AU621552B2 - Dispersion polymers comprised of vinyl monomers having an acetal group, their manufacture and their use as carriers of immunologically active materials - Google Patents

Description

To the Commissioner of Patents BEHRINGWERKE AKTIENGESELLSCH okuris Prokurist ppa. Bg ppa. Stein rj .L ~a 621552 Fori COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Cl Application Number: Lodged: *Sian d Complete Specification Lodged: Accepted: m ass Piority e Published:

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S S Related Art: A S S

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Name of Applicant: Address of Applicant:

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Actual Inventor: Address for Service: BEHRINGWERKE AKTIENGESELLSCHAFT D-3550 Marburg, Federal Republic of Germany WOLFGANG KAPMEYER and MICHAEL DENGLER EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the invention entitled: 'DISPERSION POLYMERS COMPRISED OF VINYL MONOMERS HAVING AN ACETAL GROUP, THEIR MANUFACTURE AND THEIR USE AS CARRIERS OF IMMUNOLOGICALLY ACTIVE MATERIALS The following statement is a full description of this invention, including the best method of performing it known to us /1.

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.rl: -r -1 la BEHRINGWERKE AKTIENGESELLSCHAFT 87/B 034 Ma 633 Dr.Ha/Bn 'DISPERSION POLYMERS COMPRISED OF VINYL MONOMERS HAVING AN ACETAL GROUP, THEIR MANUFACTURE AND THEIR USE AS CARRIERS OF IMMUNOLOGICALLY ACTIVE MATERIALS The invention relates to dispersion polymers composed of latex particles which are a polymer of vinyl monomers which have an acetal group or have a copolymer of such monomers with other vinyl monomers, and to a process for Sthe preparation thereof. These polymers can be used, by binding to them biologically active substances having free amino groups, for the preparation of biologically 10 active latex conjugates suitable for serological or immuno- S I logical determination methods.

*0 It is known to increase the sensitivity of serological or immunological determination methods by use of indicator or carrier particles loaded with the appropriate immuno- 15 logical reagent. Latex particles with a diameter of 0.02 to 5 can be used as carrier particles.

European Patent Application EP-A 0,080,614 (USP 4,448,908) discloses latex particles which contain acetal groups bonded via amide groups. Latex cores previously prepared in an aqueous medium are swollen with vinyl monomers which contain acetal groups bonded via amide groups.

These vinyl monomers, which must be sufficiently insoluble in water, are then copolymerized together with other monomers which can be hydrophilic or ionic in nature. Reagents of this type can be employed for nephelometric determinations of serum proteins, for example of -i C-reactive protein.

The process described in EP-A 0,080,614 is elaborate because the latex particles are prepared in two stages, with cores being prepared first. In addition, the swell- 'O not with all vinyl monomers but only with those which are n of te ~lr (3 2sufficiently insoluble in water.

Furthermore, the current core-shell polymerization process results only in Latex particles of particular sizes, starting from available latex cores.

Hence there has been a need for a one-stage copolymerization process for the preparation of dispersion polymers of any desired size using vinyl compounds having acetal groups.

It has now been found, surprisingly, that the above- 10 mentioned disadvantages of the state of the art can be overcome by using carrier particles prepared by polymerization in an aqueous medium of acrylic or methacrylic monomers which contain acetal groups bonded via amide groups, of together with other vinyl monomers.

15 Thus the invention relates to a dispersion polymer composed of a compound or of several compounds of the formula I

CH

2 =CR -CO-NH-(CH) n-CH(OR 2 )OR I in which 20 n is 1 6, and R 1 is H or CH 3 and

R

2 and R 3 are identical or different and denote

-(CH

2 )m-CH 3 with m denoting 0 7 or with X, Y and Z denoting (CH 2 )pCH 3 and p denoting 1 3, where X, Y and Z can be identical or different, and, where appropriate, of another vinyl derivative or of other vinyl derivatives in an aqueous medium.

