CN102951600B - Micro-beam preparation method that antibody fragment is modified and micro-beam immune sensing detection system of modifying based on antibody fragment - Google Patents
Micro-beam preparation method that antibody fragment is modified and micro-beam immune sensing detection system of modifying based on antibody fragment Download PDFInfo
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- CN102951600B CN102951600B CN201110240250.9A CN201110240250A CN102951600B CN 102951600 B CN102951600 B CN 102951600B CN 201110240250 A CN201110240250 A CN 201110240250A CN 102951600 B CN102951600 B CN 102951600B
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Abstract
The invention provides the micro-cantilever that a kind of antibody fragment is modified, its gold-plated surface is fixed with antibody F (ab ')
2fragment, by reducing quality and the volume of other parts beyond antigen molecule binding site on antibody, improve the effective modification density of antigen binding site on micro-beam surface, and intermolecular reaction force transmission is to the efficiency on micro-beam, reach the sensitivity object improving micro-beam immunologic detection method.The immune sensing detection system of the micro-cantilever the invention also discloses the preparation method of described micro-cantilever, modifying based on described antibody fragment and detection method.
Description
Technical field
The present invention relates to antibody fragment, specifically antibody F (ab ')
2the micro-cantilever (hereinafter also referred to as micro-beam) that fragment is modified, and preparation method thereof.The invention also discloses immune sensing detection system and the detection method of the micro-beam modified 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 reacted based on the specific pairs of detectable antigens antibody, namely for certain target molecules (antigen) needing to detect, by biological immune (what produce in 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 goes to detect the target molecule in sample.Existing immune sensing technology is as EUSA (ELISA, principle utilizes 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), label is all needed to carry out the reaction result of labelled antigen antibody, in other words only antibody is had, corresponding detection method can not be set up, also need enzyme mark thing or fluorescent marker or albumen composition.And the label of the target molecules of some difficult preparations is expensive, or may prepare or buy hardly.The detection of enzyme linked immunological kit and protein chip simultaneously all requires that operate every single step reaction cleans after complete, and labeling process is loaded down with trivial details consuming time, is detection afterwards, can not be real-time, original position, online detection.
The micro-cantilever immune sensing technology detected based on surface stress is a kind of new immune biochemical method for sensing in recent years occurred
[1]its principle is: the direct or indirect mode of probe (antigen or antibody) molecule is fixed (modification) in the Gold plated Layer of Wei Liang side, when the probe molecule generation immune biochemical on the target molecule in detected sample liquid and micro-beam gold surface reacts, micro-beam surface stress can be made to change, thus cause micro-beam deformed, detected the process of this distortion by optics or electrical method, the real time information of immune biochemical reaction can be obtained.Based on immunologic opsonin identification set up micro-beam immune sensing technology compared with traditional immuno-sensing method needing label, it is without the need to using any enzyme mark, fluorescent material and radioactivity as reaction tracer, eliminate the impact of labeling process, highly sensitive (than enzyme linked immune assay high several times
[2-4]), the course of reaction of real-time, quantitative monitoring antigen-antibody can also be carried out by monitoring the distortion of micro-beam, obtaining the information of abundanter immune biochemical reaction.Through development these years, micro-cantilever sensing is by as a kind of emerging technology, in bioengineering and environmental pollution monitoring technology etc., carry out comparative study with traditional method, 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 is also not enough to the huge advantage obtained in cost relative to conventional ELISA, if namely the sensitivity of micro-cantilever immune sensing technology or detectable limit are only than ELISA high several times, and the equipment cost of micro-cantilever immune sensing technology and enzyme linked immunological relatively high, make micro-cantilever immune sensing technology be difficult to commercialization.
At present, the method for domestic and foreign literature report mainly utilizes the thin base (-SH) of the thin base reagent with bifunctional group to catch the gold surface on micro-beam surface, and another functional group catches antibody, realizes antibody fixing on micro-beam gold-plated surface.Such as first sulfhydrylization reagent 11-thiol carboxylic acid is attached to the gold surface of micro-beam, activates the carboxyl on it, the amino made it on antibody is combined and fixes antibody (Fig. 2).Or by a kind of sulfhydrylization reagent hydrochloric acid mercaptan imine, by with antibody response, make antibody connect the molecular radical of a band sulfydryl, then by this sulfydryl, antibody is attached to (Chinese patent CN101407548) (see Fig. 3) in the gold surface of micro-beam., there is problem common as follows in these method sessile antibodies, the Fc section of (1) Y-shaped antibody (see Fig. 1) or the top ends of Fab section, can equiprobablely be fixed on a gold surface, not have directionality
[5].And when the top of Fab section is fixed on a gold surface, binding site is blocked, limits the antigen binding site of antibody and the abundant combination of antigen, thus reduce micro-cantilever sensing sensitivity; (2) there is the single chain molecule of different number C atom between antibody from micro-beam, reduce the Stress transmit of antigen-antibody reaction generation to the efficiency on beam.As can be seen here, the sensitivity of method disclosed in Chinese patent CN101407548 only improves several times compared with ELISA, also cannot reach commercialization or the detection for denier analyte.
