CN102951599A - Preparation method of microcantilever modified by antibody fragments, and microcantilever immune sensing detection system based on antibody fragment modification - Google Patents
Preparation method of microcantilever modified by antibody fragments, and microcantilever immune sensing detection system based on antibody fragment modification Download PDFInfo
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Abstract
The invention provides a microcantilever modified by antibody fragments. Antibody Fab fragments are fixed on a gold-plated surface of the microcantilever. Through the reduction of mass and volume of parts other than antigen molecule binding sites on the antibody, effective modification density of the antigen-binding sites on the surface of the microcantilever is improved, and the efficiency of the transferring of intermolecular reaction force onto the microcantilever is improved, such that microcantilever immunoassay method sensitivity is improved. The invention also discloses a preparation method of the microcantilever, and the immune sensing detection system and a detection method based on the microcantilever modified by antibody fragments.
Description
Technical field
The present invention relates to antibody fragment, the micro-cantilever (following also referred to as little beam) of specifically Fab fragment modification, and preparation method thereof.The invention also discloses immune sensing detection system and the detection method of little beam of modifying based on described antibody fragment, described system and method can be applicable to monitoring and the detection in the fields such as food security, environmental pollution, biomedicine, scientific research and the manufacturing.
Background technology
The principle of immune sensing technology is based on the specificity match reaction of detectable antigens antibody, certain target molecules (antigen) that namely detects for needs, by biological immune (what produce in the antigen injection petty action object) method, produce corresponding probe molecule (antibody), extract, be purified into this probe molecule, the specific binding of recycling antigen-antibody removes the target molecule in the test sample.Existing immune sensing technology is such as EUSA (ELISA, principle is to utilize the specific reaction of antigen and antibody and the catalysis of enzyme target to amplify to carry out) and immunofluorescence technique (IF, principle is with fluorescence light segments labelled antigen or antibody), all need label to come the reaction result of labelled antigen antibody, antibody is only arranged in other words, corresponding detection method be can not set up, enzyme mark thing or fluorescent marker or albumen composition also needed to have.And the label of the target molecules of some difficult preparations is expensive, or may prepare or buy hardly.The simultaneously detection of enzyme linked immunological kit and protein chip is cleaned after all requiring each step to react on the operate, and labeling process is loaded down with trivial details consuming time, is detection afterwards, can not be real-time, and original position, online detection.
The micro-cantilever immune sensing technology that detects based on surface stress is a kind of new immune biochemical method for sensing that in recent years occurs
[1]Its principle is: probe (antigen or antibody) molecule is fixed (modification) on the Gold plated Layer of little beam one side with direct or indirect mode, when the target molecule in the detected sample liquid and the reaction of the probe molecule generation immune biochemical on little beam gold surface, little beam surface stress is changed, thereby cause little beam deformed, detect the process of this distortion by optics or electrical method, can obtain the real time information of immune biochemical reaction.Little beam immune sensing technology that identification is set up based on immunologic opsonin is compared with traditional immuno-sensing method that needs label, it need not to use any enzyme mark, fluorescent material and radioactivity as the reaction tracer, eliminated the impact of labeling process, highly sensitive (than the high several times of enzyme linked immune assay
[2-4]), can also by monitoring the course of reaction of next real-time, the quantitative monitoring antigen-antibody of little beam distortion, obtain the information of abundanter immune biochemical reaction.Through development these years, micro-cantilever sensing is used as a kind of emerging technology, compare research at the aspect such as bioengineering and environmental pollution monitoring technology and traditional method, such as the rna transcription factor, enzyme, mercury emissions and volatile compound etc., be better than conventional enzyme-linked immunoassay method.But even so, the detection sensitivity of micro-cantilever immune sensing technology also is not enough to aspect cost to obtain the huge advantage with respect to conventional ELISA, if namely the sensitivity of micro-cantilever immune sensing technology or detectable limit are only than the high several times of ELISA, and the equipment cost of micro-cantilever immune sensing technology and enzyme linked immunological are relatively high, so that micro-cantilever immune sensing technology is difficult to commercialization.
At present, the method for domestic and foreign literature report mainly is to utilize the thin base of the thin baseization reagent with bifunctional group (SH) catch the gold surface on little beam surface, and another functional group to be caught antibody, realization antibody fixing on little beam gold-plated surface.For example first sulfhydrylization reagent 11-thiol carboxylic acid is attached to the gold surface of little beam, activates the carboxyl on it, make it amino on antibody and be combined and fix antibody (Fig. 2).Or with a kind of sulfhydrylization reagent hydrochloric acid mercaptan imine, by with antibody response, make antibody connect the molecular radical with sulfydryl, antibody is attached on the gold surface of little beam (Chinese patent CN101407548) (seeing Fig. 3) by this sulfydryl again.There is following common problem in these method sessile antibodies, the Fc section of (1) Y-shaped antibody (seeing Fig. 1) or the top ends of Fab section, and equiprobable being fixed on the gold surface of meeting do not have directionality
[5]And when the top of Fab section was fixed on the gold surface, binding site was blocked, and had limited the abundant combination of antigen binding site and the antigen of antibody, thereby had reduced micro-cantilever sensing sensitivity; (2) there is the single chain molecule of different number C atoms between antibody and the little beam, reduced stress that antigen-antibody reaction produces and be delivered to efficient on the beam.This shows that the sensitivity of disclosed method is compared with ELISA and only improved several times among the Chinese patent CN101407548, also can't reach commercialization or be used for the detection of denier analyte.
Therefore, thus exist in the art little beam surface and the combination of antibody and the demand that design further improves the sensitivity of little beam immune sensing and efficient improved.
