NO146747B - PROCEDURE FOR INSULATION AND FURTHER CLEANING OF PLACENT-SPECIFIC PROTEIN PP5 - Google Patents

Description

The invention relates to a method for isolating and purifying a placental protein that occurs in the human

equal placenta, and which was found by H. Bohn 1972 in aqueous placental extract and designated as PP^ as well as antigen-related proteins.

As discussed by H. Bohn in Archiv fur Gynåkologie, volume 212,

pages 165-175, 1972, with the help of antisera which had been obtained by immunization of placental fractions, a number of soluble anti-

genes. For the protein denoted by PP^, it could be shown that it is clearly placenta-specific, because attempts to detect this protein using standard immunological techniques in a series of embryonic and adult human organs or also in human plasma and erythrocyte lysates did not lead to a positive reaction.

In the electrophoretic migration in agar, PP^ appears as 3-^-globulin. In a polyacrylamide gel it migrates compared to albumin = 100 with a relative mobility of 75. On

due to its condition by gel filtration on cross-linked dextran, a molecular weight for PP^ of approx.

50,000. Even in placenta extract, PPg is present at a relatively low concentration, so that its pure preparation has not been possible until now, despite laborious methods from bio-

chemical preparative technique, especially because this process

way is not sufficiently selective. In placental fraction V, PP5 is enriched, and this fraction can benefit

serve as starting material for the method according to

the invention.

It was surprisingly found that PP^ can be isolated

from solutions in which it is contained, preferably from placental fraction V (Archiv der Gynakologie (1972), 212, 165),

by means of immunoadsorption and can be obtained clean by subsequent purification according to usual fractionation methods or also

in a further immunoadsorption method, in which the residual

affecting serum proteins and placental proteins can be removed.

The object of the invention is therefore a method for isolating and further purifying protein PPj- from its solutions, especially from the placental fraction V.

The placenta fraction V is prepared as follows:

Placentas are extracted with an aqueous saline solution. The extract is mixed with a solution of diaminoethoxyacridine lactate and the precipitate is separated. From the above, the diaminoethoxyacridine lactate still contained therein is precipitated with sodium chloride and separated. Ammonium sulphate is added to the above, the precipitate containing PP^ is separated, dissolved in water and dialysed against a buffer of approx. pH 7, for example 0.01 molar trisoxymethylaminomethane and centrifuged. This solution is placenta-Eraksjon V and is further enriched if necessary. The method is characterized in that this solution is brought into contact at a pH between 4 and 5 with an antibody against PP5 bound on an insoluble carrier, the carrier containing bound PP5 is separated from the liquid phase and treated with an aqueous medium of pH value 2-4 or with a solution of a substance that dissociates protein bonds at a pH value of 4-9 and purifies by isolation methods known per se after immunoadsorption to a purity of over 99%.

It is within the scope of the invention that according to this can

not only PP^, but also with PP^ antigen-related proteins, are isolated resp. enriched. Conversely, it is applicable antileg-

mers that are serologically related to PP^ for adsorptive production, both of PP^ and also of antigen-related proteins.

Human placenta is preferably used as starting material,

from which 1-2 mg PP^ can be extracted per placenta of average size (500-600 g). To prepare the aqueous extract, crushed placenta is treated with water or a suitable salt solution and the liquid phase is recovered.

Suitable salts for saline solution are mostly inert salts, as they are known as constituents of solutions for tissue extraction. Neutral salts and buffer substances are used for this purpose, especially sodium bicarbonate or tris-hydroxymethyl-aminomethane, further alkali salts of phosphoric or citric acid. Appropriate salt concentrations are at 0.1-5%.

In the method according to the invention, PP^ from commonly used placenta extracts, in which it is present in a concentration of 0.1-0.4 mg per 100 ml, enriched to a concentration of 100-1000 mg/100 ml. The specific enrichment is at 25,000-50,000.

