CN101358979A - Chloramphenicol immune detecting system marked by magnetic bead - Google Patents
Chloramphenicol immune detecting system marked by magnetic bead Download PDFInfo
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- CN101358979A CN101358979A CNA200810120219XA CN200810120219A CN101358979A CN 101358979 A CN101358979 A CN 101358979A CN A200810120219X A CNA200810120219X A CN A200810120219XA CN 200810120219 A CN200810120219 A CN 200810120219A CN 101358979 A CN101358979 A CN 101358979A
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Abstract
The present invention relates to a method which adopts the chloramphenicol immunoassay system labeled by the magnetic bead to determine the chloramphenicol residue; the complex of the anti-CAP monoclonal antibody and the magnetic bead (immunomagnetic beads) are prepared through the preparation of the anti-CAP monoclonal antibody (monoclonal antibody for short) of high specificity; the immunomagnetic beads are used for absorbing the CAP in the sample absorbed by the immunomagnetic beads, the immunomagnetic beads and the supernatant are separated with the magnetic separation technology, and then the CAP absorbed by the immunomagnetic beads are extracted with ethyl acetate, so as to achieve the purpose of gathering and concentrating the CAP; and the gathered and concentrated CAP sample are detected in the method of direct competitive inhibition. When the method is used for detecting the chloramphenicol, the stability is good, the sensitivity of the detection is simultaneously improved, and the limit value reaches to 0.002ng/mL.
Description
Technical field
The present invention relates to a kind of magnetic separation technique and enzyme linked immunosorbent detection technology, a kind of specifically marked by magnetic bead that utilizes cooperates enzyme-linked immune detection method to treat that chloramphenicol residue carries out method for measuring in the sample product.
Background knowledge
(Chloramphenicol is a kind of broad-spectrum antibiotic that 20 middle of century are found CAP) to chloromycetin, and Gram-negative bacteria and positive bacteria are all had the good restraining effect.Because its good antibiotic property, the stable property of medicine and cheap price, once in one period, use with humans and animals clinically as the medicine of bacteriosis, and be widely used in animal husbandry production as feed addictive.Yet, it is found that in use chloromycetin has toxicity, the residual health and lives safety that directly endangering people of CAP in animal food.Particularly human body contact back consequence is serious, can produce toxic and side effects such as alpastic anemia, other malignant hematologic diseases, and human beings'health is constituted huge potential threat.The U.S. stipulates not allow to use CAP the earliest in home poultry raising, and the poultry of sale must be cooked the CAP residue detection.In view of healthy reason, FAO (Food and Agriculture Organization of the United Nation) (FAO), The World Health Organization (WHO) and many national regulations are forbidden CAP is used for food animal, China has equally banned use of CAP, and the detectability value of regulation CAP: milk 0.25ng/mL, meat 1.5ng/g, urine 1.0ng/mL, serum 0.5ng/mL, honey (fast inspection) 1.5ng/g, honey (C18 post) 0.1ng/g, and China's agriculture and animal husbandry is sent out [2001] No. 24 literary compositions and is stipulated equally, and the residual chloromycetin in horse liver, chicken, porgy muscle is limited the quantity of to detecting; The same regulation chloromycetin content of the imported food hygienic standard standard of European Union must not be and detected; U.S. food and FAD (FDA) and Finland stipulate that further the maximum residue limit(MRL) (RML) of CAP is 0 at present, promptly must not detect, but the detection method of various CAP all have detection limit, can not reach 0.
