FR2676451A1 - Process for preparing a latex with surface functional groups and latex obtained - Google Patents

Abstract

Process for preparing an aqueous emulsion of a copolymer exhibiting functional groups at the surface, consisting in performing a copolymerisation between at least one monomer of vinyl or styrene-based type and a monomer bearing at least one cationic precursor group, in the presence of an initiator, in hydrolysing the cationic precursor groups in a neutral or basic medium to release the functional groups, between the copolymerisation and hydrolysis stages or not later than during the hydrolysis, an addition of at least one surface-active agent being performed to obtain, after hydrolysis, a copolymer in the form of stabilised latex. The invention also covers a dispersion of an intermediate cationic copolymer bearing at the surface precursor groups of functional groups, as well as a dispersion of the final copolymer comprising functional groups at the surface.

Description

PROCESS FOR PREPARING A GROUP LATEX
FUNCTIONAL SURFACE AND LATEX OBTAINED
The invention relates to a process for preparing a copolymeric latex having surface functional groups in a stabilized form. It also relates to the latices obtained by this method and their use for immobilizing biologically active compounds.

 By latex, within the meaning of the present invention, is meant an emulsion of a natural or synthetic polymer or copolymer, in particular an elastomer, in an aqueous medium, constituting a colloidal system.

 By surface functional group according to the invention is meant a reactive group, of the thiol, amine, imido-ester or amidine type, which is accessible and capable of interacting by physical and / or chemical bonds with a biologically active compound, the interface between the elastomer and the medium comprising said compound.

 For the purposes of the present invention, the term "biologically active compound" means any material, product or molecule having a utility, or a technical function or result, in a process, process or method, of a biochemical, biological, therapeutic, pharmacological or other nature. ..

In particular, this definition
the medicinal active principles,
biological material such as enzymes, antibodies, antigens, DNA or RNA fragments,
- nutritional cosmetics,
- phytosanitary products.

 The processes currently used to prepare a synthetic polymer having surface functional groups consist in carrying out a copolymerization, in the presence of an initiator, between at least one monomer of vinyl and / or styrenic type and a monomer having said precursor groups, that is to say groups which, in a suitable medium, release reactive functional groups.

 The biologically active compounds are then immobilized on the copolymer via its functional groups at the surface or after activation of the latter.

 The invention relates more particularly to a process for preparing a copolymer, an intermediate step of which consists in obtaining a copolymer with cationic precursor groups.

 The work of K.YAMAGUCHI et al. which are the subject of the Makromol publication. Chem., Rapid Commun., 10, 397 (1989) relate to the preparation of surface-bearing copolymers of thiol groups S-H, which find particular application in the immobilization of enzymes.

 The synthesis method described is in two stages, in which an emulsion copolymerization of styrene with isothiouronium chloride is carried out in the presence of a cationic or anionic initiator agent, and then the precursor groups of the cationic copolymer obtained are hydrolyzed in a basic medium. to release functional thiol groups. However, the process for the preparation of copolymers with surface functional groups in the presence of an anionic initiating agent leads to low yields and is not very reproducible, so that the industrial application of this process is of little interest.

 The final copolymer obtained by this process in the presence of a cationic initiator has a low thiol group content and much lower than the sulfide content of the cationic copolymer carrying the precursor groups, that is to say before hydrolysis.

This experimentally determined result by titration is the consequence of two disadvantages, essentially
the hydrolysis medium creates a destabilization of the latex resulting in an aggregation of the copolymer particles whose thiol groups become inaccessible, especially to the titration reagent
- The hydrolysis medium has difficulty accessing the isothiouronium groups inside the latex, and these are little or no hydrolysed.

 The present invention provides a solution by proposing a process for preparing an aqueous emulsion of a copolymer with functional groups at the surface, resulting in a stabilized latex in which the functional groups are obtained in high yield and are directly accessible, in particular by compounds biologically active.

