IE44824B1 - Process for the isolation of a placenta-specific glycoprotein - Google Patents

Abstract

In order to concentrate the placenta-specific protein PP5, a PP5-containing protein solution is brought into contact, at a pH of between 4 and 9, with an antibody against PP5 which is bound to an insoluble support. The support with the PP5 bound to it is separated off from the liquid phase. In order to release the PP5, this support is treated with an aqueous medium of a pH of from 2 to 4, or with a solution of a substance which dissociates protein bonds at a pH of from 4 to 9. The PP5 which is obtained can be used in a diagnostic agent for detecting PP5 and antibodies directed against PP5.

Description

This invention relates to a process for the isolation of a glycoprotein called PPg which was discovered in an aqueous extract of placentate by H. Bohn in 1972, and of proteins related antigenically thereto.

In Archiv fur Gynakologie 212 (1979) pages 165-175, H. Bohn describes a series of soluble antigens that were detected immunologically in an extract of placentae by means of antisera which had been obtained by immunizing animals with crude fractions of placentae.

It was deduced that the protein called PP^ is placentaspecific because standard immunological tests for the detection of this protein in a number of embryonic and adult human organs, in human plasma and in erthrocyte lysates did not give any positive results.

The electrophoretic migration behaviour of PPg in agar identifies it as a β-globulin. In a polyacrylamide gel in which the mobility of albumin-is taken as 100, PP^ has a relative mobility of 75. A molecular weight of approximately·, 50,000 was deduced for PP^ on the basis of its behaviour during gel filtration on cross-linked dextran.

*. The present invention provides a process for isolating PPg and/or antigenically related proteins, which comprises bringing a placental extract solution comprising PPg and/or antigenically related proteins into - 3 contact with an insoluble carrier to which are bound antibodies directed against PPg and/or against antigenically related proteins, at a pH above 4, preferably within the range of from above 4 to 9, separating the resulting carrier complex, which comprises PPg and/or antigenically related proteins, from the liquid phase and, to release the PP5 and/or antigenically related proteins, treating the carrier complex with an aqueous medium having a pH of 4 or less, preferably within the range of from 2 to 4, or with a substance capable of dissociating antigen-antibody complexes at a pH of above 4, preferably within the range of from above 4 to 9, advantageously 7.

Human placentae are preferably used as the starting material. Approximately 10 to 20 mg of PPg can be extracted from an average-sized placenta (approx. 500600 g). It will be understood that the term placental extract includes any placental extract that has been pre-purified and/or enriched with regard to the PPg content thereof.

To prepare an aqueous extract, comminuted placentae are treated with water, or a suitable salt solution, and the liquid phase is isolated. Suitable salts are those that are as inert as possible under the conditions of extraction, especially those conventionally used in the preparation of tissue extracts. In addition neutral salts and/or buffer substances is or are preferably used, especially sodium chloride, tris-hydroxymethylaminomethane, or an alkali metal salt of phosphoric or citric acid. The salt concentration is preferably from 0.1 to 5%.

The antibody required for the isolation of PPg is preferably that directed against PP5 and obtained by immunizing animals with PPg. If there is no pure PPg available, however, as is the case when the isolation -4-. procedure is ^carried out for the first time, a placental fraction in.which PPg is concentrated to a certain extent, produced by conventional precipitation processes, for example, as described by H. Bohn in 1972, may be used to immunize animals.

When immunization is carried out using a crude placental fraction, the resulting anti-serum contains multi-specific anti-bodies. The antibodies not directed specifically against PP^ are removed by treating the anti10 serum with suitable antigens obtained from serum and placental fractions and removing the resulting antibodyantigen complexes. The resulting immunoglobulin fraction r which contains antibodies directed against PPg and/or against antig-enically related proteins (called collectiv15 ely hereafter PP^-antibodies), may be obtained by conventional methods.

