MXPA98004040A - A particle resistant to storage, in particular a carrier for united reactions to carrier and the processes for the production of the - Google Patents

Abstract

A storage resistant particle of at least one first and at least one second component is described, wherein - the second component of at least one crosslinkable polymer as a cover or protective layer, envelops and / or at least partially encloses the first component as a core and - the first component has at least one valuable property, obtainable - by reaction of the first component with the crosslinkable polymer, and subsequently the reaction of the formed product with a crosslinking agent, such that the first component with storage resistance remains within of the second make up

Description

A PARTICLE RESISTANT TO STORAGE, IN PARTICULAR A CARRIER FOR UNITED REACTIONS TO CARRIERS AND THE PROCESSES FOR THE PRODUCTION OF SAME DESCRIPTION OF THE INVENTION The object of. The present invention is a storage-resistant particle, especially a carrier for carrier-bound reactions, to the detection and / or isolation processes, a process for the production of this particle resistant to storage, and the uses of the particle of according to the invention.

Particulate carriers consisting of a solid core and a polymer are known - as such, they have already been used for many purposes. In particular, such particles have been used for immunoassay markers, for linking to a solid core by means of polymers, for example, biopolymers, to an affinity component. As particulate material, especially the metal particle sols have been used, for example, REF: 27550 magnetic iron particles or colorful gold particles, non-metallic particles, for example, selenium, coal dust, Si02 or ceramic particles, corpuscles or even latex particles of another polymer with different properties (in it).

Thus, US Pat. No. 4,230,685 describes an improvement in the binding of the specific binders to the magnesium particles, whereby a particle is coated with an acrylate polymer or a polysaccharide, whereby protein A is bound to this coating by glutaraldehyde.

U.S. Patent 4,452,773 describes the production of magnetic iron microparticles-dextran. These particles have a size of 100 to 700 A, in particular 300 to 400 A. A greater number of the particles are colloidal and ferromagnetic, with a cover or protective layer of dextran. The particles obtained are functionalized by oxidation with periodate.

European Patent EP-B-452,342 relates to super-magnetic particles coated with polysaccharides having colloidal size. The particles can be attached to the additional groups. By selecting the mixture of particles in their size fractions having uniform magnetizations, compositions have been obtained which have homogeneous properties with respect to the delay thereof within a magnetic field. The possibility to separate with respect to size is also mentioned. Coatings from polysaccharides or proteins are preferred. Polysaccharides are regulated by oxidation with periodate. In addition, functionalization with bromocyanogen is possible. The binding of the protein shell or protective layer to particular molecules can be effected by side chain amino groups or sulfhydryl groups.

German Patent DE-A-40 37 724 relates to devices for immunoassays in which direct or indirect markers are used. Direct markers are preferred, since they do not require additional steps to visualize the test results. Examples for direct labels are metallic sols, coloring sols, latex particles, color indicators, dyes located in liposomes, and non-metallic sols such as a carbon sol. Yet another aspect of German Patent DE-A-40 37 724 relates to an immunochemically active marker. Subsequently, a particle of mineral carbon powder is adsorptively bound to a ligand or a ligand conjugate. The sensitivity of the test, for example, human chorionic gonadotropin hCG is denoted as 25 mIU / ml (Ul = international unit).

European Patent EP-A-0, 410, 893 describes a process for the determination and recognition of an antibody within biological fluids, directed against a specific antigen. The listed insoluble carrier particles comprise cells, gelatin particles, microcapsules, organic polymers, inorganic fine particles, or colloidal metal particles or metal compounds that are finely dispersed with bovine serum albumin or cholesterol.

Also European Patent EP-A-0, 032, 270 describes a quantitative and / or qualitative determination of an immunological component using one or several labeled compounds that are obtained by a direct or indirect coupling of such component or components to particles originating from a aqueous dispersion of a hydrophobic dye or pigment or polymeric cores that are coated with such dyes or pigments.

European Patent EP-0,321,008 relates to a process for the determination of one or several components of a reaction between a specific binding protein and the corresponding bindable substance, within a sample, the mutual activity of such components and of at least one marked component that is obtained, by coupling or adsorption of the sol particles of the marking directly or indirectly to the component. The preferred sol particles are phosphorus, carbon and / or silicon. Agglutination and aggregation can be prevented by coating the particles with macromolecules containing polar groups such as proteins, poly (ethylene glycol), polymeric carbohydrates, poly (vinyl alcohols), and the like. Suitable protective proteins are antigens, antibodies, and anti-antibodies. Immunochemically inert materials such as for example albumin, poly (ethylene glycol), or other polar macromolecules can also be used. The sensitivity of the test, for example, with respect to rabbit immunoglobulin G, is specified as 1.5 ng absolute.

