DE10249608A1 - Device and method for structural analysis and detection of complex glycostructures - Google Patents

Abstract

The invention relates to a microarray and a binding assay for the detection and structural analysis of complex glycoconjugates, the glycoconjugate interacting with at least two different lectins.

Description

The invention relates to a device and a method for the determination and analysis of complex glycostructures.

Carbohydrates can be divided into monosaccharides, disaccharides, oligosaccharides and polysaccharides. The oligo- and polysaccharides can in turn be divided into homoglycans or heteroglycans, the heteroglycans with the representatives of the proteoglycans, peptidoglycans, glycolipids and glycoproteins being among the biologically important structural units. Compound carbohydrates that include conjugated compounds, such as glycolipids and glycoproteins, are also referred to as glycoconjugates. Glycoconjugates are one of the central biomolecules in the cell and are involved in many physiological processes. Among other things, they have functions in immune defense, cell differentiation, in inflammatory processes and in general in cell-cell interactions. Due to their properties and diverse functions in the organism, the structure of glycoconjugates plays a major role in the areas of basic research, diagnostics, pharmaceutical research and for drug development. The term glycoconjugate refers to molecules in which a carbohydrate portion (mono-, oligo- or polysaccharide) is covalently bound to a protein or a lipid. The diversity within this class of substances is very large, because the glycoconjugates include, in addition to the glycoproteins and glycolipids, in which the protein or lipid component predominates, also the lipopolysaccharides and proteoglycans, in which the carbohydrate component predominates. The carbohydrate content in glycoproteins and glycolipids typically consists of up to 20 monosaccharides, in lipopolysaccharides and proteoglycans this can be considerably larger. In the biosynthesis of these complex glycoconjugates, nature uses a wide variety of linkage and branching options in addition to the selection of different monosaccharide building blocks, so that there is a huge variety of structures within this class of substances. Carbohydrates potentially have the greatest number of possible variations in a short sequence within the biological binding partners of proteins. Using a common library of 20 different monosaccharides, theoretically 10 ¹⁵ different structures result for the formation of a hexasaccharide (including branched isomers).

If the oligosaccharide chains are branched, one speaks of oligoantennaren Compounds, usually being di- to pentaantennary structures available. The resulting "multivalency" of the molecules in relation on interaction with a binding partner is an important one Aspect for the specificity and strength the bond. The oligosaccharide structures can be found at each glycosylated site of a protein vary. The differently glycosylated variants are called one as glycoforms of this protein or as micro heterogeneity.

In contrast to monosaccharides or Disaccharides or other simple molecules the quick, easy and safe structural analysis of glycoconjugates only possible to a limited extent with previous devices and methods. In the areas mentioned, in which research is carried out on and with glycoconjugates are analytical methods for clarifying the identity of attachment behavior and however, the structure of these components is essential. The mono and disaccharides successfully applied physicochemical Analytical methods can not successfully transferred to the field of glycoconjugates because the methods are very time-consuming due to the complexity of the target structures, are complicated and not very specific. Because of the great importance the glycoconjugate, there have been several efforts in the prior art, to detect their structure safely and effectively.

For example, attempts have been made to use mass selective Procedure a statement about to hit the structure. However, such procedures provide no information about that biospecific binding behavior of a substance. This is how Identification and quantification of glycosylation patterns of glycoproteins through mass selective processes. The glycoproteins first split up, that is, the structure to be examined is first destroyed and the glycans or glycopeptides become after chromatographic separation using mass spectroscopy characterized. Another characterization method glycoconjugate, affinity chromatography, with the help of lectins, which are bound to gel particles, saccharides or glycoconjugates for cleaned up the use of further analysis steps. To mixtures of substances to fractionate or get structural information about glycoconjugates, stands above all the method of serial lectin affinity chromatography to disposal, in which the sample components in the flow method over a Combination of lectin adsorbents be fractionated.

With the techniques mentioned, it is disadvantageous that a purification of the glycoconjugates is necessary, since any impurities impede the analysis. For example, it is necessary to work with large amounts of the purified glycoconjugate - often in the milligram range - which requires a great deal of preparation. So far it has not been possible to detect glycostructures in a uniform procedure, so that the analysis of glycoconjugates with conventional methods always consists of several parts steps and is therefore very time-consuming. A central disadvantage of the conventional analysis methods, however, is that the glycostructures cannot be analyzed non-destructively, i.e. the sample is no longer in its native state, which means that once a sample has been analyzed and it includes glycostructures, it cannot be used as a rule , Complex structures, such as glycoproteins with more than two or three more or less branched sugar chains (glycans) are generally not covered by the known methods.

The object of the invention is therefore to provide a device with which complex carbohydrate structures, like glycoconjugates, detected quickly, easily, safely and efficiently and can be determined in their structure.

The invention solves this technical problem by providing a microarray comprising at least one two different immobilized against the basic structure of a glycoconjugate directed lectins, the basic structure at least one glycan containing at least two glycosidically linked monosaccharides, includes.

The invention is accordingly surprising Understand that complex glycoconjugates, such as glycoproteins or glycolipids, not just interact specifically with a lectin, but that they are able to measure so with multiple lectins Interaction that an analysis of the structure of the glycoconjugates - the analyte - is possible. Through this interaction of one and the same analyte, with several different lectins, multiple areas or arise on the array Places with a special lectin-sugar interaction that with various methods known to the person skilled in the art can be detected can. These locations of specific interaction result on the microarray a particular pattern, especially when the lectins are on the microarray in a certain geometric order - for example in several Lines or in a grid - immobilized are. With well-known algorithms from bioinformatics and structural analysis Is it possible, from this pattern to the structure and binding behavior of the glycoconjugate or to conclude the glycosylation of a structure. It can be provided, for example, that two, three, four or several dozen different lectins on an ordered grid - immobolized are. If the areas where lectin-carbohydrate interactions or lectin-sugar binding events take place with a marker can be detected or if by this interaction for example an electrical pulse is released that can be measured forms for each glycoconjugate to be examined has a specific pattern or an impulse algorithm, based on which analog or homolog Structures can be determined. While Monosaccharides, disaccharides and simple oligosaccharides and simple, not complex polysaccharides with physical or chemical methods can be characterized sufficiently this is not possible with complex glycoconjugates. But that's an advantage possible, complex glycoconjugates easily, safely with the microarray according to the invention and to detect and analyze quickly.

