NO145405B - PROCEDURE FOR PURIFICATION OF THE PREGNANCY-SPECIFIC BETA-1 GYCLOPROTEIN - Google Patents

Description

In German application no. 21 57 610, an i

glycoprotein occurring in the placenta, which is called "pregnancy-specific 3-^-glycoprotein" (SP-^). Furthermore, the aforementioned patent application describes a method for isolating this glycoprotein. A further such procedure is described in Norwegian application no. 761 306.

The object of the invention is an adsorption process which makes it possible to further purify and practically purify the pregnancy-specific B^-glycoprotein, which is produced in a purity of 60-95% by the aforementioned methods.

The procedure is characterized by bringing

an aqueous phosphate-containing solution, which contains the pregnancy-specific g^-glycoprotein in contact with hydroxy-

lapatite, separates hydroxylapatite from the solution and from this recovers SP-^.

It is mentioned in Norwegian interpretation document no. 123 235

a method for the enrichment of aspergillopeptidase by chromatography using hydroxylapatite. In "Chemical Abstracts" (1968), volume 69, 94332j, it is stated that hydroxylapatite was indeed used for the fractionation of human pregnancy serum, but not with opti-

painted result.

Despite this known state of the art, it was surprising that hydroxylapatite could be used for the purification of SP^ with the good results obtained.

The pregnancy-specific g^-glycoprotein SP^ is adsorbed together with the impurities on the hydroxylapatite, but it is eluted from the hydroxylapatite with 0.005 M phosphate buffer,

while the impurities are not eluted by phosphate buffers and remain adsorbed on the hydroxylapatite.

The invention therefore relates to a method for purifying the pregnancy-specific non-g^-glycoprotein, SP^,

for the manufacture of a product containing over 99% SP-^,

in that the method is characterized by the fact that a phosphate-containing aqueous solution containing the pregnancy-specific 6-^-glycoprotein and whose purity is 60-95% with a pH of 5-8.5,

and a phosphate content of 0.001-0.01 M per litres, brought into be-

stirring with the hydroxylapatite, separates the solution from the hydroxylapatite, which is eluted with an approx. 0.005 M phosphate buffer and recover SP^ from the elution solution.

The adsorption of the pollutants can be carried out in a batch method as well as a column chromatography.

If the above-mentioned conditions are observed, the pregnancy-specific non-6-^-glycoprotein is obtained in a purity of over 99%.

The purity of the preparation is advantageously determined by immuno- ■

nological methods using antisera, which both contain antibodies against the pregnancy-specific 3-^-glycoprotein, as well as against one or more in placenta extract, in blood or urine from pregnant women occurring anti-

j genes.

For high-purification of pregnancy-specific 3^-glycoprotein, dissolve 50 mg of the 60-95% pure preparation in 10 ml

j 0.005 molar sodium phosphate buffer (pH 6.0) and applied to a column filled with hydroxylapatite (2x8 cm), which had previously been equilibrated with a 0.005 molar phosphate buffer pH 6.8.

In the evaluation of the column with 0.005 molar phosphate buffer

if the pregnancy-specific 8^-glycoprotein does not travel through the - column, the pollutants, on the other hand, are adsorbed on hydroxylapatite.

The proteinaceous passage contains pregnancy-specific 3-^-glycoprotein in pure form; it is dialyzed to remove the salts against 10 times the volume of water and then lyophilized.

Yield approx. 40 mg pregnancy-specific B^-glycoprotein.

j The purity amounts to over 99%, i.e. according to immunological methods, none of the admixtures that were present before the purification operation are detectable.

Claims (1)

Process for purifying the pregnancy-specific g-^-glycoprotein, SP1, for the production of a product containing over 99% SP.^ characterized in that a phosphate-containing aqueous solution containing the pregnancy-specific non-B-^-glycoprotein and whose purity is 60- 95% with a pH of 5-8.5 and a phosphate content of 0.001-0.01 mol/l, is brought into contact with hydroxylapatite, separates the solution from the hydroxylapatite which is eluted with an approx. 0.005 molar phosphate buffer and recover SP.^ from elution the resolution.