European Patent EP-0,298,368 relates to a process for carrying out a diagnostic immunoassay by using particles containing colloidal metal iron, which have a conjugate coupled thereto, which is capable of specifically recognizing the analyte to be determined . The sensitivity of the test for hCG is in the range of mUI / ml.

European Patent EP-A-0, 280, 560 discloses antibodies against Streptococcus A antigen that are covalently bound to the core / shell polymer particles, the shell being formed from poly (mp-chloromethylstyrene) and the nucleus from poly (styrene-co-2-acetoacetoxyethyl) methacrylate containing Oil Red EGN within the core, to obtain an agglutination reagent.

U.S. Patent 4,452,886 describes the polymerization of lysine with glutaraldehyde and Congo red to colored particles. The sensitivity of the test for hCG is in the range of 1000 Ul / l.

.P. Collins describes in his book "Alternative Immunoassays," John Wiley & Sons, Chichester, New York, etc., in the chapter "Disperse Dye Immunoassay (DAY)" on page 48 - 49 the advantages of particulate markers and points out the significance of essential parameters such as particle size, distribution of the form, the solubility in organic solvents, the stability of the colloid, and the binding capacity, ppr example, for antibodies. The sensitivities of the tests mentioned by him are in the range of mUI / ml. The conjugates are stable for at least 15 months if they are stored in the lyophilized state at -20 ° C or 4 ° C; the aqueous conjugates must be consumed within 6 days after the preparation.

Japanese Patent Jp-A-0686771 refers to the encapsulated organic pigments and the production thereof.

The inert cores described in the art have suffered from obvious drawbacks. • In the case where the nuclei are polymerized in these, the desired property of the nucleus is deteriorated too much. In the case that the polymer is absorbed onto the nucleus with a subsequent covalent bond or other type of binding of the affinity component, the bond of the core in the majority of cases is too weak, so that the stability is not secured to a longer term. Furthermore, by using the methods according to the technique only the binding of one or a small amount of reagent and the affinity components is achieved.

The object of the invention is to provide stable particles with characteristic usable properties, especially marking properties, which allow, if necessary, numerous reactive components at the same time to be also stably bonded thereon. Stability must also include, in particular, storage at longer terms.

The present invention provides a storage resistant particle, especially a particulate carrier according to the features of claim 1. Dependent claims 2-18 refer to the preferred embodiments according to the particle of the invention. Claim 19 together with the appropriate dependent claims 20-22 relate to a process for the production of the particle according to the invention, resistant to storage. Claims 23-31 relate to the uses of the particle according to the invention.

The particle according to the invention, resistant to storage is especially intended for reactions linked to a carrier, for detection and / or isolation processes. This consists of at least one first and second components, the particle being suitable to be provided with a reactive component, especially a specific reactive component. The second component consists of a crosslinkable polymer and forms more or less a protective sheath or layer and / or encloses the first component as the core, at least partially.

The first component has at least one assessable property. Additional reactive components can be accommodated on the second component. The storage resistant particle is characterized by its manufacturing process and is available by reaction of the first component with the crosslinkable polymer, after which the formed product is reacted with a crosslinking agent such that the first component is accumulated within the second component resistant to storage.

Preferably, the reaction conditions are adjusted such that the first component is wrapped and / or enclosed by the second component in a crosslinked manner.

The second component is formed the carrier according to the invention is preferably a polymer having active or activatable functional groups, which are capable of reacting in one aspect with the crosslinking agent or even with the reactive components or both. The polymer forming the second component of the carrier according to the invention can also be produced from various crosslinkable polymers and / or crosslinkable monomers. Subsequently, the polymer can be produced from proteins and / or polyamides with functional groups. In principle, other polymers such as biopolymers or monomers are also possible, as long as the crosslinking can be achieved. Preferably, the second component is crosslinkable with at least bifunctional compounds.

Preferably, polymers are used which stabilize the aqueous suspension of component 1 adsorptively, i.e., especially polar polymers.