Under immobilization in the sense of Invention are all methods of restricting mobility and solubility from lectins to chemical, biological and / or physical Because of understanding. The immobilization can be different Methods take place, such as the binding of the lectins to each other or to carrier, by sticking to the network of a polymer matrix or by immobilization on a membrane. Due to the immobilization there is a washout prevents lectins during the various washing steps. Furthermore can after the process of interaction the biological sample easily again be separated from the immobilized lectin. The bond or the Immobilization of the lectins on the carrier or microarray can be done by direct carrier connection and done by cross-linking. Carrier binding or cross-linking takes place according to the invention especially ionic / adsorptive or by covalent bonding. The Cross-linking in the sense of the invention is a cross-linking of the detection molecules - that is to say the lectins - with one another or with other polymers. When immobilizing by inclusion the lectins enclosed in gel structures or in membranes in such a way that interaction with the glycoconjugates is possible.

Lectins can be placed on the microarray e.g. by dropping round spots or by calling Lines are immobilized, being planar as well as microarray non-planar structures such as spheres, cuboids or fibrils and rods are used can be which are impregnated or immobilized with different lectins and so in a spatial Order to be brought.

Numerous possibilities are known to the person skilled in the art to immobilize the lectins on the microarray. The immobilization should take place in such a way (i) that each lectin is assigned a defined position on the support or microarray and (ii) that each position on the microarray or support can be evaluated independently. However, it can also be advantageous that the application sites of different lectins overlap partially or completely or that lectin mixtures are applied in the form of a detection spot. The immobilization can take place, for example, using a method based on semiconductor technology. Printing processes also enable the lectins to be defi ned areas or locations on or on the surface of the microarray, whereby a stable bond with high coupling efficiency can take place. All immobilization measures of biomolecules known to the person skilled in the art - for example on column materials - can also be used to immobilize the lectins on the array.

Selected methods for immobilization are, for example, contact tip printing, ring and pin printing, nanoelectric printing and nanopipetting, bubble jet printing, top spot printing, micro contact printing, micro fluidic networks methods, photolithographic activation processes, photoresist lithography, electrochemical focusing and micro Wet printing. However, it is also possible to use comparatively simple sample application devices from thin-layer chromatography or autosamplers for HPLC as well as pipettes and micropipettes (piston-stroke pipettes). With the help of these methods, a lectin spot size of approximately 1 .mu.m to more than 6 mm can be generated. According to the invention, the lectin density per cm ² can be, for example, in the range from approximately 0.01 to 1000 pmol / cm ² . The methods mentioned enable simple handling and great precision of the volumes created, as well as very homogeneous lectin spots, which are applied with a high degree of parallelization. The methods that enable a direct synthesis of the lectins on the support have in particular a high integration density and a simple combination of immobilization and coupling. It is possible to prepare the lectins, which can also be referred to as probes, with further methods in order to keep the proportion of unspecificly synthesized or bound lectins low.

However, the lectins can be on the microarray can also be immobilized by a blot process. To do this the lectins or cells or tissues comprising them by means of a Electrophoresis separated. The electrophoretic separation can e.g. by means of one-dimensional or multidimensional electrophoresis, in particular 2D electrophoresis.

The electrophoretically separated or separated lectins are then either transferred to the carrier or microarray or, on a membrane, which in a next step on the support or Microarray is applied.

In a preferred embodiment of the invention, the microarray comprises at least two different immobilized lectins, the lectins being isolable, selected from the group consisting of Abrus precatorius, Adenia digitata, Agaricus bisporus, Aleuria aurantia, Amaranthus caudatus, Amphicarpaea biacteata, Anguilla anguilla, Arachis hypogaea, Artocarpus integrifolia, Bauhinia purpurea alba, Brachypodium sylvaticum, Canavalia ensiformis, Carcino scorpius rotunda canda, Cicer arietinum, Codium fragile, Crotalaria juncea, Cytisus sessilifolius, Datura stramonium, Dioclea grandifhrinausiallichristinauria, dendella erifone, Erythema, dolomized erythematosus, dolomized erythrocyte, erythrocyte, erythrocyte, erythrocyte, erythrocyte, erythrocyte, erythrocyte, erythrocyte, erythrocyte, erythrocyte, erythrocyte, erythrocyte, erythrocyte, erythrocyte, erythropoiesis Galanthus nivalis, Glycine max, Griffonia (Bandeiraea) simplicifolia, Helix aspersa, He1ix pomatia, Hippeastrum hybrid, Hordeum vulgare, Hura crepitaus, Latyrus odoratus, Latyrus sativus, Latyrus tingitanus, Lens culinaris, Limax glavus, Lotus tetracopagonus es tum, Maackia amurensis, Maclura pomifera, Macrotyloma axillare, Momordica charantia, Narcissus pseudonarcissus, Oryza sativa, Phaseolus coccineus, Phaseolus limensis, Phaseolus vulgaris, Phytolacca americana, Pisum sativum, Phosphocarpus tetromonosumosa nasolomonosus, pseudonomacillus, pseudonarcissus, sosa Solanum tuberosum, Sophora japonica, Triticum vulgaris, Tritrichomonas mobilensis (Protozoe), Ulex europaeus, Vicia cracca, Vicia ervilia, Vicia graminea, Vicia faba, Vicia sativa, Vicia villosa, Viscum album, Wisteria floribunda, the following can also be produced by humans, especially reombinant human , Lectins are used: E-selecin (human, recombinant), L-selectin (human, recombinant) and / or P-selectin (human, recombinant) and galectins (human). Specific lectins which are suitable for binding complex glycostructures or glycoconjugates in such a way that detection and structural analysis are possible can advantageously be isolated from the organisms mentioned. For example, the lectin ABA from Agaricus bisporus, from Aleuria aurantia AAL, from Amaranthus caudatus ACL (ACA), from Anguilla (Eel) anguilla AAA, from Arachis hypogaea PNA, from Artocarpus integrifolia AIL (Jacalin), from Griffonia (Bandeiraea) simplicifolia GSL BSL, from Bauhinia purpurea alba BPL (BPA), from Canavalia ensiformis Con A, from Datura stramonium DSA, from Dolichos biflorus DBA, from Erythrina coralldendron Ecor A, from Erythrina cristagalli ECL (ECA), from Euonymos europaeus nivalis, from Galanth GNL, from Glycine max SBA, from Helix aspersa HAA, from Helix pomatia HPA, from Hippeastrum hybrid HHL (AL), from Lens culinaris LCA, LcH (Lentil), from Lotus tetragonolobus LTL, from Lycopersicon esculentum LEA (Tomato), from Maackia amurensis MAA, from Maclura pomifera MPA, from Narcissus pseudonarcissus NPL, NPA, from Phaseolus coccineus PCA, from Phaseolus limensis LBA, from Phaseolus vulgaris PHA, from Phytolacca americana PWM, from Pisum sativum PSA, from Pseudomonas aeruginosa , from Ricinus communis RCA ₆₀ and RCA ₁₂₀ , from Sambucus nigra SNA, from Solanum tuberosum STA (Potato), from Sophora japonica SJA, from Triticum vulgaris WGA (Wheat Germ Agglutinin), from Tritrichomonas mobilensis (Protozoe) TML, from Ulex europaeus I UEA-I, from Vicia faba VFA, from Vicia villosa VVA, from Viscum album VAA and from Wisteria floribunda WFA (WFL), the human (recombinant, from a CHO cell line) lectins E-selecin (human) CD62E, ELAM-1, CD62L, LAM-1, L-Selectin (human) LECAM-1, MEL-14, CD62P, GMP-140, P-selectin (human) LECAM-3 and / or PADGEM and galectins can be obtained, where "L" corresponds to a lectin and "A" corresponds to an agglutinin.