The detection method of CAP is a lot of at present.Wherein vapor-phase chromatography and liquid phase chromatography are highly sensitive, and its detection limit can reach 0.7ng/mL, but the sample pretreatment process relative complex, and the instrumentation degree is high and cost an arm and a leg, and analysis speed is slow, is not suitable for promoting the use of and the great amount of samples screening in basic unit; Microbial method economy, simple, but sensitivity is limited, and lack specificity; Radio immunoassay is applicable to meat, eggs and milk, and detectability is about 200ng/kg, but has isotope half life period weak point, has shortcomings such as radioactive contamination.And that the ELISA method has is highly sensitive, and high specificity, instrument and equipment require low relative advantage such as simple with the sample pre-treatment, are suitable for on-site supervision and great amount of samples and screen.According to present bibliographical information, conventional ELISA detection limit is 0.1ng/mL.But, along with people to improving constantly that health status requires, the maximum residue limit(MRL) of CAP (RML) equally also will be followed the same 0ng/mL that reaches with Finland of the U.S..So present various detection architecture all can not reach this requirement, therefore need more high assay method of a kind of detection sensitivity.
(Immunomagnctic beads is that immunology and magnetic carrier technology combine and the class new material that grows up IMB) to immunomagnetic beads.IMB is the magnetic microsphere that is coated with monoclonal antibody, can combine specifically with the target material that contains corresponding antigens and form new compound, effect by externally-applied magnetic field, the magnetic ball is separated fast with supernatant, can be concentrated at short notice, pure testing sample, shorten detection time, improve and detect benefit and sensitivity.At present, the MB-ELISA detection method is that immunomagnetic beads is in the topmost application of field of immunodetection, it adopts immunomagnetic beads to cooperate conventional ELISA method to be mainly used in biological macromolecule purifyings such as separating of immune detection, cell and microorganism and protein, DNA, RNA and mRNA and detects, its basic step is: one, carry out coupling with antigen and magnetic bead, the preparation immunomagnetic beads.Two, test sample and first kind of antibody (being called for short one resists) are mixed by a certain percentage, add the immunomagnetic beads that the first step of an amount of volume is prepared then therein, make antigen and the antigenic competition in the sample on the immunomagnetic beads anti-, by magnetic resolution, remove supernatant then in conjunction with one.Three, peroxidase labelling two anti-that adds certain volume was placed under 37 ℃ of conditions incubation 1 hour then, by magnetic resolution, removed supernatant again.Four, add substrate colour developing 15 minutes, colour developing liquid is moved in the 96 hole ELISA reaction plates, put into the microplate reader reading.
This method has good detection effect to big molecular antigen, but micromolecular detection examples such as relevant chloromycetin are not appeared in the newspapers as yet.Show through experiment, with MB-ELISA detection method detection sensitivity than general direct competitive ELISA method height, but detect repeated bad, poor stability, and be not suitable for micromolecule such as chlorine detection mycin.
Summary of the invention
At above-mentioned present situation, the object of the present invention is to provide a kind of and conventional enzyme-linked immune detection method to compare, it is higher to detect the sensitivity of CAP sample, and good reproducibility, detects the high chloromycetin detection method of data stability.
For achieving the above object, the technical solution adopted in the present invention is: a kind of chloramphenicol immune detecting system of marked by magnetic bead, it is characterized in that, anti--CAP the monoclonal antibody of preparation high specificity is further prepared the immunomagnetic beads that is coated with anti--CAP monoclonal antibody with itself and magnetic bead coupling then; The immunomagnetic beads that utilization is prepared adsorbs the CAP molecule in the CAP sample to be checked, remove supernatant by magnetic separation technique again, then with the CAP molecule that adsorbs in the volatile organic solvent extracting immunomagnetic beads, and it is equipped to concentrated CAP sample, with the direct competitive ELISA method CAP sample that concentrates is detected at last and obtain a result.
This method utilizes magnetic separation technique will be lower than the CAP sample that is equipped to high concentration in the low concentration CAP sample to be checked of conventional sense method sensing range after the enrichment of CAP molecule again.And then with direct competitive ELISA detection method it is detected, effectively raise detection sensitivity, and it is very good to detect benefit to CAP content.This than direct employing direct competitive ELISA method can test sample the lowest limit 0.1ng/mL of concentration high about 50 times.
