Affiliations: [a] Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran | [b] Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran | [c] Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
Correspondence:
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Corresponding author: Leili Aghebati-Maleki and Jafar Majidi, Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Tel.: +98 413 3364665; Fax: +98 41 3364665; E-mail: [email protected],[email protected].
Abstract: In present study an optimized protocol for the separation of antibodies into antigen-binding fragments F(ab’)2 using pepsin digestion was investigated. The production of these fragments is a consequential step in the development of medical research, treatment and diagnosis. For production of polyclonal antibody rabbit received antigen in four steps. The rabbit serum at 1/128000 dilution showed high absorbance in reaction with human IgG at the designed ELISA method. Rabbit IgG was purified by Ion-Exchange Chromatography (IEC) method. Purity was assessed by SDS-PAGE method. In non-reduced condition only one band was seen in about 150 kDa MW position and in reduced form, two bands were seen in 50 and 25 kDa MW positions. Rabbit IgG was digested by pepsin enzyme. The antibody fragments solution was applied to Gel filtration column to isolate the F(ab’)2. Non-reduced SDS-PAGE for determining the purity of F(ab’)2 fragment resulted in one band in 100 kDa corresponds to F(ab’)2 fragment and a band in 150 kDa MW position corresponds to undigested IgG antibodies. The activities of FITC conjugated F(ab’)2 fragment and commercial ones were compared using flowcytometry method. The activity results implied that the FITC conjugated- anti human F(ab’)2 fragment worked as efficiently as the commercial one.
