FI60875C - PROOF OF SOLUTION FOR VIDEO RENING AV PLACENTASPECIFIKT PP5 GLYCOPROTEIN - Google Patents

Description

RS ^ l ΓβΊ ANNOUNCEMENT / nflnr lBJ (11> UTLÄGGNINGSSKRIFT b (J0 / 5 C Patent granted on 13 04 1902

Patent meddelat - '(51) Kv.ik? /Int.ci.3 C 07 G 7/00 // G 01 N 33A8 FINLAND - FINLAND (21) p »« "« lh »k.mu.-P« * nttn »eknlf> f 771166 (22) H« ktml * pllvl - Ansttknlngsdag lU.0U.77 '' (23) Alkupllvi — Glltlghetsdag lU.0U.77 (41) Become Public - Bllvlt offtntllg 18.10.77

Patent and Registration Office .... .... .... ....

_. . . . . , (44) Date of publication and date of publication. -

Patent and registration authorities in the field of trade and services 31.12.8l (32) (33) (31) Pyydetty etuoikeus — Begird prloritet 17.0U.76

Federal Republic of Germany-Förbundsrepubliken

Germany (DE) P 261698U.6 (71) Behringwerke Aktiengesellschaft, D-3550 Marburg / Lahn, Federal Republic of Germany-Förbundsrepubliken Tyskland (DE) (72) Hans Bohn, Marburg / Lahn, Federal Republic of Germany-Förbundsrepubliken Tyskland (DE) 7U) Oy Kolster Ab (5U) Method for isolation and further purification of placental-specific PP ^ glycoprotein - Förfarande for isolering och vidare rening av placentaaspecifikt PP ^ glycoprotein

The invention relates to a method for isolating and further purifying a placenta-specific PP1 glycoprotein.

H. Bohn discovered PP5 glycoprotein in aqueous placental extract in 1972, and as he has reported in Archiv fiir Gynäkologie, 212 (1972) 165-175, a number of soluble antigens can be detected immunologically in placental extract by means of antisera. PP5 glycoprotein was clearly found to be placental-specific, as detection of this protein by standard immunological methods in several fetal and adult human organs or in human plasma and erythrocyte lysates did not result in a positive reaction.

In an electrophoretic mobility experiment on agar, the PP 1 glycoprotein proves to be β 3 globulin. On a polyacrylamide gel, it moves at a relative rate of 75 at an albumin rate of 100. Its behavior in gel filtration with crosslinked dextran was inferred to a molecular weight of ΡΡς: η of about 50,000. has not been possible as these methods are not selective enough.

It has now surprisingly been found that PP5-glycoprotein can be isolated from solutions containing it by immunoadsorption and further purified by conventional fractionation methods. In the process according to the invention, the plates are extracted with brine, diaminoethoxyacridine lactate solution is added to the extract, the precipitate formed is separated, the remaining diaminoethoxyacridine lactate is removed by precipitation with sodium chloride, then the pH is dissolved in discipline, after which the dialyzed PP1-containing solution is centrifuged.

The method of the invention is characterized in that a PP4-containing protein solution having a pH of 4-9 is contacted with an antibody to PP1 bound to an insoluble carrier, the carrier containing bound PP1 is removed from the liquid phase and treated with PP4. to be separated by an aqueous solution having a pH of 2 to 4, after which the product is purified to a purity of more than 99% by methods known per se.

Thus, human placenta is preferably used as the starting material, from which about 10 to 20 mg of PP 2 glycoprotein can be extracted per medium-sized (about 500 to 600 g) placenta. To prepare the aqueous extract, the comminuted chunks are treated with water or a suitable saline solution and the liquid phase is recovered.

Suitable salts for use in saline are those which are as inert as possible, which are known from the saline solutions used in the extraction of tissues. Neutral salts and buffers can be used, in particular common salt or tris-hydroxymethylaminomethane, further alkali metal salts of phosphoric or citric acid. A suitable salt content is 0.1 to 5%.

By the method of the invention, PP 2 glycoprotein can be isolated from conventional placental extracts at a concentration of about 1 to 2 mg / 100 ml to a concentration of about 100 to 1000 mg / 100 ml, i.e. a specific degree of enrichment of about 2,500 to 5 ml. 000-fold.

The antibody required in the method of the invention, which is bound to a solid support in a known manner, is obtained by immunizing 3,60875 animals with PP1. If pure protein is not available, for the immunization of vertebrates, especially rabbits, a PP1-enriched placenta fraction is prepared by a precipitation method, for example according to the method described by H. Bohn in the above-mentioned publication.

Immunization with such crude fractions yields antisera containing multispecific antibodies. Antibodies that are not specific for PPI are treated with appropriate antigens from blood serum and placental fractions, whereby non-specific antibodies can be removed by an antigen-antibody reaction.