The process according to the invention consists
to perform a copolymerization between at least one monomer of vinyl or styrenic type and a monomer bearing at least one cationic precursor group of a surface functional group, in the presence of a cationic or nonionic initiator,
to hydrolyze in a neutral or basic medium, the cationic precursor groups to release the functional groups, and between the copolymerization and hydrolysis stages or at the latest during the hydrolysis stage, an addition of at least one surfactant; is carried out to obtain after hydrolysis a copolymer in the form of stabilized latex.

 Surprisingly, it is observed that accessibility to the functional groups by biologically active compounds is not hindered by the presence of the surfactant on the surface of the latex.

 According to the invention, the surfactant is nonionic or cationic. It is also possible to use a mixture of nonionic and / or cationic surfactants.

The nonionic surfactant is preferentially chosen
among surfactants comprising an ester function such as glycol and fatty acid esters, esters of glycerol and of fatty acid, polyglycerol esters of fatty acids, esters of polyethylene glycol and of fatty acid sucrose esters of fatty acids, such as those commercially available under the trade names SPAN and MONTANE, sucrose and triglyceride esters, sorbitan and fatty acid esters and polyoxyethylene sorbitan esters, such as than those marketed under the brands TWEEN and MONTANOX
among surfactants comprising an ether function, such as ethers of polyoxyethylene glycol and of alkyl phenol, such as those sold under the tradenames TRITON, IGEPAL, SURFONIL and NONIDET, and ethers of polyoxyethylene glycol and of fatty alcohol, such as those known as BRIJ, LUBROL and AL
- Among the amide functional surfactants
- among glycosylated sugars such as alkyl glycosides and alkylthioglycosides
among the surfactants of general formula (I)
H- (OCH2CH2) a (OCH2CH (CH3)) b (OCH2CH2) c (I) where a, b, c, represent integers and a and c are statistically identical integers.

 According to a preferred implementation of the process, the surfactant is a polyoxyethylenated sorbitan ester and in particular polyoxyethylene sorbitan monolaurate, sold under the trademark TWEEN 20.

 According to another advantageous implementation of the process, the surfactant is t. octylphenoxypolyethoxyethanol marketed under the tradename TRITON X-405 or octylphenolpolyethyleneoxide marketed under the trademark NONIDET P 40.

dans laquelle r1, r2, et r3 chacun indépendamment représente des radicaux alkyles à chaîne courte tels que Cl, C2, C3 ; R représente un radical alkyle à longue chaîne linéaire ou ramifiée, de préférence de C6 à C25, un aryle substitué ou non substitué, tel que phényle, benzyle ou un hétérocycle ;X représente un halogène tel que iode, brome, fluor ou chlore, un groupement sulfonyle tel que éthyl et aryl sulfonyles ; parmi ces dérivés, on choisira préférentiellement l'iodure de n-octyltriméthylammonium, le bromure de n-décyltriméthylammonium, le bromure de n-dodécyltriméthylammonium et le bromure de n-hexadécyltriméthylammonium
- parmi les dérivés à azote hétérocyclique, notamment
les sels d'alkylpyridinium, de formule générale (III)

dans laquelle R représente un radical alkyle à longue chaîne linéaire ou ramifiée, de préférence de C6 à C25, un aryle substitué ou non substitué, tel que phényle, benzyle ou un hétérocycle ;X représente un halogène tel que iode, brome, fluor ou chlore, un groupement sulfonyle tel que éthyl- et arylsulfonyles
- parmi les sels d'alkylquinolinium, de formule générale (IV)

dans laquelle R représente un radical alkyle à longue chaîne linéaire ou ramifiée, de préférence de C6 à C25, un aryle substitué ou non substitué, tel que phényle, benzyle ou un hétérocycle ; X représente un halogène tel que iode, brome, fluor ou chlore, un groupement sulfonyle tel que éthyl- et aryl s ulfonyles
- parmi les oxydes d'amines, de formule générale (V):

