 1993; 262(5138):1448-51.
40. Heydari Zarnagh H, Hassanpour K, Rasaee M J. Constructing Chimeric Antigen for Precise Screening of HTLV-I Infection..Iran J Allergy Asthma Immunol 2015; 14(4):427-36.
41. Heydari Zarnagh H, Ravanshad M, Pourfatollah A A, Rasaee M J. Expression and Purification of a Novel Computationally Designed Antigen for Simultaneously Detection of HTLV-1 and HBV Antibodies .. Iran J Allergy Asthma Immunol 2015; 14(2):168-78.
42. Abbas AK, Lichtman AH, Pillai S. Cellular and molecular immunology: Elsevier Health Sciences; 2014.
43. Jirkalova V, Cap J, Strakova H, Pribysova J, Plicka J, Lomsky R. Immunoradiometric and luminescence immunoenzymometric assay of human thyrotropin from dried blood spots for screening of neonatal hypothyroidism. Eur J Clin Chem Clin Biochem 1996; 34(10):823-7.
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| Issue | Vol 17, No 2 (2018) | |
| Section | Original Article(s) | |
| Keywords | ||
| Cross reactivity Fusion protein immunization Immunoassay | ||
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