The immunoglobulin fraction containing the PP 1 antibody is recovered in a known manner. Accordingly, the anti-PP 2 obtained thereby is covalently bound to a suitable support by known methods. Such methods are described, for example, in Ann. Rev. Biochem, 40 (1971) 259, Naturwissenschaften 58 (1971) 383 or Nature 314 (1967) 1302. Suitable carriers are high molecular weight carbohydrates such as cellulose and agar, and synthetic resins such as polyacrylamides and ethylene / maleic anhydride can also be co-polymeric anhydride. used as a carrier. A particularly preferred carrier is purified agarose in small spheres with a diameter of 20 to 200.

To isolate PP 1 glycoprotein from solutions, especially hair extracts, which additionally contain other proteins and carbohydrates, the PP 1 -containing solution is contacted with an antibody-bearing adsorbent and the pH is adjusted to> 4. To avoid denaturation of the proteins, it is recommended to select pH less than 9. The salinity of the solution is not critical. However, conditions in which the bond between the antigen and the antibody is easily broken should be avoided. The aqueous solution preferably contains neutral salts and / or buffers commonly used in biochemical reactions, especially sodium chloride, phosphate buffer, tris-hydroxymethylaminomethane and other known buffers (see Biochem. 5 (1966) 472 and Handbook of Biochemistry, published in The Chemical Rubber). , Cleveland, Ohio, USA, second edition, part J, page 238). Concentrations of these substances are suitably between 0.01 and 2 mol / l. The ratio of protein solution to adsorbent is suitably in the range 1: 1 to 10: 1, preferably 2: 1. The adsorbent should remain in contact with the protein solution 4 60875 for 0.1 to 5 hours to form an antigen-antibody bond.

The adsorption and elution of PP 2 can be carried out either by the so-called batch method or in a chromatography column. In the batch process, the adsorbent is removed from the solution by centrifugation or filtration, washing several times with neutral brine to remove excess salts and adsorbed impurities. In the column method, the solution is removed from the column by rinsing thoroughly with a buffer solution.

Elution of PP 1 from the antibody-containing carrier can be performed in a known manner, whereby the antigen-antibody bond is broken. The elution can be carried out, for example, by treating the support with an aqueous solution having a pH <4, preferably between 2-4. The solution should contain salts and suitable buffering agents in suitable concentrations, preferably 0.2 to 8 mol / l.

In the batch process, it is recommended to keep the dispersion of the adsorbent in this solution for 0.1 to 2 hours, whereby PP ,. is desorbed by immunoad sorption. In the column method, the elution solution is allowed to flow through the column. After removal of the adsorbent, the pH of the PP-containing solution is adjusted to approximately neutral (pH = 6 to 8). The solution can be further purified if desired.

The purity of the PP2 obtained according to this method is 50 to 80%. With further cleaning methods, it can be increased to more than 99%.

Conventional fractionation methods can be used to further purify PP1-enriched PP1. In this case, it is advantageous to carry out the fractionation by means of a molecular sieve, in which case those molecules having a molecular weight of about 50,000 are separated. Another preferred method is ion exchange chromatography. In this case, the charge properties of PP 1 are exploited, with PP 2 being known to behave in the same way as β-globulin. The PP5 glycoprotein can also be prepared in pure form by an immunoadsorption method using not PP1 antibodies but antibodies to the impurities to be removed as carrier-bound adsorbents. PP ^ remains in the unbound portion when using this method.

The simplest way to determine the effectiveness of the purification method is with the corresponding antisera. From each fractionation method, those fractions are recovered in which an immunological reaction, in particular immunoprecipitation, is observed with the antiserum which reacts specifically against PP1.

The PP prepared by the process of the invention is particularly suitable for the preparation of antisera specific for it. In this case, the animals are immunized with PP1 by known methods and the antiserum is recovered from the animals' blood. These new antisera can be used to detect PPC in body fluids by immunological methods such as hemagglutination inhibition assay, complement fixation reaction, radioimmunoassay, latex agglutination, and the like.

Thus, it can be shown that PP1 is present during pregnancy in low blood levels. Furthermore, PP 1 plays a role in the diagnosis of tumors. It can be demonstrated in the blood and tumor tissue of patients with trophoblastic tumors. Corresponding diagnostic agents generally contain predominantly an anti-PP 1 agent, and in certain methods, for example radioimmunoassay methods, PP 1 is used as such as a reference agent. In diagnostic agents for the detection of antibodies to PP1, PP1 is an essential component of the reagent.

The following example illustrates the invention.

1. Preparation of immunoadsorbent

Rabbits are immunized for 6 weeks with purified PP 2 using aluminum hydroxide as an adjuvant. PP 2 is dissolved in physiological saline (3 ml of solution contains 0.06 mg NaCl), aluminum hydroxide is added and stirred to form a suspension. Rabbits received 0.06 mg of protein in 3 ml of suspension per animal for injection for five consecutive days. This is followed by a 9-day break. The animals are then re-immunized for five consecutive days with the same amount of antigen, taken again for a 9-day break, and finally injected for another five consecutive days with 0.06 mg of antigen each time. After 7-9 days, the animals are bled. Serum is separated from blood by centrifugation.

