A kind of covalent coupling agent and containing the chemically modified covalent coupling solid phase carrier of this covalent coupling agent and the preparation method and application of this carrierTechnical field
The present invention relates to technical field of immunoassay, refer to a kind of covalent coupling agent especially, the invention still further relates to a kind of chemically modified covalent coupling solid phase carrier and its preparation method and application.
Background technology
The many kinds of substances such as plastics, cellulose membrane or glass can be used as solid phase material, but polystyrene plastic is widely used owing to having excellent optical transparence, higher protein adsorption performance, good processing technology and cheap price usually.For polystyrene plastic, backbone structure is carbochain, and side chain is with non-polar group, and the solid phase surface therefore formed by it is hydrophobic in nature.The profile of solid phase also can be diversified, can make the shapes such as test tube, microwell plate, embedded hexagonal column, microballoon, film.What shape of concrete selection will be determined according to the requirement of specific immunoassay, but total be that to increase solid phase specific surface and improve immune response speed be principle.
Early stage and present solid phase isolation technique is utilize physical adsorption principle mostly, make binding label the effect of hydrophobic bond only can be relied on to be adsorbed on solid phase surface thus produce with free label to be separated, we are referred to as solid-phase physical sorbent technology, and this respect technology is comparatively ripe.
Bioactive molecules in solid phase material and immunoassay is mostly the higher compound of molecular weight.Generally speaking, there is four interactions between macromole: Van der Waals force (comprising hydrogen bond), ionic linkage, hydrophobic bond and covalent linkage.
(1) Van der Waals force is the interaction produced due to directed, induction and dispersion effect between macromole, and it is a kind of very weak reactive force, and changes with six power inverses of non covalent bond bonded atom or intermolecular distance and 1/R6.
(2) ionic linkage is a kind of electrostatic attraction effect between positive and negative electric charge. but charged ion is more prone to form hydrated ion with water molecules in aqueous, and the ionic linkage therefore between them weakens greatly.
(3) to be non-polar group be hydrophobic bond avoids aqueous phase and be clustered in reactive force together.Close in protein molecule and have the amino-acid residue of the nonpolar jack of multiple band. between these hydrophobicity jacks and the α-CH base of they and protein main chain skeleton ask and tend to form hydrophobic bond.Generally believe, in immunoassay system protein when without covalent linkage and solid phase material in conjunction with, hydrophobic bond is that it is adsorbed in the predominant intermolecular forces on hydrophobic solid phase surface (as PS surface).But for the biomolecules of parent's property forever, the reactive force forming hydrogen bond due to water molecules in they and solution is greater than the reactive force by hydrophobic bond and solid phase, is thus difficult to be adsorbed on hydrophobic nature solid phase surface; Same for small molecule bioactive molecule, because the possibility of their molecular weight little formation hydrophobic bond reduces greatly. therefore also more difficult in solid phase surface absorption.
(4) covalent linkage is the strong interaction relying on the chemical reaction of active group between differing molecular to produce.As long as with suitable active group, macromole, small molecules and hydrophihc bioactive molecule can be combined in solid phase surface securely by covalent linkage.This point has more significance for small-molecule substance because covalent linkage may be between they with solid phase directly and the unique channel that is surely combined of fringe.
Contrast this four interactions, be not difficult to find, in immunoassay system, solid phase and bioactive molecules are difficult to be produced by Van der Waals force and ionic linkage combine, and its limitation of the adsorption produced by hydrophobic bond is obvious. so covalent linkage becomes the preferably selection that solid phase is combined with bioactive molecules.On the other hand, because the analysis of bioactive molecules and feature thereof is known, most of biomolecules as immunological assay reagents has-NH2,-COOH or-SH isoreactivity group, this between they and solid phase covalent coupling provide may and prerequisite.
In recent ten years, along with the development of immuno analytical method, increasing small molecules or wetting ability biological substance, as polypeptide, haptens, polysaccharide or polynucleotide become immunological assay reagents, and these biomolecules are difficult to or can not be adsorbed on plastic solid phase surface by physical action at all, therefore the separation method of physical adsorption is relied on can not to have met new development and the new demand of immuno analytical method, seek to utilize covalent linkage biomolecules and solid phase material to be combined into securely an important selection, solid state chemistry coupling technology arises at the historic moment in immunoassay practice.