Summary of the invention
In sum, in existing little beam immune sensing technology report, distinct issues are exactly detection sensitivity.The factor that affects the sensitivity of little beam immunosensor mainly contains three aspects: the 1. sensitivity of antibody (or antibody be combined with antigen compatibility), the 2. fixing means of antibody and 3. design and the signal of little beam are read on little beam.Because the specification of the sensitivity of antibody and little beam and signal playback mode just can't change after the moulding of little beam immunosensor system, unique variable be the fixing means of little beam surface antibody.A kind of method of modifying of suitable little beam surface antibody can make the antibody and the abundant combination of the antigen in the sample solution that are fixed on little beam surface, and can efficiently antigen-antibody be delivered to little beam surface in conjunction with the STRESS VARIATION that produce.The length that connects molecule and the rigidity sensitivity that all might affect final detection of antibody between fixing directionality, density, activity and the antibody in little beam surface and little beam surface.
In view of above-mentioned the problems of the prior art, the technical scheme that the present invention is used for addressing the above problem is quality and the volume by other parts beyond the antigen molecule binding site on the minimizing antibody, improve antigen binding site in effective modification density on little beam surface, and the efficient of intermolecular reaction force transmission to little beam, reach the sensitivity purpose that improves little beam immunologic detection method.
Antibody I g molecular structures schematic diagram such as Fig. 1 comprise two identical Fabs (Fab) and a FC (Fc), and Fab is connected middle hinge area and connects with Fc, consist of the Y-shaped structure.The top of Fab is the binding site with antigen molecule.In " Y " of immune globulin antibody molecule font tetrapeptide chain structure (Fig. 1), be formed by connecting with disulfide bond by two identical heavy chains and two identical light chains.When being modified on the gold-plated surface of little beam for the Fc section (directionality of antibody modification is good), two upper end antigen binding sites of antibody symmetrical (Fab section) are combined the stress of rear generation with antigen, it is surperficial that the shank (Fc section) by the antibody Y-shaped is delivered to gold-plated little beam.The about 150kDa of molecular weight of antibody (IgG), if detected antigen molecule molecular weight is hundreds of Da, antibody antigen mass ratio nearly 1000.The site that participates in the antigen molecule combination is made of the hypervariable region (VH and VL) of heavy chain and light chain, and the mass ratio of itself and antigen molecule is about 150, and does not participate in the Fc section of being combined with antigen molecule on the antibody molecule, the mass ratio nearly 400 of itself and antigen molecule.Therefore we imagine, if can reduce antibody molecule quality and volume beyond the binding site, keep the binding site of antibody towards the exterior normal direction of fixed surface, just can carry heavily stressed transmission efficiency and antibody in effective modification density on little beam surface, thus the sensitivity that improves little beam immune sensing.
For this reason, aspect first, the invention provides a kind of method for preparing micro-cantilever, may further comprise the steps:
1) provides antibody and with the micro-cantilever of Gold plated Layer;
2) enzymolysis step: with protease described antibody is cracked into Fab fragment and a plurality of small molecule segment, described protease is for being cracked into antibody any enzyme of Fab fragment, preferred papain;
3) fixing step: the sulfhydrylization reagent that will comprise mercapto functional group and carboxyl or amido functional group is bonded on the Gold plated Layer surface of micro-cantilever;
4) connect step: with step 2) the Fab fragment and the step 3 that obtain) sulfhydrylization reagent that is combined on the micro-cantilever of acquisition is bound up, and obtains being fixed with on the Gold plated Layer surface micro-cantilever of Fab fragment.
Aspect second, the present invention also provides a kind of method for preparing micro-cantilever, may further comprise the steps:
1) provides antibody and with the micro-cantilever of Gold plated Layer;
2) enzymolysis step: with protease described antibody is cracked into Fab fragment and a plurality of small molecule segment, described protease is for being cracked into antibody any enzyme of Fab fragment, preferred papain;
3) connection step: with step 2) the Fab fragment that obtains and the sulfhydrylization reagent that comprises mercapto functional group and carboxyl or amido functional group are bound up;
The other end of the described sulfhydrylization reagent that an end that 4) fixing step: with step 3) obtains and Fab fragment connect is bonded on the Gold plated Layer surface of micro-cantilever, obtains being fixed with on the Gold plated Layer surface micro-cantilever of Fab fragment.
Aspect the 3rd, the invention provides a kind of stress sensing element, it comprises the micro-cantilever based on stress, is fixed with the Fab fragment of antibody on the Gold plated Layer of described micro-cantilever.Described micro-cantilever preferably is prepared from by method of the present invention.
Aspect the 4th, the invention provides a kind of micro-cantilever immune sensing detection system based on stress, it comprises stress sensing element of the present invention.
Aspect the 5th, the invention provides a kind of method of using micro-cantilever sensing and detecting system immune sensing to detect testing sample, may further comprise the steps:
1) provide antibody and the micro-cantilever sensing and detecting system special to target antigen, described micro-cantilever sensing and detecting system comprises reaction tank and with the micro-cantilever based on stress of Gold plated Layer;
2) enzymolysis step: with protease described antibody is cracked into Fab fragment and a plurality of small molecule segment, described protease is for being cracked into antibody any enzyme of Fab fragment, for example papain;
3) fixing step: the sulfhydrylization reagent that will comprise mercapto functional group and carboxyl or amido functional group is bonded on the Gold plated Layer surface of micro-cantilever;
4) connect step: with step 2) the Fab fragment and the step 3 that obtain) the described sulfhydrylization reagent that is combined on the micro-cantilever of acquisition is bound up, and obtains being fixed with on the Gold plated Layer surface micro-cantilever of Fab fragment.
5) with step 4) micro-cantilever that is fixed with the Fab fragment that obtains is fixed in the reaction tank of micro-cantilever sensing and detecting system;
6) testing sample is added to step 5) in the micro-cantilever sensing and detecting system that obtains, detect in the testing sample whether have described target antigen.