dans laquelle R représente un radical alkyle à longue chaîne linéaire ou ramifiée, de préférence de C6 à C25, un aryle substitué ou non substitué, tel que phényle, benzyle ou un hétérocycle ;X représente un halogène tel que iode, brome, fluor ou chlore, un groupement sulfonyle tel que éthyl- et arylsulfonyles.The cationic surfactant is preferably chosen:
among the aliphatic nitrogen derivatives, such as the quaternary ammonium salts of general formula (II)

wherein r1, r2, and r3 each independently represents short-chain alkyl radicals such as Cl, C2, C3; R represents a linear or branched, preferably C6 to C25 long-chain alkyl radical, a substituted or unsubstituted aryl, such as phenyl, benzyl or a heterocycle; X represents a halogen such as iodine, bromine, fluorine or chlorine, a sulfonyl group such as ethyl and aryl sulfonyls; among these derivatives, preference will be given to n-octyltrimethylammonium iodide, n-decyltrimethylammonium bromide, n-dodecyltrimethylammonium bromide and n-hexadecyltrimethylammonium bromide.
among the heterocyclic nitrogen derivatives, in particular
the alkylpyridinium salts of general formula (III)

wherein R represents a linear or branched long chain alkyl radical, preferably C6 to C25, a substituted or unsubstituted aryl, such as phenyl, benzyl or a heterocycle; X represents a halogen such as iodine, bromine, fluorine or chlorine; a sulfonyl group such as ethyl- and arylsulfonyls
of the alkylquinolinium salts of general formula (IV)

wherein R represents a linear or branched long chain alkyl radical, preferably C6 to C25, a substituted or unsubstituted aryl, such as phenyl, benzyl or a heterocycle; X represents a halogen such as iodine, bromine, fluorine or chlorine, a sulphonyl group such as ethyl- and arylsulfonyls;
among the amine oxides of general formula (V):

wherein R represents a linear or branched long chain alkyl radical, preferably C6 to C25, a substituted or unsubstituted aryl, such as phenyl, benzyl or a heterocycle; X represents a halogen such as iodine, bromine, fluorine or chlorine; a sulfonyl group such as ethyl- and arylsulfonyls.

 According to the invention, it is possible to add several surfactants to stabilize the latex and in particular a mixture of cationic surfactants, a mixture of nonionic surfactants or a mixture of cationic and nonionic surfactants.

The amount of surfactant added for one liter of copolymer represented by QT is defined by the equation
SL
QT = Tr ~~~~~~
100.Ao in which
QT is the number of molecules of the surfactant
Ao is the specific area occupied in 2 / molecule
SL is the area developed by one liter of copolymer, expressed in m2, and defined by the following equation
6P
SL = ~~~~~~
DP with:. P equal to the weight of the copolymer in a liter of suspension, in m2
DD equal to the diameter of the copolymer particle, in μm
pp equal to the density of the copolymer, in g / l.

 The amount of surfactant is therefore a function of the size of the particles of the copolymer, the nature of the monomer carrying the precursor groups, the latex solids content, the nature of the surfactant and the ionic strength of the medium. .

 The preferential amount of surfactant to be added is in the range of QT values, for which Tr is at least 15%, preferably between 25 and 500% and especially between 25 and 75%.

 Another subject of the present invention is a dispersion of an intermediate cationic copolymer having, on the surface, precursor groups of functional groups, comprising a nonionic or cationic surfactant, the latter being preferentially chosen from the surfactants mentioned above. The amount of surfactant (s) used for one liter of copolymer is represented by QT having the same definition as above.

 By hydrolysis in a neutral or basic medium, the precursor groups of this intermediate product may be hydrolysed, releasing the desired functional groups.

 The invention also relates to a dispersion in a neutral or basic medium, of a final copolymer having on the surface, free functional groups, comprising a nonionic or cationic surfactant, preferably selected from the surfactants mentioned above. The amount of surfactant (s) used for one liter of copolymer is represented by QT having the same definition as above.

 The present invention also provides a dispersion in a neutral or basic medium of a final copolymer, that is to say carrying functional groups at the surface, supporting or attaching a biologically active compound coupled to the functional groups of the copolymer. The latter can intervene in an immunological process and be in particular an antibody fragment.