The PPg’-antibodies, obtained by the above described process or by immunization with purified PPg obtained according to the invention, may be bonded to a suitable carrier by known methods, that is to say, methods in actual use in the art or described in the literature of the art, for example, in Ann. Rev. Bioehem. 40, (1971) page 259, Naturwissenschaften 58 (1971) page 389 and . Nature 314 (1967) page 1302.

Particularly suitable carriers are high-polymer carbohydrates, for example, cellulose and agar, and synthetic resins, for example, polyacrylamide and ethylene/maleic anhydride copolymers. Glass particles may also be used as carriers. An especially preferred carrier material is a purified agarose in the form of particles having a diameter within the range of from 20 to 200 pm, to which the PP^-antibodies can be bonded covalently according to the last of the above methods.

To isolate the PPg and/or antigenically related proteins (called collectively hereafter PPg), the - 5 PPg-containing solution is brought into contact with the carrier-bound PP^-antibody at a pH of more than 4.

To avoid denaturation of the proteins it is preferable to work at a pH not exceeding 9. The weight ratio of the solution to the adsorbent is preferably in the range of from 1:1 to 10:1, and is advantageously 2:1. The adsorbent is preferably allowed to remain in contact with the PPg-containing solution for 0.1 to 5 hours to enable the antigen-antibody complex to form.

As mentioned above, it is advantageous to incorporate neutral salts and/or buffer substances in the PP^containing solution. These components are preferably selected from those generally used in tissue extractions, for example, those described in the Handbook of Biochemistry published by The Chemical Rubber Co., Cleveland, Ohio, U.S.A., 2nd Ed. S.J. 238. The salt and buffer concentrations are not critical, but are preferably in the range of from 0.01 to 2 mol/1. In general, there should be avoided any conditions that would interfere with the formation of the antigen-antibody complex or which would lead to its premature breakdown.

The adsorption of the PPg and its subsequent separation from the complex can be carried out either in a batch process or in a chromatographic column. In the batch process, the carrier-PP5, complex may be separated from the solution by centrifugation or filtration, and washed several times with a neutral salt solution to eliminate excess salts and non-specifically absorbed impurities. In the column method, the solution is removed from the column by thorough rinsing with a buffer solution.

The PPg can be separated from the carrier complex by any method which dissociates an antigen-antibody complex, for example, by treating the carrier complex with an aqueous medium having a pH of 4 or less than 4, 8 preferably from 2 to 4. In the batch process it is. preferable to disperse the carrier-complex in the medium and. to leave the dispersion for 0.1 to 2 hours. In the column process, the acidic solution is allowed to run through the column containing the carrier complex. After removing any adsorbent present, the pH of the resulting PPg-solution is preferably adjusted to approximately neutral (pH 6-8), and the solution may be subjected to further purification, if desired.

Any aqueous medium having a suitable pH may be used, especially dilute, weak organic acids, and buffers.

The PPg can alternatively be separated from the carrier complex by means of a substance capable of breaking inter-and intra-protein interactions including dissociating antigen-antibody complexes, for example, urea or guanidine, or a so-called chaotropic salt, for example, NaNO^, NaBr, NaClOg, CF-jCOONa, NaSCN or CCljCOONa.

(A chaotropic salt is a salt that enhances the partitioning of nonpolar molecules from a nonaqueous to an aqueous phase as a result of the disruptive effect that the salt has on the structure of water.).

The concentration of the substance in the medium in which the dissociation will occur is preferably from 2 to 8 mol/litre. To remove the dissociation substance from the solution after the PPg has been desorbed from the complex and the adsorbent has been separated the resulting mixture may'be dialysed against a neutral salt or buffer solution, preferably having a concentration of 0.1 to 1 mol/litre.

By the process of the invention, concentration of PPg can be increased from 1 to 2 mg/100 ml in the initial extract to about 100-1,000 mg/100 ml ie. it can be concentrated by a factor -of 2,500 to 5,000, and the degree of purity is about 50 to 80%. This value can be - 7 increased to 99% by further purification processes. (Processes for purifying β-globulin are known.) Fractionation processes are suitable for further purification of the resulting PP5 solution, the preferred process involving the use of a molecular sieve, the PPg being found in the range of molecules having a molecular weight of approximately 50,000.