The antibody required for the enrichment of PP^, which can be bound in a known manner to a solid carrier, is obtained by immunizing animals with PP^- As long as the pure protein is not available, it is prepared for the immunization of vertebrates, especially rabbits, with a by means of the precipitation method with respect to PPp. enriched placental fraction, for example according to the method discussed by H. Bohn 1972.

By immunization of such crude fractions, antisera with multispecific antibodies are obtained. The non-specific antibodies directed against PP^ are treated with suitable antigens from blood serum and placental fractions, after which the non-specific antibodies can be removed by means of the resulting antigen-antibody reaction.

The preparation of the immunoglobulin fraction, in which the PP^ antibody is found, takes place in a known manner. The anti-PP^ thus formed is bound to suitable carriers according to known methods. Such methods are, for example, described in Ann. Fox. Biochem. 40, 1971, page 259, Natur-wissenschaften 58 (1971), page 389 or Nature 314 (1967), page 1302. Particularly suitable carriers are highly polymeric carbohydrates such as cellulose or agar, synthetic resins,

such as polyacrylamide or copolymers of ethylene/maleic anhydride, glass particles can also be used as a carrier.

Particularly preferred as a carrier material is a purified agarose in the form of small spheres with a diameter of 20-200 µm, to which, according to the above-mentioned method, antibodies against PP^ can be covalently bound.

For the isolation of PP^ from solutions, especially from placental extracts, which also contain other proteins and carbohydrates, the PP^ solution is brought into contact with the antibody-containing adsorbent, the pH value being set above 4. To avoid denaturation of the proteins it pays to choose a pH value no higher than 9. The salt concentration of the solutions is not critical. Meanwhile, conditions should be avoided which are suitable to dissolve the binding of the antigen to the antibody. Preferably, the aqueous solution contains neutral salts and/or buffer substances as they are generally used in biochemical reactions, in particular sodium chloride, phosphate buffer, tris-hydroxymethylaminomethane and other known buffer substances, as for example described in the Handbook of Biochemistry, published by The Chemical Rubber Co., Cleve-land, Ohio, USA, second edition, page J 238. The concentration of these substances is conveniently in the range between 0.01 and 2 mol/liter. The ratio protein solution: adsorbent is suitable in the range 1:1-10:1, preferably 2:1. The adsorbent first remains in contact with the protein solution for 0.1-5 hours, so that sufficient time is available to form the antigen-antibody bond.

Adsorption and elution of PP^ can be carried out both in so-called batch methods and also in a chromatography column. To this end, the adsorbent is separated from the solution, washed in the batch method by centrifugation or filtration several times with a neutral salt solution to eliminate excess salts and non-specific adsorbed contaminants. In the column method, the solution is removed from the column by thorough flushing with buffer solution.

The elution of PP,- from the antibody-containing carrier can take place in a known manner by precautions which cause the antigen-antibody bonds to dissolve. The elution can take place by treating the carrier with an aqueous medium of pH between 2 and 4. The corresponding solution should contain salts of suitable buffer substances in a suitable concentration, preferably 0.2-8 mol/litre.

In the batch method, it pays to leave the dispersion

of the adsorbent be in this solution 0.1-2 hours, PP, being desorbed by the immunoadsorbent. In the column method, the elution solution is allowed to flow through the column. After separation of the adsorbent, the pH value of the solution containing PPj- is adjusted to approx. neutral (pH 6-8) . The enriched PP^ solution can, if desired, be subjected to further purification.

The elution of PP^ from the antibody-containing carrier can also take place with the help of substances that dissociate protein bonds at pH 4-9, for example with urea or guanidine solutions or solutions of so-called chaotropic salts such as NaN03, NaBr, NaC104, CF3C00Na, NaSCN or CCl3COONa . The concentration of these salts in the elution medium is suitably 2-8 mol/litre. To remove the chaotropic salts from the solution after desorption of PP^ and to separate the adsorbent, the solution can be dialyzed against a neutral salt solution or buffer solution of concentration 0.1-1 mol/litre.

The PP^ formed by this method has a degree of purity of 50-80%. With further cleaning methods, it can be enriched to over 99%.