Figure 1 is a plot of the results of CRP determinations using a polymer according to the invention in a turbidimetric assay (abscissa) in comparison with the classical nephelometric assay on the BNA system (NA-Latex-CRP assay, Behringwerke AG) on the ordinate.

Figure 2 shows the results of IgE measurements using a r 1 -3polymer according to the invention in a turbidimetric assay (abscissa) and the classicaL enzyme immunoassay (REnzygnost IgE, Behringwerke AG) on the ordinate.

Figure 3 depicts the results of AFP measurements using a poLymer according to the invention in a turbidimetric assay (abscissa) and the classical enzyme immunoassay (REnzygnost IgE, Behringwerke AG) on the ordinate.

The compound of the formula I which is preferably used is acryl- or methacrylamidoalkanaL dialkyl acetal with C 2 to 10 C 8 -alkyl in the acetaL moiety. Particularly suitabLe 0*SS are acryl- or methacrylamidoacetaldehyde di-n-pentyl acetal.

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Examples of suitable vinyl polymers not embraced by the formula I are styrene, vinyLnaphthalene, vinyltouene, methacrylic acid, acrylic acid or crotonic acid.

It is also advantageous to use additionally water-soluble

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monomers from the group comprising hydroxyl-substituted acrylic or methacrylic esters or amides such as, for example, N-(2,3-dihydroxypropyl)methacrylamide, N-(2-hydroxypropyl)methacryamide or 2-hydroxypropy methacrylate. It may also be advantageous to use additionally water-soLuble monomers with a betaine structure such as, for example, N-(3-sulfopropyL)-N-methacryloxyethyL-N,N-dimethyLammonium betaine or N-(3-suLfopropyL)-N-methacryLamidopropyL-N,Ndimethylammonium betaine.

The dispersions ("Latices") according to the invention can be prepared by polymerization or, where appropriate, copolymerization of one or more vinyl monomers of the formuLa I and, where appropriate, other vinyl monomers in the presence of ionic and/or non-ionic detergents.

The invention therefore also relates to a process for the preparation of a polymer as defined above, which comprises a compound or several compounds of the formula I and, I11 -1 4 where appropriate, another vinyL derivative or other vinyL derivatives being polymerized in an aqueous medium in the presence of an emulsifier and of a radicaL-forming initiator ExampLes of suitable emusifiers (detergents) are poLyglycoL ethers with long-chain aliphatic alcohols, which preferabLy have 10 20 carbon atoms, or aLkylphenoLs whose aLkyl radical preferably contains 6-12 carbon atoms, or diaLkyLphenoLs or triaLkyLphenols whose aLkyL radicaLs are preferably branched aLkyL radicals each having 3-12 carbon atoms. Examples of these are the products of the reaction of ethyLene oxide with Lauryl alcohol, stearyL j alcohoL, oLeyL alcohol, coconut fatty alcohoL, octylphenoL, nonylphenoL, diisopropylphenol, triisopropyLphenol, di-t- S 15 butyLphenol and tri-t-butylphenol. Products of the reaction of ethylene oxide with polypropylene glycol or polybutylene glycol are likewise suitable.

*5 5 Of the ionic emusifiers, particularly suitable are anionic emulsifiers, especially alkali metal or ammonium salts of 20 alkyLsulfonates, arylsulfonates or aLkylarylsufonates, as well as of the appropriate sulfates, phosphates or phosphonates, which optionally have oxyethylene units between the particular hydrocarbon radical and the anionic group.

Examples of these are sodium dodecyl sulfate, sodium lauryl sulfate, sodium octylphenoL glycol ether sulfate, sodium dodecyLbenzenesulfonate, sodium lauryl diglycol sulfate, ammonium tri-t-butylphenol pentaglyco sulfate and ammonium tri-t-butyphenol octaglycol sulfate. A mixture of sodium dodecyl sulfate and octylphenoxypoLyethoxyethanol (RTriton X 405) is preferably used.

