CN101676727A - Method for detecting recin - Google Patents
Method for detecting recin Download PDFInfo
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- CN101676727A CN101676727A CN200810222339A CN200810222339A CN101676727A CN 101676727 A CN101676727 A CN 101676727A CN 200810222339 A CN200810222339 A CN 200810222339A CN 200810222339 A CN200810222339 A CN 200810222339A CN 101676727 A CN101676727 A CN 101676727A
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- monoclonal antibody
- collaurum
- magnetic nanoparticle
- ricin
- antigen
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Abstract
The invention provides a new method for detecting recin, comprising the following steps: capturing recin in the solution using magnetic nano particle coupled with anti-recin monoclonal antibody (6A6),adding colloidal gold nano particle coupled with anti-recin monoclonal antibody (7G7), using colloidal gold to catalyze the oxidation reduction reaction of silver nitrate to hydroquinone, in order tomake silver particle deposit on the surface of the colloidal gold. The magnetic nano-particle-antigen-colloidal gold-silver particle composite is transferred to a combined micro-interdigital electrode and dried and then the capacitance variance between the electrodes due to the composite is measured. The probe molecule can be replaced, thus the new method is widely used for detecting various toxin or virus. Compared with the traditional double-antibodies sandwich ELISA, the capacitance detection system based on two nano particles has advantages of high sensitivity and quick detection speed.
Description
Technical field
The present invention relates to a kind of new method that detects ricin (WA), specifically, the present invention has set up a kind of capacitive detection system based on magnetic nanoparticle and collaurum nano particle, is used for the detection of ricin (WA).By changing antibody molecule, this new detection method can be applied to the detection of multiple toxin or virus.
Background technology
Ricin (WA) (Ricin) is a kind of plant glycoprotein that contains in the castor bean, molecular weight 60-65kDa.Toxin powder colorless and odorless, dry product are at room temperature stable, heat up also not decompose, even xeothermic also very little to toxic effect during to 100 ℃, with the stability decreases that increases of moisture, boil in the water and can make its inactivation.Ricin (WA) is made up of A and two polypeptied chains of B, and two interchains are connected by a disulfide bond.Contain two galactoses on the toxin B chain, can with cell surface contain the combination of galactose binding site, enter tenuigenin by the effect of caving in, the performance toxic action.Ricin (WA) belongs to protein synthesis inhibitor or ribosomes deactivator.
Ricin (WA) is with its extremely strong toxicity and fatal rate and be subjected to the generally attention of various countries.In the Chemical Weapons Convention that came into force in 1997, ricin (WA) has been listed in the one-level control inventory of pact.U.S. counter terrorism expert organizes 18 experts and once united the issue white paper, thinks that ricin (WA) most possibly becomes the chemical and biological weapons that future war or terrorist at first use.According to this white paper, one of toxic agent of most probable use is listed ricin (WA) in calendar year 2001 U.S.'s anti-terrorism planning.The research that detects ricin (WA) has at present become the whole world and has defendd following biological war or terroristic Critical policies.Therefore, set up the toxin detection system, the tachnical storage of reinforcement detection ricin (WA) is not only very necessary, and presses for.
Capacitive transducer is a kind of sensing technology that is based upon on the capacity plate antenna model basis, by measuring some characteristics that changes in capacitance reflects system.
The capacitance of plate condenser can by under be to calculate:
Wherein ε is the specific inductive capacity of medium between pole plate, ε=ε
0ε
r
ε
0Be the specific inductive capacity of air, ε
rIt is dielectric specific inductive capacity
S be two-plate over against area
D is the distance of two-plate
K is a constant coefficient
By formula as can be seen, capacitance and DIELECTRIC CONSTANT, pole plate are relevant with polar plate spacing d over against area S, and electric capacity all can change thereupon when in these three parameters any one changes.Therefore according to changing the capacitor parameters difference, capacitive transducer can be divided three classes: variable spacing formula, variable area type and variable dielectric formula, can select different sensor types according to different physical quantity needs.For example the variable spacing formula is used for Displacement Measurement, acceleration etc. more; The variable area type variation that is used for taking measurement of an angle; The variable dielectric formula is measured and is used for measuring liquid level, substance classes or the like more.