Solid state chemistry coupling technology focus on first with chemistry or physical method make solid phase surface produce suitable active group, and then allowing bioactive molecules be connected to solid phase surface with covalent linkage by ad hoc fashion, it comprises the content of two aspects: solid phase material synthesis/process for modifying surface and bioactive molecules advocate connection technology at solid phase surface.This technology relates to the multi-disciplinary knowledge such as immunology, polymer material science, biological chemistry and organic chemistry.
Just propose in the solid phase synthesis of polypeptide and poly rib thuja acid and applied, the granules of polystyrene of such as surface chemical modification is as the carrier of solid phase synthesis, but these methods mostly use chemical reagent that toxicity is very large and complex operation step is complicated, polystyrene is swelling in solution used simultaneously, its optical property (transparency) reduces greatly, and obviously these methods are unsuitable for being used in immunoassay field.So foreign study person from the seventies with regard to seek new chemical conjugation methods for immunoassay practice in. obtain remarkable progress so far.
How to introduce suitable active group at solid phase surface is the problem that solid state chemistry coupling technology primarily solves, and is also the step that difficulty is larger.Developing direction is the synthesis modification technology adopting solid phase material, select the monomer with active group to be all polymerized or copolymerization can synthesize people expect the solid phase material of performance.The designability of synthesis of polymer material is that solid state chemistry coupling technology provides wide development space, and synthesis modification is one of the most promising method in this field.U.S.'s Seradyn company vinylbenzene and vinylformic acid carry out the microballoon (business is called CM-MP) that surface carboxyl groups modification is produced in letex polymerization.This microballoon is identical with ordinary polystyrene microballoon specific refractory power, and according to adding the difference of monomer ratio. carboxyl can from the change of 5% ~ 100% in the ratio of microsphere surface.With the carbodiimide of dissolubility forever by the activated carboxylic on microballoon, then react with the amino of protein and generate amido linkage, proteinaceous solid fixes on solid phase surface the most at last.
Another developing direction is the surface modification of solid phase material, mainly contains following several mode:
(1) photochemistry surface modification: photochemical reaction object styrene plastic(s) modifying surface obtains by the patented product Covalink NH on-gauge plate of NUNC company of Denmark exactly.In this product, secondary amine group is connected to solid phase surface by " the space arm " that 2nm is long, and " space arm " is 10 in (face) density of solid phase surface14/ cm2.The secondary amine on Covalink NH surface is different from other high reactivity groups, and existing sufficiently high activity can keep certain stability again, so non-specific binding not only but also can be down to minimum with bioactive molecules covalent attachment by it.In addition, because protein molecule is connected with solid phase surface by sufficiently long " space arm ", the appearance of protein inactivation problem can so just be avoided.When using this product, the carboxyl of bioactive molecules, by reacting with the secondary amine of Covalink NH after the activation of water-soluble carbodiimide (EDC), is finally connected to solid phase surface again.
The preparation of Covalink NH on-gauge plate: first use 8 one methoxypsoralens through organic synthesis, in connection, " space arm " and secondary amine group obtain psoralene and derive mixture, and then irradiate a few hours under the UV-light this mixture being greater than 350nm at wavelength and can obtain the finished product.
(2) radiation modification: due to gamma-rays, there is very strong penetrance and cause the ability of molecular ionization, thus available it radiation modification is carried out to solid phase material.Use Co60as source of radiation by vinyl monomer, as butenoic acid, vinylformic acid etc., radiation grafting on polystyrene surface, thus introduces carboxyl-reactive group at solid phase surface, then with water-soluble carbodiimide by protein bound in solid phase.With gamma-rays by hydrazides group radiation grafting at solid phase surface, then hydrazides group is connected with the carboxyl of protein.Solid phase surface radiation modification causes protein to be mainly connected with the solid phase surface of radiation modification by covalent linkage not have ample evidence to illustrate.Gamma-rays can cause plastic molecules ionize thus produce a large amount of secondary electron, and most of secondary electron all can cause material inside that series of physical and chemical transformation occur, and makes solid phase inside and surface produce a large amount of polarity or chemical active radical.So protein adsorption is at solid phase surface, may be physical action and the coefficient result of covalent linkage.Bioactive molecules still needs further discussion at the absorption mechanism of the solid phase surface of radiation modification.