A further method suitable for additional purification of PPg is ion exchange chromatography. For this it is necessary to assess the charge properties of PP5, which is known to behave like a β^-globulin.

Another suitable way of purifying the PP^ is to Use the principle of immuno-adsorption in the reverse sense to that described above i.e. the antibodies bonded to the carrier are not directed against PP,- but against the impurities it is desired to remove. When such an adsorbent is used, the PPg remains in solution, the impurities being adsorbed.

The progress of the process of the invention and of any subsequent purification process can be followed most easily by the use of a suitable antiserum, for example, in a fractionation process, those fractions are selected that exhibit an immunological reaction, especially a precipitation, with an antiserum directed against PPg.

The ΡΡ^ in solution as obtained directly after the process of the invention or after further purification can be lyophilized to give the PPg per se.

Due to· its purity, PPg obtained according to the invention is particularly suitable for preparing antisera directed against PP^. The present invention accordingly provides, an .antiserum directed against PP^ obtained by the process of the invention and a process for its preparation, which comprises immunizing an animal with PP5 obtained by the process of the invention, bleeding the animal after a suitable time has elapsed, and 44834 - 8 extracting the antiserum from the blood.

The antiserum of the invention may be used to determine the presence of PPg or anti-PPg in a fluid, for example, in a body fluid by an immunological method, for example, the haemagglutination inhibition test, the compliment fixation reaction, the radioimmunological test, the latex agglutination test and similar sensitive methods.

It has been found that PPg appears in low con10 centrations in the blood of pregnant women, so the invention also provides a method of pregnancy testing. It has also been found that PPg is -present in the blood and the tumerous tissue of subjects with trophoblastic tumours, so PPg itself and its antiserum are useful in the diagnosis of such tumours.

The invention accordingly provides a method of determining PPg in a sample of a fluid, especially a body fluid, which comprises subjecting the sample to an immunological test utilizing the antiserum of the invent20 ion as the antibody component and, in the case of a test involving competitive binding, a labelled form of PPg obtained according to the invention. The sample is especially a blood sample from a woman or girl.

Conversely, PPg may be used to determine anti-PPg in a sample of fluid. Labelled anti-PPg is also used if the test involves competitive binding.

Labelling, especially with a radio-isotope or a fluorescent dye, is carried out in known manner.

The following Example illustrates the invention.

Sepharose and Sephadex are Trade Marks. - 9 1. Preparation of the immuno-adsorbent 150 ml of a PP^-antiserum obtained from rabbits were dialysed against 0.02 M phosphate buffer (pH 7.0) and chromatographed over DEAE-cellulose to separate the immunoglobulin fraction (1.3 g protein) which was then reacted with 258 g of especially purified agarose in pellet form (Sepharose 4B made by Pharmacia Uppsala, Sweden), which had been activated with 16.1 g of bromocyanogen, and was thus bonded covalently to this carrier. This process is described by Axen R., Porath J.

Ernbach S., Nature 214, 1302, (1967). By means of an immuno-adsorbent prepared in this manner, PP^ can be isolated from a solution thereof, especially from a pretreated placental extract as described below. 2. Isolation of PP,. from a placental extract 2.1 Pretreatment of placentae 150'kg of deep-frozen placentae were comminuted and then extracted with 150 litres of a 0.5 % aqueous sodium chloride solution. The extract was adjusted to pH 8 with 2N sodium hydroxide and mixed with 50 litres of a 3% aqueous solution of diaminoethoxyacrldine lactate (Rivanol, Trade Mark). After the mixture had been allowed to stand for 1 hour the supernatant, which contains the PP^ together with gamma globulin, was siphoned off, mixed with 5 % solid sodium chloride (11 kg) in order to separate the Rivanol still in solution, filtered and mixed with 26.5 %, calculated on the weight of the liquid, of solid ammonium sulphate, and stirred thoroughly. After 1 hour the precipitate was filtered off. 500 g of the precipitate collected on the filter were dissolved in 500 ml of distilled water and dialysed against a 0.01 molar tris(hydroxymethylaminomethane) (hereinafter abbreviated to tris )-hydrochloric acid buffer solution having a pH of 7.0 and containing 0.05 % sodium azide. The dialysed solution was centrifuged and the supernatant was made up to 2000 ml with the same buffer solution, adjusted to pH 8.0 with 0.1 N sodium hydroxide solution, and stirred for one hour with 250 g of moist DEAE- cellulose.