Aspect the 6th, the present invention also provides a kind of method of using micro-cantilever sensing and detecting system immune sensing to detect testing sample, may further comprise the steps:
1) provide antibody and the micro-cantilever sensing and detecting system special to target antigen, described micro-cantilever sensing and detecting system comprises reaction tank and with the micro-cantilever based on stress of Gold plated Layer;
2) enzymolysis step: with protease described antibody is cracked into Fab fragment and a plurality of small molecule segment, described protease is for being cracked into antibody any enzyme of Fab fragment, for example papain;
3) connection step: with step 2) the Fab fragment that obtains and the sulfhydrylization reagent that comprises mercapto functional group and carboxyl or amido functional group are bound up;
The other end of the described sulfhydrylization reagent that an end that 4) fixing step: with step 3) obtains and Fab fragment connect is bonded on the Gold plated Layer surface of micro-cantilever, obtains being fixed with on the Gold plated Layer surface micro-cantilever of Fab fragment;
5) with step 4) micro-cantilever that is fixed with the Fab fragment that obtains is fixed in the reaction tank of micro-cantilever sensing and detecting system;
6) testing sample is added to step 5) in the micro-cantilever sensing and detecting system that obtains, detect in the testing sample whether have described target antigen.Beneficial effect of the present invention:
Importance and the sulfhydrylization reagent fixing in view of antibody in little beam immune sensing detection technique participate in the fixing complexity of antibody, and the present invention's proposition modified antibodies fragment Fab method on little beam gold-plated surface realizes little beam immune detection.The result shows that method of the present invention is compared with existing little beam immunization method and had the following advantages:
1) the Fab fragment is with respect to complete antibody, and its volume and quality are little, so after being fixed to little beam surface, has the density of higher antigen binding site.2) reduce antibody combining site quality and volume in addition, thereby the antigen binding site that has reduced antibody is put forward heavily stressed transmission efficiency to the beam surface distance.
Description of drawings
Fig. 1 is the antibody molecule structural representation.
Fig. 2 is thiol carboxylic acid compounds and bi-functional cross-linking agent method sessile antibody schematic diagram.
Fig. 3 is hydrochloric acid mercaptan imine method sessile antibody schematic diagram.
Fig. 4 is that papain hydrolysis antibody formation antibody fragment obtains the Fab schematic diagram.
Fig. 5 illustrates first Fab is connected a mercapto groups with the reaction of hydrochloric acid mercaptan imine at Fab, fixes Fab to the schematic diagram of gold surface by this sulfydryl again.
Fig. 6 illustrates first the thio-alcohol sulfhydrylization reagent is connected on the beam, connects the schematic diagram of Fab by this thio-alcohol sulfhydrylization reagent again.
Fig. 7 is the little beam of anti-human ginseng saponin(e GS-Re sulfhydrylation antibody modification, the time-displacement curve at little beam tip when detecting variable concentrations antigen.
Fig. 8 modifies little beam for modifying anti-human ginseng saponin(e GS-Re Fab fragments with mercaptan, the time-displacement curve at little beam tip when detecting variable concentrations antigen.
The specific embodiment
Definition
Antibody: antibody (antibody) refers to the immune system of body under antigenic stimulus, that the thick liquid cell that is become by bone-marrow-derived lymphocyte or memory cell Proliferation, Differentiation produces, can with the immunoglobulin (Ig) of corresponding antigens generation specific binding.Typical antibody molecule has the symmetrical structure of 4 polypeptide chains, comprises 2 identical heavy chains (H chain) long, that relative molecular weight is larger; Article 2, the identical light chain (L chain) that lack, relative molecular weight is less.Interchain forms a monomer molecule that is made of 4 polypeptide chains by disulfide bond and non-covalent bond connection.Light chain has two kinds of κ and λ, and heavy chain has five kinds of μ, δ, γ, ε and α.Whole antibody molecule can be divided into constant region and variable region two parts.In given species, the constant region of different antibodies molecule all has identical or almost identical amino acid sequence.The variable region is positioned at the two arms end of " Y ".There have the sub-fraction amino acid residue to change in the variable region to be strong especially, and these amino acid whose residues form and the easier zone of morphing that puts in order claims the hypervariable region.The hypervariable region is positioned at molecular surface, is made of 17 amino acid residues at most, only has at least 2～3.The hypervariable region amino acid sequence has determined the specificity of this antibody conjugated antigen.Two antigen-binding sites on antibody molecule are identical, are positioned at the terminal Fab (antigen-binding fragment, Fab) that claims of two arms.The shank of " Y " claims crystallizable fragment (crystalline fragment, Fc), and sugar is combined on the Fc.
From a structural point, in the present invention, all refer to whole antibody when mentioning antibody, namely comprise the antibody structure (seeing Fig. 1) of 4 chains and Fc section.In addition, on function, " antibody " among the present invention or " antibody fragment " refer to antibody or the antibody fragment special to target antigen, the antibody or the antibody fragment that namely have the specific bond ability with antigen, as not specifying, antibody among the present invention refers generally to monoclonal antibody, and antibody fragment comprises incomplete antibody fragment, F (ab ')
2Fragment, Fab ' fragment and Fab etc.
The incomplete antibody fragment: by disulfide bond reducing agent whole antibody is split into the fragment of the symmetry that respectively carries sulfydryl, each incomplete antibody fragment comprises a complete light chain and complete heavy chain and Fc fragment.
F (ab ')
2: Ig (immunoglobulin, immunoglobulin (Ig)) is cut off by the pepsin hydrolysis nearly C of disulfide bond place between the hinge area heavy chain, forms a bivalent Fab abbreviation F (ab ')
2Fragment and some small fragment pFc '.Because F (ab ')
2Fragment has kept the BA in conjunction with corresponding antigens, the side effect of having avoided again the antigenicity of Fc fragment to cause, thereby be widely used as biological products.Such as diphtheria antimycin and lockjaw antimycin, behind pepsin hydrolysis, reduced the generation of hypersensitivity because of the antigenicity of having removed the Fc fragment.PFc ' fragment finally is degraded, and abiology is active.
Fab (fragment of antigen binding): papain cuts off the Ig nearly N end of disulfide bond place between the hinge area heavy chain, form two identical monovalent antigen binding fragments and be called for short Fab section (as shown in fig. 1), a crystallizable fragment is called for short Fc (fragment crystallizable) section.
Fab ': be the monovalent antigen binding fragment with sulfydryl.
