Purification of Thyrotropin and Other Glycoprotein Hormones by Immunoaffinity Chromatography*

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Abstract
Immunoaffinity methods for separation of TSH and other glycoprotein hormones as well as their common asubunit are described. Partially purified antibodies to TSH and the a-subunit of CG were coupled to agarose and packed in columns (15 × 0.9 cm). The anti–TSH column had a capacity of 31 μg TSH/ml agarose, and the anti-a had a column capacity of 3.6 μg TSH/ml agarose. Material that had been applied and adsorbed to the columns was eluted with 0.1–4.0 m guanidine- HC1, pH 3.2, at 4 C. This procedure yielded high recovery of TSH immunoactivity and adenylate cyclase-stimulating activity. Less than 5% dissociation of the hormone into its subunits occurred during such immunoaffinity purification. For separation of the glycoprotein hormones and the free asubunit, advantage was taken of their different affinities for the antibodies. When adsorbed radiolabeled material was exposed to a linear guanidine gradient, substances with lower affinities for the antibodies eluted earlier than substances with higher affinities. Thus, from the anti–TSH column, peak elution of CG, FSH, and LH occurred at 0.5 m guanidine, well separated from the TSH peak at 1.2 m. Similarly, CG and TSH eluted from the antia column at 0.4 m guanidine, clearly earlier than the a-peak at 0.9 m. The affinity chromatography columns were also used for copurification of TSH with other glycoprotein hormones and the a-subunit from hypothyroid serum. The fractions of serum that bound to the columns were eluted with a constant concentration of guanidine (the anti-TSH column with 3 M and the anti-a column with 2 M) in a volume approximately 1.5 times the column bed. Such a one-step elution allowed a 62–91% recovery of serum TSH with a 100- to 200-fold purification (TSH activity/total protein). Simultaneously, 41–58% of FSH, 39–51% of LH, and 64-89% of free a-subunit were recovered. These immunoaffinity methods provide a convenient tool to improve the sensitivity and specificity of TSH and other glycoprotein hormone assays by prepurification and concentration of serum samples. The methods are also of potential value for large scale isolation and purification of glycoprotein hormones. (Endocrinology106: 1327, 1980)