The DEAE-cellulose was then separated from the mixture by filtration, washed twice with one litre, in each case, of 0.01 molar tris-hydrochloric acid buffer, pH 8, and afterwards treated three times with l 500 ml, in each case, of 0.02 molar tris-hydrochloric acid buffer, pH 6.5, containing 0.85 % sodium chloride and 0.05 % sodium azide. % of ammonium sulphate, calculated on the weight of the liquid was added to the combined extracts and the mixture was stirred. The precipitate containing the PP,. was dissolved in 100 ml of distilled water and dialysed against a 0.1 molar tris-hydrochloric acid buffer pH 8.0, containing 1.0 mol of NaCl per litre and 0.1 % sodium azide. 200 ml of a solution containing approx. 6 % of ovalbumin and about 30 mg of PPg was obtained. The PPg can be isolated from this fraction by immuno-adsorption.

The pre-treatment described under 2.1 is not essential to the invention, and immuno-adsorption can be carried out directly on a crude placental extract. 2.2. Immuno-adsorption of PPg The immuno-adsorbemt was suspended in 0.1 M trisHCl buffer (pH 8.0) containing 1.0 mol/1 NaCl and 0.1 % NaNg (hereinafter buffer solution I), then poured into a chromatographic column (3 X 20 cm) and rinsed with buffer solution I. 100 ml of PPg-containing solution was then allowed to flow slowly through the column and the PPg was bound by immuno-adsorption. The column was washed thoroughly with buffer I and the adsorbed protein was then eluted with 0.5 M glycine-HCl buffer (pH 2.5). 4482 4 - 11 The combined PP,.-containing eluates were adjusted to pH 7.0 with 1 N NaOH and concentrated in an ultrafilter to approx. 10 ml. Yield per adsorption ~ 2 mg PP^.

The adsorbent in the column was neutralised again with buffer solution I immediately after the elution of PPg and washed thoroughly? it can then be used again for the immuno-adsorptive binding of PP^.

The protein obtained by immuno-adsorption is frequently contaminated by non-specifically-bound serum proteins (mainly IgG and, in addition, a little IgA).

These accompanying proteins were removed by gel filtration, molecular sieve fractionation by means of crosslinked dextran. (Sephadex G - 100). In some oases these serum proteins were removed by immuno-adsorption, i.e. by means of carrier-bonded antibodies directed against these serum proteins. 3. Preparation of PP^-antiserum.

Rabbits were immunized over a period of 6 weeks with the purified PPg using aluminium hydroxide as adjuvant (the PPg was dissolved in a physiological sodium chloride (concentration 0.06 mg/3 ml) and, after the addition of aluminium hydroxide, stirred to form a suspension).

For each of 5 consecutive days, each rabbit was' injected i.v. with 0.05 mg. of protein in 3 ml of suspension. Afterwards, the rabbits were allowed to rest for 9 days. They were then immunized again on each of 5 consecutive days with the same, above-mentioned amount of antigen, allowed to rest again for 9 days and finally injected once again on each of 5 consecutive days with 0.06 mg of the antigen. After waiting for a further 7 to 9 days, the animals were bled, and after the blood had coagulated, the serum was centrifuged from the blood clot and collected.