Adopt the concrete steps of CAP content in the method test sample of the present invention to comprise:
The first step, the preparation of immunomagnetic beads
With ready resisting-CAP monoclonal antibody and the terminal magnetic bead of xMag isosulfocyanate radical, prepare the immunomagnetic beads of the anti--CAP monoclonal antibody that is coated with high specificity through magnetic bead activation → antibody coupling → steps such as magnetic bead washing → magnetic bead sealing → magnetic bead preservation;
Second step, the CAP in the immunomagnetic beads adsorption sample
In proportion the immunomagnetic beads for preparing in the first step is joined in the sample to be checked, utilize magnetic separation technique to remove supernatant behind the incubation, obtain to be adsorbed with the immunomagnetic beads compound of CAP molecule; In this step, described incubation process suits to carry out in the constant temperature shaking table of 37 ℃ of 180rpm, and the incubation time is advisable with 40min.
The 3rd step, the extraction of the CAP that immunomagnetic beads is adsorbed
Immunomagnetic beads compound after utilizing volatile organic solvent to removal supernatant in second step carries out the extracting of CAP molecule, then extract is put into water-bath and volatilizees, till the organic solvent-free smell; In this step, described volatile organic solvent is an ethyl acetate, and the number of times that the immunomagnetic beads compound is carried out the extracting of CAP molecule is twice, can be 5min each interval time.
In the 4th step, dissolve the precipitate after the organic solvent volatilization in the 3rd step, the CAP sample that preparation is concentrated with the PBS solution of 110ul;
In the 5th step, detect the sample concentrated to be checked that obtains in the 4th step with direct competitive ELISA detection method;
In the 6th step, calculate CAP content in the sample according to the CAP typical curve;
Outstanding advantage of the present invention is: the magnetic immuno carrier specificity is good, solid-liquid can be easier to separate through magneticaction, elution requirement is than the harmful effect of environment-protecting asepsis, concentrating the back for the chloromycetin sample that is lower than detection limit by the immunomagnetic beads enrichment detects, thereby change detection limit indirectly, improve the sensitivity that detects, avoid omission.
Description of drawings
Fig. 1 is the CAP typical curve.
Fig. 2 is the scanning curve of coupling antibody.Curve pre is a detection of antibodies curve before the coupling among the figure; Curve post is the detection curve of coupling supernatant; Curve wash1 is the detection curve of the cleansing solution first time; Curve wash2 is the detection curve of the cleansing solution second time.
Embodiment
Experiment (one)
One, manufactures the ELISA typical curve of CAP
Preparation CAP standard items, begin doubling dilution from 50ng/mL, choose the sample that concentration is respectively 50ng/mL, 12.5ng/mL, 3.125ng/mL, 0.781ng/mL, 0.195ng/mL, 0.097ng/mL, 0.049ng/mL, the PBS blank is set, make typical curve, as shown in Figure 1.
Two, the magnetic bead coupling resists-the CAP monoclonal antibody, the preparation immunomagnetic beads
This process is with reference to the terminal magnetic bead antibody coupling of the xMag isosulfocyanate radical kit instructions of Shanxi North America Gene Co., Ltd, promptly magnetic bead and anti-CAP-IgG coupling.Concrete steps are as follows:
The first step, the pre-service of magnetic bead: the terminal magnetic bead solution of the xMagTM isosulfocyanate radical of 1ml is removed supernatant after magnetic resolution is separated, add 500uL coupling liquid BP, magnetic bead is carried out pre-service.After magnetic resolution, remove supernatant again.Repeat pre-service again 1 time.
Second step, magnetic bead coupling: an amount of antibody is dissolved among an amount of coupling liquid BP, is mixed with the antibody-solutions that concentration is 0.3-1.0ug/mL, get in the magnetic bead after the 1mL antibody-solutions adds first step processing, surplus solution is labeled as pre, gives over to coupling efficiency and detects usefulness.The magnetic bead that is added with antibody-solutions is placed oscillating reactions on 37 ℃, the constant temperature shaking table of 180rpm.After magnetic resolution, remove supernatant then, and liquid is labeled as post, gives over to coupling efficiency and detect and use.