In a further preferred embodiment of the invention, the microarray comprises at least two different immobilized lectins, the lectins having a binding affinity of Kd zu 10 ⁻⁶ mol / l to the glycostructures to be detected. With a binding affinity of Kd ≤ 10 ^-6 mol / l, it is advantageously possible to analyze and detect the interaction between glycoconjugate and immobilized lectins so precisely that a qualitative detection of the glycoconjugate and a structural analysis is possible efficiently.

In a further advantageous embodiment In the invention, the immobilized lectins comprise a binding linker. Binding linkers in the sense of the invention are all structures that are associated with the lectins in such a way that they are covalent or complex on a support of the microarray or immobilized on the microarray itself can. The Can linkers for example, peptide fragments in the range of 0 to 50 amino acids. The lectins comprising linkers are accordingly advantageously not via a non-specific adsorption immobilized on the microarray. The left can also be designed so that an interaction between the individual Lectins or lectins and the carriers is excluded, that interference or non-specific interactions, or only to a very small extent Scope occur. In such a case, the linkers can, for example can be used as a spacer. Linker in the sense of the invention can also be short synthetic DNA double strands his. You can also be used to start certain lectins immobilize, then using specific, e.g. DNA double strand degrading enzymes to split off again. For example, it is possible, the complex glycoconjugate structures in a first step to let several specific lectins interact, with selected lectins then by a cleavage step using cleavable linkers from Microarray solved so that the glycoconjugate structures in a sample are now included other lectins can interact, e.g. have a different binding affinity than the split lectins.

In a further preferred embodiment of the invention, the linker is in particular streptavidin and / or biotin. Streptavidin in the sense of the invention is a protein from Streptomycis avidinii which binds to biotin with an affinity of 10 ⁻¹⁵ M ⁻¹ and which has a molecular weight of approximately 60,000 Da. It advantageously has a lower non-specific response than avidin. Biotin in the sense of the invention are coenzymes of the enzymes that catalyze carboxylations. The very high affinity of biotin for streptavidin as well as for avidin can advantageously be used to immobilize biotinylated reagents.

In a further advantageous embodiment of the microarray, the lectins are immobilized with a packing density of> 50 ng / cm ² . The resulting surface coverage of> 10% enables an optimal interaction with the glycostructures. The variants of the possible functionalizations of carrier materials are of course diverse. A requirement for the binding of lectins is the presence of appropriate functional groups on the support material with which the lectin can react. The following variants can be used, among others: functionalization with carboxyl groups, functionalization with amino groups, functionalization with aldehyde groups, functionalization with phenyl groups, functionalization with thiol groups, functionalization with divinylsulphones (resulting bond: ether, thioether, secondary amines), functionalization with carbonyldiimidazole, functionalization with Epichlorohydrins (resulting bond: ether, thioether, secondary amines), functionalization with glutaraldehyde (resulting bond: Michaels Adduct, Schiff base; advantage: good stability of the bond), functionalization with hydrazines (resulting bond: amide bond), functionalization with periodate ( resulting bond: alkylamine), functionalization with diazonium (resulting bond: azo), functionalization with trichloro-S-triazine (resulting bond: triazinyl), bisoxirane (resulting bond: alkylamides, ethers, thioethers), cyanogen bromide (resulting bond: Is Urea, imide, (carbamate), functionalization with 4-nitrophenychloroformate (resulting bond: anhydride, carbamate, urethane), functionalization with epoxy groups and functionalization with organic sulfonyl chlorides (tosyl chloride, tresyl chloride) (resulting bond: alkylamine).

Functionalization is an advantage possible in a quick reaction the immobilization of the lectins using the methods described in particular is advantageous since a covalent bond is formed here. This prevents the lectins from being washed out and thus a loss of binding activity or the desired one supersede the sample is contaminated by lectin molecules.

Immobilization takes place under individual reaction conditions and is often to be carried out using other chemicals. The immobilization on epoxy groups requires, for example, 8-48 h at pH 8.5-12. The binding of proteins to matrices activated with carboxyl groups takes place at pH 4-6 in 24-48 h. The use of carbodiimide is advantageous for the reaction. Proteins are also immobilized on supports substituted with amino groups using other chemicals such as carbodiimide or NaCNBH ₃ .

Functionalization with tresyl chloride (2,2,2-trifluoroethane sulfonyl chloride) is used. The functionalization of carrier materials with tresyl chloride is particularly suitable for the binding of proteins, since the reaction takes place under mild conditions (pH 7-9) and does not damage the proteins. The binding takes place via primary amino groups or thiol groups of the amino acids of the protein. The reaction is comparatively quick (a few hours) and takes place without the use of additional chemicals. Tresyl activation is stable for a few months with dry material or when stored in dilute hydrochloric acid.