Therefore, there is the combination improving micro-beam surface and antibody and design in the art thus improve the demand of the sensitivity of micro-beam immune sensing and efficiency further.
Summary of the invention
In sum, in existing micro-beam immune sensing technology report, most distinct issues are exactly detection sensitivity.The factor affecting the sensitivity of micro-beam immunosensor mainly contains three aspects: on sensitivity (or antibody be combined with antigen compatibility), 2. micro-beam of 1. antibody antibody fixing means and 3. the design of micro-beam and signal read.Because the sensitivity of antibody and the specification of micro-beam and signal playback mode just cannot change after micro-beam immunosensor system is shaping, uniquely variable is the fixing means of micro-beam surface antibody.A method of modifying for suitable micro-beam surface antibody, can make the antigen of antibody in sample solution being fixed on micro-beam surface fully be combined, and can efficiently antigen-antibody be combined the STRESS VARIATION produced be delivered to micro-beam surface.Antibody all likely affects with the length connecting molecule between micro-beam surface and rigidity the sensitivity finally detected at directionality, density, activity and the antibody that micro-beam surface is fixing.
In view of above-mentioned the problems of the prior art, the present invention is quality by reducing other parts beyond antigen molecule binding site on antibody and volume for the technical scheme solved the problem, improve the effective modification density of antigen binding site on micro-beam surface, and intermolecular reaction force transmission is to the efficiency on micro-beam, reach the sensitivity object improving micro-beam immunologic detection method.
The structural representation of antibody I g molecule, as Fig. 1, comprises two identical Fabs (Fab) and a FC (Fc), Fab with Fc is connected by middle hinge area, formation Y-shaped structure.The top of Fab is the binding site with antigen molecule.In " Y " font four peptide chain structure (Fig. 1) of immune globulin antibody molecule, be formed by connecting with disulfide bond by two identical heavy chains and two identical light chains.Time on gold-plated surface Fc section being modified to micro-beam (directionality of antibody modification is good), two upper end antigen binding sites of antibody symmetry (Fab section) are combined the rear stress produced with antigen, be delivered to gold-plated micro-beam surface by the leg (Fc section) of antibody Y-shaped.The molecular weight of antibody (IgG) is about 150kDa, if detected antigen molecule molecular weight is hundreds of Da, and antibody antigen mass ratio nearly 1000.Participate in the site that antigen molecule combines to be made up of the hypervariable region (VH and VL) of heavy chain and light chain, the mass ratio about 150 of itself and antigen molecule, and antibody molecule is not participated in the Fc section be combined with antigen molecule, the mass ratio nearly 400 of itself and antigen molecule.Therefore we imagine, if the antibody molecule quality that can reduce beyond binding site and volume, keep the exterior normal direction of binding site towards fixed surface of antibody, just can put forward heavily stressed transmission efficiency and the effective modification density of antibody on micro-beam surface, thus improve the sensitivity of micro-beam immune sensing.
For this reason, in first, the invention provides a kind of method preparing micro-cantilever, comprise the following steps:
1) antibody and the micro-cantilever with Gold plated Layer are provided;
2) enzymolysis step: described antibody is cracked into F (ab ') with protease
2fragment and multiple small molecule segment, described protease is selected from pepsin, papain or its combination;
3) fixing step: the sulfhydrylization reagent comprising mercapto functional group and carboxyl or amido functional group is bonded to the Gold plated Layer of micro-cantilever on the surface;
4) step is connect: by step 2) F that obtains (ab ')
2with step 3) the described sulfhydrylization reagent be combined on micro-cantilever that obtains is bound up, and obtain Gold plated Layer and be fixed with F (ab ') on the surface
2micro-cantilever.
In second, present invention also offers a kind of method preparing micro-cantilever, comprise the following steps:
1) antibody and the micro-cantilever with Gold plated Layer are provided;
2) enzymolysis step: described antibody is cracked into F (ab ') with protease
2fragment and multiple small molecule segment, described protease is selected from pepsin, papain or its combination;
3) step is connect: by step 2) F that obtains (ab ')
2be bound up with sulfhydrylization reagent;
4) fixing step: by step 3) one end of obtaining and F (ab ')
2the other end of described sulfhydrylization reagent of connection is bonded to the Gold plated Layer of micro-cantilever on the surface, obtains Gold plated Layer and is fixed with F (ab ') on the surface
2micro-cantilever.