The invention is now described in more detail by the following examples, given by way of illustration but which are in no way limiting, and with reference to the appended drawing in which
FIG. 1 is a representation of the colorimetric intensity (OD) developed by the reaction between a latex referenced hereinafter SC14 and the Ellman's reagent, as a function of time, the curve a) corresponding to the SC14 latex stabilized with a surfactant. nonionic and curve b) corresponding to latex SCl4 stabilized with a cationic surfactant.

 FIG. 2 is a representation of the efficiency of the coupling reaction according to the Ellman method as a function of time, from the SC14 latex stabilized with a nonionic surfactant namely TRITON according to curve a), NONIDET P40 according to curve b) and TWEEN according to curve c).

 Figure 3 is a representation of the efficiency of the coupling of antibody fragments on SC14 latex as a function of fragment concentration.

EXAMPLE 1
Preparation of latex containing surface functional groups a) General protocol
A mixture consisting of water, monomer carrying precursor groups, styrene and, if appropriate, a mixture of water, is introduced into a reactor equipped with a heating jacket, a condenser, a mechanical stirrer and a temperature probe. magnesium sulfate heptahydrate, in appropriate amounts, and the mixture is allowed to stir until the temperature stabilizes at about 70 ° C. A specified amount of a cationic initiator is then introduced into the reactor which causes the start. polymerization and the reaction continues until complete consumption of styrene The kinetics of polymerization is followed by successive samplings over time.

b) Preparation of intermediate latex containing precursor groups
thiol function
0.5 g of monomer bearing precursor groups, namely vinylbenzylisothiouronium chloride, are dissolved in 175 g of water and 40 g of styrene (Janssen Chimica, France) are added to the mixture obtained. The mixture is introduced into a reactor of the type described above and stirred at 350 rpm until the temperature reaches 70 ° C. Once the temperature is stabilized at 70 ° C., a solution of 0.14 g of 2,2'-azobis (2-amidinopropane) dihydrochloride as initiator in 5 ml of water. After two hours of polymerization, particles of 66.4 nm are obtained. The conversion is 100% and the solids content of the latex is about 20%.

The protocol as described above is repeated by halving the amount of monomer carrying precursor groups and adding 0.1 g to 0.7 g of magnesium sulfate heptahydrate. At the end of the polymerization, particles whose diameter increases with the ionic strength, from 100 nm to 274 nm, and a latex having a solids content of approximately 20%, are obtained.

c) Preparation of intermediate latexes containing precursor groups
amine function
175 g of water are dissolved in 0.37 g of monomer bearing precursor groups, namely aminomethyl-styrene hydrochloride and 0.02 g of magnesium sulfate heptahydrate. The mixture is introduced into a reactor and stirred at 350 rpm until the temperature has stabilized at 70 ° C. 40 g of styrene (Janssen Chimica, France) are then introduced and the temperature of the mixture stabilizes at 70 ° C. 0.14 g of initiator dissolved in 5 ml of water are then introduced to trigger the polymerization reaction. At the end of the polymerization, particles of 265 nm are obtained and the latex has a solid content of about 20%.

EXAMPLE 2
Obtaining stabilized end-latex after addition of surfactants and hydrolysis
Latexes prepared by copolymerization of styrene and a monomer bearing precursor groups, according to the method described in Example 1, were reduced by concentration to a solid percentage of 0.1 to 20%.

To these latex suspensions were added various amounts of surfactants depending on the size of the particles, the nature of the monomer bearing precursor groups, the level of solid to obtain a recovery rate ensuring the stability of the final latex . The surfactant used is a nonionic surfactant, namely t-octylphenoxypolyethoxyethanol marketed under the Trade Mark TRITON X-405 (TX405), in the form of a 70% aqueous solution of Sigma.
Chimica Company. The solutions were stirred for four hours at room temperature. The results are shown in the tables below.