Antigen binding site: as shown in fig. 1, the position that antibody molecule combines with antigen, CDR1, CDR2 and CDR3 light by Ig, heavy chain form.
Antigen: be class energy induction of immunity system generation immune response, and the material of specific binding can occur with the product (antibody or effector cell) of immune response.Antigen has immunogenicity and two kinds of character of reactionogenicity.Be divided into two classes according to antigenic property: comlete antigen and incomplete antigen.Comlete antigen (complete antigen) is the existing immunogenicity of a class, and immunoreactive material is arranged again.All be comlete antigen such as most protein, bacterium, virus, bacterial exotoxin etc.Incomplete antigen, namely haptens (hapten) is only to have immunoreactivity, and the material of non-immunogenicity, so claim again incomplete antigen.
Sulfhydrylization reagent: the difunctional cross-linking reagent that can connect antibody and gold with sulfydryl.
Disulfide bond reducing agent: disulfide bond claims again the S-S key, is the key between the sulphur atom of the oxidized and formation-S-S-form of 2 SH base.In the presence of the sulphur compound of mercaptoethylmaine (2-MEA), 2 mercapto ethanol, dithiothreitol (DTT) etc., can have an effect with it, be reduced into sulfydryl (SH).These sulfide are exactly said disulfide bond reducing agent among the present invention.Disulfide bond between the heavy chain of antibody in the situation that the disulfide bond reducing agent of trace exists is reduced and other disulfide bond is not destroyed.
Micro-cantilever (little beam): typical micro-cantilever is made by silicon nitride, such as commercial triangle micro-cantilever (Veeco Instruments) (size: long 200um, the wide 20um of leg, thick 0.6um), the one-sided gold that is coated with 60nm; Antibody usually the sulfydryl by sulfhydrylization reagent (SH) with the covalent bond of gold and an other end of sulfhydrylization reagent (contain-COOH or-NH
2The isoreactivity group) is combined with antibody and is secured to little beam surface.
Micro-cantilever immune sensing system based on the surface stress detection: the micro-cantilever immune sensing system mainly is comprised of laser instrument, spectroscope, micro-cantilever, photoelectric position sensor (PSD), temperature control system, peristaltic pump, reaction tank and data analysis treating apparatus.The step of typical micro-cantilever immunologic detection method is as follows: micro-cantilever is fixed in the reaction tank, flows buffer solution by reaction tank with peristaltic pump control, pass through reaction tank with the mobile buffer solution of the speed of 0.1mL/min after the bubble emptying in the question response pond.The temperature of reaction tank is controlled at 37 ± 0.01 ℃, and room temperature is controlled at 27 ± 0.01 ℃.Laser instrument sends beam of laser is radiated at micro-cantilever behind spectroscope tip, through impinging upon on the target surface of PSD after the spectroscope reflection after the micro-cantilever reflection again.After the displacement signal of micro-cantilever is stable, add the sample solution of buffer solution dilution, the most advanced and sophisticated displacement of PSD real time record micro-cantilever.
The preferred embodiments of the invention
In the present invention, Antibody types can comprise IgM, IgG, IgA, IgD, IgE.In addition, antibody can by any method preparation as known in the art, include but not limited to immunization, hybridoma method, chemical synthesis, gene engineering research etc.Described antibody can also be the hybrid antibody that genetic engineering is modified, and such as humanized antibody, camel source antibody etc. are for the antibody of certain mammal transformation, such as people-mouse hybrid antibody etc.
The testing sample of mentioning among the present invention can be biological sample, for example come from especially people's sample of mammal, comprise tissue sample (such as histopathologic slide, biopsy, hair, swab etc.), cell sample (such as cell smear, blood smear etc.), humoral sample (such as blood, urine, cerebrospinal fluid, saliva etc.), excreta (such as vomitus, sweat, ight soil etc.).Described testing sample can also be environmental sample, such as pedotheque, water sample, floating dust etc.; The sample that obtains in other production fields, such as sewage sample, food samples etc.
Mentioned antigen includes but not limited to comlete antigen and haptens among the present invention, and it can be the antigen of any type of detecting of the needs of dawn known in the art that may exist in the testing sample of mentioning in the present invention.
In the present invention, the protease that uses in enzymolysis step can at random be selected according to kind or other factors of the antibody that will be hydrolyzed, as long as comprise the Fab fragment in its hydrolysate.Optional protease is such as papain, because the reaction condition of papain is relatively gentle, so the impact of the antigen-binding activity of antagonist is less.
In the present invention, it is well known in the art adopting method and the reaction condition of protease hydrolytic antibody I g, and those of ordinary skills can determine suitable protease and corresponding hydrolysising condition according to the kind of the antibody that will be hydrolyzed.For example, when selecting papain, papain can be brought into play its active environment, generally refers to pH 3～9.5 and the environment under 25～60 ℃.The optimal pH of papain and optimum temperature are decided according to its source, are generally about pH 6.0-8.0 and about 37～55 ℃ (can determine according to the requirement of commercially available prod).The source of operable papain is unrestricted in the present invention, as long as it can be cracked into antibody molecule the Fab fragment.In the present invention, the mass ratio of antibody or antibody fragment and papain is unrestricted, as long as be enough to antibody or antibody fragment is hydrolyzed to the Fab fragment fully.Both mass ratioes can be selected according to conventional methods, also can simultaneously with reference to factors such as the activity of selected papain, selected Antibody types, generally can select in 10: 1～200: 1 scope.
In the method for the invention, the sulfhydrylization reagent that uses among the present invention is unrestricted, as long as it can connect with the Fab fragment of antibody, and can be fixed on the Gold plated Layer of little beam.The step of the connection of sulfhydrylization reagent and Fab and to be fixed to the order of the step on the Gold plated Layer of little beam unrestricted, can be first fix with gold surface after with the antibody sulfhydrylation with the sulfhydrylization reagent bind antibody, connect with antibody again after also can being fixed on sulfhydrylization reagent on the gold surface first.