In the 3rd, the invention provides a kind of stress sensing element, it comprises the micro-cantilever based on stress, the Gold plated Layer of described micro-cantilever is fixed with the F (ab ') of antibody
2fragment.Described micro-cantilever is prepared from preferably by method of the present invention.
In 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.
In the 5th, the invention provides a kind of method using micro-cantilever sensing and detecting system immune sensing to detect testing sample, comprise the following steps:
1) provide the special antibody of target antigen and micro-cantilever sensing and detecting system, described micro-cantilever sensing and detecting system comprises reaction tank and the micro-cantilever based on stress with Gold plated Layer;
2) described antibody is cracked into F (ab ') with protease
2fragment and multiple small molecule segment, described protease is selected from pepsin, papain or its combination;
3) fixing step: the sulfhydrylization reagent comprising mercapto functional group and carboxyl or amido functional group is bonded to the Gold plated Layer of micro-cantilever on the surface;
4) step is connect: by step 2) F that obtains (ab ')
2fragment and step 3) the described sulfhydrylization reagent be combined on micro-cantilever that obtains is bound up, and obtain Gold plated Layer and be fixed with F (ab ') on the surface
2the micro-cantilever of fragment.
5) by step 4) obtain be fixed with F (ab ')
2the micro-cantilever of fragment 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 testing sample whether there is described target antigen.
In the 6th, present invention also offers a kind of method using micro-cantilever sensing and detecting system immune sensing to detect testing sample, comprise the following steps:
1) provide the special antibody of target antigen and micro-cantilever sensing and detecting system, described micro-cantilever sensing and detecting system comprises reaction tank and the micro-cantilever based on stress with Gold plated Layer;
2) described antibody is cracked into F (ab ') with protease
2fragment and multiple small molecule segment, described protease is selected from pepsin, papain or its combination;
3) step is connect: by step 2) F that obtains (ab ')
2fragment and the sulfhydrylization reagent comprising mercapto functional group and carboxyl or amido functional group are bound up;
4) fixing step: by step 3) one end of obtaining and F (ab ')
2the other end of described sulfhydrylization reagent of fragment connection is bonded to the Gold plated Layer of micro-cantilever on the surface, obtains Gold plated Layer and is fixed with F (ab ') on the surface
2the micro-cantilever of fragment;
5) by step 4) obtain be fixed with F (ab ')
2the micro-cantilever of fragment 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 testing sample whether there is described target antigen.
Beneficial effect of the present invention:
The importance of fixing in view of antibody in micro-beam immune sensing detection technique and sulfhydrylization reagent participate in the fixing complexity of antibody, and the present invention proposes modified antibodies fragment Fab method on micro-beam gold-plated surface and realizes micro-beam immune detection.Result shows, method of the present invention has the following advantages compared with existing micro-beam immunization method:
1) the present invention is with antibody fragment F (ab ')
2alternative whole antibody molecule is fixed to the method in micro-beam gold surface, reaches the object improving the sensitivity of micro-beam immune sensing.Compared with existing micro-beam immunization method, this antibody fragment fixing means decreases excess mass on antibody beyond antigen binding site and volume, reduces the antigen binding site of antibody to beam surface distance, can effectively put forward heavily stressed transmission efficiency;
2) improve the modification density of antigen binding site on micro-beam surface, it also avoid Fc section blocking other antigen binding site unnecessary on antibody simultaneously, effectively improve the possibility that antigen-antibody combines, thus improve the sensitivity of micro-beam immune sensing.
Accompanying drawing explanation
Fig. 1 is 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 pepsin hydrolysis antibody forms antibody fragment and obtains F (ab ')
2fragment, is then fixed to the schematic diagram on micro-beam by sulfhydrylization reagent.
Fig. 5 is the micro-beam of anti-ginsenoside GS-Re sulfhydrylation antibody modification, detects the time-displacement curve at micro-beam tip during variable concentrations antigen.
Fig. 6 is anti-ginsenoside GS-Re antibody F (ab ')
2fragment modifies micro-beam, detects the time-displacement curve at micro-beam tip during variable concentrations antigen.
