CN102951598A - 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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- CN102951598A CN102951598A CN2011102388821A CN201110238882A CN102951598A CN 102951598 A CN102951598 A CN 102951598A CN 2011102388821 A CN2011102388821 A CN 2011102388821A CN 201110238882 A CN201110238882 A CN 201110238882A CN 102951598 A CN102951598 A CN 102951598A
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- G—PHYSICS
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention provides a microcantilever modified by antibody fragments. Semi-antibody fragments or antibody Fab' fragments are directionally fixed on a gold-plated surface of the microcantilever, such that the sensitivity of a microcantilever immunoassay method based on surface stress effect 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 the micro-cantilever (following also referred to as little beam) that antibody fragment is modified, 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 Layer.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, and the Fc section of (1) Y-shaped antibody (seeing Fig. 1) or the top ends of Fab section can equiprobable being fixed on the gold surface (be seen Fig. 2,3), 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, the sensitivity of disclosed method is compared with ELISA and is 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 problems of the prior art, the technical scheme that the present invention is used for addressing the above problem is by 1) minimizing antibody molecule binding site antibody molecule quality and volume in addition, 2) the transition molecule layer (sulfhydrylization reagent) that connects little beam gold-plated surface and antibody molecule is used in cancellation, 3) provide a kind of directed method that is fixed with little beam of antibody fragment on the gold-plated surface for preparing, 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 molecular weight of antibody (IgG) is 150kDa approximately, if detected antigen molecule molecular weight is hundreds of Da, and 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, the mass ratio of itself and antigen molecule approximately 150, and do 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) disulfide bond reduction step: use disulfide bond reducing agent to process described antibody, so that the disulfide bond reduction of described antibody hinge region becomes sulfydryl, thereby obtain the incomplete antibody fragment with sulfydryl of two symmetries;
3) the incomplete antibody fragment that fixing step: with step 2) obtains is combined with the micro-cantilever that is coated with the gold layer, obtains being fixed with on the gold-plated surface micro-cantilever of described incomplete antibody 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: described antibody is cracked into F (ab ') with protease
2Fragment and a plurality of small molecule segment, described protease are selected from pepsin, papain or its combination;
3) disulfide bond reduction step: use disulfide bond reducing agent to process described F (ab ')
2Fragment so that the disulfide bond reduction of described antibody hinge region becomes sulfydryl, thereby obtains the Fab ' fragment with sulfydryl of two symmetries;
4) Fab ' fragment that fixing step: with step 3) obtains is combined with the micro-cantilever that is coated with the gold layer, obtains being fixed with on the gold-plated surface micro-cantilever of described Fab ' fragment.
Aspect the 3rd, 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) disulfide bond reduction step: use disulfide bond reducing agent to process institute's antibody, so that the disulfide bond reduction of described antibody hinge region becomes sulfydryl, thereby obtain the incomplete antibody fragment with sulfydryl of two symmetries;
3) enzymolysis step: with protease described incomplete antibody fragment is cracked into Fab ' fragment and a plurality of small molecule segment, described protease is selected from pepsin, papain or its combination;
4) Fab ' fragment that fixing step: with step 3) obtains is combined with the micro-cantilever that is coated with the gold layer, obtains being fixed with on the Gold plated Layer micro-cantilever of described Fab ' fragment.
Aspect the 4th, the invention provides a kind of stress sensing element, it comprises the micro-cantilever based on stress, is fixed with incomplete antibody fragment or 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 5th, 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 6th, 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) use disulfide bond reducing agent to process described antibody, so that the disulfide bond reduction of described antibody hinge region becomes sulfydryl, thereby obtain the incomplete antibody fragment with sulfydryl of two symmetries;
3) with step 2) the incomplete antibody fragment that obtains is combined with the micro-cantilever that is coated with gold layer, obtains being fixed with on the Gold plated Layer micro-cantilever of described incomplete antibody fragment;
4) with step 3) micro-cantilever that is fixed with described incomplete antibody fragment that obtains is fixed in the reaction tank of micro-cantilever sensing and detecting system;
5) testing sample is added to step 4) in the micro-cantilever sensing and detecting system that obtains, detect in the testing sample whether have described target antigen.
