WO1989008257A1

WO1989008257A1 - Determining reactants and combinations of reactants involved in biospecific reactions

Info

Publication number: WO1989008257A1
Application number: PCT/SE1989/000078
Authority: WO
Inventors: Jerker Porath; Sven Oscarsson
Original assignee: Jerker Porath; Sven Oscarsson
Priority date: 1988-02-26
Filing date: 1989-02-24
Publication date: 1989-09-08
Also published as: SE462165B; SE8800676L; SE8800676D0

Abstract

A desired immunoglobulin, enzyme inhibitor, lectin, nucleic acid or nucleic acid derivative is separated from a protein complex through adsorption on a polymer carrier such as a polyhydric organic polymer, silicon gel, organic polyamine or polyamide or the like, into which have been introduced ligands containing an aliphatic group consisting of a thio-ether and a sulphone group or a thio-ether and an aromatic group or a thio-ether and a heterocyclic nitrogen or sulphur compound, or that the ligand instead of the thio-ether contains an amine or a sulphone group in combination with an aromatic or heterocyclic nitrogen or sulphur compound. The adsorption takes place in a salt environment, using antichaotropic salts such as sulphate, phosphate, chloride and the salts of polybasic acids, e.g. citrate, which promote adsorption at mol concentrations exceeding 1/5 of their solubility in water. Low-molecular substances in complex with proteins can be separated in accordance with the invention, but it is preferably used to separate protein-protein complexes in which both components have a molecular weight exceeding 10,000. However, the invention can also be applied to molecular weights as low as >100. The separation is preferably performed in a pH interval not exceeding 3 pH units from the isoelectric point of the complex. The barrier may be in the form of a thin layer, such as individual fibres, weave or paper, or it may be in the form of a solution in order to permit rapid adsorption, in which case it is precipitated out after the adsorption.

Description

Determining reactants and combinations of reactants involved in biospecific reactions

The present invention relates to the immobilization and separation of combined protein complexes for analysis and diagnosis of components in the complex having properties resulting in adsorption to an adsorbent so that the adsorption can be used for analysis and diagnosis. It is based on a constellation of several phenomena, known per se, which have never previously been combined, and enables determination of reactants and combinations of reactants participating in biospecific reactions.

The method is based on one of the proteins in the complex having certain properties which will result in adsorption to an adsorbent of a certain characteristic composition and the method is characterised in that the conjugate formed or the marked reactant not bound to the conjugate is adsorbed or absorbed to an insoluble carrier or to a carrier which can be made insoluble, with the type of bonds usually occurring at adsorption and absorption with or without the presence of salts in concentrations which increase adsorption of the conjugate formed or of the marked reactant not bound to the conjugate. Ligands consist of atoms or atom groups which have a specific bond to a certain protein and can adsorb this. If the adsorption is encouraged to become as complete as possible, the procedure permits a method of being able to quickly desorb this complex from the carrier in order to facilitate measurements with indicator systems if required, and the carrier can then be re-used. This is desirable in many cases since ligands can be extremely expensive and it is therefore advantageous to be able to utilize them to the full.

Re-use of the carrier also permits simplifications in automizing systems for diagnosis where, for instance, surfaces coated with a suitable polymer to which a ligand has been connected, can either be used in a flow system with one measuring point or be expanded to several measuring points. Several reactants are used for determining methods in biospecific affinity reactions, one of these reactants being marked with at least one analytically indicatable atom or atom group, and being soluble in the water solution in which the reaction takes place.

The reactants in biospecific affinity reactions form a conjugate in which the marked reactant is included and the analytically indicatable atom or atom group can be determined in the conjugate and/or in the marked reactant not bound to the conjugate.

In such determination methods for biospecific affinity reactions, e.g. immuno-chemical reactions, a reactant which is marked and is soluble in the water solution in which the reaction takes place, is reacted with a reactant with biospecific affinity, and possibly with a second reactant which has biospecific affinity to the first and/or to the second reactant, to a conjugate or complex. Several reactants may be included in the reaction chain, one of which must have affinity to the carrier and a second must be marked with a marker.

The reaction process can be described schematically as follows:

P + B_i A+A^x → P B_i A + P B_i A^x, where P is the carrier, the polymer

B₁; B₂; ... B_i; ... B_n are affinity components to the polymer

A is the unmarked test substance, A^x denotes a marked test substance,

AB, AB₁., A B_i, A B_n... are conjugates, as are also

A^xB, A^xB₁, A^x B_i, A^xB_n..

