Tehran University of Medical Sciences Iranian Journal of Allergy, Asthma and Immunology 1735-1502 17 2 2018 04 28 158 170 EN Maysam Mard-Soltani Department of Clinical Laboratory Sciences, Dezful University of Medical Sciences, Dezful, Iran Mohamad Javad Rasaee Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Saeed Khalili Department of Biology Sciences, Shahid Rajaee Teacher Training University, Tehran, Iran Abdol-Karim Sheikhi Cellular and Molecular Immunology Research Laboratory, Immunology Department, Dezful University of Medical Sciences, Dezful, Iran Mehdi Hedayati Cellular and Molecular Endocrine Research Center, Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran Hossein Ghaderi-Zefrehi Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Milad Alasvand Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran 2017 04 16 2017 08 01 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.  https://ijaai.tums.ac.ir/index.php/ijaai/article/view/1416 https://ijaai.tums.ac.ir/index.php/ijaai/article/download/1416/827