Sometimes it makes sense to wear the carrier Integration of a so-called spacer, a spacer functionalize. This spacer increases the distance between the carrier surface and Ligand and can therefore contribute to better accessibility of the ligand. Usual spacers exist from two terminal functional groups and one that can be selected in length C chain such as 6-aminocaproic acid. The linker used, that is the carrier functionalization used, can already be a spacer itself.

In a further advantageous embodiment of the invention, the microarray comprises metal, polypropylene, Teflon, silicon, polyethylene, polyester, polystyrene, nitride, ceramic, quartz and / or glass. Metals in the sense of the invention are all compounds whose cohesion is created by a crystal lattice. The boundary between metals and non-metals is fluid, so that the elements Ce, Sn, As and Sb are also metals in the sense of the invention. The metals according to the invention also include the metallic glasses, that is to say materials which are in a metastable, largely amorphous state. Of course, metallically conductive polymers are metals in the sense of the invention. For the purposes of the invention, metals advantageously have, in particular, good strength, good hardness and wear resistance, high toughness and good electrical and thermal conductivity. Polypropylenes in the sense of the invention are thermoplastic polymers of propylene. Polypropylenes are particularly characterized by high hardness, resilience, stiffness and heat resistance. However, the microarray can also include Teflon. Teflon according to the invention are polytetrafluoroethylenes which advantageously have good thermoplastic properties. Polyethylenes are produced in particular by polymerizing ethylene using essentially two different methods, the high-pressure and the low-pressure process. Polyethylenes which are produced in the high-pressure process advantageously have a low density. The properties of microarrays comprising polypropylene are essentially determined by the character of the polyethylene as a partially crystalline hydrocarbon. Advantageously, polyethylenes are practically insoluble up to 60 ° in all common solvents. Advantageously, polar liquids such as alcohol, esters and ketones hardly cause polyethylene to swell at room temperature. Polyethylenes are advantageously completely indifferent to water, alkalis and salt solutions as well as inorganic acids. For example, microarrays or carriers that include polyethylenes have very low water vapor permeability. However, the microarray can expediently also comprise polyester. For the purposes of the invention, polyesters are compounds which are prepared by ring-opening polymerization of lactones or by polycondensation of hydroxycarboxylic acids or of diols and dicarboxylic acids or dicarboxylic acid derivatives. For the purposes of the invention, polyesters also include polyester resins, polyester imides, polyester rubbers, polyester polyols and polyester polyuretanes. Polyesters are advantageously thermoplastics and have a distinct material character. For example, they are characterized by high thermal stability and can be processed into alloys with metals such as copper, aluminum and magnesium. However, it can also be provided that the microarray comprises ceramic. Ceramic in the sense of the invention is a collective name for a class of substances composed of in particular inorganic and predominantly non-metallic compounds and elements and in particular comprising more than 30% by volume of crystalline materials. Various ceramics or ceramic materials are known to the person skilled in the art, which he can use as a microarray. It can be, for example, so-called pottery, earthenware, split plates, laboratory porcelain, tableware, bone china, aluminum oxide ceramics, permanent magnet materials, silica stones and magnesia stones. In the case of clay ceramic materials, a distinction is made between coarse and fine materials in the sense of the invention, fine clay ceramic materials comprising earthenware, earthenware, stoneware and porcelain. However, special ceramic materials such as glass, oxide ceramics, SiC stones and molten stones can also be used as a microarray with advantage. The microarray can preferably also comprise glass. Glass in the sense of the invention are substances in the amorphous, non-crystalline solid state, that is, the glass state can be understood in the sense of the invention as frozen, supercooled liquid or melt. Glasses are therefore inorganic or organic, mostly oxidic melt products, which have been converted into a solid state by an insertion process without crystallization of the melt phase components. In the context of the invention, crystals, quartz, melts and supercooled melts are of course also to be understood as glasses. The glasses can be, for example, flat glass, container glass, industrial glass, laboratory equipment glass, lead crystal glass, fiber glass, optical glass fibers and others. Of course, it is also possible to use silicate-free glasses, for example phosphate glasses. The microar However, ray can also be such that optical glasses, that is to say, glasses with special optical refractive indices, for example, are used.

The invention also relates to a Binding assay for the determination of glycostructures in a sample, the sample being immobilized with at least two different ones Lectins contacted to form lectin-sugar complexes and the lectin-sugar complexes are detected in a manner known per se and the lectins can be isolated, selected from the group comprising Abrus precatorius, Adenia digitata, Agaricus bisporus, Aleuria aurantia, Amaranthus caudatus, Amphicarpaea biacteata, Anguilla anguilla, Arachis hypogaea, Artocarpus integrifolia, Bauhinia purpurea alba, Brachypodium sylvaticum, Canavalia ensiformis, Carcino scorpius rotunda canda, Cicer arietinum, Codium fragile, Crotalaria juncea, Cytisus sessilifolius, Datura stramonium, Dioclea grandiflora, Dolichos biflorus, Erythrina coralldendron, Erythrina cristagalli, Euonymos europaea, Galanthus nivalis, Glycine max, Griffonia (Bandeiraea) simplicifolia, Helix aspersa, Helix pomatia, Hippeastrum hybrid, Hordeum vulgare, Hura crepitaus, Latyrus odoratus, Latyrus sativus, Latyrus tingitanus, Lens culinaris, Limax glavus, Limulus polyphemus, Lotus tetragonolobus, Lycopersicon esculentum, Maackia amurensis, Maclura pomifera, Macrotyloma axillare, Momordica charantia, Narcissus pseudonarcissus, Oryza sativa, Phaseolus coccineus, Phaseolus limensis, Phaseolus vulgaris, Phytolacca americana, Pisum sativum, Phosphocarpus tetragonolobus, Pseudomonas aeruginosa, Ricinus communis, Sambucus nigra, Secale cereale, Solanum tuberosum, Sophora japonica, Triticum vulgaris, Tritrichomonas mobilensis (Protozoe), Ulex europaeus, Vicia cracca, Vicia ervilia, Vicia graminea, Vicia faba, Vicia sativa, Vicia villosa, Viscum album, Wisteria floribunda, can continue the following human, in particular recombinantly produced, lectins are used: E-Selecin (human, recombinant), L-Selectin (human, recombinant) and / or P-selectin (human, recombinant) and galectins (Human). The determination of glycostructures by the binding assay according to the invention relates on the one hand, detection, that is, qualitative detection of glycostructures in a sample, as well as the detection of certain patterns on the microarray that allow conclusions about which ones are immobilized different lectins interact with one and the same glycostructure, the structure of the complex oligosaccharide or polysaccharide or of the glycoconjugate. A sample in the sense of the invention is the name for a biological or chemical taken by sampling Good or a part or a small amount of such a Chemical, biological, clinical or similar properties can be checked should. Sampling takes place in such a way that the partial quantity taken corresponds to an average of the total amount. That through investigation Characteristics determined in the sample are used to assess the characteristics of the sample detected amount, the conclusions the total amount, e.g. Drinking water, food, tissue, cells, Cell cultures, cell culture supernatants, transplanted Organs and other. For the investigation can the samples by mixing, dividing, separating, pre-fractionating, Crushing, adding enzymes or markers or otherwise pretreated become. Various options for pretreatment are known to the person skilled in the art of the samples known. Of course it can also be provided that the sample is taken in such a way that it doesn't correspond to an average of the total amount. A Can sample all biological and non-biological materials, such as biological tissues and liquids, e.g. Blood, lymph, urine, brain fluid and others, as well Environmental effluents, Bioreactor liquids, Food ingredients, hazardous substances, aerosols, lipid mixtures and many more. more.