Aspect the 7th, 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) with protease described antibody is cracked into F (ab ')
2Fragment and a plurality of small molecule segment, described protease are selected from pepsin, papain or its combination;
3) use disulfide bond reducing agent to process described F (ab ')
2Fragment so that the disulfide bond reduction of described antibody hinge region becomes sulfydryl, thereby obtains the Fab ' fragment with sulfydryl of two symmetries;
4) with step 3) Fab ' fragment that obtains is combined with the micro-cantilever that is coated with gold layer, obtains being fixed with on the Gold plated Layer micro-cantilever of described Fab ' fragment;
5) with step 4) micro-cantilever that is fixed with described 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 8th, 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) use disulfide bond reducing agent to process described antibody, so that the disulfide bond reduction of described antibody hinge region becomes sulfydryl, thereby obtain the incomplete antibody fragment with sulfydryl of two symmetries;
3) with protease described incomplete antibody fragment is cracked into Fab ' fragment and a plurality of small molecule segment, described protease is selected from pepsin, papain or its combination;
4) with step 3) Fab ' fragment that obtains is combined with the micro-cantilever that is coated with gold layer, obtains being fixed with on the Gold plated Layer micro-cantilever of described Fab ' fragment;
5) with step 4) micro-cantilever that is fixed with described 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, the present invention splits into incomplete antibody fragment with the symmetry of sulfydryl with disulfide bond reducing agent with antibody, thereby or before or after splitting, obtain Fab ' fragment by protease digestion, then incomplete antibody fragment or the Fab ' fragment that obtains is fixed on the gold surface of little beam by the two sulfydryls that carry.The result shows, method of the present invention is compared with existing little beam immunization method and had the following advantages:
1) incomplete antibody fragment or Fab ' the fragment two sulfydryls by carrying directly are fixed to little beam gold-plated surface, and a step finishes, and is simple to operate;
The molecule of other that 2) does not add between incomplete antibody fragment or Fab ' fragment and the little beam surface is conducive to the stress transmission;
3) reduced antibody antigen-binding site antibody molecule quality and volume in addition;
4) antigen binding site that has reduced antibody is put forward heavily stressed transmission efficiency to the beam surface distance;
5) rigidity that connects of the fixing more single thiol molecule chain of the two sulfydryls between incomplete antibody fragment or Fab ' fragment and the little beam surface wants large, more be of value to the force transmission of antigen-antibody reaction to beam;
6) finish fixingly by self, fixing antigen binding site density is high, good stability in two one steps of sulfydryls;
7) good directionality of fixing antibody on the gold-plated surface is easy to be combined with antigen.
Description of drawings
Fig. 1 is typical 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 based on self sulfydryl (SH) directed fixedly incomplete antibody fragment schematic diagram.
Fig. 5 pepsin hydrolysis antibody formation antibody F (ab ')
2The fragment schematic diagram.
Fig. 6 is for passing through self sulfydryl (SH) directional immobilizing antibody Fab ' fragment schematic diagram.
Fig. 7 forms Fab ' Fragment with the pepsin hydrolysis incomplete antibody again for mercaptoethylmaine antibody being split into incomplete antibody first, at last by self sulfydryl (SH) directional immobilizing antibody Fab ' fragment schematic diagram.
Fig. 8 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. 9 is that anti-human ginseng saponin(e GS-Re incomplete antibody fragment is modified little beam, 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, shorter, identical light chain (L chain) that 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 ".Have the sub-fraction amino acid residue to change in the variable region strong especially, these amino acid whose residues form and the title hypervariable region, zone that more easily morphs that puts in order.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: as shown in Figure 4, whole antibody is split into the fragment of the symmetry that respectively carries sulfydryl by disulfide bond reducing agent, each incomplete antibody fragment comprises a complete light chain and complete heavy chain and Fc fragment.
F (ab ')
2: as shown in Figure 5, 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 (as shown in Figure 6, F (ab ') with sulfydryl
2The fragment that respectively carries sulfydryl that is split by disulfide bond reducing agent).