Summary of the invention
Monoclonal antibody 7G7,8C2 and the 6A6 of antiricin have been used in the present invention.Hybridoma 7G7, the 8C2 of secrete monoclonal antibody 7G7,8C2 and 6A6 and 6A6 are purchased from Sheng source, Beijing maple development in science and technology company limited respectively.
The preparation process of hybridoma following (referring to Kohler and Milstein, Nature 256:495,1975; Yeh et al, Proc.Natl.Acad.Sci.USA, 1979; Yeh et al., Int.J.Cancer, 1982): as immunogene BALB/C mice is carried out immunity inoculation through the ricin (WA) holotoxin of formalin-inactivated with natural purifying, each hypodermic injection 20 μ g albumen/every mouse, every fortnight once, totally three times.Before the extracting spleen cell booster immunization once, every mouse of hypodermic injection 20 μ g albumen.Booster immunization three days is afterwards got spleen, and splenocyte is suspended in the RPMI nutrient culture media.In the presence of polyglycol (PEG), splenocyte and SP2/0-Ag14 rat bone marrow tumour cell are merged, and with HAT selective medium (nutrient culture media that contains hypoxanthine (hypoxantin), aminopterin (aminopterin) and thymidine (thymidin)) hybridoma is screened, obtain hybridoma.Method with ELISA is screened the antibody that have strong binding ability with natural ricin, obtains three strain antibodies, difference called after 7G7,8C2 and 6A6, and the hybridoma of these antibody of acquisition secretion simultaneously is 7G7,8C2 and 6A6 successively.
The inventor has identified that this three strain antibody all belongs to the IgG1 hypotype.ELISA has confirmed the specificity combination of these antibody and ricin (WA).
Utilize the method for Western blotting (Western blotting), confirm the A chain of 6A6 identification ricin (WA), the B chain of 7G7 and 8C2 identification ricin (WA).
The present invention utilizes the antibody of identification A chain simultaneously, as 6A6; With the antibody of identification B chain, as 7G7, developed the method for the sandwich ELISA of a cover, be used to detect ricin (WA).
Utilize 6A6 and 7G7 two strain antibodies, set up a kind of capacitive detection system based on two kinds of nano particles, this system utilizes coupling to have the magnetic nanoparticle of antiricin monoclonal antibody (6A6) to catch ricin (WA) in the solution, add coupling again the collaurum nano particle of antiricin monoclonal antibody (7G7) is arranged, utilize the redox reaction of collaurum catalysis silver nitrate and p-dihydroxy-benzene, make silver-colored particle deposition on the collaurum surface.Magnetic nanoparticle-antigen-collaurum-Yin particle composites is transferred on the little interdigital electrode of combination, and the variation that this compound causes the interelectrode capacitance value is measured in oven dry.
The present invention realizes ricin (WA) is detected by changing interelectrode dielectric.When having ricin (WA) in the solution, can form magnetic nanoparticle-ricin (WA)-collaurum-Yin particle composites.Because silver-colored particle has good electrical conductivity, specific inductive capacity is much larger than air, thereby when having silver-colored particle to exist between pole plate, can cause the remarkable increase of capacitance.
In one embodiment of the invention, described magnetic nanoparticle is silicon dioxide embedded formula ferroferric oxide magnetic nanoparticle, and diameter is 1 μ m.
In another embodiment of the invention, the monoclonal antibody of described coupling magnetic nanoparticle is 6A6.
In another embodiment of the invention, described collaurum nano particle diameter is 10nm.
In another embodiment of the invention, the monoclonal antibody of described coupling colloid gold particle is 7G7.