P- B_i is also adsorbed and A B_i + B_i exists freely in the solution.

Characteristic of the invention is that B_i and B_iA and B_iA are adsorbed specifically to the polymer in a water solution and that this adsorption is increased by chlorides, sulphates, phosphates or salts of polybasic organic acids. When performing the invention, one or more of these salts is/are added to a concentration suitably exceeding half or one third of the saturation concentration for the salt and so that the ratio B_i + AB_i/A_free exceeds 1000. The salt forces the complex out of the solution so that the complex is collected at the phase boundary surface between solution and adsorbent. Ligands penetrate into a surface region of the complex where the dielectricity constant is deemed to be greatly reduced and lies within the interval 2 - 15.

The analysis system can be generally described as follows:

A mixture of Ig molecules and test substance molecules will, besides an excess of free substances, also give two sorts of immune complexes: marked and unmarked

When adsorbing particles:

are added, Ig molecules, free and bound, are bonded to the particle. The greater the concentration of unmarked substance, the less will be the marking-in of the particle.

Ig molecule which will not bind the test substance.

The particles are isolated and the marking-in is measured. The reaction process at separation of an antigen, using antigen β -2- μ as model is described schematically below.

Using β -2- μ as model, a substance is obtained which is present in high concentration in urine and serum, 1.10 M, and thus also gives a high antibody concentration, total y-globulin concentration 1.10^-6 M. Parts of the structure of antibodies are almost identical to β -2- μ and this is therefore an extremely suitable antigen to illustrate the general principle and provides important information as to the polymer's ability to bind specific immune complexes and specific immunoglobulins. β-2-μ is commercially available from Pharmacia, Uppsala.

= antibody directed specifically to the reactant A

which in this case is antigen β-2-μ (Mv. 1150), antigen β-2-μ marked with ¹²⁵I or enzyme. = unspecific antibodies, antibodies normally present

in serum.

The solution also contains

+

+ β-2- μ(in test) + β-2- μ¹²⁵I

↓ whereupon immune complex is formed

↓ The addition of polymer with ligands which under specific conditions bind immunoglobulins, free or in complex ↓

+ free proteins

Washing of gel, and simultaneous separation of free antigen marked with marker

(freed from free proteins)

↓

Measurement of radioactivity.

The reactant A and marked A consist of a protein. However, they may equally well be some other substance, e.g. a nucleic acid which, with at least one other reactant B which is a protein binding A and forming a complex AB which, like B, is itself adsorbed to a polymer P in an environment containing a water-structuring salt (antichaotropically) such as alkali, earth alkali and ammonium salts of, for instance, the anions chloride, sulphate, phosphate, citrate and of other water- soluble bi- and tri-basic acids, preferably in a concentration exceeding half or one third (ca.20 %) of the saturation concentration, after which the adsorbate and the adsorbent (the polymer to which AB has been adsorbed) are separated from the solution and the measurement is performed.

When performing the determining method, a carrier is used to which only one of the reactants in the conjugate can be adsorbed or absorbed. The carrier used contains covalently bonded ligands having the structures X-S, X-CH₂S, X-CH₂NR-, in which X is an isocyclic or heterocyclic ring system containing u-electrons and/or ligands containing structures -SO₂-CH₂-CH₂-Y where Y is S, N< or NR- and R is H or an alkyl. The carrier used contains either one or several sulphur atoms with or without combination of one or more nitrogen atoms in the type of combination existing in mercapto-pyridine, thio-phenol, thio-cyanate, ethyl sulphate, pyridyl sulphide or mercapto-ethanol, for instance.

The antichaotropic salts in Hofmeister's series which promote adsorption of the conjugate are used in combinations with the above-mentioned carriers and reactants in concentrations which increase the specific adsorption of the conjugate.

The carrier may consist of a polymer which is soluble or insoluble in aqueous liquids, in the first case preferably with a molecular weight greater than that of the protein complex. For chromatographic purposes the polymer must be in particle form, but for diagnostic purposes either of the forms can be used although only insoluble, adsorbing particles for immuno-diagnostic application are described here. Both synthetic and natural polymers can be used and are provided with various ligands, all of which however must contain at least one sulphur atom, either alone or in combination with at least one nitrogen atom. The polymer may be cross-linked to a water-insoluble network which may also swell in water.