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, therefore claim again incomplete antigen.
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 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 that may exist in the testing sample of mentioning in the present invention of dawn known in the art.
The disulfide bond reducing agent of mentioning among the present invention can be mercaptoethylmaine (2-MEA), 2 mercapto ethanol, dithiothreitol (DTT) etc.Thereby method and the condition of using the disulfide bond reducing agent Reduction of Disulfide to split antibody are as known in the art, for example, can be according to type, size and the character etc. of target antigen, the specification of abideing by known method in this area or commercially available related kit is selected concrete disulfide bond reducing agent and concentration, AC, both consumptions and mass ratio, incubation conditions and incubative time etc.In a preferred embodiment of the invention, after processing antibody or antibody fragment with disulfide bond reducing agent, can dialyse and remove unreacted disulfide bond reducing agent, in order to avoid impact is with the combination of the antibody fragment of sulfydryl and little beam Gold plated Layer with fix.
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 that thereby the antigen-antibody binding fragment with sulfydryl can directly be fixed on little beam gold surface in its hydrolysate, described antigen-antibody binding fragment comprises the antigen-antibody binding fragment that is not limited to unit price or divalence, such as F (ab ')
2, Fab ' etc.Optional protease is such as pepsin, and papain etc. also can use both combinations.
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 pepsin, pepsin can be brought into play its active environment, generally refers to pH 2～5 and the environment under 25～60 ℃.Pepsic optimal pH and optimum temperature are decided according to its source, are generally pH about 2 and about 37 ℃ (can determine according to the requirement of commercially available prod).Operable pepsic source is unrestricted in the present invention, as long as it can be cracked into antibody molecule F (ab ')
2Fragment or the incomplete antibody fragment is cracked into Fab ' fragment.In the present invention, antibody or antibody fragment and pepsic mass ratio are unrestricted, as long as be enough to antibody or antibody fragment are hydrolyzed to Fab ' fragment fully.Both mass ratioes can be selected according to conventional methods, also can simultaneously with reference to factors such as selected pepsic activity, selected Antibody types, generally can select in 10: 1～200: 1 scope.
In addition, can also select papain.Papain also can be hydrolyzed to antibody F (ab ') under suitable condition
2Fragment, for example, at document
[6]In can be with mouse IgG at 37 ℃ of lower 18 hours papains in the environment of slant acidity (pH5.5)
1Be hydrolyzed into F (ab ')
2Fragment.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 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.
When using the mixture of pepsin and papain, because both active environment are acidity, therefore suitably adjust both mass ratioes according to concrete application, thereby can adjust as required required hydrolysing activity and hydrolysis time.In the present invention, enzymolysis step can be carried out before or after splitting antibody.When before splitting antibody, using protease hydrolytic, when namely using protease hydrolytic antibody, obtain a F with two Fab ' fragments (ab ')
2Fragment and a plurality of small molecule segment.When after splitting antibody, using protease hydrolytic, when namely using protease hydrolytic incomplete antibody fragment, a Fab ' fragment and a plurality of small molecule segment have been obtained.Two kinds of processing modes finally all can obtain the Fab ' fragment with two sulfydryls.
In the present invention, the combination of the Gold plated Layer of antibody or antibody fragment and little beam can be used method well known in the art and condition, for example incubation a period of time combination (referring to the specification of the following examples or commercially available prod) under the condition that is suitable for both combinations.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 little beam of incomplete antibody fragment fixedly
Experiment 1 utilizes mercaptoethylmaine that antibody is split into incomplete antibody fragment with sulfydryl
1. accurately taking by weighing ginsenoside GS-Re monoclonal antibody (available from U.S. USBiological company) 7.0mg is dissolved among the 1.0mL PBS (contain 0.15M NaCl and 5.0mM EDTA, pH 6.5) and obtains the antibody-solutions that concentration is 7.0g/L;
2. taking by weighing 1mg mercaptoethylmaine (available from U.S. Sigma company) is dissolved in and obtains the antibody resolution reagent that concentration is 1.0g/L in the 1.0mL water;
3. the antibody-solutions of 1.0mL above-mentioned steps 1 and the antibody resolution reagent of 291.2 μ L above-mentioned steps 2 are mixed, reaction is 1.5 hours under the room temperature.
