CN1158658A - Process of investigating the interaction between biomolecules by means of surface plasmon resonance - Google Patents
Process of investigating the interaction between biomolecules by means of surface plasmon resonance Download PDFInfo
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- CN1158658A CN1158658A CN95195182A CN95195182A CN1158658A CN 1158658 A CN1158658 A CN 1158658A CN 95195182 A CN95195182 A CN 95195182A CN 95195182 A CN95195182 A CN 95195182A CN 1158658 A CN1158658 A CN 1158658A
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- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/26—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having more than one amino group bound to the carbon skeleton, e.g. lysine
Abstract
The invention concerns a process, a biosensor unit which can be regenerated and suitable kits for investigating the interaction between biomolecules by means of surface plasmon resonance (SPR). One of the reagents, a (poly)peptide, is coupled to the surface of the biosensor unit by means of a metal chelate. Nitrilotriacetic acid derivatives to which proteins with an affinity peptide containg histadine groups can be bonded are preferably used as chelate formers.
Description
The present invention relates to the interactional detection method of intermolecular biologic specificity.
Analyze macromolecular intermolecular interaction in the past, for example (gather) peptide-(gathering) peptide-or the interaction of (gathering) peptide-DNA generally under equilibrium condition, carry out.Up to date except a few exceptions, still can not or be difficult to measure its kinetic parameter, for example affinity chromatography or immunization are not enough to the interaction of fast monitored biologic specificity because these measure to require a large amount of and highly purified big molecule and/or existing method.
Yan Zhi method is based on the optical phenomena of surperficial plasmid gene group resonance effect (SPR) in recent years.Can carry out this analysis in time by measuring optical signalling at present, described optical signalling is relevant with biologic specificity variations in refractive index at the interface, and combine with the variation of big molecular conecentration and can obtain optical signalling, the variation of big molecular conecentration can draw by the reactive material of combination to each other.In the method, can use the not any radioactivity of carrier band or fluorescently-labeled a small amount of big molecule, and macromolecular purity is not had strict requirement.PCT application WO90/05295 and WO90/05303 disclose this method.
The most widely used; industrial system (the BIACore TM that is easy to carry out based on surperficial plasmid gene group resonance effect method; medicinal organism sensor (pharmacia Biosenor)) also have biosensor unit except the transfer equipment of optical system and sample, it is as one of its main element with alleged biology sensor substrate.The biology sensor substrate is made of glass slide, and a side of slide glass has Gold plated Layer, and the hydrogel matrix layer that is made of Sensor Chip CM 5 is to be covalently bound to this layer by the restraining barrier, and the restraining barrier is provided by the linker group.The hydrogel matrix is a kind of reactive material that is used for fixing wherein on the one hand, is on the other hand to be used to provide analyze through the required medium of the interaction of fixing big molecule and the biologic specificity between its reactive material people such as (, 1991) Stenberg.
Up to the present with following method fixing a kind of material wherein on the hydrogel matrix, this material is (many) peptides normally:
1) adopt chemical method direct, uniaxially is fixed on the hydrogel surface of biology sensor substrate,
2) make biotinylated reactive material direct, uniaxially be fixed on the streptavidin that is attached in the hydrogel or the avidin and
3) by a kind of mode that is attached to the antibodies reactive material in the hydrogel directly, uniaxially fixes.
But, these methods have various shortcomings: method 1) since between the material of hydrogel and (many) peptides the formation indirect chemical react, this reaction according to the method that is adopted preferentially in primary amino radical group, carry out in carbohydrate group or (many) peptides free thiol group, thereby has biological nature and/or the affected danger of biophysical properties that makes the latter, make it to limit or to be difficult to limit, be can not or not exclusively can be consequently by its character through fixing (many) peptides, biologically active form carries out, this be because to be subjected to the molecular domains that reactive material reacts to each other chemical group obstruction or since the conformational change in the molecule become can not be approaching due to.Because it is similar with the method that is used for coupling (many) peptide basically to be used for macromolecular these methods of biotinylation, so above-mentioned method 2) also exist these shortcomings, thus method is restricted.These two kinds of methods also have other shortcoming, although when using identical (many) peptides to carry out a series of analysis, ability with resurgent water gel surface can make methods simplification, is quite difficulty but will implement to regenerate when keeping biologically active and biophysics activity.
If in for example ELISA (immunosorbent assay of enzyme combination) technology, use the method identical in principle, so by adopting antibody (method 3) can overcome these shortcomings with the interlayer immunoassays.But adopt this method to be subjected to the restriction of necessary use to the antibody of the suitable monoclonal high affinity of (many) peptide specifics.Another restriction be this antibody not should with the interaction of epitope order, this epitope is important for interacting with the reactive material that will measure.
Fundamental purpose of the present invention provides between a kind of mensuration (many) peptides and the reactive material carries out interactional method, and this method is used SPR and overcome the shortcoming that known method exists.
The whole bag of tricks can be used for adopting the protein of gene approach preparation explanation sequence, as has the fused protein (so-called affinity peptide) that ligand is presented the sequence fragment of high affinity.Through fixing metallo-chelate affinity chromatography (IMAC) is a kind of method that is widely used in purification of protein and peptide, and wherein this class fused protein is incorporated on fixing metallo-chelate by the affinity peptide.Be selected from iminodiacetic acid derivatives, the different sequestrants of nitrilotriacetic acid(NTA) derivant have particularly advantageous characteristic, comprise some metallic ion, for example Cu
2+, Ni
2+Or Zn
2+Show high affinity.At present, people have extensively adopted this method to come the purifying recombinant fused protein, and make the affinity peptide with at least one histidine residues directly or indirectly in conjunction with thereon, wherein use nickel as metallic ion, and nitrilotriacetic acid(NTA) is as complexing agent.This method that is used for the purification of Recombinant body protein has been disclosed in EP184355 (wherein iminodiacetic acid is used as complexing agent) and EP282042 (wherein described nitrilotriacetic acid(NTA) and had a kind of protein that has the affinity peptide of two adjacent histidine residues at least).
In the SPR method, adopt the principle of metallo-chelate affinity chromatography to form the basis that reaches the object of the invention.