In another embodiment of the invention, the little interdigital electrode of described combination is to utilize the electronic circuit processing technology to process, and thickness of electrode is 0.1 μ m.Substrate is an epoxide resin material, and thickness is 1.48mm.
In another embodiment of the invention, the little interdigital electrode of described combination is characterized in that: wherein adjacent interdigital spacing is 100 μ m, and finger widths is 0.3mm, and the length of cross section is 4mm.
In another embodiment of the invention, by changing antibody molecule, described method can be used for the detection of multiple toxin or virus.
More specifically, the present invention relates to the following:
1. sandwich ELISA method that utilizes magnetic Nano material and collaurum nano material to detect ricin (WA) comprises:
Coupling there is a kind of magnetic nanoparticle of antiricin monoclonal antibody mix, to catch ricin (WA) with testing sample;
The adding coupling has the collaurum nano particle of another kind of antiricin monoclonal antibody;
Add the reaction of silver nitrate and p-dihydroxy-benzene, form magnetic nanoparticle-antigen-collaurum-Yin particle composites;
Above-mentioned magnetic nanoparticle-antigen-collaurum-Yin particle composites is transferred in the capacitive detection system that comprises electrode the change of capacitance between detecting electrode.
2. according to above 1 method, wherein said magnetic nanoparticle is silicon dioxide embedded formula ferroferric oxide magnetic nanoparticle, and this material is to be nuclear with the ferroferric oxide magnetic nanoparticle, and the outside has coated the layer of silicon dioxide material and made.
3. according to above 1 method, wherein said collaurum nano particle is the synthetic gold nano grain of gold chloride-sodium citrate method.
4. according to above 1 or 2 method, the monoclonal antibody of wherein said magnetic nanoparticle coupling is 6A6.
5. according to above 1 or 3 method, the monoclonal antibody of wherein said collaurum nano particle coupling is 7G7.
6. according to above 1 method, wherein said electrode is the little interdigital electrode of combination, and this electrode is to be formed by stacking side by side by traditional plate electrode.
7. according to above 1 method, this method is carried out in solution, the monoclonal antibody of the monoclonal antibody of preferred described magnetic nanoparticle coupling and the coupling of described collaurum nano particle is the IgG of identical or different hypotype, the same chain or the different chain of identification ricin (WA), more preferably mouse resource monoclonal antibody.
8. sandwich ELISA method that utilizes antigen in magnetic nanoparticle and the collaurum nano particle test sample comprises:
There is a kind of magnetic nanoparticle of the monoclonal antibody at described antigen to mix coupling, to catch ricin (WA) with testing sample;
Add coupling the collaurum nano particle of another kind at the monoclonal antibody of described antigen arranged;
Add the reaction of silver nitrate and p-dihydroxy-benzene, form magnetic nanoparticle-antigen-collaurum-Yin particle composites;
Above-mentioned magnetic nanoparticle-antigen-collaurum-Yin particle composites is transferred in the capacitive detection system that comprises electrode the change of capacitance between detecting electrode.
9. according to above 8 method, wherein said antigen is virus or toxin, and preferred described magnetic nanoparticle is that silicon dioxide embedded formula ferroferric oxide magnetic nanoparticle and described collaurum nano particle are the synthetic gold nano grain of gold chloride-sodium citrate method.
10. sandwich ELISA method that detects ricin (WA) is characterized in that:
Monoclonal antibody 6A6 is used as capture antibody; With
Monoclonal antibody 7G7 is used as detection antibody.
Should be pointed out that above-mentioned embodiment and feature can combination in any.
Characteristics of the present invention are: (1) utilizes the monoclonal antibody of one group of antiricin, has set up the detection that double antibodies sandwich ELISA method is used for ricin (WA); (2) set up a kind of capacitive detection system based on two kinds of nano particles, this system sensitivity height, detection speed is fast.
