CN101776691A - Method for immunoaffinity solid-phase extraction based on gold-coated ferroferric oxide nano-particles - Google Patents
Method for immunoaffinity solid-phase extraction based on gold-coated ferroferric oxide nano-particles Download PDFInfo
- Publication number
- CN101776691A CN101776691A CN201010110743A CN201010110743A CN101776691A CN 101776691 A CN101776691 A CN 101776691A CN 201010110743 A CN201010110743 A CN 201010110743A CN 201010110743 A CN201010110743 A CN 201010110743A CN 101776691 A CN101776691 A CN 101776691A
- Authority
- CN
- China
- Prior art keywords
- antibody
- gold
- epitestosterone
- ferroferric oxide
- conjugate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for the immunoaffinity solid-phase extraction based on gold-coated ferroferric oxide nano-particles. The method comprises the following steps: 1) mixing a coupling product with a sample to conduct conjugation reaction, so as to obtain a solution containing the conjugate of the coupling product and antigen materials which can be conjugated with each other; 2) separating the conjugate from the solution containing the conjugate, so as to obtain the conjugate; and 3) eluting the antigen materials from the conjugate, so as to obtain the antigen materials. The invention has wide application prospect, i.e., the detection range of the invention can be extended to blood samples according to the requirements, moreover, as long as the antibody of the target to be detected is obtained, the method of the invention is capable of coupling antibody fragments to the surface of gold-coated ferroferric oxide nano-particles, so as to prepare the immunoaffinity solid-phase extraction material and achieve the purification, enrichment and detection of the target compound to be detected; and furthermore, the invention is capable of converting the off-line detection method into the on-line detection method according to the requirements, thereby further improving the automation level.
Description
Technical field
The present invention relates to a kind of immune affinity solid phase extracting process based on the gold-coated ferroferric oxide nano particle.
Background technology
Testosterone is a kind of important anabolism male sex hormone steroid medicine, belongs to a kind of excitant.After taking testosterone, athletic muscle oxygen carrying capacity can be improved, exercise tolerance and achievement can be improved.Therefore, testosterone is prohibited by the International Olympic Committee and international anti-excitant tissue.Epitestosterone is the enantiomter of testosterone, does not have direct physiological action though take epitestosterone, still can pass through to change the ratio of " testosterone concentration/epitestosterone content " in the body, thereby shelter taking of testosterone; Therefore, thereby also need to detect the value that the amount of testosterone and epitestosterone in sportsman's body obtains " testosterone/epitestosterone " content, and synthesis result is judged.In addition, set up be suitable for the epitestosterone drug abuse easily and fast, high specificity and highly sensitive method have important meaning for the metabolic process of this medicine of research in human body.
In actual detected, the impurity of testing sample is more, has influenced the accuracy of testing result, need carry out pre-treatment to sample, but existing pre-treatment step is all more numerous and diverse, takes time and effort.
Summary of the invention
An object of the present invention is to provide a kind of from sample the method for immune affinity extraction antigenic substance.
Provided by the present invention from sample the method for immune affinity extraction antigenic substance, comprise the steps:
1) with the conjugate and the sample mix of gold-coated ferroferric oxide nano particle and antibody, carries out association reaction, obtain containing the solution of the bond of described conjugate and antigenic substance; Described antibody and described antigenic substance can mutually combine;
2) with magnet described bond is separated from the solution that contains described bond, obtained described bond;
3) described antigenic substance is eluted from described bond, obtain described antigenic substance.
In the said process, the temperature of described association reaction is 20 ℃-37 ℃, specifically can be 25 ℃, and the time of described association reaction is 0.5 hour-1.5 hours, specifically can be 1 hour.
In the said process, the eluent that uses in the described wash-out is a methyl alcohol; Described antigenic substance is an epitestosterone; Described sample is urine or stripped blood.
Another object of the present invention provides the method for antigenic substance in a kind of test sample.
The method of antigenic substance comprises the steps: in the test sample provided by the present invention
1) according to above-mentioned from sample method immune affinity extraction from testing sample of immune affinity extraction antigenic substance obtain antigenic substance;
2) antigenic substance that step 1) is obtained carries out detection by quantitative with high performance liquid chromatography.
Another object of the present invention provides the conjugate of a kind of gold-coated ferroferric oxide nano particle and antibody.
The conjugate of gold-coated ferroferric oxide nano particle provided by the present invention and antibody is the conjugate that antibody fragment and the coupling of gold-coated ferroferric oxide nano particle form; Described coupling is to realize by the golden self assembly in free sulfhydryl groups in the antibody fragment and the gold-coated ferroferric oxide nano particle;
Described antibody fragment is that the disulfide bond between the heavy chain of antibody is cut and is reduced to the antibody fragment that obtains behind the free sulfhydryl groups; Described antibody is made up of heavy chain and light chain.
Above-mentioned gold-coated ferroferric oxide nano particle prepares according to the method that comprises the steps with the conjugate of antibody: the gold-coated ferroferric oxide nano particle is mixed with the solution that contains the capacity antibody fragment, react, obtain the conjugate of gold-coated ferroferric oxide nano particle and antibody fragment;
The temperature of described reaction is 2 ℃-6 ℃, is preferably 4 ℃; The time of described reaction is 10 hours-20 hours, is preferably 15 hours;
Described antibody fragment is prepared as follows: with EDTA2H
2The solution of O, described antibody and mercaptoethylmaine mix, and the cutting reduction reaction obtains described antibody fragment;
The temperature of described cutting reduction reaction is 35 ℃-40 ℃, is preferably 37 ℃, and the time of described cutting reduction reaction is 80 minutes-100 minutes, is preferably 90 minutes;
Described EDTA2H
2The proportioning of O, described antibody and described mercaptoethylmaine is 2mgEDTA2H
2O: 14mg antibody: 11mg mercaptoethylmaine.
In the above-mentioned conjugate, described antibody is to be that the mouse source hybridoma cell strain ET-3D7 secretion of CGMCC No.3592 obtains by deposit number.
Another object of the present invention provides a kind of method for preparing the conjugate of gold-coated ferroferric oxide nano particle and antibody.
The method of the conjugate of preparation gold-coated ferroferric oxide nano particle provided by the present invention and antibody, comprise the steps: the gold-coated ferroferric oxide nano particle is mixed with the solution that contains the capacity antibody fragment, react, obtain the conjugate of gold-coated ferroferric oxide nano particle and antibody fragment;
Described antibody fragment is that the disulfide bond between the heavy chain of antibody is cut and is reduced to the antibody fragment that obtains behind the free sulfhydryl groups; Described antibody is made up of heavy chain and light chain;
The temperature of described reaction is 2 ℃-6 ℃, is preferably 4 ℃; The time of described reaction is 10 hours-20 hours, is preferably 15 hours;
Described antibody fragment is prepared as follows: with EDTA2H
2The solution of O, described antibody and mercaptoethylmaine mix, and the cutting reduction reaction obtains described antibody fragment;
The temperature of described cutting reduction reaction is 35 ℃-40 ℃, is preferably 37 ℃, and the time of described cutting reduction reaction is 80 minutes-100 minutes, is preferably 90 minutes;
Described EDTA2H
2The proportioning of O, described antibody and described mercaptoethylmaine is 2mgEDTA2H
2O: 14mg antibody: 11mg mercaptoethylmaine;
Among the above-mentioned conjugate preparation method, described antibody is to be that the mouse source hybridoma cell strain ET-3D7 secretion of CGMCC No.3592 obtains by deposit number.
Deposit number is that the mouse source hybridoma cell strain ET-3D7 of CGMCC No.3592 also belongs to protection scope of the present invention.
By deposit number is that the monoclonal antibody of the epitestosterone that obtains of the mouse source hybridoma cell strain ET-3D7 secretion of CGMCC No.3592 also belongs to protection scope of the present invention.
The application of gold-coated ferroferric oxide nano particle in immune affinity extraction also belongs to protection scope of the present invention.
Above-mentioned gold-coated ferroferric oxide nano particle is prepared as follows and obtains: with Fe
3O
4Particle and HAuCl
4Solution mixes, and makes Fe
3O
4Uniform particles is scattered in HAuCl
4In the solution, to wherein adding reductive agent, reaction obtains the gold-coated ferroferric oxide nano particle again;
Described Fe
3O
4Particle is 2: 1 with the mol ratio of gold;
Described reductive agent is glucose H
2O or glucose; Described reductive agent is excessive.
The temperature of described reaction is 85 ℃, and the time of described reaction is 1-2h.