Therefore; the present invention relates to adopt biosensor unit to measure the method for (many) peptides and reactive material interphase interaction; wherein the surperficial plasmid gene group resonance effect that produces by interacting is to determine in two media metal level at the interface; this medium is permeable to electromagnetic radiation; and has different refractive indexes; the medium that refractive index is lower is a kind of water-bearing media, and wherein (many) peptides exist with fixing form, and contact with reactive material.
The method is characterized in that (many) peptides are fixing by metal-chelator.
Another aspect of the present invention relates to be used for measure by the means of the surperficial plasmid gene group resonance effect of measuring two media metal level at the interface the biosensor unit of (many) peptides and reactive material interphase interaction; two media wherein is permeable to electromagnetic radiation; and having different refractive indexes, the medium that refractive index is lower is a kind of water-bearing media.Biosensor unit is characterised in that the sequestrant with appropriate format and complexing of metal ion is to be attached to lip-deep in the face of the biosensor unit of water-bearing media.
In preferred scheme, containing water is biocompatibility, porous matrix, especially hydrogel.In principle, to the character of hydrogel constituting body without limits,, particularly relevant with the desired diffusion of biomolecule in hydrogel matrix as long as they are applicable to the SPR method basically.The example of suitable hydrogel constituting body is for example agarose, glucosan, Irish moss, alginates, starch or a cellulose of polysaccharide, or the derivant of these polysaccharide, carboxymethyl derivant for example, or water-swellable organic polymer, for example polyvinyl alcohol (PVA), polyacrylic acid, polyacrylamide or polyglycol.
Specially suitable hydrogel is made up of relevant with the covalent bond of (many) peptides glucosan that is provided by reactive group, and described reactive group comprises for example hydroxyl, carboxyl, amino, aldehyde, carbonyl, epoxy radicals or vinyl.The PCT that the combining of the character of hydrogel layer and itself and metal level (suitable time produced by organic restraining barrier) is described in nineteen ninety Lofas and Johnsson applies among the WO90/05303.In embodiment preferred of the present invention, sequestrant is to be incorporated on the reactive group of hydrogel.
In an especially preferred embodiment, the hydrogel constituting body is to have the glucosan of carboxymethyl group as reactive group.
In another preferred embodiment, (many) peptides are to merge (many) peptides, except its bioactive fragment, have the affinity peptide that comprises at least two adjacent histidine residues.In this case, sequestrant is that general formula is Y-R-CH (COOH)-N (CH
2COOH)
2Nitrilotriacetic acid(NTA) (NTA) derivant, wherein R represents type (CH
2) n
-Alkylidene bridge, they can be that replace or unsubstituted, but condition is a substituting group effect of sequestrant are not had injurious effects, n represents 1,2,3,4,5,6,7,8,9 or 10 integer, if when alkylidene is sufficiently big, they also can comprise the part-structure of one or more alkene or alkynes.
Perhaps R wherein can represent an aromatic radical bridge of being made up of one or more monokaryons or polynuclear aromatic compound, and aromatics also can be a kind of aromatic heterocycle in case of necessity,
Perhaps R wherein can represent an aralkyl bridge, and wherein the aromatic radical part can directly or be passed through (CH
2) n
-Alkyl be connected to that Y goes up or the α-C atom adjacent with carboxyl on, wherein n represents 1,2,3,4 or 5 integer,
And Y is a reactive group, especially NH
2Or SH group.
The example of R comprise methylene, ethylidene, positive propylidene or isopropylidene and neighbour-,-, right-phenylene or β, β '-naphthylene.
The NTA derivant also can have general formula Y-R
1-CH (COOH)-N (CHR
2COOH)
2,
R wherein
1The group that representative defines R,
And R wherein
2Can be CH
3(CH
2) n
-Alkyl, they can be to replace with OH for example or Cl, or unsubstituted, n represents 1,2,3,4 or 5 integer,
Perhaps R wherein
2Can be the alkyl isopropyl for example of side chain, the tert-butyl group or isobutyl,
Perhaps R wherein
2Can be aromatic radical bridge with R definition,
And Y wherein is a reactive group, especially NH
2Or SH group.
R or R
1Selection be to make complexing power and the unconjugated NTA relatively should be as much as possible little on the one hand, be to make the surface distance that arrives biology sensor be enough to little on the other hand to not disturbing the SPR phenomenon.
R
2Selection be to make sequestrant not be subjected to negatively influencing with the ability of wanting the chelated metal ion complexation with the complexing power of material to be determined.
Substituent R or R
1Or R
2Applicability for example can be by adopting suitable ligand, generally be that the polypeptide of His (6) modification carries out combination and studies and test.The affinity that complexing power is had the substituting group reduction ligand of negatively influencing; The substituting group that influences the non-specific binding ligand has increased and has been used for not and the combining of the ligand of the sequestrant of kation complexing.Y is a reactive group, by this group sequestrant is attached on the surface of biosensor unit, particularly is attached on the reactive group that is included in the hydrogel matrix.Therefore, the reactive group of sequestrant is connected on the reactive group on biosensor unit surface, especially is connected on the reactive group of hydrogel matrix; Particularly preferably, reactive group Y is NH
2Group, this group is attached on the ethyloic of the glucosan of modification.Being suitable for covalently bound other Y reactive group is the SH group, and this group can be transformed into stable thioether bond.Through be usually used in purifying or the reductive agent of synthetic (many) peptides for example mercaptoethanol in the presence of, the stability of this thioether bond is better than the key of disulfide.
Adopt the known method of coupling (many) peptide sequestrant can be connected on the hydrogel by its reactive group, for example use N-hydroxy-succinamide (NHS) and N-ethyl-N '-(dimethylaminopropyl) carbodiimides (EDC) (referring to for example Cuatrecasas and Parikh, 1972).
The sequestrant that is applicable to the scope of the invention is described in US-A-4877830, and people such as EP-A-253303 and WO90/12803 and Hochuli and Piesecki 1992 and Yokoyama are in 1993 the article.Transition metal ion, those ions in the period 4 preferably, they are particularly preferably as metallic ion.Particularly preferred have manganese, cobalt, nickel or copper ion, especially a Ni
2+
In the present invention, N-(5-amino-1-carboxylic amyl group) iminodiacetic acid and the N that describes by people such as Hochuli (1987)
α, N
α-two (1-carboxyethyls)-2,6-diaminocaproic acid are particularly preferably as the NTA derivant, particularly preferably use nickel as with the metallic ion of its complexing.