The reactive components accommodated in the second component of the particle according to the invention, are especially molecules or molecular groups that possess affinity properties towards other substances. These include especially enzymes and substances that interact with the enzymes. Subsequently, for a. side the substrate or on the other hand, the enzyme, can be accommodated in or on the second component, as the reactive component. Antibody / antigen, biotin / streptavidin behave in the same way, Streptavidin or biotin in the form of a specific reaction component, stably bindable to the dye, has the advantage that the dye is universally usable as a co-reactant for biotinylated components, or those coupled with streptavidin. These can, on the other hand having completely different functions, for example, acting again specifically against another component or as a catalyst. In addition, the nucleic acids of the RNA or DNA types are usable within the meaning of the carrier according to the invention. The nucleic acids are capable of hybridizing with the corresponding complementary or partially complementary strands, depending on the requirement of the reaction conditions and to form stable complexes. Consequently, it is possible to identify the specific strands of nucleic acids within a sample to be investigated and the additional process based on the same. In addition, the combinations of. the reactive components can be accommodated in or on the second component. Subsequently, according to the investigations, we obtain carriers that are applicable for different problems.

Through the selection of the crosslinking agent and the concentration thereof and the adjustment of the specific processing conditions, the crosslinking reaction can be controlled such that the second component surrounds the first component more or less in a molecular network, the meshes of the they have a narrow mesh size distribution.

At the same time, by means of the crosslinking processing conditions, the number of free binding sites on the surface of the particle can be adjusted, which remain free due to the binding of at least the bifunctional crosslinking agent with only one functional group to the surface by an additional bond, especially of reactive components. The created number of binding sites can be used completely or partially with a defined portion after a partial saturation.

In particular, by means of the processing conditions the thickness of the coating layer can be adjusted, with which the homogeneity thereof is further increased, by means of a fractionation. A multi-step coverage process can also improve the inclusion of the first component.

The first component of the particle according to the invention does have at least one component 2 of evaluable property and / or the reactive components joined thereon do not possess, with respect to the quality thereof, such an absorptivity or emissivity of the electromagnetic waves. , mass, magnetism, dielectricity, radioactivity, size and / or density.

The core as the first component of the carrier according to the invention may possess evaluable properties, there also being a pharmacological-biological effect as well as a catalytic effect or combinations of these. Thus, for example, the absorptivity or emissivity of electromagnetic waves can be produced by respective chromophores or fluorophores. The properties of mass, size and density are physically interconnected, and can be adjusted accordingly by, for example, particles with a higher specific density. The property of size can be used advantageously for the agglomeration and the property of mass for gravimetry. The agglomeration is advantageous for the specific precipitation of the components in solutions or microemulsions. The first component can be provided with the property of radioactivity by radioactively labeled structures. Similarly, the other specified properties may be related to the first component. The first component can also be a capsule containing liquids or particles that can not be sufficiently enclosed (eg particles that are extremely small or unstable in solution).

In an advantageous design of the carrier according to the invention, the carrier is such that the first and second components are separately connected to one another. The connection between the first and second components is effected by enclosing the first component within the second. Basically, it is possible to expose the first component by eliminating, if necessary, the second component to facilitate a detection of properties related to the core, by means of measurement techniques. The removal of the second component can be carried out, for example in the case of proteins, by appropriate proteolytic enzymes. These degrade the cover or protective layer such that the core remains. The latter can also be treated according to their property to be measured. In addition, it is possible to solve the core of the connection of the first / second component by treating it with a medium where the core preferably dissolves, so that in the end an "empty" protective layer or layer remains, or dissolving the complete particle by means of a total solvent. Thus, for example, if an ethanol-soluble dye is enclosed by the second component, it is possible to treat with the solvent and measure the ethanolic solution obtained spectroscopically. If the conditions are standardized and, if necessary calibrated, a corresponding discoloration can be used as an amount for quantitative determinations.

In addition to the individual particulate carriers resistant to storage, the embodiments of the present also have a great significance in the realization of application methods. Then, the particulate carriers resistant to storage are mostly in the form of populations. For the application of the particles according to the invention, it is preferred that the carriers are present in populations that are distinguished by a narrow size distribution of the individual carrier particles according to the invention.

