Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54326—Magnetic particles
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
  • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
  • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
  • Y10S530/813—Carrier is a saccharide
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
  • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
  • Y10S530/813—Carrier is a saccharide
  • Y10S530/814—Cellulose or derivatives thereof
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
  • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
  • Y10S530/815—Carrier is a synthetic polymer
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
  • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
  • Y10S530/815—Carrier is a synthetic polymer
  • Y10S530/816—Attached to the carrier via a bridging agent
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
  • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
  • Y10S530/815—Carrier is a synthetic polymer
  • Y10S530/817—Entrapped within the carrier, e.g. gel, hollow fibre
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T428/00—Stock material or miscellaneous articles
  • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
  • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
  • Y10T428/2984—Microcapsule with fluid core [includes liposome]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T428/00—Stock material or miscellaneous articles
  • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
  • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
  • Y10T428/2984—Microcapsule with fluid core [includes liposome]
  • Y10T428/2985—Solid-walled microcapsule from synthetic polymer
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T428/00—Stock material or miscellaneous articles
  • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
  • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
  • Y10T428/2984—Microcapsule with fluid core [includes liposome]
  • Y10T428/2985—Solid-walled microcapsule from synthetic polymer
  • Y10T428/2987—Addition polymer from unsaturated monomers only

Definitions

• Particle that is stable on storage in particular carrier for carrier-bound reactions and method for its production
• the present invention relates to a storage-stable particle, in particular a particulate carrier for carrier-bound reactions, detection and / or isolation methods, a process for the production of this storage-stable particle and uses of the particle according to the invention
• Particulate carriers consisting of a solid none and a polymer
• Particulate carriers consisting of a solid none and a polymer
• such particles have been used for markers for immunoassays in order to connect a solid core to an affection component via polymers, for example biopolymers
• brine of metal particles for example magnetic iron particles or colored gold particles
• non-metal particles for example selenium, coal dust, SiO 2 or ceramic particles
• blood cells or even latex particles of another polymer with different (binding) properties were used as the particulate material
• No. 4,230,685 describes an improvement in the attachment of specific binders to magnetic particles, wherein a particle is coated with an acrylate polymer or a polysaccharide, protein A being bound to this coating by means of glutaraldehyde
• No. 4,452,773 describes the production of magnetic iron-dextran microparticles. These particles have a size of 100 to 700 ⁇ , in particular 300 to 400 ⁇ . Many of the particles are colloidal and ferromagnetic with one. Dextran shell The particles obtained are functionalized by periodate oxidation
• EP-B-452 342 relates to super-paramagnetic particles coated with polysaccharides, colloidal particles.
• the particles can be connected to other groups.
• compositions are obtained which have homogeneous properties with regard to their retardation in have a magnetic field
• Gioße The possibility of separation according to Gioße is mentioned.
• Preferred are coatings of polysaccharides or proteins.
• Polysaccharides are functionalized by peilodate oxidation. Functionalization with cyanogen bromide is also possible.
• the piotein shell can be bound to certain molecules via side-chain amino groups or sul fhydiyl groups take place
• 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 any additional steps to make test results visible. Examples of direct markers are metal sols, dye sols, latex particles and color indicators , colored substances, which are located in liposomes, and non-metal sols such as a carbon sol.
• EP-A-0 410 893 describes a method for determining and recognizing an antibody in biological fluids which is directed against a specific antigen.
• the insoluble carrier particles mentioned are composed of cells, gelatin particles, microcapsules, organic polyme, anoiganic fine particles or colloidal of Metal or metal compounds that are finely dispersed with bovine serum albumin or cholesterol.
• EP-A-0 032 270 also describes a quantitative and / or qualitative determination of an immunological component, in which one or more labeled compounds are used, which by direct or indirect coupling of such a component or components to particles from an aqueous dispersion ⁇ sion of a hydrophobic dye or pigment or polymeric cores, which smd coated with such dyes or pigments, is or will be obtained.
• EP-A-0 321 008 relates to a method for determining one or more components of a reaction between a specifically binding protein and the corresponding bindable substance in a sample, the mutual activity of such components and at least one labeled component being obtained by coupling or adsorption of sol particles of the label directly or indirectly onto the component.
• sol particles smd phosphorus, carbon and / or silicon. Agglutination and aggregation can be prevented by coating the particles with macromolecules which contain polar groups, such as proteins, polyethylene glycols, polymeric carbohydrates, polyhydric alcohols and the like. Suitable protection pro- tems are antigens, antibodies and anti-antibodies.