The accompanying drawing summary
Fig. 1. the affinity of antiricin monoclonal antibody 7G7,8C2 and 6A6 (8B11,9H7 and 4A5 are purchased the corresponding antibodies of containing other antiricin monoclonal antibody hybridoma cell secretion of source maple development in science and technology company limited from Beijing among the figure);
Fig. 2. the purifying of antiricin monoclonal antibody 7G7,8C2 and 6A6, (A) ascites (B) is passed, (C) antibody purified;
Fig. 3. antiricin monoclonal antibody subgroup identification;
Fig. 4. the antiricin monoclonal antibody is active to be detected;
Fig. 5. antiricin monoclonal antibody epitope is identified;
Fig. 6. antiricin monoclonal antibody various combination detection sensitivity is relatively;
Fig. 7. double antibodies sandwich ELISA detects ricin (WA);
Fig. 8. double antibodies sandwich ELISA detects the ricin (WA) typical curve;
Fig. 9. transmission electron microscope (TEM) observing colloid gold nano grain (A) and magnetic nanoparticle (B) and the design (C) of making up little interdigital electrode;
Figure 10. based on the synoptic diagram of the ricin (WA) capacitive detection system of two kinds of nano particles;
Figure 11. the specificity of ricin (WA) capacitive detection system;
Figure 12. the sensitivity of ricin (WA) capacitive detection system.
Embodiment
Below further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
The activity identification of embodiment 1 monoclonal antibody 7G7,8C2 and 6A6
Antiricin monoclonal antibody hybridoma cell 7G7,8C2,6A6,4A5,9H7 and 8B11 contain source maple development in science and technology company limited available from Beijing.Utilize the method for ELISA to measure monoclonal antibody 7G7,8C2,6A6,4A5,9H7 and 8B11 affinity to ricin (WA).Concrete grammar is as follows: use ricin (WA) (being purchased from Sigma-Aldrich), as envelope antigen, detect the culture supernatant of hybridoma 7G7,8C2,6A6,4A5,9H7 and 8B11.Concrete way is, the bag that at first spends the night on elisa plate is by the 5 μ g/ml ricin (WA) albumen of 50 μ l, gives a baby a bath on the third day after its birth time with PBS.Add 3%BSA sealing 1h.The hybridoma culture supernatant that adds secretory antibody is then hatched 1h, adds the anti-mouse antibodies of HRP labelled goat and hatches 1h, adds substrate (200ng/ml tetramethyl biphenyl diamines (TMB), 0.03%H at last
2O
2, pH4.5) colour developing, 50 μ l/ holes, 37 ℃ of reaction 15min add 50 μ l/ hole 2M H
2SO
4Cessation reaction, microplate reader 450nm reading (as Fig. 1).Can see that from the result of ELISA the affinity of 7G7,8C2 and 6A6 is higher.
A large amount of amplified hybridization oncocyte 7G7,8C2 and 6A6 of cultivating make cell suspension respectively, are used to prepare antibody ascites.The preparation method of ascites is summarized as follows: six week BALB/C mice in age (Beijing Vital River Experimental Animals Technology Co., Ltd.) lumbar injection norphytane (Sigma-Aldrich) 0.5ml/ only.After 10 days, with the hybridoma suspension inoculation in BALB/C mice abdominal cavity, 1 * 10
7/ ml/ only after about ten days, collects ascites, the centrifuging and taking supernatant.
By albumin A affinity chromatography (Roche), monoclonal antibody purification from ascites (as Fig. 2).With monoclonal antibody purification aseptic filtration, and refrigeration or freezing preservation.
Utilize murine antibody hypotype identification kit (BD Pharmingen), identify antibody 7G7,8C2 and 6A6 and all belong to the IgG1 hypotype, as shown in Figure 3.
The ELISA method is used for 7G7, the 8C2 of purification Identification and three kinds of antibody of 6A6 binding specificity for ricin (WA) albumen.The ricin (WA) of natural purifying is as envelope antigen.ELISA result as shown in Figure 4,6A6,7G7 and 8C2 all can well discern ricin (WA).
The epitope of embodiment 2 monoclonal antibody 7G7,8C2 and 6A6 is identified.