It is especially preferred that the population possess carrier particles that also have a narrow range of size distribution of protective layer, as well as a narrow range of core size distribution and consequently a narrow range of the size distribution of the protective layer. core. Also for other properties a homogeneous distribution is desired, for example, of the intensity of color or of the magnetization capacity, for the core, especially for the whole particle. In the practical use of the particle according to the invention as a carrier, it is advantageous to accommodate a high concentration of reactive components in or on the second component. Because of this, relatively high binding constants can be achieved for the corresponding complementary structures in the immunoassays, although the individual reactive component and its corresponding complementary structure possibly possess only an average binding constant. However, due to the multiplicity of link sites, the effectiveness of the link (avidity) is increased. A process for the production of the carrier according to the invention comprises the steps of reacting the first component with a crosslinkable polymer at least once. Subsequently, the polymer can be physically adsorbed on the surface of the first component. The crosslinkable polymer then, subsequent to the additional processing steps, will form the second component. This is effected by a treatment with the crosslinking agent. Possible bi-functional molecules such as glutardialdehyde, dicarboxylic acids, acrylates, methacrylates are possible crosslinking agents. Subsequently, crosslinking agents having olefinic groups are activatable by, for example, photoreactions or other chain reactions by free radicals. Functional molecules can crosslink the polymers in particular via condensation or addition reactions.

Optionally, the first component before the treatment and / or the intermediate product together with the second component and / or the particle, can be subject to a separation by size and / or by another property, to achieve a size and / or another property as homogeneous as possible. It is preferred to carry out the treatment of the first component with the polymers two to three times, and it is especially preferred to perform a size separation.

The process is particularly advantageous since it is possible to homogenize the first component and adjust the concentration thereof within the suspension initially, independently of a reaction. Depending on the first component used, standard processes for the homogenization and stabilization of the suspension can be used. Preferably, the second component is used to stabilize the suspension if it is a polar component with sufficiently good adsorption in the first component. The proportion of the first and second concentrations is selected such that an adequate thickness of the protective layer is achieved by adsorption. In the multi-step process the thickness of the cover or protective layer is preferably selected thin. Other parameters such as time and temperature can also be used to influence adsorption. After that, the intermediate product can be re-homogenized. In order to achieve a good crosslinking of the cover or protective layer, it is especially within the internal area close to the first component, the crosslinking component is added in an excess of several orders of magnitude. In addition, the effect of the crosslinking component can be influenced by other parameters, in particular the contact time as well as by the number of reactive groups formed on the surface, due to * the crosslinking molecules joined by a single side, whose number can be reduced by subsequent partial blocking.

The carriers according to the invention are preferably used in assay techniques such as immunoassay, solid phase assays and / or chromatographic assays. In addition, these can be used as the vehicle for affinity transport of effective substances, if for example pharmacologically active substances such as the first component are enclosed by the second component. By affinity transport one must understand, for example, the transport of pharmacologically active substances by means of antibodies which are located in the second component and which specifically bind to a particular target structure.

They can also be advantageously used particles with a radioactive core, since these act or concentrate due to affinity transport, above all, at the application site, and therefore lower total radioactivity doses can be used, with which a systemic tension can be maintained within narrow limits.

The use of the storage-resistant particulate carriers, according to the invention, as a catalyst for chemical reactions is also advantageous. Accordingly, for rapid intermixing of the catalyst bound to the carrier (biocatalyst) with the reaction mixture, a magnetic core can be used, the weight of which subsequently provides a rapid separation.

The invention will be further illustrated by the following examples.

EXAMPLE 1 Preparation of a Sudan IV dye particle having a high streptavidin load as the specific reactive component for the biotinylated components Peptization .0 g of Sudan IV were added to 100 ml of PBS buffer (Phosphate Buffer Solution) with 2% BSA (Bovine Serum Albumin) sterilized by filtration above 2 μm and stirred at high agitation speed to produce a suspension homogeneous After that, the suspension was filtered on wide pore paper (Biorad filter paper, model 543).

The suspension was distributed over two tubes for centrifuge and centrifuged in a centrifuge previously cooled at 4 ° C at 1000 rpm, at 2000 rpm, and at 3000 rpm for 5 minutes, respectively. Subsequently a second filtration was performed. 2. Cross-linking and activation 50 ml of a 50% aqueous solution of glutaraldehyde were brought to room temperature (hood) and adjusted to pH 7.3 with approximately 11 ml of 0.3 M Na2HP0 buffer (verification with potentiometer). Subsequently 25 ml of PBS buffer and 5 ml of a 1: 1 mixture of PBS buffer and bidistilled water were added (in the reaction solution approximately 1.5 M of glutaraldehyde or 2.8 M of aldehyde groups, approximately 1.5 x 10"4 M of BSA or with 300 amino acids per BSA, approximately 4.5 x 10 ~ 2 M reactive amino acid, ie approximately 60 aldehyde groups per amino group.) The complete solution (pH 7.3) was sterilized by filtration (0.2 μm).