• Immunochemical inert materials such as albumin, polyethylene glycol or other polar macromolecules can also be used. The test sensitivity is given for example for rabbit immunoglobulin G with 1.5 ng absolute.
• EP-A-0 184 710 relates to magnetic microsphere (MIMS), a process for its production from water-in-oil emulsion and its use for the separation and isolation of biological particles, in particular natural or artificial cells.
• the magnetic microspheres obtained by the process according to the invention are stabilized by crosslinking and carry functional groups on the surface which are the covalent ones Allow binding of biologically active ligands
• the magnetic microspheres described above consist of a magnetic component which is incorporated in a coagulated matrix protein.
• the surface of the matrix protein is activated by functional groups for binding biological particles.
• the matrix protein is a globular protein such as alumni the matrix protene is preferably activated by aldehyde groups
• WO-A-92/08134 relates generally to colloidal particles with a crosslinked coating on which there are functional groups.
• Magnetic or non-magnetic particles with biodegradable crosslinked gelatin coating smd are preferred, to which biological substances or molecules are covalently bound. These include, in particular, monoclonal ones Anticoipei The monoclonal antibodies, which are bound to the particles in this way, are used in various positive and negative biological assays
• EP-A-0 298 368 relates to a method for carrying out a diagnostic immunoassay using colloidal iron-metal-containing particles with conjugates bound to them, which are able to specifically recognize the analyte to be determined.
• the test sensitivity for hCG is in the mlU / ml - Area
• EP-A-0 280 560 discloses Streptococcus A antigen antibodies which have been covalently bound to core / shell polymer particles, the shell made of poly (mp-chloromethylstyrene) and the core made of poly (styrene-co-2-acetoacetoxydethyl methylacrylate) ) was built up containing Oil Red EGN in the core in order to obtain an agglutination reagent
• the US-A-4, 452, 886 describes the polymerization of lysine with glutaraldehyde and Congo red to color particles.
• the test sensitivity for hCG is in the 1000 lU / ml range
• DIA Dispose Dye Immunoassay
• JP-A-0686771 relates to encapsulated toners and their production
• the inert cores described in the prior art have significant disadvantages. If the cores are polymerized in, the desired core property is affected very badly. When the polymer is adsorbed onto the core with subsequent covalent or other binding of the affinity component, the binding of the core is usually weak , so that long-term stability is not guaranteed. In addition, the methods according to the prior art only bind one or a few reactive or affmitate components
• the technical problem on which the invention is based is to provide stable particles with characteristic properties which can be used, in particular marker properties, to which, if appropriate, many reactive components can also be bound at the same time.
• the stability should in particular also include long-term storage stability
• a storage-stable particle in particular particle-shaped carrier according to the features of claim 1.
• the subclaims 2 to 18 relate to preferred embodiments of the invention appropriate particle.
• Claim 19 with the associated subclaims 20 and 22 relates to a method for producing the storage-stable particle according to the invention.
• Claims 23 to 31 relate to uses of the particle according to the invention.
• the storage-stable particle according to the invention is particularly intended for carrier-bound reactions, detection and / or isolation processes. It consists of at least a first and a second component, the particle being suitable for being provided with a reactive component, in particular a specific reactive component.
• the second component consists of a crosslinkable polymer and virtually forms a shell which at least partially envelops and / or envelops the first component as a core.
• the first component has at least one detectable property.
• Reactive components can be arranged on the second component.
• the storage-stable particle is characterized by its production process and can be obtained by reacting the first component with the crosslinkable polymer, then reacting the product formed with a crosslinking agent, so that the first component is arranged in the second component in a stable manner.
• the feature of the storage-stable arrangement of the first component m of the second component means that even in the case of prolonged storage, under possibly unfavorable conditions, the first component remains in the casing. In an embodiment of the method according to the invention, this means, for example, that the first component is an elutable dye, that this dye remains essentially quantitatively in the shell which is formed from the second component. Bleeding of the dye is thus prevented.
• the sheath, formed from the second component encloses the first component sufficiently tightly.
• the condition to be observed according to the invention that the first component can be arranged in the second component in a stable manner is achieved, for example, by that the crosslinking agent is used in a large excess.
• the crosslinking agents can typically be present in ten times the concentration of the respective reactive group of the second component.