The preparation of relevant fusion (many) peptides is referring to EP-A-282042, this fusion (many) peptide has at least two adjacent histidine residues, guaranteeing that they can combine with preferred metallo-chelate, and be used to (described " His-Tag protein ") in the embodiment preferred of the present invention; The example of this class His-Tag protein is (His) 6-protein, and wherein the affinity peptide has 6 histidine residues each other abreast.
In fact, also sequestrant directly can be coupled on the metal surface of biology sensor substrate, can be undertaken by organic restraining barrier in the time of suitably with reactive group.Relevant this class restraining barrier can be disclosed referring to WO90/05303.But the scheme of fixing (many) peptides is normally preferred in hydrogel matrix, and this is because the structure of matrix is in close proximity to intracellular physiological condition, thereby approaches to measure the physical environment of bio-molecular interaction.
Major advantage of the present invention is that the surface of biology sensor is reproducible fully.This makes and carry out a series of experiments under the condition that can contrast.The new bio sensor unit satisfies the following requirement of regenerating:
1) (many) peptides that are attached on the metallo-chelate can fully be removed.
2) when loading further, the ability of (many) peptides that any combination will fix is not lost on the surface of biosensor unit.
3) binding characteristic through fixing (many) peptides is not affected.
There are several selections to can be used for regeneration and combined for example saturated sequestrant surface of metallic ion of His (6) protein of ligand.On the one hand, ligand can be removed by acid treatment (for example 10 mM acetate), and the loading of metallic ion depends on employed metallic ion, and most of metallic ion is stable.In case of necessity, with conjugated protein with metallic ion by other strong sequestrant of adding for example 100 mMEDTA from through fixing in conjunction with removing the sequestrant.The stability that metallic ion is attached on the sequestrant must be measured under each independent situation, and they depend on the stability of ligand/metal ion complex.When considering the repeatability of method, using the method on other sequestrant regeneration chelating surface is preferred for first method.
Under each situation, also need the regeneration on sandwich-like surface is experimentized, for example (wherein ligand 1 representative has (many) peptides of high affinity to metal-chelator-ligand 1-ligand 2 to the metal-chelator surface, ligand 2 representative has the big molecule of affinity to ligand 1 rather than metal-chelator surface), can not influence removing ligand 2 under the condition that ligand 1 is attached to the sequestrant surface and regenerate.The solution of high salt concentration can be used for conventional affinity chromatography, is particularly useful for this purpose.
The present invention also can be advantageously used in biological chemistry and purify except using in the conventional field of SPR method, comprises the fraction of desired protein with detection.This method has bigger superiority than conventional method of purification, not only about its degree of accuracy and speed, and relates to its simplification.
Others of the present invention relate to the biology sensor complete equipment that adopt SPR to measure (many) peptides and reactive material interphase interaction.This device is included in the surface plasmon resonance biosensor unit in first container, sequestrant in another container, in other container, be applicable to slaine with the sequestrant complexing, the reagent that is used to activate the biosensor unit surface in one or more other containers, in another container, be used to make the reagent of surperficial inactivation in case of necessity, in another container, be used in case of necessity the to regenerate reagent on surface, and one or more comparison proteins in one or more other containers in case of necessity.
A surface of biosensor unit preferably is made of hydrogel layer.Sequestrant is preferably to have certain density and its buffer solution is to be coupled to the lip-deep a kind of solution form of freezing deeply of sensor unit to exist.Nitrilotriacetic acid(NTA) derivant, especially N-(5-amino-1-carboxylic amyl group) iminodiacetic acid and N
α, N
α-two (1-carboxyethyls)-2, the 6-diaminocaproic acid is preferred sequestrant.
The slaine nickelous sulfate is preferred, preferably exists with stock solution form that can be diluted, and the degree that requires that the surface loads is depended in dilution.
In the preferred embodiment of the invention, a surface of biosensor unit is made up of the hydrogel with reactive group, especially carboxymethylated glucosan, the reagent that is used for the active bio sensor surface is N-hydroxy-succinamide and N-ethyl-N '-(dimethylaminopropyl) carbodiimides, and they preferably exist with the form of freezing solution deeply that is suitable for activation relevant with buffering solution with concentration.In preferred embodiments, reagent for retention in the N-carboxyl succinimide inactivation on the biosensor surface behind coupling (many) peptide is monoethanolamine, it has suitable concentration and in suitable buffer solution, and preferably exists to freeze form deeply equally.
The reagent that is used for the regeneration biological sensor surface is sequestrant, especially EDTA preferably.
The test protein that randomly is present in other container preferably has the affinity peptide that contains several histidine residues, and is being suitable for testing the concentration of loading biosensor surface and is existing as standard solution in buffer solution.
In carrying out test of the present invention, according to embodiment preferred of the present invention and consider simplification, coupling is carried out in hydrogel matrix, this is because the biosensor unit itself of buying from market has glucosan matrix, and when needs directly carry out coupling in reproducible mode, as if it becomes difficult to remove hydrogel layer from biosensor unit.
Adopting the couling process of N-hydroxy-succinamide is to be used for iminodiacetic acid or N with sequestrant N-(5-amino-1-carboxylic amyl group)
α, N
α-two (1-carboxyethyls)-2, the 6-diaminocaproic acid is attached on the biology sensor hydrogel surface.In the method, the sequestrant that contains free primary amino radical is a covalent coupling on the glucosan hydrogel surface of the biosensor unit of buying from the market (BIACore) of modification.The derivant that can be directly used in fixing nitrilotriacetic acid(NTA) as sequestrant is synthetic the preparation.Derivant synthetic mainly is to show and carry out (people such as Hochuli, 1987 by disclosed side; And EP-A-339389), in the presence of palladium carbon, carries out sour cracking, replace described hydrogenation, to remove blocking group but its difference is employing trifluoroacetic acid/trifluoromethane sulfonic acid.The material of gained exists owing to essentially no inorganic impurity and can be directly used on the surface that is coupled to biosensor unit in the method.
The method that employing is recommended by the biology sensor substrate manufacturer is sequestrant fixedly.Because the restriction of the instrument relevant with the lower molecular weight of nitrilotriacetic acid(NTA) derivant can not directly be measured through fixing amount, so by measuring the relative capacity indirect determination relative quantity of conjugated protein.Have been found that commercially available bovine serum albumin (BSA) is suitable for this purpose.