Claims (27)

1. A process for isolating PPg and/or antigenically related proteins, which comprises bringing a placental extract comprising PPg and/or antigenically related proteins into contact with an insoluble carrier to which are bound antibodies directed against PPg and/or against antigenically related proteins, at a pH above 4, separating the resulting carrier complex, which comprises PPg and/or antigenically related proteins, from the liquid phase and, to release the PPg and/or antigenically related proteins, treating the carrier complex with an aqueous medium having a pH of 4 or less, or with a substance capable of dissociating antigen-antibody complexes Eft a pH above 4.

2. A process as claimed in claim 1, wherein the PPg-containing solution is a placental extract that has been pre-purified and/or enriched with regard to its PPg content.

3. A process as claimed in claim 1, wherein the placental extract is an extract of human placentae.

4. A process as claimed in claim 1 or claim 3, wherein the placentae are extracted with an aqueous medium comprising neutral salts and/or buffer substances.

5. A process as claimed in any one of claims 1 to 4, wherein the carrier is a high-polymer carbohydrate, a synthetic resin or glass particles.

6. A process as claimed in claim 5, wherein the carrier is a purified agarose in the form of particles having a diameter within the range of from 20 to 200 pm.

7. A process as claimed in any one of claims 1 to 6, wherein the PPg-containing solution is treated with the carrier-bound antibody at a pH of from above 4 to 9. *4824 - 13

8. A process as claimed in any one of claims 1 to 7, wherein the weight ratio solution i adsorbent is within the range of from 1:1 to 10:1.

9. A process as claimed in claim 8, wherein the weight ratio is 2:1.

10. A process as claimed in any one of claims 1 to 9, wherein the PPg is released from the carrier complex by means of an aqueous medium having a pH of from 2 to 4.

11. A process as claimed in any one of claims 1 to 9, wherein the PPg is released from the carrier complex by treatment with urea, guanidine or a chaotropic salt (as hereinbefore defined).

12. A process as claimed in any one of claims 1 to 11, wherein the resulting PPg is further purified by a process suitable for purifying a β-globulin.

13. A process as claimed in claim 12, wherein the further purification is by any one or more of the following methods : fractionation, ion exchange chromatography and immuno-adsorption.

14. A process as claimed in claim 13, wherein the fractionation method is molecular sieving.

15. A process as claimed in any one of claims 1 to 14, wherein a resulting solution is lyophilised.

16. A process as claimed in claim 1, carried out substantially as described in the Example herein.

17. The placenta-specific glycoprotein PP^ and/or antigenically related proteins whenever prepared by a process as claimed in any one of claims 1 to 16.

18. A method of preparing an antiserum directed against PP 5 and/or antigenically related proteins, which comprises immunizing an animal with a protein as claimed in claim 17, bleeding the animal after a suitable time has elapsed, and extracting the antiserum from the blood.

19. A process as claimed in claim 18, carried out substantially as described in the Example herein.

20. An antiserum against PPg and/or antigenically related proteins whenever prepared by a process as claimed in claim 18 or claim 19.

21. A method of determining PPg in a fluid, wherein a sample of the fluid is subjected to an immunological test utilizing an antiserum as claimed in claim 20 as the antibody component, and also a labelled form of PPg as claimed in claim 17 in the case of a test involving competitive binding. i

22. I A method of determining PPg-antibodies in a fluid, wherein a sample of the fluid is subjected to an immunological test utilizing PPg as claimed in claim 17 as the antigen component, and also a labelled form of an antiserum as claimed in claim 20 in the case of a test involving competitive binding.

23. · A'Sprocess as claimed in claim 21 or claim 22, wherein the fluid is a body fluid.

24. A process as claimed in claim 23, wherein the sample of“body fluid is a blood sample from a female human being.

25. A diagnostic agent which comprises a solution of PPg as claimed in claim 17.

26. PPg as claimed in claim 17, which has been labelled. f [

27. PPg as claimed in claim 26, which has been labelled with a radioisotope or a fluorescent dye.