Detailed description of the invention
Definition
Antibody: antibody (antibody) refers to that the immune system of body is under antigenic stimulus, that the thick liquid cell be divided into by bone-marrow-derived lymphocyte or memory cell prolifera produces, can with the immunoglobulin (Ig) of corresponding antigens generation specific binding.Typical antibody molecule has the symmetrical structure of 4 polypeptide chains, comprises the identical heavy chain (H chain) that 2 longer, relative molecular weight is larger; Article 2, the identical light chain (L chain) that shorter, relative molecular weight is less.Interchain forms a monomer molecule be made up of 4 polypeptide chains by disulfide bond and non-covalent bond connection.Light chain has κ and λ two kinds, and heavy chain has μ, δ, γ, ε and α five kinds.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.Variable region is positioned at the two-arm end of " Y ".In variable region, have sub-fraction amino acid residue to change strong especially, these amino acid whose residues compositions and putting in order region of more easily morphing claims hypervariable region.Hypervariable region is positioned at molecular surface, is made up of at most, only has 2 ~ 3 at least 17 amino acid residues.Hypervariable region amino acid sequence determines the specificity of this antibodies bind antigen.Two antigen-binding sites on an antibody molecule are identical, are positioned at two-arm end and claim Fab (antigen-bindingfragment, Fab).The shank of " Y " claims crystallizable fragment (crystallinefragment, Fc), and sugar is combined on Fc.
From a structural point, in the present invention, when mentioning antibody, all refer to whole antibody, namely comprise the antibody structure (see Fig. 1) of 4 chains and Fc section.In addition, functionally, " antibody " or " antibody fragment " in the present invention refers to the special antibody of target antigen or antibody fragment, namely there is with antigen antibody or the antibody fragment of specific bond ability, if not otherwise specified, antibody in the present invention refers generally to monoclonal antibody, and antibody fragment comprises incomplete antibody fragment, F (ab ')
2fragment, Fab ' fragment and Fab etc.
Incomplete antibody fragment: the fragment by disulfide bond reducing agent, whole antibody being split into the symmetry respectively carrying sulfydryl, each incomplete antibody fragment comprises a complete light chain and an entire heavy chain and Fc fragment.
F (ab ')
2: Ig (immunoglobulin, immunoglobulin (Ig)) is cut off at the nearly C place of the heavy interchain disulfide bond of hinge area by pepsin hydrolysis, forms a bivalent Fab abbreviation F (ab ')
2fragment and some small fragments pFc '.Due to F (ab ')
2fragment remains the BA in conjunction with corresponding antigens, the side effect that the antigenicity that turn avoid Fc fragment may cause, and is thus widely used as biological products.As diphtheria antimycin and lockjaw antimycin, after pepsin hydrolysis, because eliminating the antigenicity of Fc fragment and decreasing the generation of hypersensitivity.PFc ' fragment is finally degraded, and abiology is active.
Fab (fragmentofantigenbinding): papain makes Ig cut off at the heavy interchain disulfide bond nearly N end place of hinge area, 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 (fragmentcrystallizable) section.
Antigen binding site: as shown in fig. 1, the position that antibody molecule combines with antigen, light by Ig, heavy chain CDR1, CDR2 and CDR3 form.
Antigen: be a class energy inducing immune system generation immune response, and the material of specific binding can be there is with the product of immune response (antibody or effector cell).Antigen has immunogenicity and reactionogenicity two kinds of character.Two classes are divided into: comlete antigen and incomplete antigen according to antigenic property.Comlete antigen (completeantigen) is the existing immunogenicity of a class, has again immunoreactive material.If most protein, bacterium, virus, bacterial exotoxin etc. are all comlete antigens.Incomplete antigen, namely haptens (hapten) only has immunoreactivity, and the material of non-immunogenicity, therefore also known as incomplete antigen.
Sulfhydrylization reagent: what have sulfydryl can connect antibody and golden difunctional cross-linking reagent.
Micro-cantilever (micro-beam): typical micro-cantilever is made up of silicon nitride, as commercial triangle micro-cantilever (VeecoInstruments) (size: long 200um, the wide 20um of leg, thick 0.6um), the one-sided gold being coated with 60nm; Antibody (contains-COOH or-NH by the sulfydryl (-SH) of sulfhydrylization reagent and other one end of the covalent bond of gold and sulfhydrylization reagent usually
2isoreactivity group) to be combined with antibody and to be secured to micro-beam surface.