<tb><SEP> (1) <SEP> (8 <SEP> r3} <SEP> [4) <SEP> 0 <SEP> (3)
<tb> Latex <SEP> M <SEP> (5o) <SEP> Function <SEP><SEP> Size of <SEP> Rate <SEP> of <SEP> SL <SEP> SO <SEP> Surfactant <SEP> T
<tb><SEP> S <SEP> particles <SEP> soften <SEP> (m2) <SEP> (m2)
<tb><SEP> SC1 <SEP> 5.50 <SEP> Thiol <SEP> 57 <SEP> 10 <SEP> 10526 <SEP> 4214 <SEP> 10 <SEP> 40
<tb><SEP> Suc14 <SEP> 1.46 <SEP> Thiol <SEP> 256 <SEP> 10 <SEP> 2344 <SEP> 842 <SEP> 2 <SEP> 36
<tb> scls <SEP>1;; 46 <SEP> thiol <SEP> 262 <SEP> 10 <SEP> 2290 <SEQ> 842 <SEP> 2 <SEP> 37
<tb> SC35 <SEP> 1.50 <SEP> Thiol <SEP> 1 <SEP> 700 <SEP> 5 <SEP> 429 <SEP> 842 <SEP> 2 <SEP> 200
<tb> NC3 <SEP> 0.92 <SEP> Amine <SEP> 265 <SEP> 10 <SEP> 2264 <SEW> 1684 <SEP> 4 <SEP> 74
<tb> NC5 <SEP> 1.50 <SEP> amine <SEP> 424 <SEP> 5 <SEP> 708 <SEP> 2107 <SEP> 5 <SEP> 300
(1) SC1, SC14, SC15 and SC35 resulting from the copolymerization of styrene with a monomer carrying thiol precursor groups
NC3 and NC5 result from the copolymerization of styrene with a monomer bearing amine functional precursor groups (2) M is the concentration of monomer with precursor groups (g / l)
S is the concentration of styrene (g / l) (3) SL and Tr have the same definitions as before (4) SO is equal to the area occupied by the surfactant (m2)
In the same manner, the SCl4 latex diluted to 10% solids was stabilized.
on the one hand, with 4 g / l of a cationic surfactant, namely hexadecyltrimethylammonium bromide (Fluka Chemica AG)
- on the other hand, with a nonionic surfactant: three different nonionic surfactants respectively t-octylphenoxypolyethoxyethanol (TRITON-405), octylphenolpolyethyleneoxide (NONIDET
P40 from BDH Chemicals Ltd) and polyoxyethylene sorbitan monolaurate (TWEEN 20 from Biorad) have been experimented.

EXAMPLE 3
Demonstration of the restoration of surface functional groups after hydrolysis of precursor groups a) Thiol group
To show the restoration of the thiol groups after hydrolysis, the Ellman method, as described hereinafter, is applied to the SC14 latex prepared according to Example 1.

To 4.5 ml of a 50 mM buffer was added 0.5 ml of the SC14 latex stabilized with a nonionic surfactant according to Example 2 and 0.1 ml of a 4 mg / ml solution of acid. 5,5'-dithiobis-2-nitrobenzoic (Ellman's reagent). To determine the kinetics of the reaction, 0.8 ml of aliquots were removed and centrifuged at 16,000 g in an Eppendorff 5415 centrifuge. The optical density (OD) of the supernatant was measured at 412 nm compared to a blank , which is a 0.1 ml dilution of Ellman's reagent in 5 ml of buffer. The yield of the coupling reaction between the thiol group and a 5-thiol-2-nitrobenzoic acid molecule is the ratio of the OD of a mixture of 0.1 ml of mercaptoethanol and 0.1 ml of reaction reagent. Ellman in 4.9 ml of buffer.

dans lequel A représente le réactif d'Ellman, B correspond au latex, C est le composé disulfure formé et D est le composé coloré.This method will make it possible to show the availability of the thiol groups and to highlight the importance of the type of surfactant used for the stabilization. In this method, the ability of the Ellman's reagent to react with the surface sulfhydryl groups of the latex is used to yield a disulfide compound and a colored compound as shown in the rational scheme below.

wherein A is Ellman's reagent, B is latex, C is the disulfide compound formed, and D is the colored compound.