If further purification of the immunoadsorption-enriched PP,- is desired, this can be done by usual fractionation methods. Fractionation procedures using a molecular sieve are therefore preferred with the aim of enriching the PP^ amount in the fractionation areas from the molecules with a molecular weight of approx. 50,000. A further commonly used method is ion exchange chromatography. Thereby, the charge properties of PPg» are utilized, which as is known behaves like a B^-globulin. A further possibility for pure production of PP^ is provided in the immunoadsorption methods, where the antibodies not against PP^, but against the contaminants to be removed, are used as carrier-bound adsorbents. PP^ is located after the application of this method in the unbound part.

The progress of the purification procedure is in any case verifiable most simply by means of corresponding antibodies.

In the possible fractionation procedures, the fractions are recovered which show an immunological reaction, in particular an immunoprecipitation, with the antisera reacting specifically against PP.

The PP5 obtainable by the method according to the invention

is particularly suitable for producing specifically targeted antisera. Animals are immunized according to known methods with PP^ and antiserum is extracted from the blood of the animals. With the help of these new antisera, PP^ can be detected in the body fluids by immunological methods, such as, for example, the hemagglutination inhibition test, the complement binding reaction, radioimmunoassay, latex agglutination and similar sensitive methods.

Thus, it can be shown that PP5 appears during pregnancy

in low concentration in the pregnant woman's blood. Furthermore, PP^

of importance in tumor diagnostics. It can be detected in blood in tumor tissue from patients with trophoblastic tumors. Usually, the corresponding diagnostic agents essentially consist of anti-PP, - and in certain methods, especially in radioimmunological test systems, PP^ itself is included as a standard substance. In diagnostic agents for detection against PP^-directed antibodies, PP^ is the essential component of the reagent.

The method according to the invention will be explained in more detail

using the following example.

150 ml of an anti-PP5 serum from rabbits is dialyzed against 0.02

molar phosphate buffer (pH 7.0) and chromatographed to separate immunoglobulins on DEAE cellulose. The immunoglobulin fraction (1.3 g of protein) is then reacted with 258 g of specially purified agarose in ball form ("Sepharose" 4B from Pharmacia Uppsala,

Sweden), which had been activated with 16.1 g of cyanogen bromide and so-

is led covalently bound to this carrier.

The procedure is described by Axen R., Porath J., Ernbach S.,

Nature 214, 1302, (1967).

With the aid of an immunoadsorbent prepared in this way, the placental protein PP^ can be isolated from its solutions,

especially from PP^-enriched placental extraction fractions.

2-i__^IiEi?S2iD2_5Y_EER_f ^.Ei^SSDtå^stretched

(Extraction of Placental Fraction V)

2.1. Enrichment of PP^ egg white fraction ion.

150 kg of deep-frozen placentas are crushed and extracted with 150

liter of a 0.5% aqueous sodium chloride solution. Ex-

the funnel is adjusted with 2 normal sodium hydroxide at pH 8 and mixed with 50 liters of a 3% aqueous solution of diaminoethoxyacridine lactate ("Rivanol"). After a residence time of 1 hour, remove the excess containing PP, - together with gamma globulin, mix with 5% solid sodium chloride (11 kg)

to separate the rivanol still remaining in the solution, filter and mix with 26.5%, referred to the liquid weight of solid ammonium sulphate and stir well. After 1 hour, the precipitate is removed.

500 g of the precipitate collected on the filter is dissolved in 500 ml of distilled water and dialyzed against a 0.01 molar tris-(oxymethyl)aminomethane (hereinafter abbreviated as "tris") hydrochloric acid buffer solution of pH value 7, 0 containing 0.05%

sodium azide. The dialyzed solution is centrifuged (placenta fraction V).

The above is fulfilled with the same buffer solution to 2000 ml, adjusted with 0.1 normal sodium hydroxide solution to pH

8.0 and stirred with 250 g of moist DEAE cellulose for one hour.