It is advantageous for up to 20 by weight, based on the monomer mixture, of dimethylformamide or other substances which reduce the viscosity to be added to the mixture composed of the monomers.

The polymerization or copolymerization can be carried out ie i, by processes customary per se. However, the preferred embodiment of the process according to the invention is the metering process in which the monomer or the monomer mixture is added dropwise, with continuous stirring, to the aqueous solution of emulsifier and the radical-forming initiator under polymerization conditions, i.e. at a temperature of +100C to +1200C, preferably +50oC tO+900C.

Subsequently excess monomers and residues of initiator and emulsifier are removed from the polymer by known processes. It is advantageous to subject the polymer to dialysis, for example against NaHCO3 buffer (0.01 to 0.05 by weight).

The invention additionally relates to the use of a polymer or copolymer according to the 1 0 invention for preparing a conjugate with a biologically active substance.

Thus, in this embodiment of the invention, a dispersion polymer containing structural elements of the formula ii -CH2-CR1i-CO-NH-(CH2)n-H2 rr e as HN-R4 S. 15 in which n is 1 6, and R, is H or CH3 and, R4 denotes the radical of a biologically active substance with a free amino group, obtained by reacting a dispersion polymer composed of a compound or of several compounds of the formula i CH2=CR1-CO-NH-(CH2)n-CH(OR2)OR3 in which n is 1 6, and R 1 is H or CH 3 and R 2 and R 3 are identical or different and denote 20 -(CH 2 )m-CH 3 with m denoting 0 7 or -C with X, Y and Z denoting

(CH

2 )pCH 3 and p denoting 1 3, where X, Y and Z can be identical or different, and, where appropriate, composed of a different vinyl derivative or of other vinyl derivatives in an aqueous medium with a biologically active substance R 4

-NH

2 we ao To prepare a conjugate dispersion of this type, also called latex conjugate hereinafter, it 25 is possible to adjust the pH of a suspension of the seed-polymerized latex particles described above to a value below 5, preferably below 3, and to incubate it with the immunologically active material which is to be bound, such as, for example, an antibody or antigen. The labile bonds between an amino group of the protein and the liberated aldehyde on the latex particle according to the invention are reduced by known processes.

A solution of sodium cyanoborohydride in a neutral buffer is preferably used for this. Any unbound immunologically active material or other impurities are removed from the reaction mixture. This is expediently carried out by centrifugation or washing on suitable membranes.

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RA4/> The seed-polymerized latices according to the invention are distinguished by high stability. They are suitable for preparing especially sensitive reagents. This is important because the disadvantages of conventional reagents are evident, inter alia, from the fact that the results of nephelometric or turbidimetric measurements carried out with them do not agree well with those of an enzyme I i

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6immunoassay. A reagent for determining C-reactive protein (CRP) has been prepared as in Examples 1 and 2. Turbidimetric measurements with this reagent were carried out as in Example 3.

The CRP reagent makes possible measurements between about and 250 mg/l CRP. Thus, the diagnostically important range of measurement is completely covered. As is evident from Figure 1, there is good agreement with the results of the nephelometric assay (Na-latex-CRP reagent, Behringwerke AG). The correlation constants are: Y (turbidimetry) 0.909 X (BNA) 5.821 mg/L, r 0.967. Using the polymers according to the invention it is also possible to prepare reagents for trace protein measurements.

A reagent for the turbidimetric determination of immuno- 15 globulin E (IgE) has been prepared as in Example 4, and a reagent for the turbidimetric determination of alpha-fetoprotein (AFP) has been prepared as in Example 6. The turbidimetric measurement of IgE was carried out as in Example 5, and the turbidimetric measurement of AFP was carried out as in Example 7. The range of measurement for the IgE reagent is between 30 and 2000 IU/ml. There is good agreement for pooled sera with the results of the enzyme immunoassay (Enzygnost IgE, Behringwerke AG), as shown in Figure 2. The correlation constants are: Y (tur-.

bidimetry) 0.962 X (EIA) 11.69 IU/ml, r 0.973.