Confirmed test (many) peptide is very low for the affinity of the biology sensor substrate surface of non-nickle ion, and the sequestrant modification has been used on the surface of this substrate; So can not observe any (many) peptides is attached on the modified surface.Shown in embodiment 4, directly relevant with the nickel ion concentration that is used to load in conjunction with the surperficial capacity of test protein.By adjusting nickel ion concentration, can make the binding capacity requirement of experiment that is content with very little.
Others of the present invention relate to formula N
α, N
α-two (1-carboxyethyls)-2, the nitrilotriacetic acid(NTA) derivant of 6-diaminocaproic acid.As known nitrilotriacetic acid(NTA) derivant, this sequestrant also can be used for fixing metal sequestrant affinity chromatography, with purification of protein and peptide.
Accompanying drawing:
Fig. 1: N-(5-amino-1-carboxylic amyl group) iminodiacetic acid is lip-deep fixing at the glucosan of biosensor unit
Fig. 2: adopt the loading of bovine serum albumin indirect determination with the glucosan surface of N-(5-amino-1-carboxylic amyl group)-iminodiacetic acid
Fig. 3: adopt EDTA regeneration to load the biosensor surface of bovine serum albumin
Fig. 4: measure the non-specific binding of protein to biosensor surface
Fig. 5: measure the influence of nickel concentration to conjugated protein ability to the biosensor surface
Fig. 6: range protein is attached to and is mounted with N-(5-amino-1-carboxylic amyl group)-iminodiacetic acid/Ni
2+Biosensor surface on ability
Fig. 7: through the protein bound of His (6) modification to being mounted with N
α, N
α-two (1-carboxyethyls)-2, the biosensor surface of 6-diaminocaproic acid
The following examples are in order to explain the present invention: embodiment 1a) N-(5-amino-1-carboxylic amyl group) iminodiacetic acid is synthetic
4.17 gram bromoacetic acids (Aldrich) are dissolved in the 15ml 2M NaOH solution, make solution be cooled to 0 ℃.In this solution, slowly add 4.2 gram N
ε-benzyloxycarbonyl-the solution of L-lysine (Fluka) in 22.5ml 2M NaOH stirs simultaneously.After 2 hours, warm solution stirs then and spends the night to room temperature.Then after in this solution, slowly adding 45ml 1M HCl, solution 50 ℃ of heating 2 hours down, is centrifugally gone out resulting sediment, 0.1M HCl washing and dry (5 gram products, theoretical yield 5.9 grams) are arranged.The derivant of the above-mentioned acquisition of 0.87 gram is dissolved in the 10ml trifluoroacetic acid (Merck).In clear solution, slowly add 1ml trifluoromethane sulfonic acid (Merck), this potpourri was at room temperature stirred 1 hour in cooled on ice.Isolate resulting potpourri, to this solution of doing, add 30ml water to concentrating almost.The aliquot of this solution of (Pharmacia) fractionation on PD 10 posts, and water is as eluent.Merge neutral fraction and freeze-drying that ninhydrin is positive.B) N
α, N
α-two (1-carboxyethyls)-2,6-diaminocaproic acid synthetic
5.35 gram 2 bromopropionic acids (35mmol) are dissolved among the 15ml 2M NaOH, make this solution be cooled to 0 ℃.In room temperature with under stirring, in this solution, slowly drip the 4.2 gram N ε-benzyloxycarbonyl-solution of L-lysine (15mmol) in 22.5ml 2M NaOH, resulting potpourri is stirred down at 70 ℃ spend the night.After cooling off, in this solution, slowly add 45ml 1M HCl.Centrifugally go out sediment, with 0.1M HCl washing and dry.In order to remove the Z blocking group, sediment is dissolved in the minimum trifluoroacetic acid, be added dropwise to the trifluoromethane sulfonic acid of 0.1 volume when on ice bath, cooling off.After at room temperature stirring 1.5 hours, drips of solution is added in the diethyl ether of 10 volumes.With ether washing precipitate three times and dry.Isolate established sediment, to the solution of doing, add 30ml water to concentrating almost.The aliquot of this solution of (Pharmacia) fractionation on PD 10 posts adopts water as eluent.Merge neutral fraction and freeze-drying that ninhydrin is positive.Analyze the characteristic of determining compound by proton N MR.Embodiment 2
The coupling on the glucosan surface of N-(5-amino-1-carboxylic amyl group) iminodiacetic acid and surface plasmon resonance biosensor element
Under 25 ℃, employing HBS damping fluid in BIACore instrument (Pharmacia) (10mMHEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethylsulfonic acid).150mM NaCl and 5mM MgCl
2, pH 7.4) and carry out all fixing step.
Use CM5 biology sensor substrate (Pharmacia biology sensor, regular grade) to fix.(this biosensor unit has the hydrogel surface of being made up of carboxymethylated glucosan).Hydrogel surface activates by injecting 35 μ l 0.05M N-hydroxyl-succinimides (NHS)/0.2M N-ethyl-N '-(dimethylaminopropyl)-carbodiimides (EDC) solution (flow velocity 5 μ l/ branches).For the described N-among the embodiment 1 (5-amino-1-carboxylic amyl group) iminodiacetic acid is coupled on the activating surface, divide flow velocity with 3 μ l/, inject the 1M NaOH solution of 35 μ, 1 15mg/ml N-(5-amino-1-carboxylic amyl group) iminodiacetic acid.Unsaturated from the teeth outwards binding site carries out saturated with 1M monoethanolamine (pH 8.5) (the flow velocity 5 μ l/ branches) solution of 35 μ 1 that inject.In order to regulate the surface, inject 10 μ l, 100 mM EDTA solution (being adjusted to pH 8) with NaOH, and with 4 μ l 0.1MNiSO
4/ 0.2 M CH
3-COONa solution (flow velocity is 5 μ l/ branches) loads the surface.Fig. 1 represents the fixing process according to surperficial plasmid gene group resonance effect variation.Each fixing step is shown among the figure.Because the molecular weight (Mr295) of preparation is lower, the surperficial charging capacity with sequestrant can not directly be measured, so restrain bovine serum albumin solution and the indirect determination charging capacity by inject 35 μ l with constant flow rate (5 μ l/ branch) at 1 of the mobile damping fluid of 100ml.Sensor figure (Fig. 2) shows protein and surperficial obvious the combination.Adopt the standard solution of this protein to determine the repeatability that the surface is fixing by a series of tests.Embodiment 3
The regeneration of N-(5-amino-1-carboxylic amyl group) iminodiacetic acid (salt) acid surfaces
In order to measure the reproducibility of N-(5-amino-1-carboxylic amyl group) iminodiacetic acid (salt) acid surfaces, follow these steps to the order and as described in embodiment 2, carry out continuously 30 times with constant flow velocity (5 μ l/ branch): use Ni
2+Ion loads, and (BSA, solution Sigma) are then regenerated with EDTA (ethylene diamine-N, N, N ', N '-tetraacethyl) at 1 gram bovine serum albumin of the mobile damping fluid of 100ml to inject 35 μ l then.In order to calculate the numerical value (baseline that is shown in Fig. 3, inject maximal value and conjugated protein t=1), be determined at and injected bovine serum albumin (baseline) preceding 30 seconds, inject bovine serum albumin and finish (injection maximal value) preceding 30 seconds and inject the back 30 seconds resonance effect of bovine serum albumin (in conjunction with albumen t=1).