To 20 ml of this glutaraldehyde buffer, respectively, 20 ml of the Sudan IV peptide suspension (4 preparations in parallel), respectively, were added with turbulent mixing (Vortex), and stirred for 4 hours at room temperature. 40 ml of the activated suspension, respectively, were stabilized in the centrifuge tube with 2.2 ml of a 25 BSA solution (0.2 μm filtration). Subsequently the preparations were centrifuged at 2500 rpm for 5 minutes and after that they were filtered on wide pore paper. 3. Column purification (gel filtration) A column with CL 4B Sepharose (Pharmacia) was well regenerated and equilibrated for at least one hour with PBS buffer at room temperature, then the round filter was fed and the volume of the supernatant buffer was suctioned or drained. The 160 ml of the stabilized suspension were fed to the column and the column was covered. After infiltration (approximately 25 minutes) the end of the column was rinsed with PBS buffer and a pump was started.

For the fractionation 12-ml conical tubes were prepared with graduations for the reception volumes: Tube No. 1 to 10 and 23 al.3-0 each 4 ml; No. 11 and 22 4.5 mi; No. 12 and 21 5 mi; No. 13 and 20 5.5 mi; No. 14-19 6 mi.

As soon as the front of the red solvent appeared at the exit, the fractions were collected. Gel filtration took approximately 30 minutes.

Each 5 μl of each fraction was diluted with every 5 ml of PBS buffer (1: 1000) and mixed turbulently Vortex) for optical-visual evaluation of color intensity. The evaluation of the fractions with respect to the color intensity thereof resulted in a wide central peak area with high color intensity and clearly diminishing the coloration in the fractions before and after it. The wide peak area of approximately 140 mi was used. The fractions thereof were combined, distributed in 3 tubes of 50 ml, centrifuged at 2500 rpm for 5 minutes, and subsequently filtered with wide pore paper.

(If necessary, steps 2 and 3 were repeated once or several times for a thicker and / or safer cover or protective layer). 4. Conjugation with streptavidin as the reactive, specific component 100 mg of streptavidin were dissolved in 6 ml of PBS, to test the quality the concentration of the protein was determined by means of the optical density at 280 nm and compared with the manufacturer's specification. The streptavidin solution was given in a 6 mm dialysis tubing and dialyzed against 1 liter of PBS buffer at 4 ° C overnight. The solution was placed in tubes, the tube set was re-rinsed with 4 ml of PBS buffer, and the protein concentration was again determined with the D028o to verify the concentration. The solutions of. Streptavidin were centrifuged at 4000 rpm for 5 minutes and the supernatant, after another determination of D028o, was used for conjugation.

The wide-pore filtered suspension of Sudan IV dye coated with crosslinked BSA, with active residual aldehyde groups, was coupled in 4 preparations with the streptavidin supernatant as follows: Approximately 3 ml of streptavidin solution (with approximately 11 mg / ml protein) were placed in 50 ml tubes. To these were added approximately 30 ml of the suspension with turbulent mixing (i.e., approximately 33 mg of streptavidin per 33 ml of suspension). The solutions were kept on the overhead agitator at room temperature overnight, and centrifuged at 2500 rpm the next morning for 5 minutes, and subsequently filtered through a wide pore filter. The total volume of the suspension was approximately 130 mi after this.

. Column purification with color test on CL-6B Sepharose The column was prepared and equilibrated as described under subsection 3. Infiltration of 130 ml of suspension took approximately 25 minutes, and gel filtration approximately 30 minutes. The tubes for the fractionation were prepared as described under item 3. The fractions were either diluted as described for part 3 or evaluated only optically and visually. In the event that a higher color quality was required, the fractions were tested by a 1:50 dilution in a test according to German Patent DE-A1-195 00 862, Example 1, with respect to the adequacy of the same. To a column with a gel bed with murine anti-human IgG monoclonal antibody was added 250 μl of a combined serum diluted to 1: 5000, which initially contained 84 IU / ml of human anti-tetanus IgG (sensitivity 1.7 mUI / ml, Absolutely approximately 0.4 mUI, International Units), followed by 250 μl of a 5 μg / ml solution of biotinylated tetanus toxoid. After adding 250 μl of the diluted suspension to 1:50 of the dye coupled to streptavidin, a different color was visible. The central combined range that had a high absorption comprised a wide peak area of approximately 10-0 mi, for lesser requirements the two shoulder areas, on the left and on the right, of 30 ml, respectively, were combined separately as well. After turbulent mixing (Vortex) and gel filtration both combined were stored at 4 ° C (if necessary, overnight) until stabilization was achieved. 6. , Stabilization A 20% solution of BSA in PBS buffer was sterilized by 0.2 μm filtration.