The range of measurement for the AFP reagent is between and 1500 ng/mL. Satisfactory agreement was found for serum and plasma samples with the results of the enzyme immunoassay (Enzygnost AFP, Behringwerke AG), as shown in figure 3. The correlation constants are: Y (turbidimetry) 0.965 X (EIA) 4.31 ng/ml, r 0.945.

The latex conjugates can be employed in all diagnostic methods which measure changes in particle size, for example in qualitative and semiquantitative determinations of substances with the aid of visual latex-agglutination tests, i S- 7 as well as in'nephelometric or turbidimetric determinations of trace proteins in a direct or competitive agglutination test or in a latex-hapten inhibition test.

The examples which follow illustrate the invention.

Example 1 Seed polymer 77 ml of distilled water (N2-saturated) were placed together with 2 mg of octyLphenoxypolyethoxyethanol Triton X 405, dissolved in 2 ml of distilled water) and 50 mg of 10 sodium dodecyl sulfate in the polymerization vessel, and S the oxygen was removed. 1 ml of a potassium peroxydisulfate solution, 16 mg/ml in distilled water, was also added.

A mixture of 0.4 ml of styrene, 0.4 ml of iethacrylamidoacetaldehyde di-n-pentyl acetal and 0.002 mL of methacry- 15 lic acid was prepared.

The mixture of the monomers was slowly added dropwise, in 60 minutes, to the vigorously stirred aqueous solution at 0 C. The mixture was then stirred at the same temperature for a further 5 hours. After the mixture had been 20 cooled to room temperature and filtered through a fluted filter, 78 ml of the polymer were obtained. It was subsequently dialyzed against NaHC0 3 buffer (0.25 g/l, pH 8- 8.2) for about 20 hours. 80 ml of a latex suspension with a solids content of 0.71 by weight were obtained.

Example 2 Binding of anti-CRP antibodies to a polymer according to the invention An antiserum was obtained by immunization of rabbits with purified C-reactive protein (CRP). The gamma-globulin fraction was obtained from this by an ion-exchange method using DE-32 cellulose. This fraction was employed with a Pf 1 -8protein content of about 10 mg/ml.

3.22 ml of the polymer described in ExampLe 1 were mixed with 0.1 mL of anti-CRP antibody solution. Then 0.05 mL of a 20 g/100 ml aqueous solution of eicosoxyethyene

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sorbitan Laurate Tween 20) was added, foLlowed by renewed mixing. To this mixture was added 0.05 ml of 1 N HCL so that a pH of about 2 was reached.

After an incubation time of 30 minutes at room temperature, S 0.25 m of saturated aqueousdisodiumhydrogen phosphate a 10 soLution (pH 6.5) and 0.25 mL of sodium cyanoborohydride (25 mg/ml) were added, foLLowed by thorough mixing.

9 ried out for one hour. This Loading mixture was then centrifuged at about 50,000 x g for 30 minutes (Beckman centrifuge, 20,000 rpm). The supernatant was discarded. The residue was resuspended in 2 ml of glycine/NaC buffer (0.1 M glycine, 0.17 M NaCL, 0.5 g/100 ml eicosoxyethylene

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sorbitan Laurate (RTween 20) pH 8.2).

An ultrasonic treatment (Bronson Sonyfier B 15) was subsequently carried out for 2 seconds. The reagent redispersed in this way was diluted in the ratio by volume 1:40 with 0.025 M imidazole buffer pH 8.2 (3 g/100 ml sucrose, 0.05 g/100 ml human albumin, 0.05 g/100 ml NaN 3 Example 3 Turbidimetric measurement of CRP concentrations in serum ti samples The CRP reagent prepared as in Example 2 by binding anti- CRP antibodies to polymers according to the invention was employed to measure CRP in patients' sera.