The result shows that EDTA (100 mM, pH 8) is used to remove the only reagent of conjugated protein.As shown in Figure 3, any reservation of conjugated protein after can not observing any surfactivity loss or regenerating afterwards (baseline) in the loading (injecting maximal value, conjugated protein t=1) afterwards.Because EDTA has removed the nickel ion of sequestrant combination, so after surface regeneration, also must use Ni again
2+Ion loads.Embodiment 4
Ni
2+Concentration is to the influence of conjugated protein ability
In order to measure the non-specific binding on protein and surface, the surface described in the described EDTA regeneration of embodiment 3 embodiment 1, and divide flow velocity to inject the solution of 20 μ l test proteins with 5 μ l/.After the surface is again with EDTA regeneration, with 4 μ l 0.1M NiSO
4/ 0.2 M CH
3-COONa solution (flow velocity 5 μ l/ branches) loads, and 20 μ l test protein solution reinject.As shown in Figure 4, there is not Ni
2+Shi Wei observes and surperficial combining, and still, is using Ni
2+After loading the surface, become as can be seen with the saturated state of test protein.In order to measure Ni
2+Concentration is to the influence of surperficial binding ability, as described in embodiment 3, with the Ni described in the EDTA regeneration embodiment 1
2+-N-(5-amino-1-carboxylic amyl group)-iminodiacetic acid (salt) acid surfaces uses the Ni of the prescribed concentration of 4 μ l in all cases
2+Solution (0.1M NiSO
4/ 0.2 M CH
3-COONa, flow velocity 5 μ l/ branches) load.After loading with nickel, inject protein, measure the resonance effect at t=1 place.The graph of a relation of resonance effect of being measured and nickel ion solution concentration (Fig. 5) shows the 100 μ M Ni that use 4 μ l
2+Solution just can reach the loading fully on surface.Lower concentration only causes the incomplete loading on surface, and the degree of loading depends on employed concentration.Embodiment 5
The binding ability of different proteins and N-(5-amino-1-carboxylic amyl group) iminodiacetic acid (salt) acid surfaces
In order to measure the different binding characteristics of different proteins, each of 40 μ l had the chick protein (its sequence is through His (6) ((His) 6-SCF) modification) of the sequence in the sequence table of being shown in, the solution of bovine serum albumin (Sigma) and egg albumen lysozyme (Sigma) (in the mobile damping fluid of 10 μ g/ml) injects on the biosensor surface, and it is synthetic that biosensor surface has been pressed embodiment 2.(preparation chick protein is after the commercially available expression factor [PQE 50, Diagen GmbH] is expressed, according to the purification scheme that system manufacturer provides purify [The QIAexpressionist, Diagen GmbH, scheme 3,7, the 45 pages of the 35th page and agreements].For it is further purified, adopt HQ/M anion-exchange column [Perseptive Biosystems, Freiburg] purification of protein, measure protein concentration by Bradford protein sample [BioRad]).Single combination of proteins characteristic is shown in Fig. 6: although its concentration is higher, but the resonance effect that lysozyme (Mr 14300) and bovine serum albumin (Mr 68000) have a little less than, on the contrary, the chick protein (Mr 22000) of His (6) modification has carried out strong combination the repeatedly with the surface.It can also be seen that opposite with the chick protein of His (6) modification, lysozyme and bovine serum albumin reach the saturated of surface.All chick protein through His (6) modification loads the surface that has reached saturation point protein is studied, for example by the supernatant of chick cell line cell nutrient culture media, the supernatant of the cell culture medium by injecting different clones and interacting with this protein.Can detect the combination of interaction material by the amplification resonance effect that after injection, occurs.Embodiment 6
(His) 6-SCF and N
α, N
α-two (1-carboxyethyls)-2, the combination on 6-diaminocaproic acid surface
With the condition of embodiment 2 preparation N-(5-amino-1-carboxylic amyl group) the described fairly similars of iminodiacetic acid under prepare N
α, N
α-two (1-carboxyethyls)-2,6-diaminocaproic acid surface.
The characteristic of the protein of warp (His) 6 modifications in order to measure with this surface combination is used Ni
2+Ion loads this surface, and the chick protein solution that on this surface, divides flow velocity to inject 40 μ l (the mobile damping fluids of 10 μ g/rl) with 5 μ l/, described chick protein has the sequence ((His) 6-SCF described in the embodiment 5) of His listed in sequence table (6) modification.Fig. 7 represents sensor record (sensorgram).This surface show can with the binding characteristic of comparing as embodiment 2 described surfaces with N-(5-amino-l-carboxylic amyl group) iminodiacetic acid modification.By using the standardization protein solution to carry out the repeatability that a series of tests determine that the surface is fixing.
Reference Cuatrecasas, P. and Parikh, 1972, journal of biological chemistry (J.Biochemistry), 11,2291.Hochuli, E., Dobel and Schacher, A., 1987, chromatogram magazine (J.Chromatognaphy) 411,177-184.Hochuli, E., and Piesecki, S., 1992, method (Methods) 4 (San Diego), 66-72.Lofas, S. and Johnsson, B., 1990, Chemical Society's periodical, chemical communication (J.Chem.Soc., Chem.Communications) 1526.Stenberg, E. etc., 1991. colloids and interface science collected works (L.of Colloid and Interface Science), 143,513.Yokoyama, T., Sigeko, A. and Masatoshi, K., 1993, chemical communication (Chem.Lett.) 2,383-386.