Examples of such polymers are derivatives of agarose or other water-insoluble polysaccharides, silicon gels or silicates, e.g. glass.

Examples of water-soluble polymers are derivatives of dextrane, starch or other water-soluble polysaccharides. Other examples of polymers are the native or modified, soluble or insoluble proteins or polypeptides showing adsorption of conjugate or of marked reactant A not bound to conjugate. There are many examples of polymer substances containing sulphur or sulphur and nitrogen in different combinations. Such polymers are described in Febs Letters, vol 185, No. 2, 11985.

Other examples are agarose substituted with groups

to which are bound

SCN and HS(CH₂)_n - CH₃, where n is 1 - 5.

Absorbents in the form of gel with ligands containing structures -SO₂-CH₂-CH₂-X, where X is S, NH or N, or other structures constituting heterocyclic or aromatic ring systems, and gels with ligand structures -NH- in combinations with nitrile groups, are previously known through Swedish patents 8402662-4, 8402663-2 and 8600641-8.

These adsorbents adsorb immunoglobulins at relatively low concentrations of water-structuring salts and/or by adding organic solvent of lower polarity, e.g. glycerol, glycol or some other lower alcohol, to the medium.

Even if the component B has previously shown that it is adsorbed to

P, adsorption need not necessarily occur after B has formed a complex with other high-molecular reactants A...A_n, and this can be exploited for the purpose of analysis and diagnosis.

Characteristic of the invention is that conjugates A...A_n can also be adsorbed in similar manner to the same type of adsorbent, and thus separated from the solution.

Those of the known methods which utilize a soluble polymer carrier for one of the reactants have the advantage that the separation of conjugate and marked reactant A^x not bound to conjugate can be performed more accurately. One drawback is that the biospecific affinity reaction occurs more slowly when one of the participating reactants is bound to an insoluble carrier than if this reactant is free in solution. When diagnosing immunoglobulins or antigens it is of value if the analysis requires as little time as possible. The complex is formed more quickly in solution than at a phase boundary surface and is therefore performed in solution, and precipitation then occurs with a high ligand concentration on the solid phase. Thanks to the invention, therefore, the time required for analysis is reduced to that required for immune complex formation in solution, since the adsorption of the immune complex to the solid phase is instantaneous. Immuno-globulin can also be diagnosed with anti-immunoglobulins. Examples of reactant A and other reactants showing biospecific affinity to each other are

a) antigens and haptens and specific antibodies directed thereto or modified and fragmented parts of antibodies,

b) complement binding factors which can adhere to immunoglobulins,

c) lectins which can adhere to specific carbohydrate structures and specific antibodies directed thereto,

d) enzymes and enzyme inhibitors which can adhere to these enzymes and specific antibodies directed to these reactants,

receptors and ligands to these and specific antibodies directed to these reactants,

f) nucleic acid or nucleic acid derivatives of these and specific reactants directed thereto.

To enable determination of the content of the analytically indicatable atom or atom group in the conjugate or of marked reactant A^x which is not bound to the conjugate, the conjugate and the marked reactant A not bound to the conjugate are separated by the precipitation method or by chromatographic methods.

One example of precipation methods is the double antibody solid phase method (DASP), according to which immune complex is formed in a solution and conjugate and marked reactant A^x not bound to the conjugate are then separated by the reactant bound to the conjugate being adsorbed on the solid phase through antibodies directed to one of the components in the conjugate.

An example of chromatographic methods is affinity chromatography. The division is now often arranged so that one of the reactants is bound to an insoluble polymer and the other reactants are bound thereto by a biospecific reaction, so that that portion of reactant A can be separated which is not presumed to be provided with an indicator group or atom which has not been bound biospecifically to the polymer. Examples of such systems can be found in Radioimmunoassay Methods (Ed. K.E.Kirkham and W.M.Hunter, Churchill Livingstone, London 1971, 405 - 412). This publication lists a large number of different determining methods using a marked reactant.

The indicatable reactant is marked with an analytical indicator group or atom, and the marking of a reactant is now a well-established technique which is carried out by coupling one of the reactants to an analytically indicatable atom or atom group either by direct binding or via a bridge. Usual markers are radioactive atoms or atom groups, fluorescent, luminescent or chromophore groups, enzymatically active groups, enzyme inhibitor groups or co-enzyme groups.