The standard used was a CRP standard serum with a CRP concentration of 26 mg/l (Behringwerke AG). The standard was i! 1 ~p~,l 9 diluted 1:10, 1:20, 1:40, 1:80, 1:160 and 1:320 with physiological saline. The patients' sera were diluted 1:100 with physiological saline.

For the measurement, 80 il of patient's serum dilution or standard serum dilution were mixed with 450 yl of a reaction buffer (0.1 M glycine, 0.17 M NaCL, 4 g/100 ml polyethylene glycol 6000, 0.5 g/100 ml RTween 20, pH 8.2) and 150 pl of CRP reagent from Example 2 in microcuvettes (1 x 0.4 cm, from Sarstedt). The extinction at a wavelength of 334 nm for the cuvettes was then measured in an S Eppendorf photometer after 10 seconds and after 3 minutes.

The measurements were calculated as the difference between the values at 10 seconds and at 3 minutes.

S The reference plot for the measurement of the standard was 15 drawn on semilogarithmic paper with concentrations on the abscissa and the extinctions on the ordinate, and the measurements for the patients' sera were evaluated thereon.

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Binding of anti-IgE antibodies to a polymer according to the invention from Example 1 An antiserum was obtained by immunization of rabbits with purified immunoglobulin E (IgE). The antibody was obtained from this by an immunoadsorption method using carrierbound IgE. It was subsequently concentrated to a protein content of about 10 mg/ml.

5.64 ml of the polymer prepared as in ExampLe 1 were mixed with 0.1 ml of anti-IgE antibody solution.

Then 0.05 ml of an aqueous solution containing 20 g/100 ml

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eicosoxyethylene sorbitan laurate Tween 20) was added, followed by renewed mixing. 0.18 ml of 1 N HCL was added to this so that a pH of about 2 was reached. After an incubation time of 30 minutes at room temperature, 0.25 ml A i

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of a saturated aqueousisodium hydrogen phosphate solution (pH 6.5) and 0.25 mL of sodium cyanoborohydride mg/ml) were added, followed by thorough mixing. An incubation at room temperature was subsequently carried out for one hour. This loading mixture was then centrifuged at about 50,000 x g for 30 minutes (Beckman centrifuge, 20,000 rpm). The supernatant was discarded.

The residue was resuspended in 2 ml of glycine/NaCI buffer (0.1 M glycine, 0.17 M NaCL, 0.5 g/100 ml eicosoxyethylene sorbitan laurate pH An ultrasonic treatment (Bronson Sonyfier B 15) was S •subsequently carried out for 2 seconds. The reagent redispersed in this way was diluted in the ratio by volume 1:20 with 0.025 M imidazole buffer pH 8.2

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15 (3 g/100 ml sucrose, 0.05 g/100 ml human albumin, 0.05 g/100 mL NaN 3 Example Turbidimetric measurement of IgE concentrations in serum samples The reagent for the determination of IgE prepared as in Example 4 by binding anti-IgE antibodies to latex preparations according to the invention was employed to measure S. g IgE in patients' sera.

The standard used was an IgE standard serum with an IgE concentration of 433 IU/mL (from Behringwerke AG). The standard was additionally diluted 1:2, 1:4, 1:8, 1:16, 1:32 and 1:64 with physiological saline. The patients' sera were diluted 1:5 with physiological saline.

For the measurement, 80 pl of patient's serum dilution or standard serum dilution were mixed with 450 pl of a reaction buffer (0.1 M glycine, 0.17 M NaCI, 4 g/100 ml polyethyLene glycoL 6000, 0.5 g/100 mL tween 20, pH 8.2) and pL of a solution of rabbit serum diluted 1:32 in NaCI solution containing 6 g/100 ml and 4 g/100 ml Tween ?0, as well as 150 pl of IgE reagent from Example 4, in 1 '9 11 microcuvettes (1 x 0.4 cm, from Sarstedt). The extinction at a wavelength of 334 nm for the cuvettes was then measured in an Eppendorf photometer after 10 seconds and after 3 minutes. The measurements were calculated as the difference between the vaLues at 10 seconds and at 3 minutes.