The preparation method of described gold-coated ferroferric oxide nano particle comprises the steps: to be cooled to 25 ℃ after described reaction finishes, and centrifugal, the collecting precipitation particle promptly obtains the gold-coated ferroferric oxide nano particle.
The preparation method of described gold-coated ferroferric oxide nano particle comprises the steps: that behind described collecting precipitation particle it is neutral to the pH value of upper strata clear liquid to wash described deposit seed with water.
Immune affinity extraction method of the present invention is to be carrier with gold-coated ferroferric oxide magnetic nanoparticle and epitestosterone monoclonal antibody conjugate, and the epitestosterone micromolecular compound in the sample is carried out purifying and enrichment; Overcome shortcomings such as other pre-treating method processes are numerous and diverse, consuming time, contaminated environment, improve the detection sensitivity of target analytes in the complex system.
The method of small-molecule substance content in the test sample among the present invention is to be the pre-treatment means with above-mentioned immune affinity extraction, again with the high-efficient liquid phase chromatogram technology coupling, to measure micromolecular compound such as epitestosterone; Specifically can comprise the steps: 1) epitestosterone MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying; 2) the gold-coated ferroferric oxide magnetic nanoparticle is synthetic; 3) monoclonal antibody of epitestosterone is coupled to the surface of nano particle; 4) use coupling the gold-coated ferroferric oxide magnetic nanoparticle of monoclonal antibody of epitestosterone to be arranged to the purifying and the enrichment of epitestosterone in the doping urine sample; 5) utilization high performance liquid chromatograph UV-detector is carried out quantitative measurement with the epitestosterone compound that purifying and enrichment obtain.
Provided by the present inventionly sample is carried out the method for immune affinity extraction small-molecule substance is highly sensitive, accuracy is high, specificity is high; Experiment showed, with the inventive method to extract epitestosterone from urine, the detection limit of epitestosterone (LOD) (at least 3 times of signal to noise ratio (S/N ratio)s) is 6ng/mL, and the lower limit of quantitation of epitestosterone (LOQ) (at least 10 times of signal to noise ratio (S/N ratio)s) is 20ng/mL; For 20mL concentration is 1,0.5 and 0.2ng/mL epitestosterone doped P BS solution, recovery result from 92% to 103%, relative standard deviation (RSD) is lower than 6%, realized from large volume solution, detecting the low concentration material, further proof the inventive method is highly sensitive, can the enriching low-concentration sample.In addition, the inventive method is quick, easy, accurate and effective, with low cost, environmental friendliness.
With the inventive method the epitestosterone in the urine is carried out enrichment, through being the immunity enrichment process of carrier based on the gold-coated ferroferric oxide magnetic nanoparticle, the interference of background material is eliminated basically, can not bring negative effect to the detection of epitestosterone.
Of the present invention having a extensive future, as required can be with extended detection range to blood sample, as long as can obtain the antibody of detected object, just antibody fragment can be coupled to gold-coated ferroferric oxide magnetic nanoparticle surface, thereby prepare immune affinity solid phase extraction material, realize purifying, enrichment and detection the detected object compound.And can change off-line checking method into online test method as required, further improve the degree of robotization.
Description of drawings
Fig. 1 is the transmission electron microscope result: (A) Fe
3O
4Nano particle; (B) Fe
3O
4The @Au nano particle.
Fig. 2 is FFIR figure: (A) Fe
3O
4Nano particle; (B) Fe
3O
4The @Au nano particle.
Fig. 3 is the MALDI-TOF mass spectrogram of antibody: (A) the molecular weight mass spectrogram of complete antibody molecule; (B) the molecular weight mass spectrogram of antibody fragment.
Fig. 4 is the immune affinity solid phase extraction pre-treating method synoptic diagram of carrier for the gold-coated ferroferric oxide magnetic nanoparticle of the monoclonal antibody of epitestosterone is arranged based on coupling.
Fig. 5 is the liquid chromatogram of the doping urine sample before and after immune affinity solid phase extraction pre-treating method is handled.What indicate ET among the figure is epitestosterone compound (Epitestosterone).
Fig. 6 is the liquid chromatography-electrospray ionization mass spectrum figure of epitestosterone.
Fig. 7 is the liquid chromatography standard working curve of epitestosterone concentration to the peak area match.
Fig. 8 is Fe
3O
4The structural representation of @Au nano particle.
Fig. 9 is antibody fragment and Fe
3O
4The preparation synoptic diagram of the conjugate of @Au magnetic nanoparticle.
Figure 10 is with antibody fragment and Fe with magnet
3O
4The synoptic diagram that the conjugate of @Au magnetic nanoparticle and epitestosterone bond separate from solution.
Figure 11 is Fe
3O
4The infared spectrum of standard substance.
Figure 12 is the epitestosterone composite diagram.
Figure 13 is synthetic for the epitestosterone haptens.
Figure 14 is synthetic for the epitestosterone comlete antigen.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Experiment reagent and raw material: epitestosterone and XAD-2 resin are bought from Sigma-Aldrich company.
FeNH
4(SO
4)
212H
2O, Fe (NH
4)
2(SO
4)
26H
2O and gold chloride (HAuCl
44H
2O) all buy (Shanghai, China) from Chemical Reagent Co., Ltd., Sinopharm Group.Triton x-100 is bought from Beijing chemical reagents corporation (Beijing, China).Mercaptoethylmaine hydrochloride (purity 98%) is bought from Acros Organics company.Chromatographically pure acetonitrile and methyl alcohol are to buy from Fisher Scientific company (Fair Lawn city, New Jersey, the U.S.).Unless otherwise noted, the pure water that uses in experimentation is prepared from by Milli-Q water purification system (Millipore company, Molsheim city, France).All the other reagent of not mentioning all are analytically pure, and need not further purifying when using.All reagent are all used 0.22 micron membrane filtration before entering liquid chromatographic detection.
0.01M the compound method of phosphate buffer solution (PBS, pH 7.4): with the NaCl of 8.0g, the Na of 2.9g
2HPO
412H
2The KH of O, 0.2g
2PO
4Be dissolved into the KCl of 0.2g and be prepared from behind the 1L deionized water.
The 80mL human urine is collected from 27 years old healthy male volunteer, through volunteer's agreement.These urines are handled with the 20g XAD-2 resin, have obtained blank urine specimen after removing impurity.
The storing solution of 1 μ g/mL epitestosterone (ET) is by methyl alcohol preparation and be kept in-20 ℃ the refrigerator.
Instrument. high performance liquid chromatography (HPLC) analyser is available from Tianjin, island company (Tokyo, Japan).This instrument has used Tianjin, island Prominence LC-20A chromatographic analysis system, and this system is by LC-20AT four-stage pump, SPD-M20A diode array detector, SIL-20A automatic sampler and DGU-20A
5Degasser is formed.Data acquisition and data processing are finished by Tianjin, island LC solution software (version 1.23).The JEM-200CX transmission electron microscopy instrument that transmission electron microscope (TEM) dispatches from the factory available from Japanese JEOL company.Fourier transformation infrared spectrometer (FTIR) and ultraviolet-visible spectrophotometer are respectively by Vector 22 photometers (Bruker, Germany) and CARY 1E UV-vis spectrophotometer (Varian, Australia) record.Substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF MS) Autoflex III model instrument is available from German Bruker Daltonics company.Avanti J-25 hydro-extractor is available from U.S. BeckmanCoulter company.Microcon YM-100 ultra filtration membrane is available from Millipore company.Bag filter provides (Beijing, China) by magnificent Science and Technology Ltd..The diameter of neodymium iron boron strong magnet is 29mm, and thickness is 6mm.
Embodiment 1, gold-coated ferroferric oxide nano particle (Fe
3O
4Preparation @Au) and gold-coated ferroferric oxide nano particle (Fe
3O
4@Au) with the preparation of antibody coupling matter
With the synthetic precursor tri-iron tetroxide of coprecipitation, use oxidation-reduction method then earlier at tri-iron tetroxide surface reduction last layer gold.
One, gold-coated ferroferric oxide nano particle (Fe
3O
4Preparation @Au)
(1) preparation Fe
3O
4Nano particle:
Ferric ion storing solution: with 0.0128mol FeNH
4(SO
4)
212H
2O and 0.0064molFe (NH
4)
2(SO
4)
26H
2O is dissolved in and obtains in the 100mL 0.4M aqueous sulfuric acid; FeNH
4(SO
4)
212H
2O and Fe (NH
4)
2(SO
4)
26H
2The mol ratio of O is 2: 1.