(3) chemical process surface modification: this method uses some chemical reagent to process solid phase material surface, makes it bring some active group.One is diazotization chemic modified method: the effective nitrosonitric acid of p-poly-phenyl vinyl plastics and Glacial acetic acid process, phenyl ring occur nitrated, add Reduction by Thiosulfate again and become aminopolystyrene, the amino on last phenyl ring and nitrite reaction obtain the plastic test tube that diazotization polystyrene activates.When adding lgG antibody in pipe, the phenolic hydroxyl group on its amino-acid residue is combined with solid phase surface.Because this method is too harsh, so less use.Xenopore company of the U.S. uses a unique wet processing process development to go out the covalently bound immunoassay plate (commodity are called Xenobind) of a kind of energy one step.This product surface need not add any coupling agent or activator and directly be connected with the amino covalence of protein or peptide molecule.This method is the best chemical modification method of the effect reported in current document, but the said firm is very tight to this technical know-how, without patent and comparatively detail file can consult.
(4) top coat modification: this method refers to cover at solid phase surface the coating that one deck has active group or good adsorption properties.Adopt PVLA (amino lactone polystyrene) method: because PVLA has amphipathic, it is adsorbed on as the derivative of polystyrene the polystyrene solid phase surface dredging property forever securely by physical action on the one hand, its amino lactone can produce aldehyde radical by periodate oxidation on the other hand, and the amino of protein can react with the aldehyde radical that produces on PVLA compared with under mild conditions, final protein is being fixed on solid phase surface by PVLA.This method of alkaline phosphatase is fixed, and unit carrier surface enzyme activity is 6 ~ 7 times of physisorphtion.
Solid state chemistry coupling technology mainly utilizes chemistry or physical method to solid phase material modifying surface or chemically modified, introduces active group, then is connected with solid phase surface by covalent linkage by biological boatman by the chemical reaction that platform is suitable.It has numerous sorrow points compared with solid-phase physical sorbent technology:
1, the binding capacity of solid phase surface bioactive molecules is improved: adopt solid state chemistry coupling technology artifact bioactive molecule to have significant increase at the binding capacity of solid phase surface than physisorphtion.Side as increased dozens or even hundreds of times for its binding capacity of peptide material; Even and if also higher combination rate can be obtained for common antibody under low concentration.
2, the scope of immunoassay object is expanded: for the biomolecules that polypeptide, glycolipid or polynucleotide etc. are less, because the water repellent region of these molecules is few, use the main physical adsorption techniques of hydrophobic interaction that relies on fully can not be adsorbed in plastic solid phase surface, and in washing step afterwards, be easy to De contamination phenomenon occurs.And applied chemistry coupling technology, these biological micromolecules can be combined with solid phase material preferably by covalent linkage, thus the biologically active substance being applied to immunoassay is increased greatly, facilitate the Study and Development that Novel immune analyzes commercial reagents box.
3, the forfeiture solid-phase physical sorbent technology of bioactive molecules activity is avoided to make protein molecule be adsorbed on solid phase surface by physical action, likely cause the change of protein molecule three, four rubbish structure, cause burying or losing (even producing unwanted new active site) of proteins react active site.The binding site that the solid phase polyclonal antibody that research announcement relies on physisorphtion to fix only has 5% ~ 10% binding site and solid single clonal antibody to be less than 3% has the ability of conjugated antigen, and chemical coupling techniques can allow protein molecule in a specific way orientation be combined in solid phase surface, reach the object avoiding protein inactivation sex change, make the more objective target molecule easily and in analytical solution of protein active point that immune association reaction occurs simultaneously.
4, the repeatability of immune analysis method is improved in physical adsorption process, bioactive molecules is attached to solid phase surface mainly with " at random " form greatly, position and the forfeiture degree of different biological molecules active site are not quite similar, simultaneously the bonding force (degree) of they and solid phase is also different, in immunoassay after washings rinses, in each experiment, the amount of solid phase knot platform biomolecules is just difficult to be consistent, separating effect is subject to the impact of the many factors such as operation, solution temperature and pH value, so the repeatability of physical adsorption solid phase immunoassays formats is poor.And solid state chemistry coupling technology makes to be combined securely by covalent linkage between biomolecules with solid phase, it is identical that different biological molecules and solid phase tie platform degree, and the immunocompetence point that they retain also can be consistent preferably, so chemical coupling solid phase immunoassays formats is reproducible.Report is had to compare the variation coefficient (CV) of physical adsorption and chemical coupling solid phase immunoassays formats, the enzyme plate of physically based deformation absorption high reactivity and middle activity, CV value is 6.7% and 16.9%, and use the enzyme plate of chemical coupling techniques, CV value is 1.9%, and visible chemical coupling techniques can improve the repeatability of immunoassay greatly.