Micro-cantilever immune sensing system based on surface stress detects: micro-cantilever immune sensing system forms primarily of laser instrument, spectroscope, micro-cantilever, photoelectric position sensor (PSD), temperature control system, peristaltic pump, reaction tank and Data Analysis Services device.The step of typical micro-cantilever immunologic detection method is as follows: be fixed to by micro-cantilever in reaction tank, controls flowing buffer solution by reaction tank with peristaltic pump, passes through reaction tank in question response pond after bubble emptying with the speed of 0.1mL/min flowing buffer solution.The temperature of reaction tank controls at 37 ± 0.01 DEG C, and room temperature controls at 27 ± 0.01 DEG C.Laser instrument sends the tip that beam of laser is radiated at micro-cantilever after spectroscope, impinges upon on the target surface of PSD after micro-cantilever reflection after dichroic mirror again.After the displacement signal of micro-cantilever is stable, add the sample solution of buffer solution dilution, the tip 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 by any method preparation as known in the art, can include but not limited to immunization, hybridoma, chemical synthesis, gene engineering research etc.Described antibody can also be the hybrid antibody that genetic engineering is modified, such as humanized antibody, the antibody that camelized antibody etc. are transformed for certain mammal, such as people-mouse hybrid antibody etc.
The testing sample mentioned in the present invention can be biological sample, such as come from the sample of mammal especially people, comprise tissue sample (as histopathologic slide, biopsy, hair, swab etc.), cell sample (as cell smear, blood smear etc.), humoral sample (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 obtained in other production fields, such as sewage sample, food samples etc.
Antigen mentioned in the present invention includes but not limited to comlete antigen and haptens, and it can be the antigen of any type that the needs of the dawn known in the art that may exist in mentioned in the present invention testing sample detect.
In the present invention, the protease used in enzymolysis step at random can be selected according to the kind of the antibody that will be hydrolyzed or other factors, as long as its hydrolysate comprises the antibody fragment F (ab ') with sulfydryl
2thus can directly be fixed in micro-beam gold surface.Optional protease such as pepsin, papain etc., also can use both combinations.
In the present invention, adopt the method for protease hydrolytic antibody I g and reaction condition to be well known in the art, those of ordinary skill in the art can determine suitable protease and corresponding hydrolysising condition according to the kind of the antibody that will be hydrolyzed.
Such as, when selecting pepsin, pepsin can play the environment of its activity, generally refers to pH2 ~ 5 and the environment at 25 ~ 60 DEG C.Pepsic optimal pH and optimum temperature are determined according to its source, are generally about pH2 and about 37 DEG C (can determine according to the requirement of commercially available prod).Operable pepsic source is unrestricted in the present invention, as long as antibody molecule can be cracked into F (ab ') by it
2fragment.In the present invention, antibody or antibody fragment and pepsic mass ratio unrestricted, as long as be enough to antibody or antibody fragment to be hydrolyzed to fully F (ab ')
2fragment.Both mass ratioes can be selected according to conventional methods, also simultaneously with reference to factors such as selected pepsic activity, selected Antibody types, generally can select in the scope of 10: 1 ~ 200: 1.
In addition, papain can also be selected.Antibody also can be hydrolyzed to F (ab ') by papain under suitable conditions
2fragment, such as, at document
[6]in in the environment of slant acidity (pH5.5), at 37 DEG C, 18 hours papains can by mouse IgG
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 to be hydrolyzed to fully F (ab ')
2fragment.Both mass ratioes can be selected according to conventional methods, also simultaneously with reference to factors such as the activity of selected papain, selected Antibody types, generally can select in the scope of 10: 1 ~ 200: 1.
When using the mixture of pepsin and papain, because both active environment are acidity, therefore suitably adjusting both mass ratioes according to concrete application, thus required hydrolysing activity and hydrolysis time can be adjusted as required.
In the method for the invention, the sulfhydrylization reagent used in the present invention is unrestricted, if its can with the F of antibody (ab ')
2fragment connects, and can be fixed in the Gold plated Layer of micro-beam.Sulfhydrylization reagent and F (ab ')
2connection step and to be fixed to the order of the step in the Gold plated Layer of micro-beam unrestricted, first can fix after antibody sulfhydrylation with gold surface with sulfhydrylization reagent bind antibody, also first sulfhydrylization reagent can be fixed on a gold surface and connect with antibody again.
The sulfhydrylization reagent used in the present invention comprises mercapto functional group and carboxyl or amido functional group, wherein the sulfydryl of this reagent one end is used for by self assembly fixing on a gold surface (see Fig. 5), and the carboxyl of the other end or amino (also can be exposed by activation) are for reacting (see Fig. 6) with the amino or carboxyl terminal of antibody.The present invention can sulfhydrylization reagent comprise sulfur alcohol compound, such as 11-thiol carboxylic acid, 2-aminoothyl mercaptan (AET) and 3-mercaptopropionic acid (MPA); Hydrochloric acid mercaptan imine; Sulfo group hydrocarbyl succinimide base-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 micro-beam used in the present invention can be prepared voluntarily according to the known method in this area, also can buy commercial goods, be not limited to them in the present invention to this.