Utilize the method for Western blotting, identified the epitope of three strain antibodies described in the present invention.Concrete grammar is that the disulfide bond that will connect ricin A, B chain with 0.1M reducing agent dithiothreitol (DTT) is opened, and utilizes Western blotting to identify the epitope of antibody recognition.Wherein 6A6 can discern ricin A chain, and 7G7 and 8C2 can discern ricin (WA) B chain (as Fig. 5).
Embodiment 3 double antibodies sandwich ELISA detect the foundation of ricin (WA) system
The present invention has set up a kind of double antibodies sandwich ELISA method, detects ricin (WA).At first screening detects the pairing antibody of ricin (WA), and concrete grammar is as follows: at first with 7G7,8C2 and 6A6 biotinylation (Pierce), three strain antibodies are matched respectively (as table 1), relatively more different pairings detect the sensitivity of ricin (WA), as Fig. 6.Confirmed to use 6A6 as capture antibody, the 7G7 (7G7-bio, bio represents biotin) of biotin (Biotin) mark is as detecting antibody, and it is the highest to detect sensitive poison.
Table 1
At definite capture antibody with after detecting antibody, as standard items, determine the range of linearity of detection system with ricin (WA) (being purchased) from Sigma-Aldrich.Experiment confirm, the sandwich ELISA of the present invention's exploitation has the higher sensitivity and the range of linearity of broad.
Concrete experimental technique is as follows:
1) elisa plate coated antibody: antibody 6A6 is diluted to 2 μ g/ml with 0.02M PBS (pH 7.25), and the 4 ℃ of bags in 50 μ l/ holes are spent the night.
2) the non-specific bond of sealing site: 200 μ l/ hole 2%BSA/PBS, incubated at room 2h.
3) sample is hatched: the ricin (WA) gradient of concentration known is used for the drawing standard curve with the confining liquid dilution, and the concentration after the dilution is respectively 0,4,8.1,16.12,31.25,62.5,125,250,500ng/ml.Each sample is established twice repetition.Incubated at room 2h.
4) PBST is 5 times, 1 flush away non-specific binding of PBS.
5) detect antibody incubation: detect antibody 7G7-bio and be diluted to 1 μ g/ml with 2%BSA/PB, 50 μ l/ pore chamber temperature are hatched 2h.
6) PBST is 5 times, 1 flush away non-specific binding of PBS.
7) ELIAS secondary antibody detection signal: add the Streptavidin-HRP (Sigma-Aldrich) of suitable concn, 50 μ l/ holes, incubated at room 1h.
8) PBST is 5 times, 1 flush away non-specific binding of PBS.
9) color reaction: add substrate (200ng/ml tetramethyl biphenyl diamines (TMB), 0.03%H
2O
2, pH4.5) colour developing, 100 μ l/ holes, 37 ℃ of 15min add 50 μ l/ hole 2M H
2SO
4Cessation reaction, microplate reader 450nm reading.
10) software statistics result: the software analysis light absorption value, according to standard ricin (WA) concentration drawing standard curve.
Interpretation of result:
Utilize the concentration production standard curve of known ricin (WA).The concentration of dilution back ricin (WA) is 0～500ng/ml, drawing standard curve such as Fig. 7.Draw this method sensing range as calculated and in 0～62.5ng/ml, meet linearity.Get ricin (WA) concentration 0～62.5ng/ml resulting typical curve (R that maps
2=0.99), as shown in Figure 8.The sensitivity of this detection system is 5ng/ml (50pM).
The making of the synthetic and little interdigital electrode of combination of embodiment 4 collaurums and silicon dioxide embedded formula ferroferric oxide magnetic nanoparticle
By gold chloride-sodium citrate reducing process (Oliver C, Methods in molecular biology, 34:299-302,1994) preparation collaurum nano particle.With 1ml, 1% gold chloride (HAuCl
4) (Sigma-Aldrich) solution add in the 100ml distilled water, be heated to boiling, add the sodium citrate solution of 5ml1% rapidly, under agitation constant temperature refluxes and makes red colloid gold particle.The size of observing colloid gold grain uniformity whether under the transmission electron microscope.Measuring the colloid gold particle diameter is 10nm, and yardstick homogeneous, dispersiveness and stability is (as Fig. 9 A) better.