Sequence table
(1) general information:
(i) applicant
(A) title: Boehringer Ingelheim International GmbH
(B) street: Binger Strasse 173
(C) city: Ingelheim am Rhein
(E) country: FRG
(F) postcode: 55216
(G) phone: 06132/772282
(h) fax: 06132/774377
(ii) application exercise question: adopt surperficial plasmid gene group covibration to measure the method for biomolecule interphase interaction
(iii) sequence number: 1
(iv) computer-reader form:
(A) media type: Floppy dish
(B) computer: IBM PC can cooperate
(C) operating system: PC-DOS/MS-DOS
(D) software: PatentIn Release#1.0, Version#1.25 (EPO)
(2) information of SEQ ID NO:1
(i) sequence signature:
(A) length: 204 amino acid
(B) type: amino acid
(D) topological structure: linearity
(ii) molecule type: protein
(iii) suppose: do not have
(vi) original source
(A) organism: the family expenses chicken belongs to (Gallus domesticus)
(G) cell type: fibroblast
(ix) feature
(A) title/theme: protein
(B) location: 1..204
(xi) sequence description: SEQ ID NO:1:Met Ala Ser His His His His His His Gly Gly Ser Ala Gln Ser Ser1 5 10 15Cys Gly Asn Pro Val Thr Asp Asp Val Asn Asp Ile Ala Lys Leu Val
20 25 30Gly?Asn?Leu?Pro?Asn?Asp?Tyr?Leu?Ile?Thr?Leu?Lys?Tyr?Val?Pro?Lys
35 40 45Met?Asp?Ser?Leu?Pro?Asn?His?Cys?Trp?Leu?His?Leu?Met?Val?Pro?Asp
50 55 60Phe?Ser?Arg?Ser?Leu?His?Asn?Leu?Leu?Gln?Lys?Phe?Ser?Asp?Ile?Ser65 70 75 80Asp?Met?Ser?Asp?Val?Leu?Ser?Asn?Tyr?Ser?Ile?Ile?Asn?Asn?Leu?Thr
85 90 95Arg?Ile?Ile?Asn?Asp?Leu?Met?Ala?Cys?Leu?Ala?Phe?Asp?Lys?Asn?Lys
100 105 110Asp?Phe?Ile?Lys?Glu?Asn?Gly?His?Leu?Tyr?Glu?Glu?Asp?Arg?Phe?Ile
115 120 125Pro?Glu?Asn?Phe?Phe?Ser?Leu?Phe?Asn?Ser?Thr?Ile?Glu?Val?Tyr?Lys
130 135 140Glu?Phe?Ala?Asp?Ser?Leu?Asp?Lys?Asn?Asp?Cys?Ile?Met?Pro?Ser?Thr145 150 155 160Val?Glu?Thr?Pro?Glu?Asn?Asp?Ser?Arg?Val?Ala?Val?Thr?Lys?Thr?Ile
165 170 175Ser?Phe?Pro?Pro?Val?Ala?Ala?Ser?Ser?Leu?Arg?Asn?Asp?Ser?Ile?Gly
180 185 190Ser?Asn?Thr?Ser?Ser?Asn?Ser?Asn?Lys?Glu?Ala?Leu
195 200
Claims (26)
1. adopt biosensor unit to measure the method for (many) peptides and reactive material interphase interaction; wherein the resonance effect of the surperficial plasmid gene group that produces by interacting is to measure in the metal level at the interface of two media; this two medium can permeate for electromagnetic radiation; and has different refractive indexes; the medium that refractive index is lower is a water-bearing media; (many) peptides wherein exist with fixed form and contact with reactive material, it is characterized in that (many) peptides are fixing by metal-chelator.
2. according to the method for claim 1, it is characterized in that water-bearing media is the porous matrix of bio-capacitivity, especially hydrogel.
3. according to the method for claim 2, it is characterized in that sequestrant is that the reactive group that has by hydrogel is attached to the lip-deep of biosensor unit.
4. according to the method for claim 2 or 3, it is characterized in that hydrogel is selected from polysaccharide for example glucosan, agarose, Irish moss, alginates, starch, cellulose or derivatives thereof, or the organic polymer of water-swellable polyvinyl alcohol (PVA) for example, polyacrylic acid, polyacrylamide or polyglycol.
5. according to the method for claim 4, it is characterized in that hydrogel is a glucosan.
6. according to the method for claim 5, it is characterized in that glucosan has ethyloic as reactive group.
7. according to the method for one of aforesaid right requirement, it is characterized in that (many) peptides are to merge (many) peptides, except its bioactive fragment, also have the affinity peptide that has a histidine residues at least, and sequestrant are a kind of iminodiacetic acid derivatives.
8. according to the method for claim 7, it is characterized in that polypeptide has the affinity peptide, this affinity peptide has at least two adjacent histidine residues, and sequestrant is general formula Y-R-CH (COOH)-N (CH
2COOH)
2The nitrilotriacetic acid(NTA) derivant,
Wherein R represents type (CH
2) n
-Alkylidene, they can be that replace or unsubstituted, but condition is a substituting group effect of sequestrant are not had injurious effects, n represents 1,2,3,4,5,6,7,8,9 or 10 integer, if when alkylidene is sufficiently big, they also can comprise the part-structure of one or more alkene or alkynes
Perhaps R wherein can represent the aromatic radical bridge of being made up of one or more monokaryons or polynuclear aromatic compound, and aromatics also can be a kind of aromatic heterocycle in case of necessity,
Perhaps R wherein can represent an aralkyl bridge, and wherein the aromatic radical part can directly or be passed through (CH
2) n
-Alkyl be connected to Y and go up or be connected on the α-C atom adjacent with carboxyl, wherein n represents 1,2,3,4 or 5 integer,
And wherein Y is a reactive group, especially NH
2Or SH group.
9. method according to Claim 8 is characterized in that, the nitrilotriacetic acid(NTA) derivant is N-(5-amino-1-carboxylic amyl group) iminodiacetic acid.