Below in conjunction with specific embodiment, the present invention will be further described, but it should be noted that the following examples do not limit the scope of the invention.
Embodiment
Embodiment 1 is sessile antibody F (ab ') in micro-beam Gold plated Layer
2fragment
Test 1 antibody F (ab ')
2the preparation of fragment
1) ginsenoside GS-Re monoclonal antibody (IgG) (purchased from American USBiological company) is dissolved to 10mg/ml with 0.1mol/L citrate buffer solution (pH3.5);
2) pepsin (pepsin) (purchased from American Sigma company) is dissolved to 1mg/ml with 0.1mol/L citrate buffer solution (pH3.5);
3) by IgG:pepsin 100: 1 mixing in mass ratio, to be placed in 37 DEG C of water baths 2 hours, then to add 3mol/LTris and make pH about 7 with cessation reaction.
4) with the PBS buffer solution (containing 0.15MNaCl and 5.0mMEDTA, pH6.5) of 20mM to above-mentioned steps 3) reaction mixture dialyse 48 hours, every 3 hours of period changed a dislysate, obtained antibody F (ab ')
2fragment.
Test 2 micro-beam surface antibody F (ab ')
2fixing of fragment
1) mercaptan self assembly: will clean and put into ELISA Plate aperture with the micro-beam (purchased from American Veeco company) being coated with layer gold that nitrogen dries up, adding 200 μ l concentration be 10mM11-thiol carboxylic acid, sealing room temperature 12 hours;
2) clean: carefully take out micro-beam with tweezers, with the micro-beam of washed with de-ionized water three times;
3) activate: added in new plate hole by cleaned micro-beam, each 75 μ l of NHS (N-hydroxy-succinamide) of the EDC (1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) and 0.05M that add 0.2M activate beam 40 minutes;
4) clean: carefully take out micro-beam with tweezers, with the micro-beam of washed with de-ionized water three times;
5) sessile antibody: put among new plate hole by cleaned micro-beam, adds the anti-ginsenoside GS-Re monoclonal antibody fragment that 150 μ L above-mentioned experiments 1 obtain, 37 DEG C of incubations 1.5 hours, has been fixed antibody F (ab ')
2micro-beam of fragment.
Test 3 direct competive ELISA method evaluation antibody F (ab ')
2fragment and micro-beam gold surface in conjunction with situation
Concrete steps are as follows:
1) clean: carefully take out with tweezers and above-mentionedly secure antibody F (ab ')
2the micro-cantilever of fragment, with cleaning solution (containing 8.0gNaCl, 0.2gKH in every 1L cleaning solution
2pO
4, 2.96gNa
2hPO
412H
2o, 1.0mLTween-20, all the other are water) rinse for several times, nitrogen dries up;
2) close: the micro-cantilever after being dried up by nitrogen puts into ELISA Plate respectively, add 100 μ L mass percentage be 5% BSA solution (dissolving with PBS) close, 37 DEG C of incubations 30 minutes;
3) clean: carefully take out micro-cantilever with tweezers, rinse for several times with cleaning solution, nitrogen dries up;
4) compete: two apertures that the micro-cantilever after being dried up by nitrogen puts into ELISA Plate respectively (carry out mark, Kong Yufei is suppressed to suppress hole), suppress hole add 50 μ L through sample diluting liquid (containing final concentration be the PBS of 0.1mol/LpH7.5, the gelatin of the Tween-20 of 0.1% volumn concentration and 0.1% mass percentage) concentration of diluting is the ginsenoside GS-Re solution (purchased from American Sigma company) of 100mg/L, non-suppression hole adds 50 μ L sample diluting liquids in contrast; And then add the ginsenoside GS-Re peroxide enzyme conjugation thing (purchased from American Sigma company) that concentration is 1.0mg/L respectively, 37 DEG C of incubations 30 minutes;
5) clean: carefully take out the micro-cantilever in above-mentioned suppression Kong Yufei suppression hole with tweezers, rinse for several times with cleaning solution, nitrogen dries up;
6) develop the color: two micro-cantilevers after being dried up by nitrogen put into two apertures of ELISA Plate respectively, every hole add 100 μ L chromophoric solutions (tmb substrate use liquid and substrate buffer solution by 1: 9 volume ratio mix, 4.0mg urea peroxide is added 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, take out micro-cantilever, 450nm wavelength place reads OD value respectively.