26mg ferroferric oxide magnetic nanoparticle (happy chromatographic technique development centre is doubly thought in Tianjin) is mixed with 40ml ethanol, add 0.5ml deionized water and 1.5ml 25% ammoniacal liquor.Constantly stir, add ethyl orthosilicate (TEOS) and (Sigma-Aldrich) react 8hr, take out, with ethanol washing three times, 60 ℃ of dryings.The diameter of transmission electron microscope observing magnetic nanoparticle is 1 μ m (as Fig. 9 B).
Make up the sensitivity of little interdigital electrode and compare traditional capacitor raising several times (Varshney M﹠amp; Li Y, Biosens.Bioelectron, 22:2408-2414,2007), be more suitable for measuring small variable.The electrode design that makes up little interdigital electrode simultaneously is convenient, and size is controlled easily, can adjust the size of electrode in actual applications according to the requirement of applied environment, and interdigital finger length, finger beam, refers to spacing and interdigital number.Fig. 9 C shows the size of interdigital electrode, and wherein adjacent interdigital spacing is 100 μ m, and finger widths is 0.3mm, and the length of cross section is 4mm.
Electrode adopts the electronic circuit processing technology to process (CAS Electrical Engineering Research Institute), and thickness of electrode is 0.1 μ m.Substrate is an epoxide resin material, and thickness is 1.48mm.This material was not only economical convenient but also can antiacid caustic corrosion, was suitable for the detection of biomolecule.
In order to set up capacitive detection system, we are at first with collaurum nano particle and antiricin monoclonal antibody 7G7 coupling, as detector probe.Concrete grammar is as follows: get the colloid gold particle of preparation among the 1ml embodiment 4, use 25mM K
2CO
3Transfer pH to 8.5, add 10 μ l, the antiricin monoclonal antibody 7G7 of 1mg/ml, mixing, room temperature leaves standstill 10min.Add 100 μ l10%BSA, mixing, room temperature leaves standstill 10min, and the centrifugal 20min of 10000rpm absorbs supernatant.With the resuspended collaurum precipitation of 50 μ l PBS, 4 ℃ of preservations.
We are with the silicon dioxide embedded formula ferroferric oxide nano granules and the antiricin monoclonal antibody 6A6 coupling of preparation among the embodiment 4, as capture probe subsequently.Concrete grammar is as follows: the silicon dioxide embedded formula ferroferric oxide magnetic nanoparticle of getting 1ml10mg/ml, the NHS (N-hydroxy-succinamide) that adds 50mg/ml, each 50 μ l of EDC (carbodiimide), incubated at room 30min, use washed with de-ionized water, remove unnecessary NHS/EDC.The sodium acetate that adds 1ml pH 6.0, the antiricin monoclonal antibody 6A6 of 100 μ g, mixing is hatched 2hr for 4 ℃, and the PBS washing adds the carboxyl that 50mM pH 7.4Tris-HCl sealing activates, and PBS is resuspended, 4 ℃ of preservations.
After preparing detector probe and capture probe, utilize coupling to have the magnetic nanoparticle (MNPs-6A6) of antiricin monoclonal antibody 6A6 to catch ricin (WA) in the solution, add coupling again the collaurum nano particle (GNPs-7G7) of antiricin monoclonal antibody 7G7 is arranged, incubated at room 30min (is with NaNO with the phosphate buffer difference with PBN
3Substitute the NaCl in the phosphate buffer) washing, remove the collaurum of non-special absorption, add silver nitrate/p-dihydroxy-benzene mixed liquor (Sigma-Aldrich), lucifuge reaction 5min, magnetic nanoparticle-antigen-collaurum-Yin particle composites is transferred on the little interdigital electrode of combination oven dry (as Figure 10).