10. according to the method for claim 7, it is characterized in that (many) peptides have the affinity peptide, this affinity peptide has at least two adjacent histidine residues, and sequestrant is general formula Y-R
1-CH (COOH)-N (R
2CHCOOH)
2The nitrilotriacetic acid(NTA) derivant,
R wherein
1Have group as defined in claim 8,
And R
2Can be CH
3(CH
2) n
-Alkyl, it can be to replace with OH for example or Cl, or unsubstituted, n represents 1,2,3,4 or 5 integer,
Perhaps R wherein
2Can be the alkyl of side chain, for example isopropyl, the tert-butyl group or isobutyl.
Perhaps R wherein
2Can be the aryl bridge that in the claim 8 R is defined,
And Y is a reactive group, especially NH
2Or SH group.
11. the method according to claim 10 is characterized in that, the nitrilotriacetic acid(NTA) derivant is N
α, N
α-two (1-carboxyethyls)-2, the 6-diaminocaproic acid.
12. the method according to one of among the claim 7-11 is characterized in that, sequestrant is and transition metal ion, especially the complexing of metal ion of period 4.
13. the method according to one of among the claim 7-12 is characterized in that sequestrant is and Ni
2+Ion complexation.
14. formula N
α, N
α-two (1-carboxyethyls)-2, the nitrilotriacetic acid(NTA) derivant of 6-diaminocaproic acid.
15. the purposes according to the nitrilotriacetic acid(NTA) derivant of claim 14 is used for by metal-chelating affinity chromatogram purification of protein and peptide.
16. measure the biosensor unit of (many) peptides and reactive material interphase interaction by the resonance effect of measuring the surperficial plasmid gene group in the two media metal level at the interface; medium wherein is permeable to electromagnetic radiation; and has different refractive indexes; the medium that refractive index is lower is a water-bearing media; it is characterized in that, be to be attached to lip-deep in the face of the biosensor unit of water-bearing media with the sequestrant that exists with the complexing of metal ion form in case of necessity.
17. the biosensor unit according to claim 16 is characterized in that, sequestrant is on the reactive group of the matrix that is attached to biocompatible porous, especially hydrogel.
18. biosensor unit according to claim 17, it is characterized in that, hydrogel is selected from polysaccharide for example glucosan, agarose, Irish moss, alginates, starch, cellulose or derivatives thereof, or the organic polymer of water-swellable polyvinyl alcohol (PVA) for example, polyacrylic acid, polyacrylamide or polyglycol.
19. the biosensor unit according to claim 18 is characterized in that, hydrogel is a glucosan.
20. the biosensor unit according to claim 19 is characterized in that, glucosan has carboxymethyl group as reactive group.
21. the biosensor unit according to one of among the claim 16-20 is characterized in that sequestrant is an iminodiacetic acid derivatives.
22. the biosensor unit according to claim 21 is characterized in that, sequestrant is general formula Y-R-CH (COOH)-N (CH
2COOH)
2The nitrilotriacetic acid(NTA) derivant,
Wherein R represents type (CH
2) n
-Alkylidene, it can be that replace or unsubstituted, but condition is a substituting group effect of sequestrant is not had injurious effects, n represents 1,2,3,4,5,6,7,8,9 or 10 integer, if when alkylidene is sufficiently big, it also can comprise the part-structure of one or more alkene or alkynes
Perhaps R wherein can represent the aromatic radical bridge of being made up of one or more monokaryons or polynuclear aromatic compound, and aromatics also can be a kind of aromatic heterocycle in case of necessity,
Perhaps R wherein can represent an aralkyl bridge, and wherein the aromatic radical part can directly or be passed through (CH
2) n
-The alkyl of type is connected to Y and goes up or be connected on the α-C atom adjacent with carboxyl, and wherein n represents 1,2,3,4 or 5 integer,
And wherein Y is a reactive group, especially NH
2Or SH group.
23. the biosensor unit according to claim 22 is characterized in that, the nitrilotriacetic acid(NTA) derivant is N-(5-amino-1-carboxylic amyl group) iminodiacetic acid.
24. the biosensor unit according to claim 21 is characterized in that, (many) peptides have the affinity peptide, and this affinity peptide has at least two adjacent histidine residues, and sequestrant is general formula Y-R
1-CH (COOH)-N (R
2CHCOOH)
2The nitrilotriacetic acid(NTA) derivant,
R wherein
1Be the group that has as defined in claim 8,
And R wherein
2Can be CH
3(CH
2) alkyl of n, this group can be that replace or unsubstituted, n represents 1,2, and 3,4 or 5 integer,
Or R wherein
2Can be the alkyl of side chain, for example isopropyl, the tert-butyl group or isobutyl,
Or R wherein
2Can be the aryl bridge that has in the claim 8 the R definition,
And Y wherein is a reactive group, especially NH
2Or SH group.
25., it is characterized in that the nitrilotriacetic acid(NTA) derivant is N according to the biosensor unit of claim 24
α, N
α-two (1-carboxyethyls)-2, the 6-diaminocaproic acid.