4. with the PBS buffer solution (contain 0.15M NaCl and 5.0mM EDTA, pH 6.5) of 20mM
To the reactant liquor of above-mentioned steps 3 dialysis 48 hours, during changed a dislysate in per 3 hours, obtain the incomplete antibody fragment with sulfydryl.With-20 ℃ of preservations after the dilution of glycerine equal-volume.
Test the combination of 2 incomplete antibody fragments and little beam gold surface
To clean and put into the ELISA Plate aperture with the little beam (available from U.S. Veeco company) that is coated with the gold layer that nitrogen dries up, adding 150 μ L concentration is the anti-human ginseng saponin(e GS-Re monoclonal antibody fragment of experiment 1 step 1 acquisition of 5.0mg/mL, 37 ℃ of incubations 1 hour namely are fixed little beam of incomplete antibody fragment.
Test the as follows in conjunction with the situation concrete steps of 3 direct competitive ELISA method evaluation incomplete antibody fragments and little beam gold surface:
1. clean: with the above-mentioned little beam of fixing the incomplete antibody fragment 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 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 50 μ L stop buffer (sulfuric acid solution of 2.0M) cessation reactions, takes out little beam, and 450nm wavelength place reads respectively the OD value.
Three repetition are established in experiment, and the result shows, the absorbance under the 450nm wavelength in inhibition hole and non-inhibition hole is respectively 0.121 and 0.727, illustrate anti-human ginseng saponin(e GS-Re incomplete antibody fragment 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: the little beam that is fixed with anti-human ginseng saponin(e GS-Re antibody fragment in the step 2 is fixed in the reaction tank of micro-cantilever sensing system, and 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.
Fig. 9 is that anti-human ginseng saponin(e GS-Re incomplete antibody fragment is modified little beam, the time-displacement curve at little beam tip when detecting variable concentrations antigen.Detect the most advanced and sophisticated displacement amplitude of little beam that the sample of three concentration 1.0ng/mL, 0.1ng/mL, 0.02ng/mL obtains and be respectively 77,58 and 32nm.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 incomplete antibody fragment can effectively be attached on the gold surface of little beam and can quantitatively detect ginsenoside GS-Re.And has very high detection sensitivity (Fig. 8 is for utilizing the hydrochloric acid mercaptan imine to come behind the sulfhydrylation antibody antibody to be fixed to the result that Wei Liangshang (Chinese patent CN101407548) detects ginsenoside as sulfhydrylization reagent).
Conclusion:
Little beam of modifying with little beam and the complete antibody of incomplete antibody fragment modification of the present invention carries out the experiment test contrast, when detecting the ginsenoside GS-Re antigen of variable concentrations, the time shifting response curve at little beam tip (Fig. 9 and Fig. 8) shows, when little beam displacement response is the 20 nanometer left and right sides, the incomplete antibody fragment is modified and the detection ginsenoside GS-Re antigen concentration of sulfhydrylation antibody modification is respectively 0.02ng/mL and 0.5ng/mL, and sensitivity has improved approximately 20 times.
Little beam that embodiment 2 Fab ' Fragments are modified
Test 1 antibody F (ab ')
2The preparation of fragment
1. dissolve ginsenoside GS-Re monoclonal antibody (IgG) (available from U.S. USBiological company) to 10mg/ml with 0.1mol/L citrate buffer solution (pH3.5);
2. dissolve pepsin (pepsin) (available from U.S. Sigma company) to 1mg/ml with 0.1mol/L citrate buffer solution (pH3.5);
3. with IgG: pepsin mixes in mass ratio at 100: 1, is placed in 37 ℃ of water baths 2 hours, then add 3mol/L Tris make pH about 7 with cessation reaction.
4. with the PBS buffer solution (contain 0.15M NaCl and 5.0mM EDTA, pH 6.5) of 20mM
To the reaction mixture of above-mentioned steps 3 dialysis 48 hours, during changed a dislysate in per 3 hours, obtain antibody F (ab ')
2Fragment.