In an advantageous embodiment In the binding assay, the lectins become physical, chemical and / or biologically by in situ synthesis or by depositing previously synthesized Lectins immobilized on the microarray. For the binding assay, regardless of the type of immobilized lectins the carrier or microarrays on two principally different ways are made:

• 1. Storage and immobilization of previously synthesized or lectins from libraries at defined positions a functionalized carrier material. Therefor can both spotting and printing processes are used. Under Spotting is a process in which drops of liquid are deposited, being through surface interaction and drying essentially round spots arise. Other printing processes enable the substrate in defined areas on the surface store.
• 2. In situ synthesis of the lectins at defined positions of the carrier or the microarray by successively coupling monomeric synthesis building blocks.

In a further advantageous embodiment become the lectins through Contact Tip Printing, Ring and Pin Printing and Nanopipetting, Buble Jet Printing, TopSpot Printing, Micro Contact Printing, Micro Fluidic Networks methods, Photolithographic Activation methods, Photoresist Lithography, Electroche mical Focusing and / or Micro Immobilized wet printing.

In a further preferred embodiment of the invention, the surface of the microarray with poly-L-lysines, aminosilanes, aldehyde silanes, epoxy groups, gold, streptavidin, polylysines, silanes, reactive groups, polyacrylamide pads, immobilized nitrocellulose, activated aldehydes, agarose Aldehyde groups and / or tresyl groups coated. Such substrate surfaces actions, it is advantageously possible to improve the durability and the binding capacity of the surface of the microarrays in such a way that the lectins can remain immobilized very well over a longer period of time. Of course, the surface of the carrier can be made very generally, in particular by the action of biological, physical and / or chemical influences. Physical effects include polishing, etching, pickling, sandblasting, but also physical processes that lead to hardening, coating, tempering, covering with protective skins and the like. A surface treatment by biological action can include, for example, overgrowth by microorganisms. A chemical modification of the surface of the carrier includes, for example, treatment with acids, bases, metal oxides and others. The surface of the carrier can be modified so that the detection molecules or lectins adhere particularly well to the carrier or so that their activity is not disadvantageously modified. A surface modification of the carrier naturally also includes a treatment which leads to increased stability and breaking strength, in particular of the microarray. Classic surface modifications from histology can of course also be carried out, such as coating with, for example, protein glycerol, polylysine, activated dextrans and / or chromium gelatin.

In a further preferred embodiment The invention will be before or after the formation of the sugar-lectin complexes a labeled or unlabeled lectin, a labeled or unlabeled Glycostructure and / or a labeled or unlabeled antibody added.

The following detection systems can be used with advantage:
Direct: The sample molecules themselves are provided with a marker (enzyme (A), fluorescent or Vis dye (B), biotin (C) ...) After the binding event and washing, either:

• - directly be read out (B)
• - or after implementation of an enzyme substrate and reading of the resulting Color signal (A)
• - or (C) after adding another marker (enzyme, fluorescent or Vis dye) coupled with avidin / streptavidin.

A simple, fast, direct result achieved because the sample itself is marked.

Indirect: After adsorption of the sample molecules this with one for the glycoconjugate specific antibody to be determined or Lectin. This is again marked. After binding the event can be read out.

The "double" specificity advantageously makes it very specifically identified a specific substance.

Sandwich: After adsorption of the sample molecules provide them with the same lectin that immobilizes on the surface is (see embodiment). This lectin binds specifically to the glycoconjugate to be determined from the opposite Page and is itself marked. After binding can the event can be read out. The same sugar structure is advantageously recognized and detected "twice". It can be a sugar specificity be determined. There are only complex systems with at least two Glycosylation sites recorded. It will only be the ones that already exist Lectins needed no more antibodies or similar ..

Competitive: After adsorption of the sample molecules provide the wells with a marked model adsorptive. This is known to bind that immobilized lectin. The model adsorptive can only be used on free Binding sites dock or compete with the sample molecule for them Binding sites. A positive signal is received if none or just a few sample molecules are connected or according to the relationship that exists in this competitive situation established.

The proof is advantageous systems with only a complex sugar structure possible. It known model systems are used. The competitive situation also provides information about the binding constants of the complex with the sample molecule.

In a further advantageous embodiment The invention uses an enzyme, a dye, for labeling Fluorescent marker, a radioisotope, a metal colloid and / or a Chelator used. Methods such as markers are known to the person skilled in the art especially through reinforcement mechanisms train more sensitive.

In a further embodiment of the invention, the lectins are immobilized on micro test plates, glass plates, membranes, braids, fibrils, in particular made of polypropylene, nitrocellulose, glass and / or PVDF. Microtiter plates, for example, can be used as microtest plates. Microtest plates advantageously have dimensions that allow their use in numerous laboratory routines. For example, numerous fluorescence measuring devices, fluorescence spectrometers and photometers such as fluorescence microscopes and the like are designed in such a way that microtest plates can be used as standard. The immobilization of the molecules on special laboratory vessels, for example microtiter plates, petri dishes, multiple dishes, multi dishes and other culture vessels and slides, therefore advantageously also enables the use of the existing laboratory means and devices for incubating, freezing, lyophilizing and similar laboratory devices in clinical or research laboratories. For example, microtiter plates with transparent, non-fluorescent flat bottoms can preferably be used as microtest plates or white or black, opaque flat bottom.