When quantitatively determining one of the reactants, it is known to use varying known quantities of this reactant in order to establish standard curves which are then used to determine the quantity of the reactant in a sample.

The invention is further explained through the following examples with reference to the accompanying drawings, in which

Figure 1 shows a standard curve of β -2- μ using peroxides as marker,

Figure 2 shows adsorption on agarose with various ligands,

Figure 3 shows adsorption of immune complex and marker as functions of the time, and

Figure 4 shows a standard curve for β-2-μ using I as marker.

Example 1

Producing a standard curve for β -2- μto determine the concentration of β-2-μ in the samples. Peroxidases are used as marker. The antibodies were non IS-cleaned (immuno-sorbent) antibodies from immunisation of β-2- μ in sheep through the precipitation of serum with 18 % Na₂SO₄ and desalinating on Sephadex® G25.

The carrier consisted of epichlorohydrin-activated agarose (Sepharose® 5B) to which mercaptopyridine had been coupled in the presence of 0.5 g NaBH₄ in 0.1 M Na-phosphate buffer, pH 7.5, by the addition of 2 g mercaptopyridine to 25 g epichlorohydrin-activated gel and reaction at room temperature for 18 hours. When the reaction was complete the carrier was washed first with distilled water, then ethanol and finally with distilled water. 30 mg dry agarose was allowed to swell in 1 ml 0.5 ml K₂SO₄ - 0.1 M Tris (trishydroxy methyl amino methane hydrochloride), pH 7.5. 25 Ail anti-β-2-μ solution diluted with 0.1 M Tris, pH 7.5 to a concentration of 1.8 x 10^-6M with respect to the total concentration of immunoglobulin was poured into tubes of make Ellerman to hold a volume of 3 ml . Thereafter 25 μl β-2-μ solution was measured into two series of tubes with concentrations decreasing from 30 μg/ml to 0.0 μg/ml. After five minutes 25 μl 1 x 10^-7M β-2-μ-peroxidase conjugate was added, and after 15 min reaction time 500 μl mercaptopyridine gel having a concentration of 30 mg gel/ml, corresponding to 1.8 mg carbohydrate/ml. After 10 minutes the tubes were centrifuged, the supernatant was suctioned off and 1000 μl 0.5 M K₂SO₄ - 0.1 M Tris was added. The tubes were centrifuged again, the supernatant suctioned off and 1 ml 5-aminosalicylic acid (80 mg 5-aminosalicylic acid in 100 μl distilled water) was added. The solution was titratedto pH 6.0 and 20 μI of a 3 % H₂O₂ solution was added to 10 μl of the solution. After 10 minutes reaction time, 100 μl 5 M NaOH was added and the absorbance was measured in the supernatant at 449 nm. The standard curve showing A 449 nm as a function of the β-2-μ concentration is plotted in Figure 1.

Example 2

The effect of various ligands cpupled to cross-linked agarose on the adsorption of immune complex.

a) Synthesis of 3-(2-pyridylsulphido)-2-hydroxy propyl agarose (PyS). 40 ml 4 M NaOH was mixed with 0.17 g NaBH₄ and 50 g washed and suctiondried Sepharose® 6B was added. Thereafter first 4.25 ml epichlorohydrin was added, followed by another 4 ml after 30 min. Over the next hour epichlorohydrin was added another four times at regular intervals, 4 ml each time. After the mixture had been allowed to react at room temperature for 18 hours, the reaction was interrupted and the gel washed with distilled water to neutral reaction. 2 g mercaptopyridine was dissolved in 100 ml 0.1 M Na-phosphate buffer, pH 7.5, with the addition of 0.5 g NaBH₄ and was added to the gel after pH adjustment.

After reacting for 18 hours at room temperature, the product was washed with ethanol and water. Analysis showed 810 Λimol S and 800 μmol N per gram of the dried product.

b) Synthesis of 3-(2-pyridyloxi)-2-hydroxy propyl agarose (PyO).

The epichlorohydrine activation of the gel was performed in the same way as for a). Out of 100 ml 1 M NaOH, half was mixed with 0.5 g NaBH₄ and half with 5 g hydroxy pyridine. The two solutions were mixed and 5 g suction-dried Sepharose® 6B was added and allowed to react at room temperature for 18 hours. Nitrogen analysis showed 1064 μmol N/g dried product.

c) Synthesis of 3-(phenyloxi)-2-hydroxy propyl agarose (PhO).