1. use deionized water dissolving mercaptoethylmaine (2-MEA) (available from U.S. Sigma company) to 1mg/ml;
2. with the F (ab ') of preparation in the step 1
2: 2-MEA mixes in mass ratio at 20: 1, is placed in 37 ℃ of water baths 2 hours;
With the PBS buffer solution (contain 0.15M NaCl and 5.0mM EDTA, pH 6.5) of 20mM to the reaction mixture dialysis of above-mentioned steps 2 48 hours, during changed a dislysate in per 3 hours, obtain Fab ' Fragment.With being kept under-20 ℃ of environment after the dilution of glycerine equal-volume.
Experiment 3 is modified Fab ' antibody fragment on little beam gold surface
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 150 μ L concentration and be Fab ' the antibody fragment solution that the above-mentioned steps 2 of 5.0mg/mL makes, be placed in 37 ℃ of water baths 2 hours, namely be fixed little beam of Fab ' Fragment.
Experiment 4ELISA direct competition method effect detection
1. clean: with the above-mentioned micro-cantilever of fixing Fab ' Fragment of the careful taking-up of tweezers, (contain 8.0g NaCl, 0.2g KH in every 1L cleaning solution with cleaning solution
2PO
4, 2.96gNa
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, the absorbance under the 450nm wavelength in inhibition hole and non-inhibition hole is respectively 0.178 and 0.853, illustrate that anti-human ginseng saponin(e GS-Re Fab ' Fragment 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. fixing little beam: will test and be modified with anti-human ginseng saponin(e GS-Re antibody passage Fab in 3 ' micro-cantilever be fixed in the reaction tank of micro-cantilever sensor-based system, the PBS that flows is by reacting the 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 1.0ng/mL, 0.1ng/mL, 0.01ng/mL obtains and be respectively 79,61 and 21nm; 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 Fab ' Fragment can effectively be attached on the gold surface of micro-cantilever and can detect ginsenoside GS-Re.
Conclusion:
Little beam of modifying with little beam and the complete antibody of Fab ' Fragment modification of the present invention carries out the experiment test contrast, when detecting the ginsenoside GS-Re antigen of variable concentrations, when little beam displacement response is the 20 nanometer left and right sides, Fab ' Fragment is modified and sulfhydrylation antibody modification detection ginsenoside GS-Re antigen concentration is respectively 0.01ng/mL and 0.5ng/mL, and sensitivity has improved nearly 40 times.
Little beam that embodiment 3 Fab ' Fragments are modified
Experiment 1 utilizes mercaptoethylmaine that antibody is split into incomplete antibody fragment with sulfydryl
1. accurately taking by weighing ginsenoside GS-Re monoclonal antibody (available from U.S. USBiological company) 7.0mg is dissolved among the 1.0mL PBS (contain 0.15M NaCl and 5.0mM EDTA, pH 6.5) and obtains the antibody-solutions that concentration is 7.0g/L;
2. taking by weighing 1mg mercaptoethylmaine (available from U.S. Sigma company) is dissolved in and obtains the antibody resolution reagent that concentration is 1.0g/L in the 1.0mL water;
3. the antibody-solutions of 1.0mL above-mentioned steps 1 and the antibody resolution reagent of 291.2 μ L above-mentioned steps 2 are mixed, reaction is 1.5 hours under the room temperature.
With the PBS buffer solution (contain 0.15M NaCl and 5.0mM EDTA, pH 6.5) of 20mM to the reactant liquor dialysis of above-mentioned steps 3 48 hours, during changed a dislysate in per 3 hours, obtain the incomplete antibody fragment with sulfydryl.With-20 ℃ of preservations after the dilution of glycerine equal-volume.
Test the preparation of 2 Fab ' Fragments
1. dissolve pepsin (pepsin) (available from U.S. Sigma company) to 1mg/ml with 0.1mol/L citrate buffer solution (pH3.5);
2. with incomplete antibody fragment and pepsin 100: 1 hybrid reactions in mass ratio of preparation in the step 1, be placed in 37 ℃ of water baths 2 hours, then add 3mol/L Tris make pH about 7 with cessation reaction.