• the crosslinking agent preferably contains more than 10 to 100 times more reactive groups than corresponding groups of the second component. who react to one another with networking
• reaction conditions are preferably set in such a way that the second component is enveloped or enveloped by the second component
• the second component which forms the carrier according to the invention is preferably a polymer which has active or activatable functional groups which can react with the crosslinking agent or with the reactive components, or both, since the second component of the polymer according to the invention can form the carrier
• the polymer can also be formed from proteins and / or polyamides with functional groups. Basically, other polymers such as biopolymers or monomers are also conceivable, as long as crosslinking is possible second component can be networked with at least bifunctional connections
• Polymers are preferably used which adsorptively stabilize the aqueous suspension of component 1, in particular polar polymers
• the reactive components arranged on the second component of the particle according to the invention are, in particular, molecules or groups of molecules which have affine properties for other substances. These include, in particular, enzymes and substrates interacting with enzymes. On the one hand, the substrate or, on the other hand, the enzyme as a reactive component on the second Component arranged
• the same applies with antibodies / antigen, Biotm / Streptavidm systems Strep tavidm or biotin as a specific reaction component firmly bound to the dye has the advantage that the dye can be used universally as a reaction partner for biotinylated or streptavidin-linked components. These in turn can have very different functions, for example, again specifically against another component or act as a catalyst.
• nucleic acids of the RNA or DNA type can be used as reactive components in the sense of the carrier according to the invention.
• the nucleic acids are able to react with the corresponding ones to hybridize complementary or partially complementary strands, depending on the stringency of the reaction conditions, and to form stable complexes. It is therefore possible to identify specific strands of nucleic acid in a sample to be examined and to process them further on the basis thereof.
• Combinations of the reactive components can also be arranged on or on the second component. Carriers according to the invention are then obtained which can be used for various problems
• the crosslinking reaction can be controlled so that the second component envelops the first component quasi in a molecular network, the mesh of which has a narrow mesh size distribution
• the number of free binding sites on the particle surface can be set, which remains free by binding the least bifunctional crosslinker with only one function to the surface for further binding, in particular of reactive components.
• the number of binding sites that are created can be full or, after partial saturation, to a defined extent
• the thickness of the shell can be determined by the process conditions, the homogeneous valley of which still leaves can be improved by a fractionation.
• a multi-stage process of the coating can further improve the inclusion of the first component.
• the first component of the particle according to the invention preferably has at least one detectable property which, at least in its quality, is not inherent in component 2 and / or the reactive components bonded to it, such as absorption or emissivity of electromagnetic waves, mass, magnetism, dielectric.
• Radioactivity, high and / or density on detectable properties which can have the core as the first component of the carrier according to the invention, is also understood to mean a pharmacological-biological effect and a catalytic effect or combinations thereof.
• the absorption or emission capability can be electromagnetic Waves are produced by appropriate chromophores or fluorophores.
• the properties of mass, size and density are physically related and can be adjusted accordingly, for example by specifically heavy particles.
• the property size can be advantageous for the Using agglomeration and the property mass for gravimetry
• the agglomeration is advantageous for the specific precipitation of components in solutions or microemulsions.
• the first component can be provided with the property radioactivity by radioactively marked structures. The other properties mentioned can be similarly done with the first component connect
• the first component can also be a capsule and contain liquids or particles that are not sufficiently enveloped (eg extremely small or unstable in solution)
• the carrier is designed such that the first and second components are separably connected to one another.
• the connection between the first and second components is made by wrapping the first component with the second principle. It is possible to remove the second component to expose the most iconic component, possibly for a mechanical engineering
• the properties associated with the core are easier to remove.
• the second component can be removed by appropriate proteolytic enzymes. This degrades the shell so that the core remains. This can then be treated further according to its property to be measured.
• the storage-stable, particle-shaped carriers appear in a plurality of populations in appearance.
• the carriers are present in populations which are characterized by a narrow distribution of the size of the individual carrier particles according to the invention
• the population has carrier particles which have both a narrow range of the size distribution of the shell and a narrow range of the size distribution of the core and thus a narrow distribution of the size ratio shell / core.
• the particle aL carrier according to the invention it is advantageous to use a or the second form component to arrange a high concentration of reactive components.
• a or the second form component to arrange a high concentration of reactive components.
• relatively high binding constants for corresponding complementary structures can be achieved, for example, in immunoassays, although the individual reactive component may have only an average binding constant for the corresponding complementary structure.
• increasing the number of binding sites the effectiveness of education (avidity) is reached
• a method for producing the carrier according to the invention comprises the steps of reacting the first component at least once with a crosslinkable polymer.
• the polymer can be physically adsorbed on the surface of the first component.
• the crosslinkable polymer will form the second component after further treatment steps treatment with crosslinking agent as crosslinking agent, in particular, bifunctional molecules such as glutardialdehyde, dicarboxylic acids, acrylates, methacrylates are suitable.