The gel was epichlorohydrine-activated in the same manner as earlier. 0.25 g NaBH₄ was mixed with 25 ml 1 M NaOH and 10 g phenol was also dissolved in 25 ml 1 M NaOH. The solutions were mixed together, 25 g epichlorohydrine-activated gel was added and allowed to react for 18 hours at room temperature. The degree of substitution was determined using NMR to 670 μmol ligand/g dried product.

d) Different ligands' adsorption of immune complex at various gel concentrations.

25 μl 1.8 x 10^-6M anti-β-2-μ-peroxidase conjugate and 25 μl 1 x 10^-7M β-2-μ-peroxidase conjugate was measured into Ellerman test tubes.

After being allowed to react for 30 min, varying concentrations of each gel were added to the tubes. The gel concentrations are stated on the abscissa in Figure 2. After reacting for 30 min at room temperature, the polymers were carefully washed with 1 M 0.5 M K₂SO₄ - 0.1 M Tris solution, pH 7.5. Washing was performed three times, with centrifuging in a table centrifuge at 3000 rpm between the washes and the supernatant was suctioned off. 1 ml 5-aminosalicylic acid solution (80 mg/100 ml distilled water) was then added and the substrate solution adjusted to pH 6.0 with 1 M NaOH. 20 μl of a 3 % solution of H₂O₂ was added to 1 ml of this substrate solution and after 15 min the reaction was interrupted by the addition of 100 μl 5 M NaOH. After centrifuging off the gel, the absorbance of the supernatant was read in the various tubes. The curves are shown in unbroken lines in Figure 2. Corresponding curves in broken lines show the unspecified adsorption of marker to gel in the absence of antibodies.

Example 3 Desorption of adsorbed immune complex.

Anti-β-2-μ-peroxidase conjugate and β-2-μ-peroxidase conjugate were measured into Ellerman test tubes in the manner described in the preceding example. The gel, mercaptopyridine-based, cross-linked agarose, was added and the immune complex formed was adsorbed in the presence of 0.5 M K₂SO₄ - 0.1 M Tris, pH 7.5. Two parallel experiment series were performed. In one of the series, the excess of β-2- μ- peroxidase not bound to the carrier was washed with 0.5 M K₂SO₄ - Tris, pH 7.5, and in the other series it was washed with 0.1 M Tris, pH 7.5.

By measuring the quantity of immune complex which had been bound in the presence of 0.5 M K₂SO₄ - 0.1 M Tris, pH 7.5, and comparing this with when the corresponding immune complex was first adsorbed in the presence of 0.5 M K₂SO₄ and then desorbed by washing the carrier with 0.1 M Tris, pH 7.5, the desorbed quantity of the adsorbed immune complex could be determined.

25 μl 1 x 10^-6M anti-β-2-μ-peroxidase and 25 μl β-2- μ-peroxidase were measured into 6 Ellerman test tubes.

In tubes 1 and 2 the immune complex formed was adsorbed in the presence of 0.5 M K₂SO₄ and washed with 0.5 M K₂SO₄ - 0.1 M Tris, pH 7.5, after which the activity was measured. In tubes 3 and 4 the carrier was washed with 0.1 M Tris, pH 7.5, and the enzyme activity was measured.

In tubes 5 and 6 a corresponding volume of 0.1 M Tris, pH 7.5, was added instead of anti-β-2-μ, and the carrier was washed with 0.5 M K₂SO₄

- 0.1 M Tris, pH 7.5.

Results of experiment

Tube No. Absorbance at 449 nm

1 1.17

2 1.06

3 0.11

4 0.12

5 0.11

6 0.12

Tubes 1 and 2 show the specific adsorption of the immune complex. Tubes 3 and 4 show the specific adsorption of the immune complex after desorption since salt has been eliminated. Tubes 5 and 6 show the undesired adsorption of marker to the polymer and also the level of enzyme activity to be found in tubes 3 and 4 if desorption of the immune complex is complete.

Example 4

Adsorption of the immune complex formed to 3-(2-pyridylsulphido)-2-hydroxypropylagarose as a function of the time.