With the PBS buffer solution (contain 0.15M NaCl and 5.0mM EDTA, pH 6.5) of 20mM to the reaction mixture dialysis of above-mentioned steps 2 48 hours, during changed a dislysate in per 3 hours, obtain Fab ' Fragment.With being kept under-20 ℃ of environment after the dilution of glycerine equal-volume.
Experiment 3 is modified Fab ' Fragment on little beam gold surface
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 150 μ L concentration and be the Fab ' Fragment solution that the above-mentioned steps 2 of 5.0mg/mL makes, be placed in 37 ℃ of water baths 2 hours, namely be fixed little beam of Fab ' Fragment.
Experiment 4ELISA direct competition method effect detection
1. clean: with the above-mentioned micro-cantilever of fixing Fab ' Fragment 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 pH 7.5, 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, the absorbance under the 450nm wavelength in inhibition hole and non-inhibition hole is respectively 0.152 and 0.763, illustrate that anti-human ginseng saponin(e GS-Re Fab ' Fragment 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 ' Fragment 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 1.0ng/mL, 0.1ng/mL, 0.015ng/mL obtains and be respectively 68,42 and 22nm; 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 Fab ' Fragment can effectively be attached on the gold surface of micro-cantilever and can detect ginsenoside GS-Re.
Conclusion:
Little beam that little beam that the Fab ' Fragment of the present invention of the method preparation by enzymolysis after splitting is first modified and complete antibody are modified carries out the experiment test contrast, when detecting the ginsenoside GS-Re antigen of variable concentrations, when little beam displacement response is the 20 nanometer left and right sides, Fab ' Fragment is modified and sulfhydrylation antibody modification detection ginsenoside GS-Re antigen concentration is respectively 0.015ng/mL and 0.5ng/mL, and sensitivity has improved nearly 33 times.
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 Baomin Wang，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 Using Microcantilever 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 antibody functionalized 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
[6] [J] Chou Kai etc., papain prepare the anti-F of anti-human liver cancer (ab ')
2And Fab fragment.Journal of the Fourth Military Medical University, 1995; 16 (6): 414-416.
Claims (10)
1. method for preparing micro-cantilever may further comprise the steps:
1) provides antibody and with the micro-cantilever of Gold plated Layer;
2) disulfide bond reduction step: use disulfide bond reducing agent to process described antibody, so that the disulfide bond reduction of described antibody hinge region becomes sulfydryl, thereby obtain the incomplete antibody fragment with sulfydryl of two symmetries;
3) the incomplete antibody fragment that fixing step: with step 2) obtains is combined with the micro-cantilever that is coated with the gold layer, obtains being fixed with on the gold-plated surface micro-cantilever of described incomplete antibody 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: described antibody is cracked into F (ab ') with protease
2Fragment and a plurality of small molecule segment, described protease are selected from pepsin, papain or its combination;
3) disulfide bond reduction step: use disulfide bond reducing agent to process described F (ab ')
2Fragment so that the disulfide bond reduction of described antibody hinge region becomes sulfydryl, thereby obtains the Fab ' fragment with sulfydryl of two symmetries;
4) Fab ' fragment that fixing step: with step 3) obtains is combined with the micro-cantilever that is coated with the gold layer, obtains being fixed with on the gold-plated surface micro-cantilever of described Fab ' fragment.
3. method for preparing micro-cantilever may further comprise the steps:
1) provides antibody and with the micro-cantilever of Gold plated Layer;
2) disulfide bond reduction step: use disulfide bond reducing agent to process institute's antibody, so that the disulfide bond reduction of described antibody hinge region becomes sulfydryl, thereby obtain the incomplete antibody fragment with sulfydryl of two symmetries;
3) enzymolysis step: with protease described incomplete antibody fragment is cracked into Fab ' fragment and a plurality of small molecule segment, described protease is selected from pepsin, papain or its combination;
4) Fab ' fragment that fixing step: with step 3) obtains is combined with the micro-cantilever that is coated with the gold layer, obtains being fixed with on the Gold plated Layer micro-cantilever of described Fab ' fragment.