• crosslinking agents carrying the olefinic groups can be activated, for example, by photoreactions or other radical chain reactions network
• the first component can be subjected to a separation according to size and / or another property before the treatment and / or the intermediate product with the second component and / or the particle in order to achieve a distribution of the size and / or another property that is as homogeneous as possible
• the treatment of the first component with polymers is preferably carried out two to three times and particularly preferably a separation according to size
• the method is particularly advantageous since, regardless of the reaction, the first component is first homogenized and its concentration in the suspension is adjusted 1 af t Depending on the first component used, SMII are common Use method to homogenize and stabilize the suspension.
• the second component is preferably used to stabilize the suspension if it is a polar compound with sufficiently good adsorption on the first component.
• the ratio of the concentrations of the first and second components is chosen so that a suitable shell thickness is achieved by adsorption In the multi-stage process, the shell thickness is chosen to be rather thin. Other parameters such as time and temperature can also be used to influence the adsorption.
• the intermediate product can be homogenized again in order to achieve good crosslinking of the shell, in particular in the inner region near the first component calibration, the crosslinking component is then used in a crossover of several orders of magnitude.
• the effect of the crosslinking component can also be influenced by other parameters, in particular the exposure time, just like the number of reactive groups formed on the surface by one-sided g bound crosslinker molecules, which can subsequently be reduced by partial blocking
• the carriers according to the invention are preferably used in assay methods such as immunoassay, solid-phase assays and / or chromatographic assay. They can also be used as vehicles for the transport of active substances if the first component, for example, is enveloped by the second component and the second component, for example Afmatate transport means, for example, transport of pharmacologically active substances by means of antibodies which are arranged on the second component and bind specifically to a specific target structure
• Particles with a radioactive nucleus can also be used advantageously because they act or are concentrated mainly at the place of use due to the transport of the afflicate and therefore overall lower amounts of radioactivity can be used, so that a systemic load can be kept within narrow limits
• the use of the storage-stable, particle-shaped supports according to the invention as a catalyst for chemical reactions is also advantageous.
• a magnetic core can be used for rapid mixing of the carrier-bound catalyst (biocatalyst) with the reaction solution, the weight of which then ensures rapid segregation
• the suspension was divided into two centrifuge tubes and centrifuged in a centrifuge precooled to 4 ° C. for 5 minutes at 1000 rpm, at 2000 rpm and at 3000 rpm, followed by a second filtration.
• Tubes No. 1-10 and 23-30 each 4 ml; No. 11 and 22 4.5 ml, No. 12 and 21 5 ml; No. 13 and 20 5.5 ml; No. 14-19 6 ml
• each 5 ⁇ l of each fraction was diluted (1 1000) with 5 ml PBS buffer for each optical-visual color strength assessment and vortexed for 5 seconds (Vorfex).
• the evaluation of the fractions according to their color strength led to a broad central peak area with high color strength and clearly falling color in the fractions before and after.
• the broad peak area of approx. 140 ml was used. Its fractions were pooled, distributed into 3 50 ml tubes, centrifuged for 5 minutes at 2500 rpm and then coarsely filtered
• Steps 2 and 3 were repeated one or more times if needed for a thicker and / or more secure shell
• streptavidm 100 mg streptavidm were dissolved in 6 ml PBS buffer, for quality control the protein concentration was determined with the optical density at 280 nm and compared with the manufacturer's specification.
• the streptavid solution was placed in a 6 mm dialysis tube and at 4 ° C. overnight against 1 1 PBS buffer dialyzed The solution was placed in a tube, the tube was rinsed with 4 ml PBS buffer, and the protein concentration was checked again with the 0D ⁇ O ( to check the concentration).
• the streptavid solutions were centrifuged for 5 minutes at 4000 rpm and the Supernatant for conjugation is used after determining the 0D 2 ⁇ C again
• the size after approx. One coating is approx. 51 to 200 nm, after a second coating at 300 to 400 nm
• the lower reaction field (comparison field) of the reaction column is loaded with protein G and bound to it a defined amount of anti-tetanus human IgG, which corresponds to adequate vaccination protection m IU (International Units) of the blood amount m of the capillaries for the blood application Serum diluted accordingly and the entire human IgG including the tetanus-specific bound in flow to the protein G of this reaction field (protein G coupled to CNBi - activated agarose, see DE-A-195 00 862, example 2 there)
• m IU International Units
• the middle negative control field contains CNBr-activated Sepha rose 4b with covalently coupled BSA
• An upper reaction field (test field) of the reaction unit contains only protein G coupled to CNBr-activated agarose
• a defined amount of blood is applied to the column with a capillary. After washing with washing buffer, 250 ⁇ l of a solution of 5 ⁇ g / m] of biotinylated tetanase toxoid (degree of breadmylation 50) are applied in washing buffer. After washing again, a 1 20 -washing buffer dilution of the streptavidm dye emulsion is applied applied and washed again
• the entire human IgG of the sample is bound in the test carrier bed during its passage.