26. the complete equipment that the biology sensor that adopts SPR to measure (many) peptides and reactive material interphase interaction is used, wherein this device is included in the surface plasmon resonance biosensor unit in first container, sequestrant in another container, in another container, be suitable for slaine with the sequestrant complexing, the reagent that in one or more other containers, is used to activate the biosensor unit surface, the reagent that in another container, is used for surperficial inactivation in case of necessity, the reagent on surface and the protein of the contrast of one or more in one or more other containers in case of necessity in case of necessity are used to regenerate in another container.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4433980.1 | 1994-09-23 | ||
| DE4433980A DE4433980C2 (en) | 1994-09-23 | 1994-09-23 | Process and biosensor hit for investigating the interaction of biomolecules by means of surface plasma resonance |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1158658A true CN1158658A (en) | 1997-09-03 |
Family
ID=6528988
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN95195182A Pending CN1158658A (en) | 1994-09-23 | 1995-09-21 | Process of investigating the interaction between biomolecules by means of surface plasmon resonance |
Country Status (12)
| Country | Link |
|---|---|
| EP (1) | EP0784792A2 (en) |
| JP (1) | JPH10505910A (en) |
| KR (1) | KR970706494A (en) |
| CN (1) | CN1158658A (en) |
| AU (1) | AU3608995A (en) |
| BR (1) | BR9509077A (en) |
| CA (1) | CA2198615A1 (en) |
| CO (1) | CO4410395A1 (en) |
| DE (1) | DE4433980C2 (en) |
| PL (1) | PL319354A1 (en) |
| WO (1) | WO1996009547A2 (en) |
| ZA (1) | ZA958024B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101261226B (en) * | 2007-03-08 | 2010-12-08 | 北京宏荣博曼生物科技有限责任公司 | Surface plasma resonance instrument chip based on polyethyleneglycol |
| CN114797804A (en) * | 2022-03-29 | 2022-07-29 | 翌圣生物科技(上海)股份有限公司 | NTA chromatographic medium with long connecting arm and preparation method thereof |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6165335A (en) | 1996-04-25 | 2000-12-26 | Pence And Mcgill University | Biosensor device and method |
| US6107080A (en) * | 1996-04-25 | 2000-08-22 | Mcgill University | Biosensor device and method |
| US6130037A (en) * | 1996-04-25 | 2000-10-10 | Pence And Mcgill University | Biosensor device and method |
| US5955379A (en) * | 1996-04-25 | 1999-09-21 | Mcgill University | Biosensor device and method |
| AU6883198A (en) * | 1997-04-24 | 1998-11-13 | American Home Products Corporation | Method for the identification and characterization of nuclear receptor ligands |
| CA2342767C (en) * | 1998-09-03 | 2009-11-03 | Trellis Bioinformatics, Inc. | Multihued labels |
| AUPP856399A0 (en) * | 1999-02-08 | 1999-03-04 | Australian Membrane And Biotechnology Research Institute | Improved compounds for protein binding |
| US6787368B1 (en) | 1999-03-02 | 2004-09-07 | Helix Biopharma Corporation | Biosensor method for detecting analytes in a liquid |
| US7804592B2 (en) | 2003-10-16 | 2010-09-28 | Nard Institute, Ltd. | Method for measuring a surface plasmon resonance and noble metal compound used for the same |
| EP1674857B1 (en) * | 2003-10-16 | 2014-04-02 | Kabushiki Kaisha Nard Kenkyusho | Method for measuring a surface plasmon resonance and noble metal compound used for the same |
| JP2010197046A (en) * | 2007-05-28 | 2010-09-09 | Tanaka Holdings Kk | Biosensor |
| EP2015071A1 (en) | 2007-07-13 | 2009-01-14 | FUJIFILM Corporation | Carrier, process for producing same, bioreactor, and chip for surface plasmon resonance analysis |
| KR100953558B1 (en) | 2007-12-12 | 2010-04-21 | 한국전자통신연구원 | PS-SPCL searching apparatus and method using surface plasmon resonance |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4569794A (en) * | 1984-12-05 | 1986-02-11 | Eli Lilly And Company | Process for purifying proteins and compounds useful in such process |
| CA1304886C (en) * | 1986-07-10 | 1992-07-07 | Heinz Dobeli | Metal chelate resins |
| IL85137A (en) * | 1987-01-21 | 1992-02-16 | Ares Serono Res & Dev Ltd | Method of assaying for a ligand using surface plasmon resonance effect |
| CA1340522C (en) * | 1987-03-10 | 1999-05-04 | Heinz Dobeli | Fusion proteins containing neighbouring histidines for improved purification |
| DE58905660D1 (en) * | 1988-04-25 | 1993-10-28 | Hoffmann La Roche | Diagnostic tools. |
| SE462454B (en) * | 1988-11-10 | 1990-06-25 | Pharmacia Ab | METHOD FOR USE IN BIOSENSORS |
| US5169936A (en) * | 1989-04-14 | 1992-12-08 | Biogen, Inc. | Protein purification on immobilized metal affinity resins effected by elution using a weak ligand |
| DE59106527D1 (en) * | 1990-11-14 | 1995-10-26 | Hoffmann La Roche | Immunosensitive transducer and method for its production. |
-
1994
- 1994-09-23 DE DE4433980A patent/DE4433980C2/en not_active Expired - Fee Related
-
1995
- 1995-09-21 CN CN95195182A patent/CN1158658A/en active Pending
- 1995-09-21 CA CA002198615A patent/CA2198615A1/en not_active Abandoned
- 1995-09-21 CO CO95043762A patent/CO4410395A1/en unknown
- 1995-09-21 PL PL95319354A patent/PL319354A1/en unknown
- 1995-09-21 EP EP95933414A patent/EP0784792A2/en not_active Ceased
- 1995-09-21 JP JP8510606A patent/JPH10505910A/en active Pending
- 1995-09-21 AU AU36089/95A patent/AU3608995A/en not_active Abandoned
- 1995-09-21 BR BR9509077A patent/BR9509077A/en not_active Application Discontinuation
- 1995-09-21 WO PCT/EP1995/003731 patent/WO1996009547A2/en not_active Application Discontinuation
- 1995-09-22 ZA ZA958024A patent/ZA958024B/en unknown
-
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- 1996-09-21 KR KR1019970701787A patent/KR970706494A/en not_active Application Discontinuation
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101261226B (en) * | 2007-03-08 | 2010-12-08 | 北京宏荣博曼生物科技有限责任公司 | Surface plasma resonance instrument chip based on polyethyleneglycol |
| CN114797804A (en) * | 2022-03-29 | 2022-07-29 | 翌圣生物科技(上海)股份有限公司 | NTA chromatographic medium with long connecting arm and preparation method thereof |
| CN114797804B (en) * | 2022-03-29 | 2023-08-04 | 翌圣生物科技(上海)股份有限公司 | NTA chromatographic medium with long connecting arm and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| DE4433980A1 (en) | 1996-03-28 |
| ZA958024B (en) | 1996-03-25 |
| WO1996009547A2 (en) | 1996-03-28 |
| JPH10505910A (en) | 1998-06-09 |
| DE4433980C2 (en) | 1996-08-22 |
| KR970706494A (en) | 1997-11-03 |
| WO1996009547A3 (en) | 1996-05-30 |
| PL319354A1 (en) | 1997-08-04 |
| CA2198615A1 (en) | 1996-03-28 |
| EP0784792A2 (en) | 1997-07-23 |
| CO4410395A1 (en) | 1997-01-09 |
| AU3608995A (en) | 1996-04-09 |
| BR9509077A (en) | 1997-11-25 |
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