The reference plot for the measurement of the standard was drawn on semilogarithmic paper with IgE concentrations on the abscissa and the extinctions on the ordinate, and the measurements for the patients' sera were evaluated thereon.

o .Example 6 Binding of anti AFP antibodies to a polymer according to the invention An antiserum was obtained by immunization of rabbits with 15 purified alpha-fetoprotein (AFP). The antibody was ob- S tained from this by an immunoadsorption method using carrier-bound AFP. It was subsequently concentrated to a S protein content of about 10 mg/mL.

5.64 ml of the polymer prepared as in Example 1 were mixed 20 with 0.1 ml of anti-AFP antibody solution.

Then 0.05 mL of a 20 g/100ml aqueous solution of eicosoxyethylene sorbitan Laurate (RTween 20) was added, followed by renewed mixing. To this mixture was added 0.18 ml of 1 N HCL so that a pH of about 2 was reached.

After an incubation time of 30 minutes at room temperature, 0.25 ml of a saturated aqueous disodiumhydrogen phosphate solution (pH 6.5) and 0.25 ml of sodium cyanoborohydride mg/ml) were added, followed by thorough mixing. An incubation at room temperatures was subsequently carried out for one hour.

This laoding mixture was then centrifuged at about 50,000 x g for 30 minutes (Beckham centrifuge, 20,000 rpm).

I' i -12 The supernatant was discarded. The residue was resuspended in 2 ml of glycine/NaCI buffer (0.1 M glycine, 0.17 MNaCI, g/100 ml Tween 20 pH 8.2).

An uLtrasonic treatment (Bronson Sonyfier B 15) was subsequentLy carried out for 2 seconds. The reagent redispersed in this way was diluted in the ratio by volume 1:40 with 0.025 M imidazoLe buffer pH 8.2 (3 g/100 mL sucrose, 0.05 g/100 ml human albumin, 0.05 g/100 mL NaN 3 Example 7 lg 10 Turbidimetric measurement of AFP concentrations in serum samples

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0 The reagent for the determination of AFP prepared as in S Example 6 by binding anti-AFP antibodies to Latex preparations according to the invention (from ExampLe 1) was 15 employed to measure AFP in patients' sera.

0* The standard used was an AFP standard serum with an AFP concentration of 300,000 ng/ml (from Behringwerke AG).

The standard was diluted stepwise to 600 ng/ml in an AFPfree pooled serum. This dilution was used to prepare a standard series with decreasing AFP concentrations in the dilutions 1:2, 1:4, 1:8, 1:16, 1:32 and 1:64 with physiological saline. The patients' sera were diluted 1:5 with physiological saline.

For the measurement, 80 il of patient's serum dilution or standard serum dilution were mixed with 450 yl of a reaction buffer (0.1 M glycine, 0.17 M NaCl, 4 g/100 ml polyethylene glycol 6000, 0.5 g/100 ml RTween 20, pH 8.2) and 10 li of a solution of rabbit serum diluted 1:8 ina solution containing 8 g/100 ml NaCI and 5 g/100 ml RTween 20 as well as with 150 pl of AFP reagent from Example 6, in microcuvettes (1 x 0.4 cm, from Sarstedt). The extinction at a wavelength of 334 nm for the cuvettes was then measured I 111C iUliUWIIig SuauXnenl IS a TUII aescription OT Inis invention, iniucing tne DeSi MMwtOU OT parTorming it Knolwnl iii I Eli A -13 in an Eppendlorf photometer after 10 seconds and after 3 minutes. The measurements were caLcuLated as the difference between the values at 10 seconds and at 3 minutes.