As the reaction continues, the yellow color of the supernatant intensifies and the OD of the supernatant can be measured on a spectrophotometer at a wavelength of 412 nm. As is apparent from the reaction scheme above, the developed color intensity is proportional to the disulfide compound formed and therefore to the free thiol groups on the surface of the latex.

The same technique was used to test the stabilized SC14 latex with a cationic surfactant, hexadecyltrimethylammonium bromide.

For each of the two stabilized latexes, one with the nonionic surfactant, the other with a cationic surfactant, the evolution of the colorimetric intensity developed as a function of time was studied.

Thus, according to FIG. 1, the curve a) represents the colorimetric intensity developed as a function of time for a SC14 latex stabilized with Triton.
X-450 nonionic. This curve thus confirms the restoration of thiol surface groups of the latex after hydrolysis. Curve b) corresponds to the same representation for a SC14 latex stabilized with hexadecyltrimethylammonium bromide (cationic).

The comparison between curves a) and b) of FIG. 1 shows that in the case of stabilization with a cationic surfactant, in this case hexadecyltrimethylammonium bromide, the signal is lower than that obtained with a nonionic surfactant such as Triton. This means that the nonionic surfactants are neutral stabilizers against the coupling reaction, whereas the cationic surfactants seem to be inhibitors of this reaction.

According to FIG. 2, are reported the results of a study on the behavior of different surfactants belonging to the same group of nonionic surfactants, on the stabilization of SC14 latex: the curve a) corresponds to the
TRITON, curve b) to NONIDET P40 and curve c) to TWEEN. This figure shows a better coupling efficiency from TRITON stabilized SC14 latex, which would allow a fast and high performance of the functional groups.

b) Amine groups
The availability of amine functions after stabilization of amino latex with Triton X 405 has been demonstrated, according to the protocol described below, which is an adaptation to latex of the method described by TT Ngo (Journal of Biochemical and Biophysical Methods 12,349 ) (1986).

To 0.5 ml of 5% latex is added 0.5 ml of carbonate buffer (pH 8.2, 0.1 mM), 10 μl of a 0.5 M solution of N-succinimidyl 3- (2%). -pyridyldithio) propionate (SPDP) in dioxane and 1 grain of 4-dimethylaminopyridine (DMAP). It is stirred for 100 minutes at room temperature, then the latex is centrifuged and the supernatant is recovered. The latexes are resuspended in 1 ml of carbonate buffer (pH 8.2, 0.05 mM) and centrifuged again. The supernatant is conserved and the pellet is resumed as before. To each supernatant and the latex suspension was added 0.1 ml of a solution of dithiothreitol (DTT) (0.5 mM). It is stirred for 30 minutes at room temperature, the latexes are centrifuged and the supernatants are recovered. The three supernatants of each latex are diluted 1/20 in a carbonate buffer (pH 8.2, 0.1 mM) and the density is read. optical at 343 nm against a blank containing the same amount of DTT.

The same treatment is carried out with SPDP previously hydrolysed with 10 μl of ethanolamine to evaluate the value of the possible adsorption of the reagent by the latex.

The reaction scheme is described below

Pyridine-2-thione is an indicator that absorbs light with a wavelength of 343 nm.

The results for the previously described latexes NC5 and NC3 are respectively 70 μg / g of dry latex and 40 μg / g of dry latex. This represents in each of these cases, about 70% availability compared to the amounts introduced during the synthesis of the latex.

c) Hypothesis on the stabilizing role of surfactant in latex
The nonionic or cationic surfactants have a hydrophobic part and a hydrophilic part. The surfactants by their hydrophobic part adsorb on the surface of the copolymer by hydrophobic interaction.