The sulfhydrylization reagent that uses among the present invention comprises mercapto functional group and carboxyl or amido functional group, wherein the sulfydryl of this reagent one end is used for being fixed on gold surface (referring to Fig. 5) by self assembly, and the carboxyl of the other end or amino (also can be exposed by activation) are used for amino or carboxyl terminal with antibody react (referring to Fig. 6).The present invention can with sulfhydrylization reagent comprise sulfur alcohol compound, 11-thiol carboxylic acid for example, 2-aminoothyl mercaptan (AET) and 3-mercaptopropionic acid (MPA); The hydrochloric acid mercaptan imine; Sulfo group hydrocarbyl succinic imide-6-(3 ' 2-pyridine, two sulphur-propionamide)-acetic acid esters (Sulfo-LC-SPDP); With 3, the two sulfosuccinimide propionic esters (DTSSP) of 3 '-two sulphur etc.
The little beam that uses in the present invention can prepare voluntarily according to the known method in this area, also can buy the commercial goods, and this is not limited to them in the present invention.
The present invention will be further described below in conjunction with specific embodiment, but need to prove that the following examples do not limit the scope of the invention.
Embodiment
Embodiment 1 is sessile antibody fragment Fab on little beam Gold plated Layer
Test the preparation of 1 antibody passage Fab
1. accurately taking by weighing ginsenoside GS-Re monoclonal antibody (available from U.S. USBiological company) 10.0mg is dissolved among the 1.0mL 0.1M Tris-HCl (containing 2.0mM EDTA, pH8.0) and obtains the antibody-solutions that concentration is 10.0g/L;
2. taking by weighing 1.5mg papain (available from U.S. Sigma company) is dissolved in and obtains the papain solution that concentration is 1.5g/L in the 1.0mL water;
3. the antibody-solutions of 1.0mL above-mentioned steps 1 and the papain solution of 1.0mL above-mentioned steps 2 are mixed, 37 ℃ were reacted 1 hour.Must fall Fab fragments.With-20 ℃ of preservations after the dilution of glycerine equal-volume.
Test the fixing of 2 little beam surface antibody Fab fragments
1. mercaptan self assembly: will clean and put into the ELISA Plate aperture with the little beam (available from U.S. Veeco company) that is coated with gold layer that nitrogen dries up, adding 200 μ l concentration is 10mM 11-thiol carboxylic acid, sealing room temperature 12 hours;
2. clean: with the little beam of the careful taking-up of tweezers, use the little beam of washed with de-ionized water three times;
3. activation: little beam that will clean adds in the new plate hole, adds the EDC (1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) of 0.2M and each 75 μ l of NHS (N-hydroxy-succinamide) of 0.05M and activates beam 40 minutes;
4. clean: with the little beam of the careful taking-up of tweezers, use the little beam of washed with de-ionized water three times;
5. sessile antibody: little beam that will clean is put among the new plate hole, adds the anti-human ginseng saponin(e GS-Re monoclonal antibody Fab fragment that the above-mentioned experiment 1 of 150 μ L obtains, and 37 ℃ of incubations 1.5 hours are fixed little beam of antibody passage Fab.
Test the as follows in conjunction with the situation concrete steps of 3 direct competitive ELISA method evaluation Fab antibody fragments and little beam gold surface:
1. clean: carefully take out little beam that antibody fragment has been fixed in above-mentioned experiment 2 with tweezers, (contain 8.0g NaCl, 0.2g KH in every 1L cleaning solution with cleaning solution
2PO
4, 2.96g Na
2HPO
412H
2O, 1.0mL Tween-20, all the other are water) the flushing several, nitrogen dries up;
2. sealing: the little beam after nitrogen dried up is put into respectively ELISA Plate, adds 100 μ L quality percentage compositions and be 5% BSA solution (dissolving with PBS) sealing, 37 ℃ of incubations 30 minutes;
3. clean: with the little beam of the careful taking-up of tweezers, with the cleaning solution flushing for several times, nitrogen dries up;
4. compete: the little beam after nitrogen is dried up is put into respectively two apertures of ELISA Plate and (is carried out mark, suppress Kong Yufei and suppress the hole), it is the ginsenoside GS-Re solution (available from U.S. Sigma company) of 100mg/L through the concentration of sample diluting liquid (containing the PBS that final concentration is 0.1mol/L pH7.5, the Tween-20 of 0.1% volumn concentration and the gelatin of 0.1% quality percentage composition) dilution that the inhibition hole adds 50 μ L, and non-inhibition hole adds 50 μ L sample diluting liquids in contrast; And then add respectively the ginsenoside GS-Re peroxide enzyme conjugation thing (available from U.S. Sigma company) that concentration is 1.0mg/L, 37 ℃ of incubations 30 minutes;
5. clean: with the careful little beam that takes out in the above-mentioned inhibition Kong Yufei inhibition hole of tweezers, with the cleaning solution flushing for several times, nitrogen dries up.
6. develop the color: two little beams after nitrogen is dried up are put into respectively two apertures of ELISA Plate, every hole adds 100 μ L chromophoric solutions, and (tmb substrate uses liquid and substrate buffer solution to press 1: 9 volume ratio mixing, add the 4.0mg urea peroxide in the above-mentioned mixed liquor of every 10mL), color development at room temperature;
7. stop: after 15 minutes, every hole adds 50uL stop buffer (sulfuric acid solution of 2.0M) cessation reaction, takes out little beam, and 450nm wavelength place reads respectively the OD value.
Experiment is established and was repeated last time, and the result shows, the absorbance that suppresses under the 450nm wavelength in hole and non-inhibition hole is respectively 0.113 and 0.841, illustrate anti-human ginseng saponin(e GS-Re Fab fragments can be effectively in conjunction with on the gold surface of little beam and have an activity.