• a corresponding amount of biotmylieitetetanus toxoid is bound during the passage of the previously bound anti-tetanus human IgG.
• Unspecifically bound proteins are released from the The subsequent puff is washed out.
• the amount of streptavid-coupled dye corresponding to the amount of tetanus toxoid bound is also bound.
• unspecific bound protein is washed out by the washing buffer.
• the semi-quantitative evaluation is carried out by visual color comparison. If the color of the test field is the same or stronger than that of the comparison field, the vaccination status is sufficient. If it is below this, vaccination should be carried out, and the greater the difference in color, the more urgent it is (fine subdivisions of comparison fields) , eg two years of protection, after five years of protection, vaccination recommended, vaccination strongly recommended, can be prepared according to medical or WHO information.
• Eme alternative is the evaluation of the depth of penetration of the colors into the test field with appropriate markings. Then a comparison point d can be omitted or serve as protection)
• test field is not required for this, but positive and negative control fields can be built in for safety
• the field is loaded with mouse anti human IgG in accordance with DE-A-195 00 862, Example 2.
• the evaluation is carried out by photometric absorption measurement at 520 nm or 492 nm after elution with alcoholic alcohol Solution
• Elution with alcohol is necessary because, owing to the high number of binding sites, the dye marker sits on the gel bed with such stability that it cannot be detached from the gel bed like other markers with the aid of pH changes or by displacement with a competition molecule. also not partially, but in this case only by means of a resolution
• test sensitivity for both evaluations is in the mlU / ml range for anti-tetanus-human IgG
• further test optimization amounts below 10 pg IgG can be determined qualitatively (visually) and quantitatively, even less when enriched with the column (pg / ml Area)
• the ongoing quality control showed that the emulsion remained stable for at least 1 .. months
• 0.1 to 2 g of carbon are added to 20 ml of BSA solution in a concentration range of 1 to 50 mg / ml in 0.01-0.2 M phosphate buffer solution, pH 6-8.5.
• the mixture is placed on a magnetic stirrer or a Vortex mixer mixed until the protein is adequately adsorbed (peptized).
• the suspension obtained is centrifuged at 6000x g for 5 to 10 minutes
• the carbon particles from 50 to 350 nm on which BSA is adsorbed are activated in the next step with glutardialdehyde.
• the glutardialdehyde concentration for this is in the range of 1 to 25%, the activation time is 20 to 120 minutes.
• the activated suspension is purified by centrifuging 3 to 4 times and by gel filtration with Sepharose 6B, 4B, 2B, CL-6B, CL-4B, CL-2B Sepharcryl S300 or with other gels (Toyoperl, Ultragel etc.) , the exclusion limit must not be less than 1 million Daltons for globular proteins.
• black dye particles are obtained.
• the production of other dye particles takes place analogously, with instead of carbon e.g. Sudan II is used for red, Sudan III for dark red, Sudan-black for gray, Tetiazolformazanvrolett for violet, Neotetrozoldiformazan for dark violet and Tetrazoldiformazan blue for blue.
• the working range for these conjugates in suspension is 0.08% based on the dry weight.
• the dye labels are produced by conjugation with anti-analytes such as protein A, streptavidin or antibodies.
• Polystyrene ELISA plates with adsorbed synthetic peptides of HIV-1 and HIV-2 were used as the solid phase.
• a Kanmchen anti-HIV serum was then added in a dilution series. After an incubation of 15 minutes and a washing step, protein A-carbon conjugate was added and the result spot was read after 15 minutes after a washing step.
• a strip of 2x16 mm nitrocellulose is glued to transparent PVC 0.5 mm. The end of the strip was glued with a 1 x 1 cm piece of chromatography paper as a suction element.
• a solution of anti-HCG was used in the middle of the nitrocellulose strip with a Hamilton syringe.
• Antibody (1 mg / ml in phosphate buffer) added. After 30 minutes, blocking with 1% case solution (phosphate buffer with Tween) (hour), then washing and drying
• the HCG standard is diluted once in phosphate / Tween puffer, then again in urine from healthy people, with up to 0.1% Tween 20 being added to 50 ⁇ l of this standard solution, 50 ⁇ l carbon conjugate with anti HCG added The free end of the strip is immersed in this solution, after 2 to 3 minutes the result is visually read as a black line.
• the sensitivity of 50 mlU / ml is sufficient for a pregnancy test after one week

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