The 50 ml tubes rinsed with BSA solution were placed with 22 ml of 20% BSA solution with turbulent mixing (Vortex) 18 ml of the coarse filtered suspension.

Subsequently, the suspensions were refiltered in thick pore. 7. Storage In well-sealed 50 ml tubes, the suspension stabilized with 0.09% sodium azide is stable at 4 ° C for at least 12 months with stable absorption value. 8. Properties Compared to an average size of approximately 50 nanometers of the Sudan IV particles, the size is approximately 51 to 200 nm after a single coating and 300 to 400 nm after a double coating.

EXAMPLE 2 Immunoassay using the particulate carrier materials of the invention, resistant to storage according to Example 1.

Semi-quantitative and quantitative request for the condition of vaccination against tetanus, that is, the concentration of anti-tetanus IgG in the blood. a) Semi-quantitative test For the semi-quantitative test indicated towards the visual effect, a reaction column was used for simultaneous multi-measurement according to German Patent DE-A1-195 00 862 with 3 areas.

The reaction area of the bottom (comparative area) of the reaction column was loaded with G protein and bound thereon with a defined amount of human anti-tetanus IgG corresponding to a sufficient inoculation in Ul (International Units) of the blood volume within of the capillary for the application of blood. This combined serum was diluted accordingly, and the total human IgG, including the tetanus specific, was bound to the G protein of this reaction area in the flow (G protein coupled with agarose activated with CNBr, see German Patent DE-A- 195 00 862, Example 2 given therein).

The intermediate negative control area contains Sepharose 4B activated with CNBr, with BSA covalently coupled.

The upper reaction area (test area) of the reaction unit contains exclusively G protein coupled with agarose activated with CNBr.

A defined amount of blood is applied to the column by means of a capillary. - After washing with a wash buffer, 250 μl of a solution of .5 μg / ml biotinylated tetanus toxoid (biotinylation level 50) is applied to the wash buffer. After an additional wash, a dilution of 1:20 wash buffer of the streptavidin dye emulsion is applied and washed again.

The total human IgG of the sample is bound within the test carrier bed during its flow. In both areas, the test area and the comparative area during the flow, an amount of the anti-tetanus human IgG is reached in advance, respectively, corresponding to the biotinylated tetanus toxoid. Non-specifically bound proteins are washed with subsequent buffer. After this, during the flow of the dye emulsion in the test area and the comparative area there is a bound amount of dye coupled with streptavidin, corresponding to the amount of tetanus toxoid. In this case too, the non-specifically bound protein is washed with a washing buffer. Nothing is attached to the negative control area.

The semi-quantitative evaluation 'takes place by visual comparison of the colors. In the case where the coloration of the test area equals or exceeds that of the comparative area, the vaccination status is sufficient, in the case in which it is more weak, it must be inoculated, that is, the more urgently, the more important is the color difference (finer gradations of the comparative areas can be produced according to medical or WHO information, for example, two years of remaining protection, five years of remaining protection, - recommended inoculation, strongly recommended inoculation. The alternative is to evaluate the penetration depth of the colors within the test area with the corresponding marking, then a comparative area can be omitted or used as protection). b) Quantitative test For this, only one test area is required, however, for certain the positive and negative control areas can be incorporated.

The area is loaded with murine anti-human IgG according to German Patent DE-A-195 00 862, Example 2. The application sequence and the reactions are as described under subparagraph (a). The evaluation is carried out by measuring photometric absorption at 520 nm Q 492 nm after elution with an alcohol solution.

Elution with alcohol is necessary since the dye marker, due to the large number of binding sites, adheres so stably to the gel bed that it can not be separated from the gel bed as other markers, by pH variations or by its treatment with a molecule of concurrence, even not partially, but in this case exclusively by dissolution.

The sensitivity of the test is in the range of mUI / ml for both evaluations or human anti-tetanus IgG. Absolutely, without further optimization of the test, the amounts of minus 10 pg of IgG can be determined qualitatively (visually) and quantitatively, with enrichment. inside of. even smaller column (pg / ml interval).

The current quality control produced an emulsion stability of at least 12 months.

EXAMPLE 3 Synthesis of an activated dye particle To 20 ml of BSA solution within a concentration range of 1 to 50 mg / ml in 0.01-0.2 M phosphate buffer, pH 6-8.5-, 0.1 to 2 g of carbon were added. The mixture is mixed by a magnetic stirrer or Vortex-type mixer until sufficient adsorption of the protein has occurred (peptization). The suspension obtained is centrifuged at 6000 x g for 5 to 10 minutes.