Test 4 micro-cantilever sensing device effect monitorings
1. clean: absolute ethyl alcohol, acetone and the PBS micro-cantilever sensing system that flows through respectively, flow velocity 0.5mL/min;
2. fix little beam: will test the little beam that is fixed with anti-human ginseng saponin(e GS-Re Fab fragments in 2 and be fixed in the reaction tank of micro-cantilever sensing system, the PBS that flows is by the reaction pond.Get rid of behind the bubble in the reaction tank PBS that flows with 0.1mL/min.
3. adjusting light path: the tip reflection that the laser that the position of adjusting laser instrument and PSD (photoelectric position sensor) is sent laser instrument impinges upon little beam is received by PSD afterwards.
4. sample determination: after little beam defection signal that PSD receives is stable, add the ginsenoside GS-Re standard sample of 2mL PBS dilution.By the most advanced and sophisticated displacement information of the little beam of PSD real time record.
Three concentration 20ng/mL, 1ng/mL, 0.05ng/mL have been detected; The displacement that obtains of sample be about respectively 80,45 and 20nm (Fig. 8).The ginsenoside GS-Re standard sample of variable concentrations can produce little beam displacement in various degree, illustrates that anti-human ginseng saponin(e GS-Re antibody passage Fab can effectively be attached on the gold surface of little beam and can quantitatively detect ginsenoside GS-Re.
Embodiment 2 is sessile antibody fragment Fab on little beam Gold plated Layer
Test the preparation of 1 antibody passage Fab
1. accurately taking by weighing ginsenoside GS-Re monoclonal antibody (available from U.S. USBiological company) 10.0mg is dissolved among the 1.0mL 0.1M Tris-HCl (containing 2.0mM EDTA, pH8.0) and obtains the antibody-solutions that concentration is 10.0g/L;
2. taking by weighing 1.5mg papain (available from U.S. Sigma company) is dissolved in and obtains the papain solution that concentration is 1.5g/L in the 1.0mL water;
3. the antibody-solutions of 1.0mL above-mentioned steps (1) and the papain solution of 1.0mL above-mentioned steps (2) are mixed, 37 ℃ were reacted 1 hour.Must fall antibody fragment.With-20 ℃ of preservations after the dilution of glycerine equal-volume.
Experiment 2 utilizes the hydrochloric acid mercaptan imine with the antibody passage Fab sulfhydrylation
1. taking by weighing 1.0mg hydrochloric acid mercaptan imine (available from U.S. sigma company) is dissolved in and obtains the sulfhydrylization reagent that concentration is 1.0g/L (now with the current) in the 1.0mL distilled water;
2. the sulfhydrylization reagent with the antibody passage Fab in the above-mentioned experiment 1 of 1.0mL and the above-mentioned experiment 1 of 45.8 μ L mixes, gentle agitation reaction 1h under the room temperature condition;
With the PBS buffer solution (containing 0.15M NaCl and 1.0mM EDTA, Ph7.2) of 20mM to the reactant liquor dialysis of above-mentioned steps 2 48 hours, during changed a dislysate in per 3 hours, obtain sulfhydrylation antibody fragment solution
Test the fixing of 3 little beam surface sulfhydrylation Fab fragments
To clean and put into the ELISA Plate aperture with the little beam that is coated with golden film (available from U.S. Veeco company) that nitrogen dries up, add the sulfhydrylation Fab fragments solution that the above-mentioned experiment 2 after 150 μ L dilute 50 times with PBS makes, be placed in 37 ℃ of water baths 2 hours, namely be fixed little beam of Fab fragments.
Experiment 4ELISA direct competition method effect detection
1. clean: with the above-mentioned micro-cantilever of fixing antibody passage Fab of the careful taking-up of tweezers, (contain 8.0g NaCl, 0.2g KH in every 1L cleaning solution with cleaning solution
2PO
4, 2.96g Na
2HPO
412H
2O, 1.0mL Tween-20, all the other are water) the flushing several, nitrogen dries up;
2. sealing: the micro-cantilever after nitrogen dried up is put into respectively ELISA Plate, adds 100 μ L quality percentage compositions and be 5% BSA solution (dissolving with PBS) sealing, 37 ℃ of incubations 30 minutes;
3. clean: carefully take out micro-cantilever with tweezers, with the cleaning solution flushing for several times, nitrogen dries up;
4. compete: the micro-cantilever after nitrogen is dried up is put into respectively two apertures of ELISA Plate and (is carried out mark, suppress Kong Yufei and suppress the hole), it is the ginsenoside GS-Re solution (available from U.S. Sigma company) of 100mg/L through the concentration of sample diluting liquid (containing the PBS that final concentration is 0.1mol/L pH7.5, the Tween-20 of 0.1% volumn concentration and the gelatin of 0.1% quality percentage composition) dilution that the inhibition hole adds 50 μ L, and non-inhibition hole adds 50 μ L sample diluting liquids in contrast; And then add respectively the ginsenoside GS-Re peroxide enzyme conjugation thing (available from U.S. Sigma company) that concentration is 1.0mg/L, 37 ℃ of incubations 30 minutes;
5. clean: with the careful micro-cantilever that takes out in the above-mentioned inhibition Kong Yufei inhibition hole of tweezers, with the cleaning solution flushing for several times, nitrogen dries up;
6. develop the color: two micro-cantilevers after nitrogen is dried up are put into respectively two apertures of ELISA Plate, every hole adds 100 μ L chromophoric solutions, and (tmb substrate uses liquid and substrate buffer solution to press 1: 9 volume ratio mixing, add the 4.0mg urea peroxide in the above-mentioned mixed liquor of every 10mL), color development at room temperature;
7. stop: after 15 minutes, every hole adds 50 μ L stop buffer (sulfuric acid solution of 2.0M) cessation reactions, takes out micro-cantilever, and 450nm wavelength place reads respectively the OD value.
Three repetition are established in experiment, and the result shows that the absorbance under the 450nm wavelength in inhibition hole and non-inhibition hole is respectively 0.156 and 0.944, illustrate that anti-human ginseng saponin(e GS-Re Fab fragments behind the sulfhydrylation is effectively on the gold surface in conjunction with micro-cantilever.