25 μl 1 x 10^-7M β-2-μ-peroxidase and 25 μl 1 x 10^-6M anti-β-2-μ-peroxidase were measured into a number of Ellerman test tubes. 500 Ail gel (30 mg swelled, suction-dried gel mixed with 1 ml 0.5 M K₂SO₄

- 0.1 M Tris, pH 7.5) was then added to each tube. The reaction was interrupted after 1, 7, 10, 15, 30, 60 and 120 minutes, respectively, by adding 1 ml 0.5 M K₂SO₄ - 0.1 M Tris, pH 7.5, to each tube. The tubes were centrifuged and the supernatant withdrawn by suction.

The procedure was repeated twice. The enzyme activity was read after

15 min reaction with substrate as described in Example 1. The enzyme reaction was stopped by the addition of 100 μl 5 M NaOH. The result of the experiment is shown in Figure 3. Example 5 PyS was used as carrier in accordance with Example 2, ¹²⁵I as marker and non-IS-cleaned antibodies in accordance with Example 1.

25 μl anti-β-2-μ-solution, diluted with 0.1 M Tris, pH 7.5, to 1.8 x 10^-6M with respect to the total concentration of immunoglobulin, was measured into Ellerman test tubes holding a total volume of 3 ml. Two series of tubes were used, 25 μl β -2- μ being measured into each tube, with concentrations falling from 31 g/ml to 0.48 g/ml. After 5 min, 25 μl β -2- μ marked with ¹²⁵I, and with a concentration of 1.5 x 10^-7 M was added to each tube. After a total reaction time of 15 min, 500 μl PyS gel having a concentration of 30 mg swelled suction-dried gel/ml 0.5 M K₂SO₄ - 0.1 M Tris, pH 7.5, was added.

After 15 min the tubes were centrifuged and the supernatant withdrawn by suction. 1 ml 0.5 M K₂SO₄ - 0.1 M Tris, pH 7.5, was added and the tubes again centrifuged. This was repeated twice. The activity in the various tubes was determined in y-counters.

Extremely good results have also been obtained with lectin, α₂-macroglobulin, trypsin, kymotrypsin, enzyme complexes of α₂-macroglobulin or sojabean trypsin inhibitor and proteolytic enzymes, inter alia. Even low-molecular compounds having a molecular weight greater than 100 have been diagnosed with good results and diagnostic tests have been performed on nortryptilin and antibodies could be obtained by coupling to a carrier molecule, resulting in the hapten becoming immunogenic.

Claims

C l a i m s

1. A method of immobilizing and separating combined protein complexes by adsorption on a carrier, characterised in that the adsorption is performed on a polymer such as a polyhydric organic polymer, silicon gel, organic polyamine or polyamide or the like, into which have been introduced ligands containing an aliphatic group consisting of a thio-ether and a sulphone group or a thio-ether and an aromatic group or a thio-ether and a heterocyclic nitrogen or sulphur compound, or that the ligand instead of the thio-ether contains an amine or a sulphone group in combination with an aromatic or heterocyclic nitrogen or sulphur compound.

2. A method as claimed in claim 1, characterised in that the adsorption is increased by being performed in a salt environment, selected from salts which promote adsorption at mol concentrations exceeding 1/5 of their solubility in water.

3. A method as claimed in claim 2, characterised in that the salt is antichaotropic and selected from alkali, earth alkali or ammonium salts of hydrochloric acid, sulphuric acid, phosphoric acid or polyvalent organic acids.

4. A method as claimed in any of the preceding claims, characterised in that one of the components in the protein complex is an immunoglobulin.

5. A method as claimed in any of claims 1-3, characterised in that one of the components in the protein complex constitutes an enzyme inhibitor, an enzyme or a lectin having high affinity to the polymer.

6. A method as claimed in any of claims 1-3, characterised in that one of the components in the protein complex consists of a nuclein acid or derivate thereof.

7. A method as claimed in claim 4, characterised in that the separated substance consists of an immunoglobulin and a hapten linked thereto, such as a low-molecular pharmaceutical, hormone or the like.

8. A method as claimed in any of the preceding claims, characterised in that the carrier is a polyhydric polysaccharide, PVA, silica gel or derivative thereof.

9. A method as claimed in any of the preceding claims, characterised in that the polymer is soluble in water to 0.1? or more.

10. A method as claimed in any of the preceding claims, characterised in that the polymer is present in the form of a substance insoluble in water, such as in the form of a particle, thread or membrane.