The present invention utilizes electrochemical workstation (Zahner) to measure the variation that this compound causes the interelectrode capacitance value.The experimental group capacitance that contains ricin (WA) is 25pF, and the control group capacitance that does not contain ricin (WA) is 3pF (as Figure 11).
In order to identify the specificity of this detection system, we with normal mouse IgG (mIgG) in contrast, prepare the magnetic nanoparticle (MNPs-mIgG) that coupling has mouse IgG as mentioned above, be used for then being used to detect ricin (WA) with above identical detection architecture.No matter can observe in the solution whether ricin (WA) is arranged, capacitance is 3pF (as Figure 11).
In order to identify the sensitivity of this detection system, with ricin (WA) gradient dilution (10
-9～10
-13M), the concentration of ricin (WA) is greater than 10
-11M all can detect (as Figure 12).
Compare with traditional sandwich ELISA, the sensitivity of capacitive detection system is higher, and is consuming time shorter, as Table 2.
The comparison of table 2 capacitive detection system and traditional sandwich ELISA
Claims (10)
1. double antibodies sandwich ELISA method of utilizing magnetic Nano material and collaurum nano material to detect ricin (WA) comprises:
Coupling there is a kind of magnetic nanoparticle of antiricin monoclonal antibody mix, to catch ricin (WA) with testing sample;
The adding coupling has the collaurum nano particle of another kind of antiricin monoclonal antibody;
Add the reaction of silver nitrate and p-dihydroxy-benzene, form magnetic nanoparticle-antigen-collaurum-Yin particle composites;
Above-mentioned magnetic nanoparticle-antigen-collaurum-Yin particle composites is transferred in the capacitive detection system that comprises electrode the change of capacitance between detecting electrode.
2. according to the process of claim 1 wherein that described magnetic nanoparticle is silicon dioxide embedded formula ferroferric oxide magnetic nanoparticle.
3. according to the process of claim 1 wherein that described collaurum nano particle is the synthetic gold nano grain of gold chloride-sodium citrate method.
4. according to the method for claim 1 or 2, the monoclonal antibody of wherein said magnetic nanoparticle coupling is 6A6.
5. according to the method for claim 1 or 3, the monoclonal antibody of wherein said collaurum nano particle coupling is 7G7.
6. according to the process of claim 1 wherein that described electrode is the little interdigital electrode of combination.
7. according to the method for claim 1, this method is carried out in solution, the monoclonal antibody of the monoclonal antibody of preferred described magnetic nanoparticle coupling and the coupling of described collaurum nano particle is the IgG of identical or different hypotype, the same chain or the different chain of identification ricin (WA), more preferably mouse resource monoclonal antibody.
8. double antibodies sandwich ELISA method of utilizing magnetic Nano material and collaurum nano material to detect antigen comprises:
There is a kind of magnetic nanoparticle of the monoclonal antibody at described antigen to mix coupling, to catch ricin (WA) with testing sample;
Add coupling the collaurum nano particle of another kind at the monoclonal antibody of described antigen arranged;
Add the reaction of silver nitrate and p-dihydroxy-benzene, form magnetic nanoparticle-antigen-collaurum-Yin particle composites;
Above-mentioned magnetic nanoparticle-antigen-collaurum-Yin particle composites is transferred in the capacitive detection system that comprises electrode the change of capacitance between detecting electrode.
9. method according to Claim 8, wherein said antigen is virus or toxin, and preferred described magnetic nanoparticle is that silicon dioxide embedded formula ferroferric oxide magnetic nanoparticle and described collaurum nano particle are the synthetic gold nano grain of gold chloride-sodium citrate method.
10. double antibodies sandwich ELISA method that detects ricin (WA) is characterized in that:
Monoclonal antibody 6A6 is used as capture antibody; With
Monoclonal antibody 7G7 is used as detection antibody.
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