Experiment established repeated last time, and result shows, the absorbance under the 450nm wavelength in suppression hole and non-suppression hole is respectively 0.166 and 0.913, and anti-ginsenoside GS-Re antibody F (ab ') is described
2fragment can effectively in conjunction with in the gold surface of micro-cantilever.
Test 4 micro-cantilever sensing device effect monitorings
1) clean: absolute ethyl alcohol, acetone and PBS flow through micro-cantilever sensing system respectively, flow velocity 0.5mL/min;
2) fixing micro-beam: anti-ginsenoside GS-Re antibody F (ab ') will be modified with in experiment 2
2the micro-cantilever of fragment is fixed in the reaction tank of micro-cantilever sensor-based system, and flowing PBS is by reaction pond.With 0.1mL/min flowing PBS after bubble in eliminating reaction tank.
3) light path is regulated: the laser that the laser instrument regulating the position of laser instrument and PSD (photoelectric position sensor) to make sends impinges upon the tip of micro-beam and received by PSD.
4) sample determination: after micro-beam defection signal of PSD reception is stable, add the ginsenoside GS-Re standard sample of 2mLPBS dilution.By PSD real time record micro-beam tip displacement information.
Have detected three concentration 1.0ng/mL, displacement that the sample of 0.1ng/mL, 0.02ng/mL obtains is about 80,60 and 20nm (Fig. 6) respectively, the ginsenoside GS-Re standard sample of variable concentrations can produce micro-cantilever displacement in various degree, and anti-ginsenoside GS-Re antibody F (ab ') is described
2the gold surface that fragment effectively can be attached to micro-cantilever can detect ginsenoside GS-Re.
Conclusion:
For ginsenoside GS-Re antigen-antibody molecule, with antibody F of the present invention (ab ')
2micro-beam that fragment is modified and micro-beam that complete antibody is modified carry out experiment test contrast, when detecting the ginsenoside GS-Re antigen of variable concentrations, time shifting response curve (Fig. 6 and Fig. 5) display at micro-beam tip, when micro-Liang Sicheng response is 20 ran, antibody F (ab ')
2fragment is modified and sulfhydrylation antibody modification detection ginsenoside GS-Re antigen concentration is respectively 0.05ng/mL and 0.5ng/mL, and sensitivity improves nearly 10 times (antibody is fixed to the result that on beam, (Chinese patent CN101407548) detects ginsenoside by Fig. 5 after utilizing hydrochloric acid mercaptan imine to carry out sulfhydrylation antibody as sulfhydrylization reagent).
By above-mentioned nonrestrictive embodiment, can find out, method of the present invention and the micro-cantilever prepared by method of the present invention can improve 20-40 doubly in immune sensing detection technique compared with micro-beam method of prior art, be equivalent to the 200-400 of conventional ELISA doubly, achieve object of the present invention, namely in the fields such as food security, environmental pollution, biomedicine, scientific research and the manufacturing, monitor and detect possibility and the practicality of denier analyte, actual commercial applications can be realized completely.
Bibliography
[1]KoutilyaR.Buchapudi，XinHuang，XinYang，HaiFengJiandThomasThundat，Microcantileverbiosensorsforchemicalsandbioorganisms.Analyst，2011，136，1539-1556
[2]WeimingTan，YuanHuang，TieguiNan，ChangguoXue，ZhaohuLi，QingchuanZhang，andBaominWang，DevelopmentofProteinAFunctionalizedMicrocantileverImmunosensorsfortheAnalysesofSmallMoleculesatpartpertrillionLevels.AnalysisChemistry，2010，82(2)，615-620
[3]HongweiZhao；ChangguoXue；TieguiNan；GuiyuTan；ZhaohuLi；QingX.Li；QingchuanZhang；BaominWang，DetectionofCopperIonsUsingMicrocantileverImmunosensorsandEnzyme-linkedImmunosorbentAssay，AnalyticaChimicaActa，2010，676，81-86，
[4]ChangguoXue，HongweiZhao，YanyunChen，BaominWang，QingchuanZhang，XiaopingWu，Developmentofsulfhydrylatedantibodyfunctionalizedmicrocantileverimmunosensorfortaxol，SensorsandActuatorsB，2011，156，863-866
[5]A.Kausaite-Minkstimiene，A.Ramanaviciene，J.Kirlyte，andA.Ramanavicius.ComparativeStudyofRandomandOrientedAntibodyImmobilizationTechniquesontheBindingCapacityofImmunosensor.AnalysisChemistry，2010，82，6401-6408
[6] [J] Chou Kai etc., papain prepares 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 (7)
1. prepare a method for micro-cantilever, comprise the following steps:
1) antibody and the micro-cantilever with Gold plated Layer are provided;
2) enzymolysis step: described antibody is cracked into F (ab ') with protease
2fragment and multiple small molecule segment, described protease is selected from pepsin, papain or its combination;
3) fixing step: the sulfhydrylization reagent comprising mercapto functional group and carboxyl or amido functional group is bonded to the Gold plated Layer of micro-cantilever on the surface;
4) step is connect: by step 2) F that obtains (ab ')
2with step 3) the described sulfhydrylization reagent be combined on micro-cantilever that obtains is bound up, and obtain Gold plated Layer and be fixed with F (ab ') on the surface
2micro-cantilever.