In addition, solid state chemistry coupling technology make the aspects such as the stability of bioactive molecules, storage capability also comparatively solid-phase physical sorbent technology improve a lot.Solid state chemistry coupling technology is subject to extensive attention and the attention of various countries immunoassay worker day by day owing to having so outstanding advantage, it all has a direct impact the automatization of the sensitivity of immunoassay, quality and operation, has nowadays become one of gordian technique impelling immunoassay integral level to reach a new high.In addition economically, along with solid state chemistry coupling technology is in the extensive utilization of commercial immunoassay kit, it will become new growth engines in immunoassay field.
Summary of the invention
An object of the present invention is to provide a kind of covalent coupling agent, can the multiple biomacromolecule of coupling, realize nucleic acid, the covalency of peptide and protein fixes, overcome the problem of the non-specific adsorption that physical adsorption process causes, substantially increase the specificity of immunology detection.
Another object of the present invention is to provide a kind of chemically modified covalent coupling solid phase carrier, overcomes the problem of the non-specific adsorption that physical adsorption process causes equally, improves the specificity of immunology detection.
Another object of the present invention is to provide a kind of preparation method of chemically modified covalent coupling solid phase carrier, and its preparation method is simple, is easy to scale operation.
Another object of the present invention is to provide a kind of application of chemically modified covalent coupling solid phase carrier.
Technical scheme of the present invention is achieved in that
A kind of covalent coupling agent, one end of described covalent coupling agent is the photochemical activity group for being fixed on surface of solid phase carriers, and the other end is connected with and nucleic acid, polypeptide, protein covalent reaction chemical group by space arm.
Preferably, described photochemical activity group utilizes ultraviolet light, and the component or biomolecules with special properties are coupled to surface of solid phase carriers, the light wave of described ultraviolet light to be wavelength be 300-420nm.
Preferably, described photochemical activity group, its base type has any one of the compounds such as furocoumarin(e) class, aryl azides class, Benzophenones and Anthraquinones.
Preferably, described space arm is the polymkeric substance of the chemical group such as ethylene glycol or vinyl.
Preferably, described covalent reaction chemical group is carboxyl (-COOH), amino (-NH2), sulfydryl (-SH), thiocyanogen (-SCN), cyano group (-CN), isothiocyano (-NCS), N-hydroxy-succinamide ester group (-NHS), chlorsulfonic acid base (-ClSO2), one in the group such as phosphoramidite base.
A kind of chemically modified covalent coupling solid phase carrier, comprise solid phase carrier and be connected to the coupling agent on described solid phase carrier, described coupling agent adopts above-mentioned covalent coupling agent.
Preferably, described solid support material is the macromolecular materials such as polystyrene, urethane, silicon rubber, polyvinyl chloride, tetrafluoroethylene, polyacrylamide, polyoxyethylene glycol, polyvinylpyrrolidone, nitrocellulose.
A preparation method for chemically modified covalent coupling solid phase carrier, comprises the steps:
1. the immobilization of coupling agent:
By the coupling agent dissolution with solvents dilution be applicable to, evenly add polymer solid phase carrier respectively, uviolizing 15-20 minute, discards coupling agent, with ultrapure water cleaning polymer solid phase carrier, for subsequent use;
2. covalent coupling:
With the antibody of suitable damping fluid in proportion needed for dissolved dilution, antigen or nucleic acid primer, after mixing, uniformly distributing is in preparing on the solid phase carrier of coupling agent, and room temperature reaction 3-4 hour, discards damping fluid, 37 DEG C of dry 30-45 minute.
