ELISA Based hTSH Assessment Using Two Sensitive and Specific Anti-hTSH Polyclonal Antibodies

Authors: Maysam Mard-Soltani, Mohamad Javad Rasaee, Saeed Khalili, Abdol Karim Sheikhi, Mehdi Hedayati

Abstract:

Production of specific antibody responses against hTSH is a cumbersome process due to the high identity between the hTSH and the other members of the glycoprotein hormone family (FSH, LH and HCG) and the high identity between the human hTSH and host animals for antibody production. Therefore, two polyclonal antibodies were purified against two recombinant proteins. Four possible ELISA tests were designed based on these antibodies. These ELISA tests were checked against hTSH and other glycoprotein hormones, and their sensitivity and specificity were assessed. Bioinformatics tools were used to analyze the immunological properties. After the immunogen region selection from hTSH protein, c terminal of B hTSH was selected and applied. Two recombinant genes, with these cut pieces (first: two repeats of C terminal of B hTSH, second: tetanous toxin+B hTSH C terminal), were designed and sub-cloned into the pET32a expression vector. Standard methods were used for protein expression, purification, and verification. Thereafter, immunizations of the white New Zealand rabbits were performed and the serums of them were used for antibody titration, purification and characterization. Then, four ELISA tests based on two antibodies were employed to assess the hTSH and other glycoprotein hormones. The results of these assessments were compared with standard amounts. The obtained results indicated that the desired antigens were successfully designed, sub-cloned, expressed, confirmed and used for in vivo immunization. The raised antibodies were capable of specific and sensitive hTSH detection, while the cross reactivity with the other members of the glycoprotein hormone family was minimum. Among the four designed tests, the test in which the antibody against first protein was used as capture antibody, and the antibody against second protein was used as detector antibody did not show any hook effect up to 50 miu/l. Both proteins have the ability to induce highly sensitive and specific antibody responses against the hTSH. One of the antibody combinations of these antibodies has the highest sensitivity and specificity in hTSH detection.

Keywords: hTSH, bioinformatics, protein expression, cross reactivity.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1315871

References:

[1] S. Rabbiosi, M. C. Vigone, F. Cortinovis, I. Zamproni, L. Fugazzola, L. Persani, C. Corbetta, G. Chiumello, G. Weber, "Congenital hypothyroidism with eutopic thyroid gland: analysis of clinical and biochemical features at diagnosis and after re-evaluation," The Journal of Clinical Endocrinology & Metabolism, vol. 98, pp. 1395-402, 2013.
[2] R. A. McPherson, M. R. Pincus. Henry's Clinical Diagnosis and Management by Laboratory Methods E-Book: Elsevier Health Sciences; 2017.
[3] L. De Groot, G. Chrousos, K. Dungan, K. Feingold, A. Grossman, J. Hershman, C. Koch, M. Korbonits, R. McLachlan, M. New, "Assay of Thyroid Hormones and Related Substances--Endotext," vol., pp., 2000.
[4] C. Ronin, M. Papandreou, S. Sergi, C. Labbe-Jullie, G. Medri, T. Hoffmann, H. Darbon, "Glycosylation-dependent epitope mapping of human TSH (hTSH) isoforms," International Journal of Radiation Applications and Instrumentation Part B Nuclear Medicine and Biology, vol. 17, pp. 651-6, 1990.
[5] J.-M. Bidart, F. Troalen, P. Ghillani, A. Puisieux, C. Bohuon, D. Bellet, "Immunochemical mapping of antigenic regions on the human thyrotropin beta-subunit by antipeptide antibodies," Journal of Biological Chemistry, vol. 266, pp. 19238-44, 1991.
[6] C. Cahoreau, D. Klett, Y. Combarnous, "Structure–function relationships of glycoprotein hormones and their subunits’ ancestors," Frontiers in endocrinology, vol. 6, pp., 2015.
[7] C. A. Spencer, M. Takeuchi, M. Kazarosyan, "Current status and performance goals for serum thyrotropin (TSH) assays," Clinical chemistry, vol. 42, pp. 140-5, 1996.
[8] A. K. Abbas, A. H. Lichtman, S. Pillai. Cellular and Molecular Immunology E-Book: Elsevier Health Sciences; 2014.
[9] M. Tiwary, N. Agarwal, A. Dinda, S. C. Yadav, "Overexpression and purification of folded domain of prostate cancer related proteins MSMB and PSA," Molecular biology reports, vol. 43, pp. 349-58, 2016.
[10] H. Schellekens, N. Casadevall, "Immunogenicity of recombinant human proteins: causes and consequences," Journal of neurology, vol. 251, pp. ii4-ii9, 2004.
[11] A. Jahangiri, I. Rasooli, P. Owlia, A. A. I. Fooladi, J. Salimian, "In silico design of an immunogen against Acinetobacter baumannii based on a novel model for native structure of outer membrane protein A," Microbial Pathogenesis, vol., pp., 2017.
[12] E. Kazemi Moghaddam, P. Owlia, A. Jahangiri, I. Rasooli, M. R. Rahbar, M. Aghajani, "Conserved OprF as a Selective Immunogen Against Pseudomonas aeruginosa," Iranian Journal of Pathology, vol. 12, pp. 165-70, 2017.
[13] S. Khalili, A. Jahangiri, Z. S. Hashemi, B. Khalesi, M. Mardsoltani, J. Amani, "Structural pierce into molecular mechanism underlying Clostridium perfringens Epsilon toxin function," Toxicon, vol., pp., 2017.
[14] S. Khalili, M. Rasaee, T. Bamdad, "3D structure of DKK1 indicates its involvement in both canonical and non-canonical Wnt pathways," Molecular Biology, vol. 51, pp. 155-66, 2017.
[15] S. Khalili, A. Zakeri, Z. S. Hashemi, M. Masoumikarimi, M. R. R. Manesh, N. Shariatifar, M. J. Sani, "Structural analyses of the interactions between the thyme active ingredients and human serum albumin," Turkish Journal of Biochemistry, vol. 42, pp. 459-67, 2017.
[16] S. Khalili, H. Mohammadpour, M. S. BAROUGH, P. Kokhaei, "ILP-2 modeling and virtual screening of an FDA-approved library: a possible anticancer therapy," Turkish journal of medical sciences, vol. 46, pp. 1135-43, 2016.
[17] R. S. Chambers, S. A. Johnston, "High-level generation of polyclonal antibodies by genetic immunization," Nature biotechnology, vol. 21, pp. 1088-92, 2003.
[18] C. A. King, M. B. Spellerberg, D. Zhu, J. Rice, S. S. Sahota, A. R. Thompsett, T. J. Hamblin, J. Radl, F. K. Stevenson, "DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma," Nat Med, vol. 4, pp. 1281-6, 1998.
[19] M. Mard-Soltani, M. J. Rasaee, A. Sheikhi, M. Hedayati, "Eliciting an antibody response against a recombinant TSH containing fusion protein," Journal of Immunoassay and Immunochemistry, vol. 38, pp. 257-70, 2017.
[20] C. A. Spencer, M. Takeuchi, M. Kazarosyan, "Current status and performance goals for serum thyrotropin (TSH) assays," Clinical chemistry, vol. 42, pp. 140-5, 1996.
[21] N. Azzam, R. Bar-Shalom, Z. Kraiem, F. Fares, "Human thyrotropin (TSH) variants designed by site-directed mutagenesis block TSH activity in vitro and in vivo," Endocrinology, vol. 146, pp. 2845-50, 2005.
[22] R. S. Chambers, S. A. Johnston, "High-level generation of polyclonal antibodies by genetic immunization," Nat Biotechnol, vol. 21, pp. 1088-92, 2003.
[23] J. C. Drees, J. A. Stone, C. R. Reamer, V. E. Arboleda, K. Huang, J. Hrynkow, D. N. Greene, M. S. Petrie, C. Hoke, T. S. Lorey, "Falsely undetectable TSH in a cohort of South Asian euthyroid patients," The Journal of Clinical Endocrinology & Metabolism, vol. 99, pp. 1171-9, 2014.
[24] S. Maxeiner, A. Glassmann, H.-T. Kao, K. Schilling, "The molecular basis of the specificity and cross-reactivity of the NeuN epitope of the neuron-specific splicing regulator, Rbfox3," Histochemistry and cell biology, vol. 141, pp. 43-55, 2014.
[25] N. Sinha, S. Mohan, C. A. Lipschultz, S. J. Smith-Gill, "Differences in electrostatic properties at antibody–antigen binding sites: implications for specificity and cross-reactivity," Biophysical journal, vol. 83, pp. 2946-68, 2002.