The NaOH aqueous solution of preparation 250mL 1M, triton x-100 aqueous solution to the ultimate density of triton x-100 in the NaOH aqueous solution that evenly drips 0.1M in this solution is 0.01M.This solution is transferred in three mouthfuls of round-bottomed flasks of 500mL, be heated to 85 ℃ in the water-bath, effectively stir with nonmagnetic mechanical stirrer simultaneously, slowly drip the ferric ion storing solution of 25mL simultaneously with dropper, remain on 85 ℃ then and continue to stir 30 minutes, after this just obtained the Fe of black
3O
4The suspension of particle.Stop and removing heating and stir (but will keep nitrogen atmosphere), allow Fe
3O
4Particle is deposited on container bottom naturally.The question response solution temperature drops to and carries out after the room temperature centrifugally, discards supernatant liquor, keeps deposit seed and successively with a large amount of deionized waters and absolute ethanol washing, puts into 70 ℃ of baking ovens then and dried 3 hours, and is standby.
(2) preparation gold-coated ferroferric oxide nano particle (Fe
3O
4@Au):
The aqueous solution of gold chloride: with 0.412g gold chloride (HAuCl
44H
2O) be dissolved in the 100mL water, obtain Au (III) solution.
1, in the 100mL conical flask with Fe
3O
4The aqueous solution of particle and 0.01M gold chloride (wherein, Fe
3O
4With the molar ratio of gold chloride be 2: 1), ultrasonic 15 minutes to quicken Fe
3O
4Particle dispersion.
2, in the system of step 1, slowly drip reductive agent glucose H
2O 0.55g (containing pure glucose 0.5g), heating is 1 hour and with gentle agitation in water-bath (85 ℃); During this time, along with Fe
3O
4Particle is wrapped up by gold grain gradually, and grain color becomes bronzing by black.
3, heating stopped heating after 1 hour, removed water-bath, naturally cooled to room temperature; Centrifuging then obtains deposit seed, and supernatant liquor is fully transparent.
4, neutral with pure water washing precipitation particle to the pH value of upper strata clear liquid, in 70 ℃ of baking ovens, dry then, promptly obtain gold parcel Fe
3O
4Particle.
Fe
3O
4The structural representation of @Au nano particle as shown in Figure 8.
(3) gold-coated ferroferric oxide nano particle (Fe
3O
4@Au) and Fe
3O
4The checking of nano particle
1, transmission electron microscope (TEM): with Fe
3O
4Nano particle and Fe
3O
4The @Au nano particle is ultrasonic dispersing 10 minutes in water respectively earlier.Get two kinds of sample solutions then respectively and drop on two preprepared copper mesh sheets that are coated with carbon, do tem analysis again after at room temperature solution evaporation being done.
The TEM experimental result proves, Fe
3O
4The size of magnetic nanoparticle is 13 ± 1nm (Figure 1A).At synthetic Fe
3O
4Add triton x-100 in the experimentation of magnetic nanoparticle, made magnetic-particle can be good at being dispersed in the solution, be not easy to take place self-polymerization.Fe
3O
4The size of @Au magnetic nanoparticle is 50 ± 5nm (Figure 1B).
2, infrared spectrum characterization
FFIR characterizes: with the infrared detection that the method for pressing potassium bromide troche is done, belong to middle infra-red range.
Fe
3O
4And Fe
3O
4The @Au magnetic nanoparticle characterizes collection of illustrative plates as shown in Figure 2 with FFIR, and A represents Fe
3O
4The infrared spectrum of nano particle, B represents Fe
3O
4The infrared spectrum of @Au nano particle.Infrared spectrogram result shows that along with the parcel of gold on the tri-iron tetroxide surface, the stretching vibration frequency of Fe-O key is from 579cm
-1Move to 586cm
-1At 636cm
-1And 586cm
-1Locate emerging infrared peak and show Fe
3O
4Nano particle is successfully wrapped up by gold.
Fe
3O
4The FFIR of standard substance (U.S. Sigma-Aldrich company) characterizes collection of illustrative plates as shown in figure 11, shows the Fe that the present invention obtains
3O
4Particle is correct.With Fig. 2 A and Fe
3O
4The standard substance spectrogram compare, difference mainly be on the intensity at peak to some extent the wave number at difference and peak can not fit like a glove.The former chief reason is in the operating process of measuring infrared data, adds Fe
3O
4The amount of particle can't be in full accord, so difference occurs on peak intensity.The latter is because each spectrogram result who measures, and is difficult to accomplish identical on the numerical value of wave number, and the error of instrument itself is 10%.Fe
3O
4In the spectrogram of particle standard substance, three peaks are all more approaching with the mapping peak of Fig. 2 A, are due to the error that exists of instrument itself, so the result of two spectrograms can mapping, the standard spectrogram that promptly provides can prove experiment synthetic be exactly Fe
3O
4Particle.
At Fe
3O
4Residual Fe in the @Au magnetic nanoparticle
3O
4Particle does not have golden parcel owing to surperficial, thereby can not influence antibody fragment at Fe
3O
4The coupling on @Au magnetic nanoparticle surface.Because the coupling of antibody fragment on particle needs the support of gold surface, there is not the Fe of gold
3O
4Particle naturally can't be fixing in its surface with antibody fragment, so Fe
3O
4Particle can not adsorb antibody fragment, and promptly non-specific adsorption is very weak.
Two, Fe
3O
4The conjugate of @Au magnetic nanoparticle and antibody and preparation thereof
1, epitestosterone MONOCLONAL ANTIBODIES SPECIFIC FOR
(1) epitestosterone synthesizes as shown in figure 12
Synthesizing of epitestosterone benzoic ether: 2.9g testosterone, 5.2g triphenyl phosphorus and 2.4g benzoic acid are dissolved in the 100mL benzene, as A liquid.4.0g the liposoluble of azoformic acid diisopropyl in 20mL benzene, and under agitation slowly adds in the A liquid, stirring at room reaction 24 hours.Reactant liquor placed to spend the night in the fuming cupboard volatilize solvent benzol, obtain the about 20mL of yellow thick product.With sherwood oil: ethyl acetate=17: 5 is as eluant, eluent, the silicagel column purified product.Target product Rf value R
f=0.5, there is an impure point on a small quantity in each down thereon simultaneously.
Synthesizing of epitestosterone: in 3.0g epitestosterone benzoic ether, add the NaOH of 1.5g and the methyl alcohol of 25mL, stirred overnight at room temperature.Thin-layer chromatography shows that the raw material point reduces, and product epitestosterone point occurs.In reaction system, add entry, use ethyl acetate extraction 4 times.Organic layer spends the night with anhydrous sodium sulfate drying.Elimination drying agent and revolving boils off and removes most of solvent, has yellow solid to separate out.Re-crystallizing in ethyl acetate obtains light yellow solid, and infrared lamp is the oven dry product down, obtains the 1.1g epitestosterone.Thin-layer chromatography shows that product purity is very high.
(2) the epitestosterone holoantigen is synthetic
ET-3-is haptenic synthetic: as shown in figure 13,0.2g epitestosterone and 0.2g hydrochloric acid ethyloic azanol are placed 40mL ethanol, add 5% NaOH 4mL, 110 ℃ of reflux 15 hours.Thin-layer chromatography shows does not almost have epitestosterone residue (ethyl acetate is developping agent).Vacuum rotary steam is removed most of ethanol, adds 5mL water, uses extracted with diethyl ether three times, removes unreacted stock chart testosterone and impurity.Add 1M HCl to acid to aqueous phase, have this moment a large amount of white precipitates to generate.The dissolution precipitation that adds diethyl ether extracts three times.Ether layer water extracting and washing three times is to neutral.In the ether layer, added anhydrous sodium sulfate drying 8 hours.The elimination drying agent revolves to boil off and removes most of solvent ether, obtains yellow oil 140mg (productive rate=56%).
Synthesizing of 3 holoantigens of epitestosterone (ET-3-BSA): as shown in figure 14,34mg ET-3-haptens, 14mg NHS and 23mg EDC are dissolved among the 2.3mL DMSO altogether, normal temperature stirred 2 hours down, as A liquid.NaHCO with 0.1M
3Transfer to pH=7.0 with 1M HCl.The BSA of 161mg is dissolved in the above-mentioned solution of 10mL as B liquid, and A liquid is being stirred in the adding B liquid.Slow stirring reaction is 2 hours under the room temperature, and avoid as far as possible in the stirring bubble produces (n (ET-3-hapten): n (NHS): n (EDC): n (Protein)=40: 50: 50: 1) as far as possible.The solution that the has reacted bag filter of packing into, successively with PBS dialysis 24 hours, deionized water dialysis 48 hours.Preserve down in-20 ℃ after the freeze-drying.