4. stress sensing element, it comprises the micro-cantilever based on stress, is fixed with incomplete antibody fragment or the Fab ' fragment of antibody on the Gold plated Layer of described micro-cantilever.
5. micro-cantilever immune sensing detection system based on stress, it comprises the stress sensing element of claim 4.
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) use disulfide bond reducing agent to process described antibody, so that the disulfide bond reduction of described antibody hinge region becomes sulfydryl, thereby obtain the incomplete antibody fragment with sulfydryl of two symmetries;
3) with step 2) the incomplete antibody fragment that obtains is combined with the micro-cantilever that is coated with gold layer, obtains being fixed with on the Gold plated Layer micro-cantilever of described incomplete antibody fragment;
4) with step 3) micro-cantilever that is fixed with described incomplete antibody fragment that obtains is fixed in the reaction tank of micro-cantilever sensing and detecting system;
5) testing sample is added to step 4) in the micro-cantilever sensing and detecting system that obtains, detect in the testing sample whether have described target antigen.
7. 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) with protease described antibody is cracked into F (ab ')
2Fragment and a plurality of small molecule segment, described protease are selected from pepsin, papain or its combination;
3) use disulfide bond reducing agent to process described F (ab ')
2Fragment so that the disulfide bond reduction of described antibody hinge region becomes sulfydryl, thereby obtains the Fab ' fragment with sulfydryl of two symmetries;
4) with step 3) Fab ' fragment that obtains is combined with the micro-cantilever that is coated with gold layer, obtains being fixed with on the Gold plated Layer micro-cantilever of described Fab ' fragment;
5) with step 4) micro-cantilever that is fixed with described 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.
8. 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) use disulfide bond reducing agent to process described antibody, so that the disulfide bond reduction of described antibody hinge region becomes sulfydryl, thereby obtain the incomplete antibody fragment with sulfydryl of two symmetries;
3) with protease described incomplete antibody fragment is cracked into Fab ' fragment and a plurality of small molecule segment, described protease is selected from pepsin, papain or its combination;
4) with step 3) Fab ' fragment that obtains is combined with the micro-cantilever that is coated with gold layer, obtains being fixed with on the Gold plated Layer micro-cantilever of described Fab ' fragment;
5) with step 4) micro-cantilever that is fixed with described 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.
9. each described method, stress sensing element or immune sensing detection system according to claim 1-8 is characterized in that in described disulfide bond reducing agent selected from mercapto ethamine, 2 mercapto ethanol, the dithiothreitol (DTT) one or more.
10. each described method, stress sensing element or immune sensing detection system according to claim 1-9 is characterized in that described antibody is any among IgM, IgG, IgA, IgD, the IgE.
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| CN101935008B (en) * | 2010-07-30 | 2012-10-17 | 中国科学院上海微系统与信息技术研究所 | Method of micro cantilever beam sensor using functional carbon nano tubes as sensitive materials |
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| US20030068657A1 (en) * | 2000-10-30 | 2003-04-10 | Sru Biosystems Llc | Label-free methods for performing assays using a colorimetric resonant reflectance optical biosensor |
| 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 |
| CN101407548A (en) * | 2008-11-25 | 2009-04-15 | 中国农业大学 | Method for preparing micro-cantilever beam modified with antibody |
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| CN103869062A (en) * | 2012-12-18 | 2014-06-18 | 中国科学技术大学 | Micro-cantilever beam array biochemical sensing apparatus based on piezoelectric scanning and method |
| CN106124105A (en) * | 2016-06-13 | 2016-11-16 | 常州大学 | Utilize the method that DNA molecular intermolecular forces is measured in flexural deformation |
| CN106124104A (en) * | 2016-06-13 | 2016-11-16 | 常州大学 | A kind of micro-cantilever device measuring genetic fragment active force |
| CN107764992A (en) * | 2017-10-16 | 2018-03-06 | 南京诺唯赞医疗科技有限公司 | The orientation coupling method of a kind of microballoon and antibody and application |
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