In a further advantageous embodiment the invention is a largely planar surface of the Microarrays coated with a polymer and following are lectins immobilized on the surface by means of the polymer. advantageously, takes place in this embodiment immobilization of the lectins directly via the polymer.

The polymer is preferably selected such that it has a hydrophobic surface trains and the surface across from a watery Medium electrically isolated. This hydrophobic can advantageously be smooth surface among other things, show a self-cleaning effect.

In a further preferred embodiment In the invention, the polymer is a polyimide or polystyrene. you choose the polymer is suitable, e.g. Polyimides, can also be more Microarrays or even additional ones Microsystems with the same inorganic surface, e.g. a semiconductor formwork, connect industrially into one system. Polyimides are special high temperature resistant polymers; they advantageously have excellent mechanical, thermal and electrical properties. The applications of the polyimide include in particular buffer layers, passivation layers, binding layers and dielectric interlayers on the carrier. Polyimides in particular liquid applied and then cured. at this curing step receives the polyimide advantageously the desired Characteristics. For the applications, the polyimide can be structured lithographically. Of course, polyimide can also as an intermediary for Potting material and used as a buffer layer. The polyimide layer For example, reduces the stress in silicon caused by the encapsulation is caused and prevents cracks on the edges. silicon can be applied to the micro array in interconnects. The polyimide especially under very uniform temperature conditions hardened to cracking in the polyimide and color unevenness to prevent. Low oxygen levels are e.g. beneficial to to achieve good liability. Polystyrene or polystyrene is a thermoplastic, which is mainly by radical polymerization of Styrene is obtained. The radical end of a growing polymer chain never attacks a double bond in the ring because the benzene ring has one extraordinarily stable structure. This has several advantages for the Use of polystyrene, for example, polystyrene is resistant to acids, bases and alcohol.

W reactive molecules are preferred by the radiation with W light is covalently immobilized. Is preferred furthermore that the polymer is only present in predefined areas the surface of the microarray is applied. This can advantageously, similar to for circuits, certain structures, a defined arrangement the lectin spots or patterns of immobilization of the lectins can be specified. Consequently are certain reactions or interactions between the lectins and glycostructures controllable.

In a further advantageous embodiment the surface, on which the lectins are to be bound directly, by plasma treatment positively and / or negatively charged electrically. That is, the surfaces are loaded differently in different places. polymers In particular, materials come in various forms. The individual forms place different demands on the machining process. Depending on the shape of the surface these e.g. accessible to the plasmas in different ways. Plasma treatment of the polymer surface can advantageously the surface energy increase sharply and enable other processing methods. With a plasma treatment especially the ions and radicals of the plasma react with the polymer surface and create functional groups there that reflect the surface properties determine the polymer with advantage. By the positive or negative Charge will in particular have better wettability and / or better binding of the biomolecules reached.

The invention also relates to the use of the Microarrays and / or the binding assay according to the invention for detection an analyte and / or a pattern analysis. In addition to the above Ellipsometry can also be used. At this Technology are adsorbed layers (monolayer and submonolayer) from one surface measured using a polarized laser beam. The reflected Laser beam with a modified Polarization can be measured.

The technology of the SPR (Surface Plasmon Resonance) works with a sensor chip that consists of a gold-plated Glass plate, layered with a polymer. A prism is installed on the glass surface. A signal arises from the change the refractive index through the prism when molecules flow from the solution flowing past the sensor adsorb. The signal indicates a mass change taking place on the gold surface.

With the microarray according to the invention or the binding assay according to the invention, unknown substances from the group of glycoconjugates can be screened, detected and analyzed. By using immobilized lectins, it is advantageously possible for co- and post-translational changes in the proteins, such as glycosylation, to be detected and analyzed. This is of particular importance since the glycosylation of proteins modulates their bioactivity and has a decisive influence on the function, availability, interaction, compartmentalization and lifespan of this substance in the organism. Glycosylation of the lipids also plays a major role in their function as receptors in signal transmission. The glycostructures to be investigated can be contained, for example, in samples which comprise natural or synthetic glycostructures as well as their derivatives and degradation products, defined substance mixtures, synthetic and natural multi-substance mixtures, such as cells, cell supernatants, nutrient solutions, extracts and / or sera. The screening function of the described microarray and the binding assay is based in particular on the parallel use of many different, or at least two, lectins or lectin systems. The targeted selection of carbohydrate-binding lectins advantageously offers a broad spectrum of binding affinities for defined sugar structures. The use of the microarray and the binding assay thus makes it possible to use lectins in the form of an array for the analysis, in particular structural analysis, of glycoconjugates. In contrast to the known screening methods, it is advantageously also possible to detect and analyze glycoconjugates based on their glycosylation. It takes into account and detects the co- and post-translational modifications, such as glycosylation, of the proteins that have a decisive influence on the physiological properties of the substances. Due to the high specificity of the selected lectins, even slightly changed sugar structures can be distinguished from each other and structural information can be obtained, above all, about unknown glycoconjugates. Through the use of different lectins with complementary binding specificities, a pattern analysis of the sample is also advantageously possible. This also enables the comparison of samples - such as cell lines - with each other, which is a common problem in basic research, medicine and clinic. The invention thus enables the detection and analysis of new, unknown glycostructures in complex multicomponent mixtures.

The invention is intended to be described below an example based on the associated Drawings explained without being limited to this example.

Show it:

1 a schematic representation of an embodiment of the lectin microarray;

2 an arrangement of immobilized lectins on a 96-well microtiter plate in array format and

3 oligosaccharide structures bound by Con A.

The 1 shows the schematic representation of an embodiment of the lectin microarray. A lectin is immobilized on the surface of a microtiter plate well by the formation of the high-affinity complex streptavidin-biotin. Complex glycans of glycoproteins can then bind to the saccharide binding sites of the lectin.

The 2 shows the arrangement of immobilized lectins on the surface in array format.