(3) MONOCLONAL ANTIBODIES SPECIFIC FOR
The key step that hybridoma technology prepares monoclonal antibody comprises immune animal (to the immunity of Balb/c mouse), Fusion of Cells, the selection of hybridoma is cultivated, the screening of positive cell, a large amount of preparation several steps of hybridoma cloning, frozen and monoclonal antibody.
(3) .1 immune animal:, be made into the solution that concentration is 280 μ g/mL with autoclaved physiological saline (0.9%) dissolving ET-3-BSA.Become water in oil emulsion with the Fu Shi Freund's complete adjuvant of equivalent is fully emulsified, inject in 5 Balb/c mouse peritoneals every 0.5mL (70 μ g).Carried out the immunity second time on the 14th day, complete anti-concentration is reduced by half, with ET-3-BSA normal saline solution and the emulsification of equivalent freund 's incomplete adjuvant of 140 μ g/mL, mouse is carried out subcutaneous multi-point injection, every 0.4mL (28 μ g) in the backbone both sides.Carried out immunity for the third time on the 24th day.Reagent and consumption are with for the second time identical, and the injection site is the subcutaneous of four limbs top.Get mouse tail blood after 10 days, centrifugal, get supernatant and survey with ELISA and tire (envelope antigen ET-3-BSA 5 μ g/mL), 5 mice serums are tired all more than 4, and are stand-by.Between whole duration of immunity, the mouse healthy growth.
(3) .2 Fusion of Cells
The shop system of feeder cells: the feeder cells of normal use are the mouse peritoneal cells.It can discharge a kind of non-species specificity growth factor, promotes the cell growth; In addition, macrophage is wherein removed the effect of dead cell in addition.Merging a few days ago, carry out the shop system of feeder cells.Get one of Kunming mouse, after dislocation of cervical vertebra is put to death, be soaked in 75% alcohol and its fur carried out disinfection in 15 minutes.Mouse is placed on the double dish, and belly at its abdominal scissors one osculum, is peeled off skin of abdomen with sterile scissors up, exposes peritonaeum, and 75% alcohol disinfecting peritonaeum dries.Dropper with sterilization injects the about 5mL of DMEM complete medium in peritonaeum, with the soft belly of needle handle, the nutrient culture media color is by the pink orange colour that becomes, and sucking-off also places the 50mL centrifuge tube, repeatedly several times to the nutrient culture media nondiscolouring.Centrifugal 5 minutes of 1000rpm, supernatant discarded is suspended from cell precipitation in the 40mL HAT nutrient solution, drops in 4 96 well culture plates every hole 100 μ L, 37 ℃ of CO behind the mixing
2Cultivate stand-by in the incubator.
The preparation of myeloma cell's suspension: (1) myeloma cell's recovery and cultivation: (190 ℃) take out one ampere of myeloma cell that bottle is freezing from liquid nitrogen container, put into also continuous stirring of 38 ℃ of water-baths immediately and injure cell to prevent intracellular moisture crystallization.Dropwise add the about 3mL of complete medium after dissolving, centrifugal 5 minutes of 1000rpm, thoroughly abandoning supernatant (including DMSO), add about 2mL complete medium, cell is blown afloat, be transferred in the culture flask, add 10% DMEM complete medium to the 5mL, place 37 ℃ of CO
2Cultivate in the incubator.The myeloma cell cultivates in the complete culture solution that contains 10% calf serum, changes liquid (1-2 days) when nutrient culture media color generation significant change.When cell is in logarithmic growth mid-term, in the time of at the bottom of being paved with bottle by 1: 3 diluted passage (2-4 days pass a generation).Merge and went down to posterity in preceding 48 hours, merge and to choose vigorous, perfectly round bright, the logarithmic phase cell 4-6 bottle that form is good of growth the same day, other is frozen.(2) preparation of myeloma cell's suspension: the Sp2/O cell that will be in exponential phase in Fusion of Cells the same day is blown down, place the 50mL centrifuge tube, centrifugal 5 minutes of 1000rpm, supernatant discarded, the full nutrient culture media that toos many or too much for use cleans twice, cell is suspended from the incomplete nutrient culture media of 40mL at last.Cell count is 10
7The individual order of magnitude.
The preparation of immune spleen cell suspension: (1) spleen booster immunization: merge first three day, the Balb/c mouse of choosing two immunity carries out the spleen booster immunization.The ET-3-BSA normal saline solution that with concentration is 150 μ g/mL directly injects in the mouse peritoneal every 0.2mL (30 μ g) without emulsification.(2) preparation of immune spleen cell suspension: merge the same day, two mouse of getting the spleen booster immunization are got its blood (about each 1mL).Supernatant packing after centrifugal, frozen is as positive control.Put to death mouse, the spleen of enlargement is carefully taken out in sterilization and cut belly open in super-clean bench, places on the stainless (steel) wire in the sterile petri dish, adds incomplete nutrient culture media immediately.Spleen is shredded and roll with needle handle, splenocyte is filtered in the double dish through mesh, extract places the 50mL centrifuge tube.Centrifugal, wash 2 rear overhangs in the incomplete nutrient culture media of 40mL.Cell count is 10
8The individual order of magnitude.
Fusion of Cells: with 10
7Individual Sp2/0 cell and 10
8Individual splenocyte is mixed in the 50mL centrifuge tube, adds incomplete nutrient culture media to 40mL.After stirring evenly gently, centrifugal 5 minutes of 1000rpm abandons supernatant, at the bottom of finger attack pipe, makes two kinds of cells be mixed into pasty state.Centrifuge tube is placed 37 ℃ of sterilized water.Add the incomplete nutrient culture media of equivalent in the sterilized centrifuge tube that the poly-second 2 1500 (PEG1500) of 0.4mL is housed, constantly suction makes liquid reach pink.Draw 50% PEG1500 solution with suction pipe, insert the centrifuge tube bottom.Left hand is coil slowly, and the right hand stirs cell precipitation gently, slowly drips PEG1500 simultaneously, adds in 1 minute, leaves standstill in the water-bath and merges in 1.5 minutes.Leave standstill finish after, add the incomplete nutrient culture media of 5mL immediately, make PEG1500 dilution and fail, the dropping method is to add 1mL in preceding 1 minute, adds remaining 4mL in back 1 minute.Add incomplete nutrient culture media 20mL, centrifugal 3 minutes of 300rpm abandons supernatant.Add the HAT nutrient solution and stir gently, make cell suspension.Add HAT to 40mL, mixing is added drop-wise in 4 96 well culture plates that are covered with feeder cells, and every hole 100 μ L are nutrient solution with HAT, 37 ℃ of CO
2Cultivate in the incubator.
(3) selection of .3 hybridoma is cultivated
Immune mouse spleen cell and murine myeloma cell are after PEG1500 handles, form the mixture of various kinds of cell, comprising the myeloma cell and the splenocyte that do not merge, the coenocyte that myeloma cell self is merged, the heterocaryon that coenocyte that splenocyte self merges and myeloma cell and splenocyte merge.Changed half HAT nutrient solution in every 2-3 days, myeloma cell and splenocyte are dead successively after several days, and the growth of the hybridoma cluster that merges forms little cell colony.At this moment, change the HT medium culture into to the fortnight, can be observed tangible colony, at this moment draw nutrient culture media and carry out specific antibody and detect, to determine to contain the hybridoma of secreting specificity antibody.
The screening of positive cell
When fused cell is cultured to two all left and right sides, select the hole that cell colony is obvious, the nutrient culture media change color is big to carry out positive test with the ELISA method.Drawing 100 μ L nutrient culture media during test detects as determinand.For the positive hole that records first, draw the upper strata nutrient culture media every other day and detect, three final definite positive cells in the positive stable back of mensuration.The ELISA process is as follows:
(1) with the CBS compound concentration be the ET-3-BSA coating buffer of 10 μ g/mL, every hole 100 μ L placed 15 hours for 4 ℃.Discard coating buffer, with PBST washing 3 times, every hole 200 μ L placed 3 minutes at every turn, got rid of, and patted dry.
(2) with the sealing of 1% gelatin solution, every hole 200 μ L, 37 ℃ of following incubations 2 hours.Discard confining liquid, with PBST washing 3 times, every hole 200 μ L placed 3 minutes at every turn, got rid of, and patted dry.
(3) add 100 μ L nutrient culture media to be measured, and with the rabbit anteserum of 100 times of dilutions as negative control, a blood of immune mouse is as positive control, 37 ℃ of incubations 1 hour.Discard the nutrient culture media reactant liquor, with PBST washing 3 times, every hole 200 μ L placed 3 minutes at every turn, got rid of, and patted dry.