The DEAE cellulose is then separated from the solution by filtration, washed twice with each time 1 liter of 0.01 molar tris-hydrochloric acid buffer of pH value 8.0 and then eluted three times with each time 500 ml of 0.02 molar tris-hydrochloric acid buffer,

pH 6.5, containing 0.85% sodium chloride and 0.05% sodium azide.

The combined eluates are added to 30% ammonium sulfate, referred to

to liquid weight and the whole thing is stirred. The precipitate containing PP^ is dissolved with 100 ml of distilled water and dialyzed against a 0.1 molar tris-hydrochloric acid buffer pH 8.0, which contains 1.0 mol of NaCl per liter and 0.1% sodium azide. You get 200 ml of a solution with approx. 6% egg white and approx. 30 mg PP^. PP^ can be isolated from this fraction by immunoadsorption.

The enrichment method set forth under 2.1 is not essential to the invention. The immunoadsorption can also be carried out directly with placental fraction V.

2.2. Immunoadsorption of PP^

The immunoadsorbent is suspended in 0.1 molar tris-HCl buffer

(pH 8.0), which contains 1.0 mol/liter NaCl and 0.1% NaN^

(buffer solution I below), is then filled into a chromatography column (3 x 20 cm) and rinsed with buffer solution I. Then 100 ml of a PP^-containing solution is slowly passed through the column, as PP^

bind immunoadsorptively. The column is washed with buffer I

and then eluting the adsorbed protein with 0.5 molar glycine-HCl buffer (pH 2.5). The PP5-containing eluates are adjusted with 1 normal NaOH to pH 7.0 and evaporated in an ultra-filter to approx. 10 ml. Dividend per adsorption around 2 mg PP5.

The adsorbent in the column is neutralized immediately after the elution of PPg again with buffer solution I and washed thoroughly, it can then be used again for immunoadsorptive binding of PP^.

The protein produced by immunoadsorption is frequently contaminated by non-specifically bound serum protein (mainly IgG and also some IgA). The separation of these companion proteins is successful, e.g. by gel filtration, molecular sieve fractionation, e.g. preferably by means of cross-linked dextran such as "Sepha-dex" G - 100, however, the serum proteins can also be removed by immunoadsorption, i.e. by means of carrier-bound antibodies against serum proteins.

The use of PP5 for the production of anti-PP5 serum is as follows: Rabbits were immunized with the purifying PP using aluminum hydroxide as adjuvant over a period of

6 weeks.

PP^ is dissolved in physiological saline solution (concentration 0.06 mg/3 ml) and with the addition of aluminum hydroxide to a suspension. For 5 consecutive days, the rabbits are each injected with 0.06 mg of protein in 3 ml of suspension per animals intravenously.

The animals are then allowed to rest for 9 days. The animals are then immunized again for 5 consecutive days with the above-mentioned equal amounts of antigen, the animals are again allowed to rest for 9 days and finally injected for 5 consecutive days each time with 0.06 mg of antigen. After a renewed waiting period of 7-9 days, the animals are bled. After coagulation of the blood, serum is filtered from the blood cake and recovered.

Claims (1)

Process for the isolation and further purification of the placenta-specific protein PP^, in which placentas are extracted with an aqueous salt solution, the extract is mixed with a solution of diaminoethoxyacridine lactate, separated from the precipitate, the remaining diaminoethoxyacridine lactate in the supernatant is precipitated with sodium chloride, the supernatant is mixed with ammonium sulfate, the precipitate separated, dissolved in water, dialysed against a buffer of pH 7 and the dialysed solution centrifuged and possibly further enriched, characterized in that this solution at a pH value between 4 and 9 is brought into contact with an antibody against PP bound on an insoluble carrier ^, separates the carrier containing bound PP,-from the liquid phase and to release PP^ treats with an aqueous medium of pH value 2-4 or with a solution of a substance that dissociates a protein bond at a pH value of 4 -9 and cleans with per se known isolation methods after the immunoadsorption until a pure t of over 99%.