Test 5 micro-cantilever sensing device effect detection
1. clean: absolute ethyl alcohol, acetone and the PBS micro-cantilever sensing system that flows through respectively, flow velocity 0.5mL/min;
2. fix little beam: will test the micro-cantilever that is modified with anti-human ginseng saponin(e GS-Re Fab fragments in 3 and be fixed in the reaction tank of micro-cantilever sensor-based system, the PBS that flows is by the reaction pond.Get rid of behind the bubble in the reaction tank PBS that flows with 0.1mL/min.
3. adjusting light path: the laser that the laser instrument that the position of adjusting laser instrument and PSD (photoelectric position sensor) makes sends impinges upon the most advanced and sophisticated of little beam and is received by PSD.
4. sample determination: after little beam defection signal that PSD receives is stable, add the ginsenoside GS-Re standard sample of 2mL PBS dilution.By the most advanced and sophisticated displacement information of the little beam of PSD real time record.
Detect the most advanced and sophisticated displacement amplitude of little beam that the sample of three concentration 20ng/mL, 1.0ng/mL, 0.05ng/mL obtains and be respectively 95,50 and 25nm (Fig. 8); The ginsenoside GS-Re standard sample of variable concentrations can produce micro-cantilever displacement in various degree, illustrates that anti-human ginseng saponin(e GS-Re antibody fab fragment can effectively be attached on the gold surface of micro-cantilever and can detect ginsenoside GS-Re.
Conclusion:
Take ginsenoside GS-Re antigen-antibody molecule as example, carry out the experiment test contrast with little beam that antibody passage Fab of the present invention is modified and complete antibody is modified, when detecting the ginsenoside GS-Re antigen of variable concentrations, the time shifting response curve at little beam tip (Fig. 8 and Fig. 7) shows, when little beam displacement response is the 20 nanometer left and right sides, the detection ginsenoside GS-Re antigen concentration that Fab fragments is modified and complete antibody is modified is respectively 0.5ng/mL and 0.05ng/mL, and sensitivity has improved about 10 times.(Fig. 7 is for utilizing the hydrochloric acid mercaptan imine to come behind the sulfhydrylation antibody antibody to be fixed to the result that (Chinese patent CN101407548) detects ginsenoside on the beam as sulfhydrylization reagent).
By above-mentioned nonrestrictive embodiment, can find out, method of the present invention and micro-cantilever by method of the present invention preparation are compared with little beam method of prior art in the immune sensing detection technique can improve 20-40 doubly, be equivalent to the 200-400 of conventional ELISA doubly, realized purpose of the present invention, namely possibility and the practicality of monitoring and detection denier analyte in the fields such as food security, environmental pollution, biomedicine, scientific research and the manufacturing can realize actual commercial applications fully.
List of references
[1]Koutilya R.Buchapudi，Xin Huang，Xin Yang，HaiFeng Ji and Thomas Thundat，Microcantilever biosensors for chemicals and bioorganisms.Analyst，2011，136，1539-1556
[2]Weiming Tan，Yuan Huang，Tiegui Nan，Changguo Xue，Zhaohu Li，Qingchuan Zhang，and BaominWang，Development of Protein A Functionalized Microcantilever Immunosensors for the Analyses ofSmall Molecules at part per trillion Levels.Analysis Chemistry，2010，82(2)，615-620
[3]Hongwei Zhao；Changguo Xue；Tiegui Nan；Guiyu Tan；Zhaohu Li；Qing X.Li；Qingchuan Zhang；Baomin Wang，Detection of Copper Ions UsingMicrocantilever Immunosensors and Enzyme-linked Immunosorbent Assay，Analytica Chimica Acta，2010，676，81-86，
[4]Changguo Xue，Hongwei Zhao，Yanyun Chen，Baomin Wang，Qingchuan Zhang，Xiaoping Wu，Development of sulfhydrylated antibodyfunctionalized microcantilever immunosensor for taxol，Sensors and Actuators B，2011，156，863-866
[5]A.Kausaite-Minkstimiene，A.Ramanaviciene，J.Kirlyte，and A.Ramanavicius.Comparative Study of Random and Oriented Antibody Immobilization Techniques on the Binding Capacity of Immunosensor.Analysis Chemistry，2010，82，6401-6408。
Claims (8)
1. method for preparing micro-cantilever may further comprise the steps:
1) provides antibody and with the micro-cantilever of Gold plated Layer;
2) enzymolysis step: with protease described antibody is cracked into Fab fragment and a plurality of small molecule segment, described protease is for being cracked into antibody any enzyme of Fab fragment, preferred papain;
3) fixing step: the sulfhydrylization reagent that will comprise mercapto functional group and carboxyl or amido functional group is bonded on the Gold plated Layer surface of micro-cantilever;
4) connect step: with step 2) the Fab fragment and the step 3 that obtain) sulfhydrylization reagent that is combined on the micro-cantilever of acquisition is bound up, and obtains being fixed with on the Gold plated Layer surface micro-cantilever of Fab fragment.
2. method for preparing micro-cantilever may further comprise the steps:
1) provides antibody and with the micro-cantilever of Gold plated Layer;
2) enzymolysis step: with protease described antibody is cracked into Fab fragment and a plurality of small molecule segment, described protease is for being cracked into antibody any enzyme of Fab fragment, preferred papain;
3) connection step: with step 2) the Fab fragment that obtains and the sulfhydrylization reagent that comprises mercapto functional group and carboxyl or amido functional group are bound up;
The other end of the described sulfhydrylization reagent that an end that 4) fixing step: with step 3) obtains and Fab fragment connect is bonded on the Gold plated Layer surface of micro-cantilever, obtains being fixed with on the Gold plated Layer surface micro-cantilever of Fab fragment.
3. stress sensing element, it comprises the micro-cantilever based on stress, is fixed with the Fab fragment of antibody on the Gold plated Layer of described micro-cantilever.
4. micro-cantilever immune sensing detection system based on stress, it comprises the stress sensing element of claim 3.