The beneficial effect that the present invention produces is: one aspect of the present invention proposes a kind of covalent coupling agent, realize nucleic acid, the covalency of peptide and protein fixes, overcome the problem of the non-specific adsorption that physical adsorption process causes, substantially increase the specificity of immunology detection; On the other hand a kind of chemically modified covalent coupling solid phase carrier is proposed, possesses the advantage of described covalent coupling agent, the invention allows for a kind of preparation method of chemically modified covalent coupling solid phase carrier, its preparation method is simple, be easy to scale operation, the invention allows for a kind of application of chemically modified covalent coupling solid phase carrier, more practical.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the space structure schematic diagram of a kind of chemically modified covalent coupling of the present invention solid phase carrier, and in figure, 1 is solid phase carrier; 2 is photochemical activity group; 3 is space arm; 4 is covalent reaction chemical group; A represents coupling nucleic acid 5; B represents coupled peptide 6; C represents coupling protein matter 7.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
The total technical scheme of the present invention is as follows:
As shown in Figure 1: a kind of photochemical activity group 2 of its one end of covalent coupling agent is fixed on solid phase carrier 1 surface under UV-light room temperature is irradiated, and the covalent coupling agent the other end is connected with and nucleic acid 5, polypeptide 6, protein 7 covalent reaction chemical group 4 by space arm 3.
In the present invention, the photochemical activity group 2 of covalent coupling agent, its base type has the one of the compounds such as furocoumarin(e) class, aryl azides class, Benzophenones and Anthraquinones; The covalent reaction chemical group 4 of covalent coupling agent is carboxyl (-COOH), amino (-NH2), sulfydryl (-SH), thiocyanogen (-SCN), cyano group (-CN), isothiocyano (-NCS), N-hydroxy-succinamide ester group (-NHS), chlorsulfonic acid base (-ClSO2), one in the group such as phosphoramidite base.The present invention through overtesting determination furocoumarin(e) class and anthraquinone analog compound best performance different.The present invention is through overtesting determination isothiocyano (-NCS), N-hydroxy-succinamide ester group (-NHS), chlorsulfonic acid base (-ClSO2), the best performance of phosphoramidite base is different, covalent coupling reaction conditions is gentle, is applicable to scale operation.
In the present invention, the light wave of ultraviolet light to be wavelength be 300-420nm, when getting 350nm, effect is best.
In the present invention, space arm 3 is the polymkeric substance of the chemical group such as ethylene glycol or vinyl.
A kind of chemically modified covalent coupling solid phase carrier, comprises solid phase carrier and is connected to the coupling agent on solid phase carrier, the covalent coupling agent during the employing of this coupling agent is above-mentioned.The present invention can pass through immunology special detector, detects the immunological response that solid phase carrier occurs.
A preparation method for chemically modified covalent coupling solid phase carrier, comprises the steps:
1. the immobilization of agent is joined:
By the coupling agent dissolution with solvents dilution be applicable to, evenly add polymer solid phase carrier 1 respectively, ultraviolet (350nm) irradiates 15-20 minute, discards coupling agent, with ultrapure water cleaning polymer solid phase carrier 1, for subsequent use;
2. valency coupling:
With the antibody of suitable damping fluid in proportion needed for dissolved dilution, antigen or nucleic acid primer, after mixing, uniformly distributing is on the solid phase carrier 1 preparing coupling agent, and room temperature reaction 3-4 hour, discards damping fluid, 37 DEG C of dry 30-45 minute.In the present invention, solid phase carrier 1 material is the macromolecular materials such as polystyrene, urethane, silicon rubber, polyvinyl chloride, tetrafluoroethylene, polyacrylamide, polyoxyethylene glycol, polyvinylpyrrolidone, nitrocellulose.
In the present invention; substance withdrawl syndrome, pH value and the components such as coupling agent diluent, covalent reaction damping fluid can be identical; can be different; it is all within protection scope of the present invention, for different components out cited by specific embodiment be the present inventor do experiment in some of them preferred embodiment.
A kind of chemically modified covalent coupling solid phase carrier, is detecting the application in biological sample, and detected object is the content of antigen, antibody and nucleic acid in blood or body fluid.