The 3 shows the advantage of this application example, which lies in the information that only complex glycoconjugates with at least two glycans are detected, which can interact with both the immobilized lectin and the detection lectin at the same time. This demonstration in the form of a sandwich technique makes the analytical result particularly specific and meaningful.

example

Material:

Manufacturing the array:

• - fluorescence spectrometer in the form of a microtiter plate reader
• - 96 well microtiter plate, planar (flat bottom), black, not transparent; the plate is evenly coated with streptavidin at the bottom of the wells
• - biotinylated Concanavalin A (lectin from Canavalia ensiformis)
• - biotinylated RCA (lectin from Ricinus communis)
• - biotinylated WGA (lectin from Triticum vulgaris)
• - buffer solution A: BisTris buffer pH 7.0; 0.15 mol / L NaCl, 1 mmol / L calcium, manganese and magnesium ions
• - Buffer solution B: Buffer A in which 250 µg / mL BSA (bovine serum albumin) are dissolved.

Use of the array:

• - buffer solution A: BisTris buffer pH 7.0; 0.15 mol / L NaCl, 1 mmol / L calcium, manganese and magnesium ions
• - Fetal Bovine serum, sterile filtered, diluted 1: 500 with buffer A.

detection:

• - buffer solution A: BisTris buffer pH 7.0; 0.15 mol / L NaCl, 1 mmol / L calcium, manganese and magnesium ions
• - Concanavalin A (lectin from Canavalia ensiformis), with FITC (fluorescein-5-isothiocyanate, Fluorescent dye) marked.
• - RCA (Lectin from Ricinus communis), with FITC (fluorescein-5-isothiocyanate, Fluorescent dye) marked
• - WGA (Lectin from Triticum vulgaris), with FITC (fluorescein-5-isothiocyanate, Fluorescent dye) marked

Preparation of the lectin array:

The biotinylated lectins Con A, RCA and WGA are each dissolved in buffer A at the following concentrations:
Con A: 5.5 μg / mL
RCA: 3.0 μg / mL
WGA: 4.5 μg / mL

100 μL each of the lectin solutions are pipetted into a well of the microtiter plate and incubated for 4 h at 8 ° C. pipetting:
A 1: ConA
A 2: RCA
A 3: WGA

The occupancy achieved is then 100-400 ng lectin / cm ² or 70-300 ng lectin / well (corresponding to 1-4 pmol / well).

The plate is then washed three times with buffer A. Then incubate with 100 μL buffer B per well for a further 4 h at 8 ° C. The plate is then washed three times with buffer A. The device thus produced consists of the arrangement of immobilized lectins on a surface in array format ( 2 ).

Use of the lectin array:

100 μL of the fetal diluted 1: 500 Bovine serums are added to wells A1, A2 and A3 (pipetted) and for 4 h at 8 ° C incubated. The plate is then washed three times with buffer A.

The lectins Con A, RCA and WGA labeled with FITC are each dissolved in buffer A at the following concentrations:
Con A: 10 μg / mL
RCA: 10 μg / mL
WGA: 10 μg / mL

100 μL each of these lectin solutions are pipetted into the wells of the microtiter plate and incubated for 4 h at 8 ° C. pipetting:
A 1: FITC-ConA
A 2: FITC-RCA
A 3: FITC-WGA

The plate is then three times washed with buffer A.

Measurement and evaluation:

The evaluation is carried out in the fluorescence spectrometer (Microplate reader). The relative fluorescence activity in the Wells determined. The excitation wavelength is 485 nm. The emission is measured at 535 nm.

Result:

In Well A 2 and A 3 respectively a positive fluorescence activity measured. Well A 1 shows no fluorescence. This indicates the Binding of a complex glycostructure from the serum to the lectins RCA and WGA out. Depending on the binding affinities of RCA and WGA, this can be used the following structures can be concluded:

• - the Glycostructure (s) in the serum each have two glycans (two different ones glycosylation sites)
• - the Glycans have ß-D-galactoses (according to the affinity from RCA see below) and terminal N-acetylglucosamines or sialic acids (corresponding of affinity from WGA see below).
• - Mannose containing Structures corresponding to the affinity of Con A (see below) do not occur on.

Meet the properties mentioned to the glycoprotein fetuin present in the serum, as is known. Beef fetuin has a carbohydrate content of around 30%. It has a total of 6 glycans, 3 of which are bi- and triantennary N-glycans with terminal sialic acids are.

Binding specificities of the lectins used: RCA: Generally, glycans are bound with terminal galactoses. The glycostructure β-D-Gal-1,4-β-D-GlcNAc-1,0-R (terminal Lactosamine) is a prerequisite for the binding of a glycan, where fixed bonds are highly specific only with branched, multivalent N-glycans come about.

WGA is a chitin binding lectin, the binding site for N-acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (NeuNAc) has. The bond strength for oligosaccharides decreases with the number of G1cNAc residues strongly too. The disaccharide N, N'-diacetylchitobiose gets 131 times stronger bound as the monomer and trisaccharide N, N ', N' 'triacetylchitotriose 3700 times stronger. WGA is binding sialic acid Glycoproteins.

Claims (19)

