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2008, Number 4

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Bioquimia 2008; 33 (4)

Isolation and purification of human thyrotropin from pituitary glands for immunodiagnostic use

Pérez-López L, Hernández-Pérez L, Melchor-Rodríguez A, Zulueta-Rodríguez O, López-Cisnero R

Language: Spanish
References: 34
Page: 137-146
PDF size: 179.52 Kb.


ABSTRACT

A simple method is describe for the efficient purification of human thyrotropin from pituitary glands by immunoaffinity chromatography, using a policlonal antibody anti bTSH. The glands were dried with acetone, and a powder of pituitary glands was obtained using a mill. The extraction operation was performed with a mixture of ammonium acetate:ethanol. The obtained extract was precipitated with 87% (v/v) ethanol. The pellet was recovered by centrifugation, suspended in diethyl ether and finally dried by evaporation. The resultant powder was resuspended in saline-phosphate buffer pH 7.4, clarified and applied to a column packed with goat IgG anti ΒTSH-Sepharose 4B. The TSH fraction was eluted with 0.2 mol/L glycine pH 2.8, lyophilized and stored at -20 °C. The purity was analyzed by SDS-PAGE using commercial TSH as control. Purified TSH showed a molecular mass of 54.1 kDa and a purity of 93.87%. Subunit characterization was carried out by SDS-PAGE under reducing conditions. Dissociated TSH migrated as two bands, corresponding to a and Β chains, with a molecular mass of 16.8 kDa and 20.2 kDa, respectively. The recovery of TSH by this procedure was 43.7%, and a purification factor of 41.7% was reached. The specific activity obtained was 21.1 UI/mg. These results indicate that immunoaffinity purification of TSH by means of this polyclonal antibody is a simple one-step procedure, which is reproducible and allows obtaining a hormone with physical-chemical characteristics, and a biological activity very similar to the commercial one. This hormone is appropriate for use as immunogens or immunoassay calibrators.


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