2. prepare a method for micro-cantilever, comprise the following steps:
1) antibody and the micro-cantilever with Gold plated Layer are provided;
2) enzymolysis step: described antibody is cracked into F (ab ') with protease
2fragment and multiple small molecule segment, described protease is selected from pepsin, papain or its combination;
3) step is connect: by step 2) F that obtains (ab ')
2be bound up with sulfhydrylization reagent;
4) fixing step: by step 3) one end of obtaining and F (ab ')
2the other end of described sulfhydrylization reagent of connection is bonded to the Gold plated Layer of micro-cantilever on the surface, obtains Gold plated Layer and is fixed with F (ab ') on the surface
2micro-cantilever.
3. use micro-cantilever sensing and detecting system immune sensing to detect a method for testing sample, comprise the following steps:
1) provide the special antibody of target antigen and micro-cantilever sensing and detecting system, described micro-cantilever sensing and detecting system comprises reaction tank and the micro-cantilever based on stress with Gold plated Layer;
2) described antibody is cracked into F (ab ') with protease
2fragment and multiple small molecule segment, described protease is selected from pepsin, papain or its combination;
3) fixing step: the sulfhydrylization reagent comprising mercapto functional group and carboxyl or amido functional group is bonded to the Gold plated Layer of micro-cantilever on the surface;
4) step is connect: by step 2) F that obtains (ab ')
2fragment and step 3) the described sulfhydrylization reagent be combined on micro-cantilever that obtains is bound up, and obtain Gold plated Layer and be fixed with F (ab ') on the surface
2the micro-cantilever of fragment;
5) by step 4) obtain be fixed with F (ab ')
2the micro-cantilever of fragment 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 testing sample whether there is described target antigen;
Described stress changes, thus cause micro-beam deformed, detected the process of this distortion by optics or electrical method, the real time information of immune biochemical reaction can be obtained.
4. use micro-cantilever sensing and detecting system immune sensing to detect a method for testing sample, comprise the following steps:
1) provide the special antibody of target antigen and micro-cantilever sensing and detecting system, described micro-cantilever sensing and detecting system comprises reaction tank and the micro-cantilever based on stress with Gold plated Layer;
2) described antibody is cracked into F (ab ') with protease
2fragment and multiple small molecule segment, described protease is selected from pepsin, papain or its combination;
3) step is connect: by step 2) F that obtains (ab ')
2fragment and the sulfhydrylization reagent comprising mercapto functional group and carboxyl or amido functional group are bound up;
4) fixing step: by step 3) one end of obtaining and F (ab ')
2the other end of described sulfhydrylization reagent of fragment connection is bonded to the Gold plated Layer of micro-cantilever on the surface, obtains Gold plated Layer and is fixed with F (ab ') on the surface
2the micro-cantilever of fragment;
5) by step 4) obtain be fixed with F (ab ')
2the micro-cantilever of fragment 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 testing sample whether there is described target antigen;
Described stress changes, thus cause micro-beam deformed, detected the process of this distortion by optics or electrical method, the real time information of immune biochemical reaction can be obtained.
5. the method according to any one of claim 1-4, is characterized in that described sulfhydrylization reagent is selected from sulfur alcohol compound; Hydrochloric acid mercaptan imine; Sulfo group hydrocarbyl succinimide base-6-(3 ' 2-pyridine two sulphur-propionamide)-acetic acid esters; One or more in the two sulfosuccinimide propionic ester of 3,3 '-two sulphur.
6. according to the method described in claim 5, wherein, described sulfur alcohol compound is selected from 11-carboxy thiol, 2-aminoothyl mercaptan and 3-mercaptopropionic acid.
7., according to the method described in claim 5, it is characterized in that described antibody is any one in IgM, IgG, IgA, IgD, IgE.
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