150 ml of the rabbit anti-PP 2 serum prepared above are dialyzed against 0.02 M phosphate buffer (pH 7.0) and chromatographed to separate the immunoglobulin with DEAE cellulose as absorbent. The immunoglobulin fraction (1.3 g of protein) is reacted with 258 g of specially purified spherical (R) agrosis (Sepharose 4B, Pharmacia Uppsala, Sweden) activated with 16.1 g of bromocyanine, whereby the immunoglobulin covalently binds to the support.

The method has been reported by Axen R., Porath J., Ernbach S., Nature 6 60875 214 (1 967) 1 302.

By means of an immunoadsobent prepared in this way, the placental protein PP1 can be isolated from solutions containing it, in particular from a PP5-enriched placental extract extract.

2. Enrichment of PPc glycoprotein from placental extract 2. Enrichment of PP 2 glycoprotein fraction 150 kg of deep-frozen placenta are comminuted and extracted with 150 liters of 0.5% aqueous sodium chloride solution. The pH of the extract is adjusted to 8 with 2N sodium hydroxide, and 50 liters of a 3% aqueous solution of (R) diaminoethoxyacridine lactate (Rivanol) are added. After 1 hour, the upper layer containing PP1 and gamma globulin is removed, 5% solid sodium chloride (11 kg) is added to separate the remaining rivanol in the solution, filtered and 26.5% solid ammonium sulfate, based on the weight of the liquid, is added and mixed well. After one hour, the precipitate is filtered off.

Dissolve 500 g of the precipitate collected on the filter in 500 ml of distilled water and dialyze against 0,01 M tris- (oxymethyl) -aminomethane (abbreviated as "tris") hydrochloric acid buffer solution, pH 7,0, containing 0, 05% sodium azidia. The dialyzed solution is centrifuged, and the upper layer is diluted to 2000 ml with the same buffer solution, and its pH is adjusted to 8.0 with 0.1 M sodium hydroxide solution, 250 g of moist DEAE cellulose is added, and stirred for one hour.

The DEAE-cellulose is then separated from the solution by filtration, washed twice with 1 liter of 0.01 M tris-hydrochloric acid buffer, pH 8.0, and then eluted three times with 500 ml of 0.02-M tris-hydrochloric acid buffer. hydrochloric acid buffer, pH 6.5, containing 0.85% sodium chloride and 0.05% sodium azide.

To the combined eluates is added 30% ammonium sulfate, based on the weight of the liquid, and mixed. Precipitate containing PP ^. is dissolved in 100 ml of distilled water and dialyzed against 0.1 M tris-hydrochloric acid buffer, pH 8.0, containing 1.0 mol / l sodium chloride and 0.1% sodium azide. 200 ml of a solution containing 6% protein and about 30 mg of PP2 are obtained. PP1 can be isolated from this fraction by immunoadsorption.

The enrichment method described in section 2.1 is not essential to the invention. Immunoadsorption can also be performed directly on the placental extract.

1 60875 2. 2 Immunoadsorption of PPr.-glycoprotein

The inuno-adsorbent is suspended in 0.1 M Tris-HCl buffer (pH 8.0) containing 1.0 M NaCl and 0.1% NaN 2 (hereinafter abbreviated as Buffer I), poured onto a chromatography column (3 x 20 cm) and rinsed with buffer solution I. Then 100 ml of the PP 2 -containing solution is allowed to drain slowly through the column, whereby PP 2 binds immunoadsorbently. The column is washed thoroughly with buffer solution I, and the adsorbed protein is eluted with 0.5 M glycine-HCl buffer (pH 2.5). The pH of the PP 2 -containing eluate is adjusted to 7.0 with 1 N NaOH and the solution is concentrated on an ultrafilter to about 10 ml. The yield per adsorption is about 2 mg PP 2.

The adsorbent in the column is neutralized immediately after elution of PP1 with buffer solution I and washed thoroughly, so that the absorbent can be reused for immunoadsorbent binding of PP1.

The protein obtained by immunoadsorption often contains non-specifically bound serum proteins (mainly IgG and also a small amount of IgA) as impurities. These co-proteins can be removed, for example, by gel filtration or molecular sieve fractionation, for example crosslinked dextran such as Sephadex G-100; however, serum proteins can also be removed by immunoadsorption, i.e. by means of antibodies to carrier-bound serum protetins.

2. 3 Gel filtration of PP 2 glycoprotein To further purify the PP (.glycoprotein by gel filtration), 10 to 20 mg of the product obtained by immunoadsorption is dissolved in 5 to 10 ml of buffer solution I and passed through a Sephadex G-100 column (2.5 x 100 mm Elute with the same buffer solution The PP4 content of the eluents is determined by the Ouchterlony gel diffusion test using PP, antiserum, the PP4 fractions are combined, evaporated to dryness on an ultrafilter, dialyzed against water and lyophilized. such as immunoglobulins (IgG and IgA), elute before PP, and thus separate from PP.