The reference pLot for the measurement of the standard was drawn on semiLogarithmic paper with concentrations on the abscissa and the extinctions on the ordinate, and the measurements for the patients' sera were evaLuated thereon.

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Claims (9)

1. A dispersion polymer containing structural elements of the formula ii -CH 2 -CR 1 -CO-NH-(CH 2 )n-CH 2 II HN-R 4 in which n is 1 6, and R 1 is H or CH 3 and, R 4 denotes the radical of a biologically active substance with a free amino group, obtained by reacting a dispersion polymer composed of a compound or of several compounds of the formula i CH 2 =CR 1 -CO-NH-(CH 2 )n-CH(OR 2 )OR3 I in which n is 1 6, and R 1 is H or CH 3 and R 2 and R 3 are identical or different and denote -(CH2)m-CH3 with m denoting 0 7 or -C with X, Y and Z denoting (CH 2 )pCH3, and p denoting 1 3, where X, Y and Z can be identical or different, and, where appropriate, composed of a different vinyl derivative or of other vinyl derivatives in an aqueous medium with a biologically active substance R 4 -NH 2

2. A method of producing biologically active latices by reacting a biologically active substance R 4 -NH 2 with a dispersion polymer composed of a compound or of several compounds of the formula i CH 2 =CR 1 -CO-NH-(CH 2 )n-CH(OR 2 )OR3 I in which n is 1 6, and R 1 is H or CH 3 and R 2 and R 3 are identical or different and denote -(CH2)m-CH3 with m denoting 0 7 or -C with X, Y and Z denoting (CH 2 )pCH 3 and p denoting 1 3, where X, Y and Z can be identical or different, and, where appropriate, composed of a different vinyl derivative or of other vinyl derivatives in an aqueous medium.

3. A polymer as claimed in claim 1, in which the compound of the formula i is acryl- or methacrylamidoalkyl-aldehyde dialkyl acetal with 2C- to C8-alkyl in the acetal moiety.

4. A polymer as claimed in claim 1, in which the compound of the formula i is acryl- or methacrylamidoalkyl-aldehyde di-n-penyl acetal.

A polymer as claimed in claim 1, wherein the different vinyl derivative is styrene, vinylnaphthalene, vinyltoluene, methacrylic acid, acrylic acid or crotonic acid. 3 C.>i h i i i~i- preparation of a poLymer as defined above, which comprises a compound or several compounds of the formula I and, "i iA K

6. A polymer as claimed in claim 1, wherein the different vinyl derivative is a water- soluble monomer selected from the group comprising hydroxyl-substituted acrylic or methacrylic esters or amides or of water-soluble monomers with a betaine structure.

7. A method of preparation of a conjugate with a biologically active substance having a free amino group comprising reacting a dispersion polymer composed of a compound or of several compounds of the formula i CH 2 =CR 1 -CO-NH-(CH 2 )n-CH(OR 2 )OR 3 I in which n is 1 6, and R 1 is H or CH 3 and R 2 and R 3 are identical or different and denote -(CH 2 )m-CH3 with m denoting 0 7 or -C with X, Y and Z denoting (CH 2 )pCH 3 and p denoting 1 3, where X, Y and Z can be identical or different, and, where appropriate, composed of a different vinyl derivative or of other vinyl derivatives, with a biologically active substance having a free amino group.

8. A method of preparation of a conjugate with an immunoglobulin comprising reacting a polymer or copolymer as claimed in claim 1 with an immunoglobulin appropriate for formation of said conjugate.

9. A method of preparation of a conjugate with antibodies against C-reactive protein (CRP), against alpha-feto-protein (AFP) or against immunoglobulin E (IgE) comprising .:.reacting a polymer or copolymer as claimed in claim 1 with a protein appropriate for formation of said conjugate. DATED this 19th day of December, 1991. BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRAUA AU002204188.WPC 2 4