Key of the present invention is the solidification of coupling agent and the preparation method of covalent coupling, is specifically implemented as follows:
Embodiment 1
The solidification of coupling agent and the preparation method of covalent coupling, comprise the steps:
Coupling agent photochemical activity group 2 is coumarins, and space arm 3 is the pentamer of ethylene glycol, and covalent reaction chemical group 4 is isothiocyano (-NCS).Be calculated in mass percent, the coupling agent dmso solution of 0.036g/L is diluted, stir evenly evenly, add in 96 hole polystyrene microwell plates respectively, every hole 100 microlitre, ultraviolet lamp (350nm) irradiates 30 minutes, discard diluent, with milli-Q water microwell plate 3 times, pat dry for subsequent use.
With the sodium bicarbonate buffer liquid of 0.05mol/L, pH9.5, dilute appropriate hepatitis B surface antigen, mixing, add respectively and solidified in 96 hole polystyrene microwell plates of coupling agent, every hole 100 microlitre, room temperature reaction 4 hours, discard damping fluid, pat dry, 37 DEG C of dryings 30 minutes.
Embodiment 2
The solidification of coupling agent and the preparation method of covalent coupling, comprise the steps:
Coupling agent photochemical activity group 2 is Anthraquinones, and space arm 3 is the pentamer of ethylene glycol, and covalent reaction chemical group 4 is chlorsulfonic acid base (-ClSO2).Be calculated in mass percent, the coupling agent dmso solution of 0.028g/L is diluted, stir evenly evenly, add in 96 hole polystyrene microwell plates respectively, every hole 100 microlitre, ultraviolet lamp (350nm) irradiates 30 minutes, discard diluent, with milli-Q water microwell plate 3 times, pat dry for subsequent use.
With the sodium bicarbonate buffer liquid of 0.035mol/L, pH9.3, dilute appropriate Procalcitonin monoclonal antibody, mixing, add respectively and solidified in 96 hole polystyrene microwell plates of coupling agent, every hole 100 microlitre, room temperature reaction 4 hours, discard damping fluid, pat dry, 37 DEG C of dryings 45 minutes.
Embodiment 3
The solidification of coupling agent and the preparation method of covalent coupling, comprise the steps:
Coupling agent photochemical activity group 2 is Anthraquinones, and space arm 3 is six aggressiveness of vinyl, and covalent reaction chemical group 4 is phosphoramidite base.Be calculated in mass percent, by the coupling agent of 0.028g/L dimethyl sulphoxide aqueous solution dissolved dilution, stir evenly evenly, point sample is in polystyrene slides respectively, each check point 50 receives liter, and ultraviolet lamp (350nm) irradiates 30 minutes, discards diluent, with milli-Q water slide glass 3 times, pat dry for subsequent use.
With the sodium carbonate buffer of 0.05mol/L, pH9.6, dilute appropriate hepatitis B virus nucleic acid primer, mixing, adds respectively and has solidified on the slide glass of coupling agent, and each check point 30 receives liter, and room temperature reaction 4 hours, discards damping fluid, 37 DEG C of dryings 30 minutes.
Embodiment 4
The solidification of coupling agent and the preparation method of covalent coupling, comprise the steps:
Coupling agent photochemical activity group 2 is coumarins, and space arm 3 is the pentamer of ethylene glycol, and covalent reaction chemical group 4 is chlorsulfonic acid base (-ClSO2).Be calculated in mass percent, the coupling agent dmso solution of 0.028g/L is diluted, stir evenly evenly, add in 96 hole polystyrene microwell plates respectively, every hole 100 microlitre, ultraviolet lamp (350nm) irradiates 30 minutes, discard diluent, with milli-Q water microwell plate 3 times, pat dry for subsequent use.
With the sodium bicarbonate buffer liquid of 0.05mol/L, pH9.5, dilute appropriate Hepatitis E virus ORF2 polypeptide, mixing, add respectively and solidified in 96 hole polystyrene microwell plates of coupling agent, every hole 100 microlitre, room temperature reaction 4 hours, discard damping fluid, pat dry, 37 DEG C of dryings 45 minutes.
One aspect of the present invention proposes the covalent coupling agent of chemically modified surface of solid phase carriers, realizes nucleic acid, the covalency of peptide and protein fixes, overcome the problem of the non-specific adsorption that physical adsorption process causes, substantially increase the specificity of immunology detection; Propose a kind of solid phase carrier preparation method based on covalent coupling method on the other hand, its preparation method is simple, is easy to scale operation.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.