(4) it is anti-to add the mark two of 5000 times of dilutions, every hole 100 μ L, 37 ℃ of following incubations 1 hour.Discard two anti-solution, with PBST washing 3 times, deionized water wash 2 times, every hole 200 μ L placed 3 minutes at every turn, got rid of, and patted dry.
(5) add substrate solution, every hole 100 μ L, black out colour developing 15 minutes.Add 2M sulfuric acid color development stopping.450nm reading on the microplate reader.
Hybridoma cloning
Select positive monoclonal 5 strains of stablizing more than three times to carry out enlarged culture.One hole is expanded to a row, finally keeps two strain monoclonal 1E5,3D7 and a few strain polyclone cell.Porose all positive through check 1E5 and 3D7 institute, further prove monoclonal.When row's cell grows to logarithmic phase, go to enlarged culture in the bottle that is covered with feeder cells when being paved with at the bottom of the hole.Nutrient culture media progressively is changed to the nutrient culture media that contains 20% serum by HT.
(3) the .4 hybridoma is frozen
Cultivate two strain monoclonals and polyclone cell in culture flask respectively, the nutrient culture media serum content slowly reduces to 17.5% by 20%, reduces to 15% at last.Frozen in batches in liquid nitrogen.Method is as follows: (1) discards and blots nutrient culture media, uses a small amount of not exclusively nutrient culture media floating dead cell of flush away gently, discards; (2) add incomplete nutrient culture media about 3mL, cell is blown down fully with the elbow dropper; (3) the incomplete media transfer that will contain cell is to the 7mL centrifuge tube, and adhesive plaster seals, and following centrifugal 3-5 minute of 1000rpm discards nutrient culture media; (4) add the 1.8mL cryopreserving liquid in the precipitation, after the pressure-vaccum dissolving, go in the ampere bottle repeatedly.(5) ampere bottle is outer wraps cotton, place-70 ℃ frozen; (6) take out the back of spending the night, frozen in-190 ℃ of liquid nitrogen.
This hybridoma cell strain ET-3D7 that secretes the monoclonal antibody of anti-epitestosterone is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on January 26th, 2010 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.3592, classification called after mouse source hybridoma cell strain, the cell line name is called ET-3D7.
(3) a large amount of preparations of .5 monoclonal antibody
The MONOCLONAL ANTIBODIES SPECIFIC FOR method of anti-epitestosterone: the hybridoma CGMCC No.3592 that will set up places cell culture medium, places 37 ℃ and 5% CO
2Cultivate in the incubator, change cell culture fluid one time, treat that cell concentration is greater than 10 every 2d
5Stop to change liquid during mg/ml, it is all dead to continue to cultivate cell.Collect culture supernatant, 1500rpm, centrifugal 10 minutes, supernatant contained high-caliber monoclonal antibody, and-20 ℃ of preservations are standby.Described cell culture medium is for to add hyclone in the DMEM nutrient culture media, making the final concentration of hyclone in cell culture medium is 15-20% (volumn concentration), and the pH of described cell culture medium is 7.4.
Can also be prepared as follows: in a large amount of preparations of antibody a few days ago, in healthy Balb/c mouse peritoneal, inject paraffin oil.The monoclonal cell of some is blown down with aseptic PBS solution, following centrifugal 3-5 minute of 1000rpm, cell precipitation is dissolved among the 0.5mL PBS, injects in the mouse peritoneal.Monoclonal cell injected mouse after 7-10 days, and as seen mouse web portion obviously swells, and gets its ascites.Concrete grammar is as follows: with the fixing mouse of left hand, with its belly of 75% alcohol disinfecting, place a test tube in the mouse below again, No. 16 injection needles of sterilization are thrust the mouse lower abdomen, allow ascites splash in vitro, when ascites stops dripping, mobile gently syringe needle, and soft its belly, make ascites continue to ooze.Once can obtain about 1-3mL ascites.Centrifugal 5 minutes of 1000rpm draws the transparent clear liquid in upper strata, freezes in-20 ℃
2, preparation Half-IgG antibody fragment
Antibody is the monoclonal antibody of epitestosterone, is to be that the mouse source hybridoma cell strain ET-3D7 secretion of CGMCC No.3592 obtains by deposit number.
With method I the antibody molecule cutting is reduced: with the EDTA2H of 2mg
2The O solid adds and contains in the PBS buffer solution of 14mg antibody, adds 11mg mercaptoethylmaine solid again in buffer solution, reacts 90 minutes down at 37 ℃; Then, reaction mixture was dialysed 15 hours to 0.01M PBS buffer solution (pH 7.4) under 4 ℃; At last, unreacted complete antibody molecule is completely removed with Microcon YM-100 ultrafiltration, obtains antibody fragment solution (solute is an antibody fragment, and solvent is a PBS buffer solution).The molecular weight of complete antibody molecule and antibody fragment (half-IgG) is measured (Fig. 3) by the MALDI-TOF mass spectrometer.Fig. 3 shows that mass spectral result shows that the complete antibody molecule and the molecular weight of antibody fragment are respectively 147798 ± 443g/mol and 73816 ± 221g/mol.
Utilize following method II and method III with antibody molecule cutting reduction, result and method I do not have significant difference.
Method II: except that temperature of reaction is that 35 ℃, reaction time are 100 minutes, all the other are all identical with method I.
Method III: except that temperature of reaction is that 40 ℃, reaction time are 80 minutes, all the other are all identical with method I.
3, preparation antibody fragment and Fe
3O
4The conjugate of @Au magnetic nanoparticle
The preparation of conjugate as shown in Figure 9.
Method I: with gold-coated ferroferric oxide nano particle (Fe
3O
4@Au) add and to contain in the PBS buffer solution of capacity epitestosterone monoclonal antibody fragment, gentle agitation spend the night (15 hours) in 4 ℃ of refrigerators; After reaction finishes, use magnet that magnetic-particle is separated from solution; According to the ultraviolet absorptivity result of supernatant before and after the reaction, the antibody fragment that draws about 1mg is coupled to the Fe of 10mg
3O
4On the @Au particle.
Supernatant is a PBS solution before the reaction, does not contain antibody;
Reaction back supernatant is the PBS solution that contains antibody, can use the concentration of UV, visible light spectrophotometric determination antibody;
The antibody coupling efficient of traditional Sepharose 4B carrier material: volume had 3.5mL approximately after 1g Sepharose 4B gel expanded, but the about 5-10mg antibody of every mL coupling.If calculate with 8mg antibody, so the 1g gel needs 3.5 * 8=28mg antibody approximately, i.e. 10mg gel energy coupling 0.28mg antibody.
Among the present invention, 10mg Fe
3O
4The @Au particle can coupling 1mg antibody, be classic method 1mg/0.28mg=3.57 doubly, so than the antibody coupling efficient height of traditional Sepharose 4B carrier material more than 3 times.
Show that of the present invention is the antibody coupling efficient that the antibody coupling efficient of the immune affine enrichment material of carrier is better than traditional Sepharose 4B carrier material with the gold-coated ferroferric oxide magnetic-particle.
Antibody fragment is to realize by sulfydryl on the antibody fragment and gold effect in the coupling of gold surface.Disulfide bond between the antibody molecule heavy chain obtains antibody fragment after being reduced by the mercaptoethylmaine cutting, and expose free mercapto groups, obtain antibody fragment with free sulfhydryl groups, by the self-assembling reaction of free sulfhydryl groups, antibody fragment can firmly directionally be coupled to the gold-coated ferroferric oxide nano grain surface then.
The coupling of antibody fragment on enrichment material not only can not be lost the immunocompetence of antibody, and can also further improve the coupling density of antibody on enrichment material, thereby improves the coupling rate of antibody.Because the antibody of coupling is the fragment of antibody, have only 1/2 of complete antibody, the antibody number of coupling is the twice of complete antibody on unit materials so.
Utilize following method II and method III to prepare the conjugate of gold-coated ferroferric oxide nano particle and antibody, result and method I do not have significant difference.
Method II: except that temperature of reaction is that 2 ℃, reaction time are 20 hours, all the other are all identical with method I.
Method III: except that temperature of reaction is that 6 ℃, reaction time are 10 hours, all the other are all identical with method I.
Embodiment 2, use Fe
3O
4The @Au magnetic nanoparticle extracts epitestosterone and detects epitestosterone content from sample solution
(1) preparation of typical curve
1, the preparation of standard solution
In the PBS damping fluid, add epitestosterone, make the concentration of epitestosterone in the PBS damping fluid be respectively 20ng/mL, 50ng/mL, 80ng/mL, 100ng/mL, 200ng/mL, promptly obtain the standard solution of each concentration.
2, from standard solution, extract epitestosterone
The synoptic diagram of extraction process as shown in Figure 4.