Microarray comprising at least two of each other various immobilized against the basic structure of a glycoconjugate directed lectins, the basic structure containing at least one glycan comprises at least two glycosidically linked monosaccharides. Microarray according to claim 1, characterized in that the lectins can be isolated selected from the group comprising Abrus precatorius, Adenia digitata, Agaricus bisporus, Aleuria aurantia, Amaranthus caudatus, Amphicarpaea biacteata, Anguilla anguilla, Arachis hypogaea, Artocarpus integrifodium albaachachuria, Bauhinia purpuria, Bauhinia purpuria, Bauhinia purpura sylvaticum, Canavalia ensiformis, Carcino scorpius rotunda canda, Cicer arietinum, Codium fragile, Crotalaria juncea, Cytisus sessilifolius, Datura stramonium, Dioclea grandiflora, Dolichos biflorus, Erythrina coralldendron, Erythrina Glyonymonia neaulum, ect. simplicifolia, Helix aspersa, Helix pomatia, Hip peastrum hybrid, hordeum vulgare, hura crepitaus, latyrus odoratus, latyrus sativus, latyrus tingitanus, lens culinaris, limax glavus, limulus polyphemus, lotus tetragonolobus, lycopersicon esculentum, maackia amurensis, maclura pomiferus, characaria, saracomacia, macrotyloma aria, macrotyloma saras, macrotyloma saras, macrotyloma saras, macrotyloma aria, macrotyloma aria, macrotyloma sarea, macrotyloma saras, macrotyloma sia , Phaseolus coccineus, Phaseolus limensis, Phaseolus vulgaris, Phytolacca americana, Pisum sativum, Phosphocarpus tetragonolobus, Pseudomonas aeruginosa, Ricinus communis, Sambucus nigra, Secale cereale, Solanum tuberosum, Sophora japonica, Triticum vulgaris, Tritrichomonas mobilensis (protozoan), Ulex europaeus, Vicia cracca, Vicia ervilia, Vicia graminea, Vicia faba, Vicia sativa, Vicia villosa, Viscum album, Wisteria floribunda, E-Selecin (human, recombinant), L-Selectin (human, recombinant), P-Selectin (human, recombinant) and / or Galectine (human). Microarray according to one of claims 1 or 2, characterized in that the lectins to the detecting glycostructures have a binding affinity of Kd ≤ 10 ^-6 mol / l. Microarray according to one of claims 1 to 3, characterized in that the lectins are a binding linker and / or a functionalization group include. Microarray according to one of claims 1 to 4, characterized in that the linker and / or the functionalization group are selected from the group consisting of streptavidin, biotin, carboxyl groups, Amino groups, aldehyde groups, phenyl groups, thiol groups, divinyl sulfones, Carbonyldiimidazole, epichlorohydrins, glutaraldehyde, hydrazines, Periodate, diazonium, trichloro-S-triazine, bisoxirane, cyanogen bromide, 4-nitropheny chloroformates, epoxy groups and / or sulfonyl chlorides. Microarray according to one of claims 1 to 5, characterized in that the lectins are immobilized in a packing density of> 50 ng / cm ² . Microarray according to one of claims 1 to 6, characterized in that that the microarray metal, polypropylene, teflon, silicon, polyethylene, Includes polyester, polyethylene, nitride, ceramic, quartz and / or glass. Binding assay for the determination of glycostructures in a sample, characterized in that the sample with at least two different immobilized lectins under training of lectin-sugar complexes is brought into contact and the lectin-sugar complexes are detected are selected from which the lectins can be isolated of the group comprising Abrus precatorius, Adenia digitata, Agaricus bisporus, Aleuria aurantia, Amaranthus caudatus, Amphicarpaea biacteata, Anguilla anguilla, Arachis hypogaea, Artocarpus integrifolia, Bauhinia purpurea alba, Brachypodium sylvaticum, Canavalia ensiformis, Carcino scorpius rotunda canda, Cicer arietinum, Codium fragile, Crotalaria juncea, Cytisus sessilifolius, Datura stramonium, Dioclea grandiflora, Dolichos biflorus, Erythrina coralldendron, Erythrina cristagalli, Euonymos europaea, Galanthus nivalis, Glycine max, Griffonia (Bandeiraea) simplicifolia, Helix aspersa, Helix pomatia, Hippeastrum hybrid, Hordeum vulgare, Hura crepitaus, Latyrus odoratus, Latyrus sativus, Latyrus tingitanus, Lens culinaris, Limax glavus, Limulus polyphemus, Lotus tetragonolobus, Lycopersicon esculentum, Maackia amurensis, Maclura pomifera, Macrotyloma axillare, Momordica charantia, Narcissus pseudonarcissus, Oryza sativa, Phaseolus coccineus, Phaseolus limensis, Phaseolus vulgaris, Phytolacca americana, Pisum sativum, Phosphocarpus tetragonolobus, Pseudomonas aeruginosa, Ricinus communis, Sambucus nigra, Secale cereale, Solanum tuberosum, Sophora japonica, Triticum vulgaris, Tritrichomonas mobilensis (Protozoe), Ulex europaeus, Vicia cracca, Vicia ervilia, Vicia graminea, Vicia faba, Vicia sativa, Vicia villosa, Viscum album, Wisteria floribunda, E-Selecin (human, recombinant), L-selectin (human, recombinant), P-selectin (human, recombinant) and / or Galectine (human). Binding assay according to claim 8, characterized in that that the lectins are physically, chemically and / or biologically in situ synthesis or by depositing previously synthesized lectins be immobilized on a microarray. Binding assay according to one of the preceding claims 8 or 9, characterized in that the lectins by contact tip printing, Ring and Pin Printing, Nanopipetting, Buble Jet Printing, TopSpot Printing, Micro Contact Printing, Micro Fluidic Networks methods, Photolithographic activation process, Photoresist lithography, electrochemical focusing and / or micro Wet printing to be immobilized. Binding assay according to one of claims 8 to 10, characterized in that a surface of the microarray with poly-L-lysines, Aminosilanes, aldehyde silanes, epoxy groups, gold, streptavidin, reactive groups, polyacrylamide pads, immobilized nitrocellulose, activated aldehydes, agarose-aldehyde groups and / or tresyl groups is coated. Binding assay according to one of claims 8 to 11, characterized in that before or after training the Sugar-lectin complexes at least one labeled or unlabeled Lectin, at least one labeled or unlabeled glycostructure and / or at least one labeled or unlabeled antibody is added become. Binding assay according to one of claims 8 to 12, characterized in that an enzyme, a Dye, a fluorescent marker, a radioisotope, a metal colloid and / or a chelator can be used. Binding assay according to one of claims 8 to 13, characterized in that the lectins on microtiter plates, Glass flakes, Membranes, braids, fibrils, in particular made of polypropylene, Nitrocellulose, glass and / or PVDF can be immobilized. Binding assay according to one of claims 8 to 14, characterized in that a largely planar surface of the Microarrays are coated with a polymer and then lectins be immobilized on the surface by means of the polymer. Binding assay according to one of claims 8 to 15, characterized in that as the polymer is a polyimide or Polystyrene can be used. Binding assay according to one of claims 8 to 16, characterized in that the polymer only in predefined Areas on the surface of the Microarrays is applied. Binding assay according to one of claims 8 to 17, characterized in that the surface by plasma treatment is positively and / or negatively charged electrically. Use of a microarray according to one of claims 1 to 7 and / or a binding assay according to any one of claims 8 to 18 for the detection of an analyte and / or for a pattern analysis.