The carbon particles of 50 to 350 nm, on which the BSA is adsorbed, are activated with glutardialdehyde during the next step. The concentration of glutardialdehyde for this is in the range of 1 to 25%, the activation time is 20 to 120 minutes.

The activated suspension is purified by centrifugation 3 to 4 times and by gel filtration with Sepharose 6B, 4B, 2B, CL-6B. CL4B, CL-2B, Sephacryl S300, or with other gels (Toyoperl, Ultragel, etc.), whereby the exclusion limit is not allowed to fall below 1 x 106 Dalton for globular proteins. Using this method, black coloring particles are obtained. The preparation of other coloring particles is done in an analogous way using instead of coal, for example, Sudan II for red, Sudan II for dark red, Sudan black for gray, tetrazolformazan violet for violet, neotetrazoldiformazan for dark violet, and tetrazoldiformazan blue for blue. The working range for these conjugates in suspension is approximately 0.08%, based on the dry weight. The dye markers are produced by conjugation with antianalytes such as protein A, streptavidin or antibodies.

EXAMPLE 4 Serological determination of anti-HIV antibodies on a non-porous solid phase, by means of a protein A-carbon conjugate As a solid phase, polystyrene ELISA plates with synthetic peptides adsorbed with HIV-1 and HIV-2 were used. Subsequently, rabbit serum anti-HIV was added in a serial dilution. After a 15 minute incubation and a purification step the protein A-carbon conjugate was added and the resulting spot or spot was read after 15 minutes following a purification step.

In parallel, analogous experiments were conducted with the colloid protein A-gold, commercially purchased (particle size 40 nm) and protein A-POD. The comparison of the visual evaluation produced a sensitivity of both of the other markers for a dilution of 1 / 12-80, for the carbon conjugate of 1/20480.

EXAMPLE 5 Determination of HCG with carbon conjugate by thin layer chromatography A strip of nitrocellulose of 2 x 16 mm joins transparent PVC of 0.5 mm. One end of the strip was attached to a piece of 1 x 1 cm of chromatography paper as the suction element. In the intermediate part of the nitrocellulose strip, an anti-HCG antibody solution (1 mg / ml in phosphate buffer) was applied with a Hamilton syringe. After 30 minutes it was blocked with a solution of 1% casein (phosphate buffer with Tween) (one hour), after which it was purified and dried.

The HCG standard is diluted once with phosphate / Tween buffer, after which once again with urine from a healthy individual adding up to 0.1 Tween 20. Add 50 μl of carbon conjugate with anti-HCG to 50 μl of this standard solution. The free end of the strip is submerged in this solution, the result is read visually as a black line after 2 to 3 minutes. The sensitivity of 50 mIU / ml is sufficient for a pregnancy test after one week.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (31)

RE IVINDICATIONS

1. A storage resistant particle, of at least one first and at least one second component, characterized in that: the second component of at least one crosslinkable polymer, such as a cover or protective layer, envelops and / or encloses the first component as a number and - the first component has at least one valuable property, obtainable by reacting the first component with a crosslinkable polymer and thereafter the reaction of the formed product with at least a tenfold excess of crosslinking agent, such that the first component is maintained with storage resistance within the second component.

2. The particle according to claim 1, characterized in that the particle is a storage-resistant particle for reactions bound to carriers, for detection and / or isolation methods, whereby the carrier is suitable to be provided with at least one reactive component , in particular a specific reaction component.

3. The particle according to claim 1 and / or 2, characterized in that the first component is wrapped or enclosed reticularly by the second component.

4. The particle according to at least one of claims 1 to 3, characterized in that the second component is at least one polymer comprising active or activatable functional groups.

5. The particle according to claim 3, characterized in that at least one polymer is produced from one or more crosslinkable polymers and / or crosslinkable monomers.

6. The particle according to at least one of claims 1 to 5, characterized in that the polymer is produced from at least one polyamide, preferably a protein, or at least one other biopolymer having functional groups.

7. The particle according to any one of claims 1 to 6, characterized in that the second component is crosslinkable with at least bifunctional compounds.

8. The particle according to claim 7, characterized in that at least the bi-functional compounds bound to the surface also have at least one reactive group, and because these reactive groups can be saturated together or partially and / or can be used together or partially, preferably to a specific portion, for the covalent bonding of the reactive components or an additional layer of the second component.