The conjugate of epitestosterone antibody fragment and gold-coated ferroferric oxide nano particle joined (amount of conjugate is 10mg in each standard solution, the amount of standard solution is 1mL), association reaction is 1 hour under room temperature (25 ℃), obtains the bond of conjugate and antibody fragment; With magnet bond is separated from solution that (magnet is placed a side of container, and bond as shown in figure 10), is outwelled the supernatant in the bottle being adsorbed under the effect of magnet near on the chamber wall of magnet, and residue is inhaled the bond on the bottle wall; Wash with water and reclaim bond 3 times, with methyl alcohol epitestosterone is eluted from bond more afterwards that (methyl alcohol elutes antigen by changing the solution environmental between antigen and the antibody from antibody.Dry up the meoh eluate that contains epitestosterone with high pure nitrogen, at room temperature be settled to 1mL with the moving phase of liquid chromatography again again, pending efficient liquid phase chromatographic analysis detects.
In the said process, at magnet and Fe
3O
4Under the interaction of @Au nano particle, bond (Fe
3O
4The bond that the conjugate of @Au nano particle and antibody fragment and epitestosterone form) is easy to from solution, separate, therefore this Fe
3O
4@Au nano particle carrier material can satisfy the needs of immune affinity solid phase extraction.Fe
3O
4The @Au magnetic nanoparticle can be reused more than 10 times at least.
3, the method for high performance liquid chromatography detects the amount of the epitestosterone in the extraction sample
Chromatographic column: C
18Reverse-phase chromatographic column (Gemini company), the Phenomenex board, column length 250mm, internal diameter 4.6mm, column packing are C
18Filler, column packing particle diameter are 5 μ m,
The aperture.Is furnished with a C
18Pre-column (column length of pre-column is 4mm, and internal diameter is 3.0mm).
25 ℃ of column temperatures;
The automatic sampler sample introduction, sample size 20l;
Eluent (moving phase) is: the equal-volume potpourri of acetonitrile and acetic acid aqueous solution; The volume ratio of acetate and water is 1: 1000 in the acetic acid aqueous solution.
Flow velocity is 1ml/min;
Detecting device is a UV-detector, detects wavelength 244nm;
The separation of high performance liquid chromatography is at room temperature carried out.
Every part of solution replicate determination 3 times.Write down the peak area value of the epitestosterone of every part of solution correspondence.
The epitestosterone standard items are epitestosterone (pure product, faint yellow solid particle), and available from U.S. Sigma-Aldrich company, the numbering on the products catalogue is E5878-100MG.
4, typical curve
Standard working curve is to get by the liquid chromatography peak area of epitestosterone and the linear relationship match between its corresponding concentration.The horizontal ordinate of standard working curve is the concentration value of epitestosterone in the standard solution, and ordinate is the peak area value that high performance liquid chromatography detects.
Typical curve as shown in Figure 7.
The equation of linear regression of standard working curve is Y=53.2 * X+837.
Extract epitestosterone with following two kinds of methods from standard solution, all the other are all same as described above, and the typical curve that the result obtains does not have significant difference.
Method II: in the step of extraction epitestosterone, the temperature of removing in conjunction with reaction is 20 ℃ from standard solution, and the time is outside 1.5 hours, and all the other are all with identical described in the experiment 2.
Method III: in the step of extraction epitestosterone, the temperature of removing in conjunction with reaction is 37 ℃ from standard solution, and the time is outside 0.5 hour, and all the other are all with identical described in the experiment 2.
Carry out wash-out with methyl alcohol, purpose is that antigen-antibody complex is dissociated, to obtain the solution of antigen.The requirement of eluant, eluent is non-salts substances, lethal by force, high volatility and material that can be harmful to the chromatographic column of liquid chromatography can not antagonist be arranged.The experiment proved that the elute effect of methyl alcohol is good.
(2) to the detection of the urine of adding epitestosterone
1, adds the preparation of the urine of epitestosterone
The epitestosterone methyl alcohol storing solution of 1g/mL: epitestosterone is dissolved in the methyl alcohol, and concentration is 1g/mL.
Dilute the epitestosterone methyl alcohol storing solution of 1 μ g/mL respectively with the urine that does not contain epitestosterone, be respectively 50ng/mL, 100ng/mL, 200ng/mL to the concentration of epitestosterone, obtain the doping urine specimen of variable concentrations.
2, from the urine of adding epitestosterone, extract epitestosterone
Method is with identical described in the experiment ().
3, the method for high performance liquid chromatography detects the amount of the epitestosterone in the extraction sample
Method is with identical described in the experiment ().
4, obtain the amount of epitestosterone in the extract according to typical curve
The concentration of epitestosterone is that the equation of linear regression of peak area numerical value substitution standard working curve that detection is obtained draws.
Experimental group 1: epitestosterone concentration is the epitestosterone doping urine specimen of 50ng/mL, detects according to the method for above-mentioned steps 1-4.
Experimental group 2: epitestosterone concentration is the epitestosterone doping urine specimen of 100ng/mL, detects according to the method for above-mentioned steps 1-4.
Experimental group 3: epitestosterone concentration is the epitestosterone doping urine specimen of 200ng/mL, detects according to the method for above-mentioned steps 1-4.
Control group 1: epitestosterone concentration is the epitestosterone doping urine specimen of 100ng/mL, without any pre-treatment, directly carries out efficient liquid phase chromatographic analysis (promptly directly carrying out above-mentioned steps 3 and 4).
Control group 2 (blank group): epitestosterone concentration is the epitestosterone doping urine specimen of 100ng/mL, after coupling does not have the gold-coated ferroferric oxide magnetic nanoparticle immunity extraction of the monoclonal antibody of epitestosterone to handle, carries out efficient liquid phase chromatographic analysis.
3 repetitions are established in experiment, and the result takes the mean ± standard deviation.Result such as table 1 and shown in Figure 5.
Among Fig. 5, (a) liquid chromatogram of control group 1; (b) liquid chromatogram of experimental group 2; (c) liquid chromatogram of control group 2.
The result of control group 1: the background signal of liquid chromatogram is very high, and this is to be caused by the impurity in the urine.Show that if do not advance any pre-treatment step, the epitestosterone molecule of directly measuring in the doping urine is very difficult.
The result of experimental group 2: the background interference signal of liquid chromatogram is eliminated, show, have the gold-coated ferroferric oxide magnetic nanoparticle of the monoclonal antibody of epitestosterone that urine specimen to be measured is carried out the immunity enrichment pre-treatment with coupling, the epitestosterone molecule is by specific enrichment.
The result of control group 2: use not coupling to have the gold-coated ferroferric oxide magnetic nanoparticle of the monoclonal antibody of epitestosterone that the doping urine is done pre-treatment, do not detect epitestosterone.Show that the epitestosterone molecule is not by specific enrichment.
Simultaneously, above-mentioned experiment also proves there is not the Fe of coupling antibody
3O
4The @Au nano particle does not have non-specific adsorption to epitestosterone, can not cause interference to the quantitative test of epitestosterone.Experiment is proof also, and coupling has the gold-coated ferroferric oxide magnetic nanoparticle of monoclonal antibody of epitestosterone very high to the specificity of the immunity enrichment of epitestosterone in the doping urine.
Table 1. is the determination of recovery rates of the immune affinity extraction experiment of carrier to epitestosterone doping urine with the gold-coated ferroferric oxide magnetic-particle
| Volume (mL) before the enrichment | Concentration (ng/mL) before the enrichment | Volume after the enrichment (mL) | The concentration (ng/mL) that detects | The recovery (%) | Relative standard deviation (%) |
| ??1 | ??50 | ??1 | ??49±3 | ??99 | ??6 |
| ??1 | ??100 | ??1 | ??92±4 | ??92 | ??4 |
| ??1 | ??200 | ??1 | ??218±6 | ??109 | ??3 |
The epitestosterone eluting peak that experimental group 2 is collected carries out liquid chromatography-electrospray ionization mass spectrum (LC-ESI-MS) analysis.
Mass spectrometer uses the positive ion mode of Bruker Esquire-LC ion trap detector (available from Billerica city, the Maryland State, the U.S.).The high pure nitrogen of 40psi is as atomization gas.The actual conditions of electrospray ionization source is as follows: capillary voltage is-4000V; End disk voltage is-3500V; Capillary outlet voltage is 104V; The temperature of nitrogen drying gas is located at 300 ℃, and flow velocity is located at per minute 12.0L.Moving phase is the equal-volume potpourri of acetonitrile and acetic acid aqueous solution; The volume ratio of acetate and water is 1: 1000 in the acetic acid aqueous solution.