5. method of using micro-cantilever sensing and detecting system immune sensing to detect testing sample may further comprise the steps:
1) provide antibody and the micro-cantilever sensing and detecting system special to target antigen, described micro-cantilever sensing and detecting system comprises reaction tank and with the micro-cantilever based on stress of Gold plated Layer;
2) enzymolysis step: with protease described antibody is cracked into Fab fragment and a plurality of small molecule segment, described protease is for being cracked into antibody any enzyme of Fab fragment, for example papain;
3) fixing step: the sulfhydrylization reagent that will comprise mercapto functional group and carboxyl or amido functional group is bonded on the Gold plated Layer surface of micro-cantilever;
4) connect step: with step 2) the Fab fragment and the step 3 that obtain) the described sulfhydrylization reagent that is combined on the micro-cantilever of acquisition is bound up, and obtains being fixed with on the Gold plated Layer surface micro-cantilever of Fab fragment.
5) with step 4) micro-cantilever that is fixed with the Fab fragment that obtains is fixed in the reaction tank of micro-cantilever sensing and detecting system;
6) testing sample is added to step 5) in the micro-cantilever sensing and detecting system that obtains, detect in the testing sample whether have described target antigen.
6. method of using micro-cantilever sensing and detecting system immune sensing to detect testing sample may further comprise the steps:
1) provide antibody and the micro-cantilever sensing and detecting system special to target antigen, described micro-cantilever sensing and detecting system comprises reaction tank and with the micro-cantilever based on stress of Gold plated Layer;
2) enzymolysis step: with protease described antibody is cracked into Fab fragment and a plurality of small molecule segment, described protease is for being cracked into antibody any enzyme of Fab fragment, for example papain;
3) connection step: with step 2) the Fab fragment that obtains and the sulfhydrylization reagent that comprises mercapto functional group and carboxyl or amido functional group are bound up;
The other end of the described sulfhydrylization reagent that an end that 4) fixing step: with step 3) obtains and Fab fragment connect is bonded on the Gold plated Layer surface of micro-cantilever, obtains being fixed with on the Gold plated Layer surface micro-cantilever of Fab fragment;
5) with step 4) micro-cantilever that is fixed with the Fab fragment that obtains is fixed in the reaction tank of micro-cantilever sensing and detecting system;
6) testing sample is added to step 5) in the micro-cantilever sensing and detecting system that obtains, detect in the testing sample whether have described target antigen.
7. each described method, stress sensing element or immune sensing detection system according to claim 1-6 is characterized in that described sulfhydrylization reagent is selected from sulfur alcohol compound, for example 11-carboxyl mercaptan, 2-aminoothyl mercaptan and 3-mercaptopropionic acid; The hydrochloric acid mercaptan imine; Sulfo group hydrocarbyl succinic imide-6-(3 ' 2-pyridine, two sulphur-propionamide)-acetic acid esters; In the two sulfosuccinimide propionic esters of 3,3 '-two sulphur one or more.
8. each described method, stress sensing element or immune sensing detection system according to claim 1-7 is characterized in that described antibody is any among IgM, IgG, IgA, IgD, the IgE.
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| US5372930A (en) * | 1992-09-16 | 1994-12-13 | The United States Of America As Represented By The Secretary Of The Navy | Sensor for ultra-low concentration molecular recognition |
| US20040058335A1 (en) * | 2002-09-24 | 2004-03-25 | Xing Su | Detecting molecular binding by monitoring feedback controlled cantilever deflections |
| US20050123563A1 (en) * | 2003-07-30 | 2005-06-09 | Doranz Benjamin J. | Lipoparticles comprising proteins, methods of making, and using the same |
| DE102006009709A1 (en) * | 2006-03-02 | 2006-10-12 | Müller, Merold, Dr. | Method for detecting microorganisms without a culturing step, useful particularly for detecting potential germ warfare agents, uses a cantilever coated with specific binding material for the target organism |
| CN1869689A (en) * | 2006-05-18 | 2006-11-29 | 张青川 | Micro-cantilever array bio-chip |
| WO2007087653A1 (en) * | 2006-02-03 | 2007-08-09 | Universität Linz | Method for detecting 5-methylcytosine |
| US20080090259A1 (en) * | 2006-06-08 | 2008-04-17 | Eric Toone | Methods, devices, systems and computer program products for stochastic, competitive, force-based analyte detection |
| US20080160638A1 (en) * | 2006-12-27 | 2008-07-03 | David Lederman | Functionalized Microcantilever Sensor and Associated Method For Detection of Targeted Analytes |
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2011
- 2011-08-19 CN CN201110238911.4A patent/CN102951599B/en not_active Expired - Fee Related
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5372930A (en) * | 1992-09-16 | 1994-12-13 | The United States Of America As Represented By The Secretary Of The Navy | Sensor for ultra-low concentration molecular recognition |
| US20040058335A1 (en) * | 2002-09-24 | 2004-03-25 | Xing Su | Detecting molecular binding by monitoring feedback controlled cantilever deflections |
| US20050123563A1 (en) * | 2003-07-30 | 2005-06-09 | Doranz Benjamin J. | Lipoparticles comprising proteins, methods of making, and using the same |
| WO2007087653A1 (en) * | 2006-02-03 | 2007-08-09 | Universität Linz | Method for detecting 5-methylcytosine |
| DE102006009709A1 (en) * | 2006-03-02 | 2006-10-12 | Müller, Merold, Dr. | Method for detecting microorganisms without a culturing step, useful particularly for detecting potential germ warfare agents, uses a cantilever coated with specific binding material for the target organism |
| CN1869689A (en) * | 2006-05-18 | 2006-11-29 | 张青川 | Micro-cantilever array bio-chip |
| US20080090259A1 (en) * | 2006-06-08 | 2008-04-17 | Eric Toone | Methods, devices, systems and computer program products for stochastic, competitive, force-based analyte detection |
| US20080160638A1 (en) * | 2006-12-27 | 2008-07-03 | David Lederman | Functionalized Microcantilever Sensor and Associated Method For Detection of Targeted Analytes |
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