9. The carrier according to at least one of claims 2 to 8, characterized in that the reactive component is a molecule or a molecular group that has properties related to other substances.

10. The carrier according to claim 7, characterized in that at least one reactive component is an enzyme, a substrate that interacts with the enzymes, an antibody, antigens, biotin or streptavidin, a nucleic acid of the RNA or DNA types that are hydro. with other nucleic acid.

11, The particle according to at least one of claims 1 to 10, characterized in that the second component surrounds the first component in a molecular network.

12. The particle according to at least one of claims 1 to 11, characterized in that the valorable property of the first component is at least one selected from the group consisting of absorptivity or emissivity of electromagnetic waves, mass, magnetism, dielectricity, radioactivity, size , density as well as the pharmacological, biological and / or catalytic effect.

13. The particle according to at least one of claims 1 to 12, characterized in that the first and second components are connected to each other detachably.

14. The particle according to at least one of claims 1 to 13, characterized in that the carrier is present in populations, and the populations are fractioned at least once by the size of the particle after the enclosure and / or by one of the valuable properties of said first component before, during and / or after the closure.

15. The particle according to claim 14, characterized in that the particle is present in a population with a narrow size distribution and / or the titratable property, in particular size and / or color.

16. The particle according to claim 14 and / or 15, characterized in that said particle is present within a narrow range of the distribution of a valuable property of the first component, in particular the size and / or the color.

17. The particle according to at least one of claims 2 to 16, characterized in that the carrier has a high coverage of the second component, with at least one reactive component, in particular specific reactive components.

18. The particle according to any one of claims 1 to 17, characterized in that the reactive component (s) is (are) bound together via. the spacer with the cover or protective layer and the core.

19. A process for the production of a particle of at least a first and at least a second component, characterized in that the first component is treated at least once with at least one crosslinkable polymer as the second component, and the respective product and / or the final product is crosslinked with at least one bifunctional compound that is present in at least an excess of ten times.

20. The process according to claim 19, characterized in that during said process at least once a fractionation of the first untreated component, an intermediate product and / p the particle, by size and / or some other valuable property is conducted.

21. The process according to claim 19 and / or 20, characterized in that at least the bifunctional compound is a compound selected from the group that. it consists of, for example, a dialdehyde, a particular glutardialdehyde, a dicarboxylic acid, an acrylate, or a divinyl compound and the crosslinkable polymer is crosslinked by condensation, addition or substitution reactions.

22. The process according to at least one of claims 19 to 21, characterized in that the treatment with the polymers and / or the crosslinking agent and / or a fractionation are carried out twice or several times.

23. The use of the particle according to at least one of claims 2 to 18 in assay techniques, in particular qualitative, semi-quantitative and quantitative solid phase column immunoassays.

24. The use of the particle according to claim 23, characterized in that the titratable property is evaluated manually and / or visually qualitatively or semi-quantitatively, via the comparative areas and / or the concentration zones.

25. The use of the particle according to claim 23, characterized in that the titratable property is evaluated by technical and quantitative measurement, if necessary after having dissolved the second component and / or the complete particle in another solvent or with different conditions, such as as a different pH and / or different salt concentration.

26. The use of the particle according to at least one of claims 23 to 25, characterized in that at least two evaluable properties are used, for example, magnetism to improve the intermingling and / or the subsequent separation of the solid phase from the fluid phase, and / or absorption to quantify.

27. The use of a particle according to at least one of claims 1 to 15, for the use of active pharmacological and / or biological substances, characterized by the dissolution of at least the second, separate component.

28. The use of the particle according to claim 27, characterized in that a narrow size distribution of the second component determines the time to dissolution, and / or a narrow distribution of at least one valuable property of the first component determines the effect in a range narrow.

29. The use of a particle according to claim 17 and / or 28 as a vehicle for affinity transport of active substances.

30. The use of a particle according to at least one of claims 2 to 15, as a catalyst for chemical and / or biochemical reactions.

31. The use of the particle according to claim 30, characterized in that at least two valuable properties are used, for example, magnetism for rapid intermixing and weight for rapid separation. R? JSUMEU OF IJÜVEUCION e describes a storage-resistant particle of -a-to us-a-pne and -ai-less a second component ^ where the second component of at least one polymer reticulabie as a cover p layer-proctor s < e ueis ^ e yVo encloses at least partial, the first component as a core and - the first component has at least a valuable property, obtainable by reaction of the first component with the crosslinkable polymer, and subsequently the reaction of the product formed with an agent of crosslinking, such that the first component with storage resistance remains within the second component.