Repetition, unanimity are as a result established in experiment 3 times.
The result as shown in Figure 6.Mass spectral result shows, carries out acidifying owing to add acetate in moving phase, so under positive ion mode, the molecular ion peak of epitestosterone [M+H]
+Mass-to-charge ratio m/z be 289, and the mass spectra peak [M+Na] of the sodion addition of epitestosterone
+Mass-to-charge ratio m/z be 311, but the former peak intensity is higher than the latter.
Above-mentioned high performance liquid chromatography experiment and mass spectrum experiment show: it is exactly epitestosterone to the material that doping urine immunity enrichment obtains that coupling has the gold-coated ferroferric oxide magnetic nanoparticle of the monoclonal antibody of epitestosterone.Therefore, the immune affinity solid phase extracting process based on the gold-coated ferroferric oxide magnetic nanoparticle is very potential sample-pretreating method.
(3) to the detection of the PBS damping fluid that adds epitestosterone
In order to investigate the enrichment performance of magnetic-particle, carry out following experiment.
1, adds the preparation of the PBS damping fluid of epitestosterone
In the PBS damping fluid, add epitestosterone, make the concentration of epitestosterone in the PBS damping fluid be respectively 1ng/mL, 0.5ng/mL, 0.2ng/mL, promptly obtain adding the PBS damping fluid of epitestosterone.
2, from the PBS damping fluid that adds epitestosterone, extract epitestosterone
Method is with identical described in the experiment (), and different is: it is 10mg that coupling has the amount of the gold-coated ferroferric oxide magnetic nanoparticle of epitestosterone antibody fragment, and the amount of adding the PBS damping fluid of epitestosterone is 20mL.
3, the method for high performance liquid chromatography detects the amount of the epitestosterone in the extraction sample
Method is with identical described in the experiment (), and different is after nitrogen dries up, to enter high-performance liquid chromatogram determination with moving phase solution constant volume behind 0.2mL.
4, obtain the amount of epitestosterone in the extract according to typical curve
The concentration of epitestosterone is that the equation of linear regression of peak area numerical value substitution standard working curve that detection is obtained draws.
3 repetitions, results averaged ± standard deviation are established in experiment.The result is as shown in table 2.
Table 2. is the result of the immune affinity extraction experiment of carrier to the enrichment of large volume low concentration with the gold-coated ferroferric oxide magnetic-particle
| Volume (mL) before the enrichment | Concentration (ng/mL) before the enrichment | Volume after the enrichment (mL) | Theoretical concentration after the enrichment (concentration before the enrichment * 100 concentration that obtain) (ng/mL) | Dense (ng/mL) that detects | The recovery (%) | Relative standard deviation (%) |
| ??20 | ??1 | ??0.2 | ??100 | ??92±4 | ??92 | ??4 |
| ??20 | ??0.5 | ??0.2 | ??50 | ??51±2 | ??103 | ??5 |
| ??20 | ??0.2 | ??0.2 | ??20 | ??19±1 | ??94 | ??4 |
As shown in Figure 7, the linear regression coeffficient R of the standard working curve of liquid chromatography
2Be 0.995, quantitative scope is between 20-200ng/mL, and accuracy good (because the peak area relative standard deviation of each concentration point all is lower than 1.5% on the typical curve, deviation is extremely low, and degree of accuracy is fine).The detection limit of epitestosterone (LOD) (at least 3 times of signal to noise ratio (S/N ratio)s) is 6ng/mL.The lower limit of quantitation of epitestosterone (L0Q) (at least 10 times of signal to noise ratio (S/N ratio)s) is 20ng/mL.50,100 and the recovery of 200ng/mL epitestosterone doping urine be 92% to 109% (seeing Table 1).In addition, use coupling to have the gold-coated ferroferric oxide magnetic nanoparticle of the monoclonal antibody of epitestosterone that the epitestosterone PBS solution of large volume, low concentration (being lower than 6ng/mL) is carried out immune affinity extraction experiment, enrichment to as if 20mL concentration be 1,0.5 and 0.2ng/mL epitestosterone doped P BS solution, experimental result sees Table 2, recovery result from 92% to 103%, and relative standard deviation (RSD) is lower than 6%.Table 3 is the result show, through immunity enrichment extraction pre-treatment step, the detection limit of epitestosterone can improve 100 times, promptly improves two orders of magnitude.
To sum up the result shows, the gold-coated ferroferric oxide magnetic nanoparticle that the epitestosterone antibody fragment arranged with coupling of the present invention carries out the method for immune affinity extraction to testing sample can the enriching low-concentration sample.With the UV-detector is example, and the detection limit LOD that detects epitestosterone with it is 6ng/mL, but has used the magnetic-particle that 100 times of concentration effects are arranged, LOD can reduce by 100 times to 0.06ng/mL, the bioaccumulation efficiency height of this method is described, has improved detection sensitivity, table 3 has just illustrated this conclusion.
Table 3. uses the lower limit of quantitation of immunity enrichment pre-treating method front and back and the comparison of detection limit
| The efficient liquid-phase chromatography method that does not have the immunity enrichment pre-treatment | The efficient liquid-phase chromatography method that the immunity enrichment pre-treatment is arranged | |
| Lower limit of quantitation (ng/mL) | ??20 | ??0.20 |
| Detection limit (ng/mL) | ??6 | ??0.06 |
Claims (11)
1. the method for an immune affinity extraction antigenic substance from sample comprises the steps:
1) with arbitrary described conjugate and sample mix among the claim 5-7, carries out association reaction, obtain containing the solution of the bond of described conjugate and antigenic substance; Described antibody and described antigenic substance can mutually combine;
2) with magnet described bond is separated from the solution that contains described bond, obtained described bond;
3) described antigenic substance is eluted from described bond, obtain described antigenic substance.
2. method according to claim 1 is characterized in that: the temperature of described association reaction is 20 ℃-37 ℃, is preferably 25 ℃, and the time of described association reaction is 0.5 hour-1.5 hours, is preferably 1 hour.
3. method according to claim 1 and 2 is characterized in that: the eluent that uses in the described wash-out is a methyl alcohol; Described antigenic substance is an epitestosterone; Described sample is urine or stripped blood.
4. the method for antigenic substance in the test sample comprises the steps:
1) obtains antigenic substance according to arbitrary described method immune affinity extraction from testing sample among the claim 1-3;
2) antigenic substance that step 1) is obtained carries out detection by quantitative with high performance liquid chromatography.
5. the conjugate of gold-coated ferroferric oxide nano particle and antibody is the conjugate that antibody fragment and the coupling of gold-coated ferroferric oxide nano particle form; Described coupling is to realize by the golden self assembly in free sulfhydryl groups in the antibody fragment and the gold-coated ferroferric oxide nano particle;
Described antibody fragment is that the disulfide bond between the heavy chain of antibody is cut and is reduced to the antibody fragment that obtains behind the free sulfhydryl groups; Described antibody is made up of heavy chain and light chain.
6. conjugate according to claim 5, it is characterized in that: described gold-coated ferroferric oxide nano particle prepares according to the method that comprises the steps with the conjugate of antibody: the gold-coated ferroferric oxide nano particle is mixed with the solution that contains the capacity antibody fragment, react, obtain the conjugate of gold-coated ferroferric oxide nano particle and antibody fragment;
The temperature of described reaction is 2 ℃-6 ℃, is preferably 4 ℃; The time of described reaction is 10 hours-20 hours, is preferably 15 hours;
Described antibody fragment is prepared as follows: with EDTA2H
2The solution of O, described antibody and mercaptoethylmaine mix, and the cutting reduction reaction obtains described antibody fragment;
The temperature of described cutting reduction reaction is 35 ℃-40 ℃, is preferably 37 ℃, and the time of described cutting reduction reaction is 80 minutes-100 minutes, is preferably 90 minutes;
Described EDTA2H
2The proportioning of O, described antibody and described mercaptoethylmaine is 2mgEDTA2H
2O: 14mg antibody: 11mg mercaptoethylmaine.
7. according to claim 5 or 6 described conjugates, it is characterized in that: described antibody is to be that the mouse source hybridoma cell strain ET-3D7 secretion of CGMCC No.3592 obtains by deposit number.