When nonionic surfactants are used, the stabilization of the copolymer particles would be due to a steric hindrance phenomenon. In other words, when the nonionic surfactant molecules are adsorbed on the surface of the copolymer particles by their hydrophobic part, their hydrophilic tail occupies a certain space of the aqueous medium, preventing any approach of other stabilized copolymer particles. This prevents the aggregation of the copolymer particles.

When cationic surfactants are used, their hydrophilic tail carries positive charges and the aggregation of the copolymer particles would be prevented by electrostatic repulsion of the positive charges therebetween.

EXAMPLE 4
Use of stabilized latexes according to the invention for immobilizing proteins
As an example for this application, we will use Fab fragments obtained from polyclonal anti-protein C reactive C (Biomérieux, France) digested with pepsin at 37 "C to give the fragment F (ab ') 2 The reduction of these fragments with mercaptoethylamine provides crude Fab fragments The purification was completed by HPLC (Kontron Instruments) after concentration.

a) Coupling anti-reactive protein C antibody fragments 0.1 ml of a 10% solids suspension of stabilized SC14 latex according to the invention was added to a solution of antibody fragments in a Tris buffer ( pH 9.2, 50 mM) to obtain a 1% solids solution. The flasks were rotated for 4 hours at room temperature.

After centrifugation, the supernatants were removed and the latexes were resuspended in 0.15 M BSA 10 g / l BSA (bovine serum albumin), and a 50 mM glycine buffer. The amount of protein remaining in the supernatants was measured using the Bradford test. The difference between the initial concentration and that in the supernatants gives the amount of Fab fragments immobilized on the latex particles.

The results of a study on the efficiency of coupling between antibody fragments and TRITON-stabilized SC14 latex as a function of fragment concentration are shown in FIG. 3.

They show that over this concentration range, the saturation of the latex is not reached.

With conventional latices, during the immobilization of antibody Fab fragments, either by covalent bonding or by passive adsorption, the latices flocculate a great deal. With the latexes of the invention, stable colloles are obtained.

b) Immunologic activity of the latices according to the invention
The evaluation was made with Fab-peroxidase conjugate and ortophenylenediamine as substrate. The detection of the enzymatic reaction was made by reading the OD at 492 nm. The immunological activity of bound antibody fragments was measured. The sensitivity corresponds approximately to a concentration of 500 μg / l of antigens (Protein
C-reactive). In practice, in diagnostic tests, it is not necessary to reach such a low level for this parameter.

Claims (13)

 1 / A process for preparing an aqueous emulsion of a copolymer having functional groups at the surface consisting of  to carry out a copolymerization between at least one monomer of vinyl or styrene type and a monomer bearing at least one cationic precursor group, in the presence of an initiator,  to hydrolyze in a neutral or basic medium, the cationic precursor groups for releasing the functional groups, characterized in that between the copolymerization and hydrolysis stages or at the latest during the hydrolysis stage, at least one agent is added; surfactant to obtain after hydrolysis a copolymer in the form of stabilized latex.  2 / A method according to claim 1, characterized in that the surfactant is nonionic and is chosen from  surfactants comprising an ester function, such as glycol and fatty acid esters, glycerol esters of fatty acid, polyglycerol esters of fatty acid esters of polyethylene glycol and of fatty acid, sucrose and fatty acid esters, sucrose and triglyceride esters, sorbitan and fatty acid esters and polyoxyethylenesorbitan esters  surfactants comprising the ether function, such as polyoxyethylene glycol ethers and alkyl phenol ethers and polyoxyethylene glycol ethers and fatty alcohol ethers;  surfactants with amide function  glycosylated sugars such as alkyl glycosides and alkylthioglycosides  surfactants of general formula (I)  H (OCH2CH2) a (OCH2CH (CH3)) b (OCH2CH2c OH (I) wherein a, b, c, represent integers and a and c are statistically identical integers.  3 / A method according to claim 2, characterized in that the surfactant is a polyoxyethylenated sorbitan ester, and in particular polyoxyethylene sorbitan monolaurate.  4 / A method according to claim 2, characterized in that the surfactant is t. octylphenoxypolyethoxyethanol or octylphenolpolyethyleneoxide.  5 / A method according to claim 1, characterized in that the surfactant is cationic and is chosen from  aliphatic nitrogen derivatives, such as quaternary ammonium salts of general formula (II)