8. method for preparing the conjugate of gold-coated ferroferric oxide nano particle and antibody, comprise the steps: the gold-coated ferroferric oxide nano particle is mixed with the solution that contains the capacity antibody fragment, react, obtain the conjugate of gold-coated ferroferric oxide nano particle and antibody fragment;
Described antibody fragment is that the disulfide bond between the heavy chain of antibody is cut and is reduced to the antibody fragment that obtains behind the free sulfhydryl groups; Described antibody is made up of heavy chain and light chain;
The temperature of described reaction is 2 ℃-6 ℃, is preferably 4 ℃; The time of described reaction is 10 hours-20 hours, is preferably 15 hours;
Described antibody fragment is prepared as follows: with EDTA2H
2The solution of O, described antibody and mercaptoethylmaine mix, and the cutting reduction reaction obtains described antibody fragment;
The temperature of described cutting reduction reaction is 35 ℃-40 ℃, is preferably 37 ℃, and the time of described cutting reduction reaction is 80 minutes-100 minutes, is preferably 90 minutes;
Described EDTA2H
2The proportioning of O, described antibody and described mercaptoethylmaine is 2mgEDTA2H
2O: 14mg antibody: 11mg mercaptoethylmaine;
9. method according to claim 8 is characterized in that: described antibody is to be that the mouse source hybridoma cell strain ET-3D7 secretion of CGMCCNo.3592 obtains by deposit number.
10. deposit number is the mouse source hybridoma cell strain ET-3D7 of CGMCC No.3592; The monoclonal antibody of the epitestosterone that obtains with the mouse source hybridoma cell strain ET-3D7 secretion that by deposit number is CGMCC No.3592.
11. the application of gold-coated ferroferric oxide nano particle in immune affinity extraction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010110743A CN101776691A (en) | 2010-02-09 | 2010-02-09 | Method for immunoaffinity solid-phase extraction based on gold-coated ferroferric oxide nano-particles |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010110743A CN101776691A (en) | 2010-02-09 | 2010-02-09 | Method for immunoaffinity solid-phase extraction based on gold-coated ferroferric oxide nano-particles |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101776691A true CN101776691A (en) | 2010-07-14 |
Family
ID=42513203
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010110743A Pending CN101776691A (en) | 2010-02-09 | 2010-02-09 | Method for immunoaffinity solid-phase extraction based on gold-coated ferroferric oxide nano-particles |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101776691A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103091465A (en) * | 2013-01-29 | 2013-05-08 | 南昌大学 | Quick detection method for food-borne pathogenic bacteria based on immune magnetic separation of Fe3O4 and Au nano-material |
| CN110632295A (en) * | 2019-09-26 | 2019-12-31 | 东南大学 | Fe3O4-Au antibody nano magnetic bead and preparation method and application thereof |
| CN112285262A (en) * | 2020-10-20 | 2021-01-29 | 武汉海关技术中心 | AAS detection method combining magnetic solid phase extraction and liquid phase mass spectrometry |
| CN113009017A (en) * | 2021-02-25 | 2021-06-22 | 杭州佰辰医学检验所有限公司 | Hormone mass spectrometry detection method based on antibody coupling magnetic bead enrichment technology |
| WO2022198925A1 (en) * | 2021-03-22 | 2022-09-29 | 深圳市亚辉龙生物科技股份有限公司 | Nano-magnetic bead coating, and preparation method therefor, detection reagent thereof and detection kit thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1542449A (en) * | 2003-04-30 | 2004-11-03 | �����ɷ� | Nuclear/shell type superparamagnetism composite particulate, preparation method and application thereof |
| CN101620910A (en) * | 2008-07-01 | 2010-01-06 | 中国科学院成都有机化学有限公司 | Preparation method and application of core-shell magnetic/gold nanocomposite particles |
-
2010
- 2010-02-09 CN CN201010110743A patent/CN101776691A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1542449A (en) * | 2003-04-30 | 2004-11-03 | �����ɷ� | Nuclear/shell type superparamagnetism composite particulate, preparation method and application thereof |
| CN101620910A (en) * | 2008-07-01 | 2010-01-06 | 中国科学院成都有机化学有限公司 | Preparation method and application of core-shell magnetic/gold nanocomposite particles |
Non-Patent Citations (3)
| Title |
|---|
| S P GUBIN等: "Magnetic nanoparticales:preparation, structure and properties.", 《RUSSIAN CHEMICAL REVIEWS》 * |
| SHUANG QIU等: "Pseudo-homogeneous immunoextrction of epitestosterone from human urine samples based on gold-coated magnetic nanoparticles.", 《TALANTA》 * |
| 崔亚丽等: "核/壳型Fe3O4/Au超顺磁性微粒的制备及机理", 《中国科学（B辑）》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103091465A (en) * | 2013-01-29 | 2013-05-08 | 南昌大学 | Quick detection method for food-borne pathogenic bacteria based on immune magnetic separation of Fe3O4 and Au nano-material |
| CN110632295A (en) * | 2019-09-26 | 2019-12-31 | 东南大学 | Fe3O4-Au antibody nano magnetic bead and preparation method and application thereof |
| CN112285262A (en) * | 2020-10-20 | 2021-01-29 | 武汉海关技术中心 | AAS detection method combining magnetic solid phase extraction and liquid phase mass spectrometry |
| CN112285262B (en) * | 2020-10-20 | 2023-07-04 | 武汉海关技术中心 | AAS detection method combining magnetic solid phase extraction and liquid phase mass spectrum |
| CN113009017A (en) * | 2021-02-25 | 2021-06-22 | 杭州佰辰医学检验所有限公司 | Hormone mass spectrometry detection method based on antibody coupling magnetic bead enrichment technology |
| CN113009017B (en) * | 2021-02-25 | 2021-10-08 | 杭州佰辰医学检验所有限公司 | Hormone mass spectrometry detection method based on antibody coupling magnetic bead enrichment technology |
| WO2022198925A1 (en) * | 2021-03-22 | 2022-09-29 | 深圳市亚辉龙生物科技股份有限公司 | Nano-magnetic bead coating, and preparation method therefor, detection reagent thereof and detection kit thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102675463B (en) | Carbendazim monoclonal antibody, preparation method and application thereof | |
| CN101776691A (en) | Method for immunoaffinity solid-phase extraction based on gold-coated ferroferric oxide nano-particles | |
| CN102967709B (en) | Detect enzyme linked immunological kit and the application thereof of zearalenone medicine | |
| CN103421072B (en) | Dexamethasone hapten and its preparation method and application | |
| CN105200013B (en) | One plant of anti-vancocin monoclonal antibody hybridoma cell strain and its application | |
| Deschamps et al. | Polyclonal and monoclonal enzyme immunoassays for picloram detection in water, soil, plants, and urine | |
| CN105838681A (en) | Anti-dexamethasone-specificity monoclonal antibody hybridoma cell strain C3 and application thereof | |
| CN105277423A (en) | Immunomagnetic bead used for vomitoxin enrichment purifying and preparation method and application thereof | |
| CN103760353B (en) | Method and special ELISA (Enzyme Linked Immunosorbent Assay) kit for detecting ustiloxin A | |
| CN101455958A (en) | Quinolone and sulpha compound extraction method from animal sample and special immuno affinity absorbent | |
| Long et al. | Simultaneous identification and quantification of the common compounds of Viscum coloratum and its corresponding host plants by ultra-high performance liquid chromatography with quadrupole time-of-flight tandem mass spectrometry and triple quadrupole mass spectrometry | |
| CN103170309A (en) | Ractopamine antibody immunoaffinity chromatographic column and application thereof | |
| CN105758846A (en) | Chemiluminescence enzyme-linked immunosorbent assay reagent kit for detecting clenbuterol | |
| Hu et al. | Development of an immunochromatographic strip test for the rapid detection of okadaic acid in shellfish sample | |
| CN102627628B (en) | Chlorantraniliprole antigen, its preparation method and application | |
| CN104031886A (en) | Method of detecting monensin by immunomagnetic bead purification-enzyme-linked immunoassay and special monoclonal antibody used therein | |
| Jiang et al. | Preparation of monoclonal antibody based indirect competitive ELISA for detecting 19-nortestosterone residue | |
| CN102295698B (en) | Cyclosporine A immunogen, cyclosporine A specific antibody, detection reagent, and detection kit | |
| CN112625119B (en) | Estrone antigen and preparation method thereof | |
| CN1967247A (en) | Detection kit for enzyme linked immunosorbent assay applied for diethylstilbestrol residue analysis | |
| CN1966523A (en) | Dursban monoclonal antibody preparing method | |
| Babakov et al. | Culture of transformed horseradish roots as a source of fusicoccin-like ligands | |
| CN104569416B (en) | Detect method and the special ELISA reagent kit thereof of rice aspergin B | |
| CN103352028A (en) | Hybridoma cell strain YNEMC3, monoclonal antibody generated thereby, and application of monoclonal antibody | |
| Wei-Wei et al. | Immuno-voltammetric determination of chloramphenicol residues in milk |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20100714 |