 wherein each of R 1, R 2, R 3 independently represents short-chain alkyl radicals such as C 1, C 2, C 3; R represents a linear or branched, preferably C6 to C25 long-chain alkyl radical, a substituted or unsubstituted aryl, such as phenyl, benzyl or a heterocycle; X represents a halogen such as iodine, bromine, fluorine or chlorine, a sulfonyl group such as ethyl and aryl sulfonyls,  heterocyclic nitrogen derivatives, in particular  the alkylpyridinium salts of general formula (III)

 wherein R represents a linear or branched long chain alkyl radical, preferably C6 to C25, a substituted or unsubstituted aryl, such as phenyl, benzyl or a heterocycle; X represents a halogen such as iodine, bromine, fluorine or chlorine, a sulphonyl group such as ethyl and aryl sulphonyls,  the alkylquinolinium salts of general formula (IV)

 wherein R represents a linear or branched long chain alkyl radical, preferably C6 to C25, a substituted or unsubstituted aryl, such as phenyl, benzyl or a heterocycle; X represents a halogen such as iodine, bromine, fluorine or chlorine; a sulphonyl group such as ethyl and aryl sulphonyls,  the amine oxides of general formula (V)

 wherein R represents a linear or branched long chain alkyl radical, preferably C6 to C25, a substituted or unsubstituted aryl, such as phenyl, benzyl or a heterocycle; X represents a halogen such as iodine, bromine, fluorine or chlorine, a sulphonyl group such as ethyl and aryl sulphonyls.  6 / A method according to claim 1, characterized in that one adds several cationic and / or nonionic surfactants.  7 / A method according to any one of claims 1 to 6, characterized in that the amount of surfactants added to one liter of copolymer is represented by QT defined by the equation  SL  QT = Tr.  SL is the surface developed by one liter of copolymer (m2) the recovery ratio Tr is at least 15%, preferably between 25 and 500% and advantageously between 25 and 75%.  Ao is the specific area occupied by a surfactant molecule (A2 / molecule)  Tr is the rate of recovery of the copolymer by the surfactant  QT is the number of molecules of the surfactant  100.Ao in which  8 / An aqueous dispersion of an intermediate cationic copolymer having at the surface precursor groups of functional groups, characterized in that it comprises a nonionic or cationic surfactant.  9 / Dispersion according to claim 4, characterized in that the amount of surfactant used for one liter of copolymer, represented by QT, is defined by the equation  SL  QT = Tr.  SL is the surface developed by a liter of copolymer, (m2) the recovery ratio Tr being at least 15%, preferably between 25 and 500%, and especially between 25 and 75%.  Ao is the specific area occupied by a molecule of surfactant (A2)  Tr is the rate of recovery of the copolymer by the surfactant  QT is the number of molecules of the surfactant  100.Ao  10 / An aqueous dispersion in a neutral or basic medium of a final copolymer having functional groups at the surface, characterized in that it comprises a nonionic or cationic surfactant.  11 / Dispersion according to claim 6, characterized in that the amount of surfactant used for one liter of copolymer is represented by QT defined by the equation  SL  QT = Tr.  SL is the surface developed by one liter of copolymer in m 2, the degree of recovery Tr being at least 15%, preferably between 25 and 500% and advantageously between 25 and 75%.  Ao is the specific area occupied by a molecule of surfactant, in R2 / molecule  Tr is the rate of recovery of the copolymer by the surfactant  QT is the number of molecules of the surfactant  100.Ao in which  12 / dispersion according to claims 10 or 11, characterized in that it supports a biologically active compound coupled to the functional groups of the copolymer.  13 / Dispersion according to claim 12, characterized in that the biologically active compound is an antibody fragment.