In the 3rd step, clean: the magnetic bead remove supernatant in second step after adds 500uL cleaning fluid WP, and the resuspended magnetic grain of jog after magnetic resolution, takes out supernatant and is labeled as wash1; Repeated washing operation 1 time is taken out supernatant and is labeled as wash2, gives over to coupling efficiency and detects usefulness.
The 4th step, sealing: add the 3mL confining liquid in the magnetic bead after cleaning, place 37 degrees centigrade, react 2h in the 180rpm shaking table.After magnetic resolution, remove supernatant again.
In the 5th step, clean: add 1mL cleaning fluid WP in magnetic bead, the resuspended magnetic bead of jog after magnetic resolution, is removed supernatant again.Repeated washing operation 3 times.
In the 6th step, preserve: in magnetic bead, add 1mL and preserve liquid SP, the resuspended particulate of jog, 4 ℃ of preservations are standby.
In said process, use ultraviolet-visible spectrophotometer, with coupling liquid BP zeroing, measure pre, post sample light absorption value respectively at the 280nm place; With cleaning fluid WP zeroing, measure wash1, wash2 sample light absorption value respectively, and note down at the 280nm place, as shown in table 1.
Table 1 coupling antibody concentration detects data
Record numerical value and according to following formula with reference to table 1: coupling efficiency=[OD280 (pre)-OD280 (post)-OD280 (wash1)-OD280 (wash2)]/OD280 (pre) * 100%, calculating the coupling efficiency of albumen/antibody on the terminal magnetic particle of xMagTM isosulfocyanate radical surface is 43.7%.
Three, immunomagnetic beads concentration and separation CAP
The CAP standard items of preparation 0.1ng/mL, get the centrifuge tube of 6 50mL, in the centrifuge tube of each 50mL, add the PBS damping fluid of 29400uL0.1M/L, wherein add the CAP standard items of 600uL0.1ng/mL in 5 centrifuge tubes, mix, promptly obtain the CAP solution of 0.002ng/mL.One pipe blank is set simultaneously, promptly in the 6th centrifuge tube, adds the PBS damping fluid of 600uL.
Add the good immunomagnetic beads of 160uL coupling in 6 50mL centrifuge tubes respectively, be placed on the interior incubation 40min of constant temperature shaking table of 37 ℃ of 180rpm, magnetic resolution, the sucking-off supernatant is preserved in order to further detecting.Wash immunomagnetic beads three times with PBST, 3min/ time.With ethyl acetate to immunomagnetic beads extracting twice, 5min/ time, utilize magnetic separation technique that extract is separated with magnetic bead, respectively corresponding the immigration in the little centrifuge tube is placed on then in 70 ℃ of water-baths and volatilizees, until no ethyl acetate smell.
Four, direct competitive ELISA detects enrichment with magnetic bead sample CAP
Add the PBS damping fluid of the 0.1M/L of 110uL in every pipe, add isopyknic enzyme simultaneously and mark anti-CAP-IgG, be placed on incubation 20min in 37 ℃ of constant temperature ovens.Mixed liquor is transferred in the good ELISA Plate of sealing (in advance by 1: 6000 times of bag by BSA-CAP), the 100uL/ hole is placed on incubation 40min in 37 ℃ of constant temperature ovens.Wash plate three times with PBST, 5min/ time.Add 100uL/ hole TMB, be placed on the 15min that develops the color in 37 ℃ of constant temperature ovens.The concentrated sulphuric acid that adds 50uL/ hole 2M stops, and puts into reading on the microplate reader.The data of reading are calculated by the typical curve formula, and it is as shown in table 2 to obtain data.
The CAP of table 2 magnetic bead separation and concentration detects data
Five, the ELISA of CAP detects in the supernatant of magnetic bead absorption back
The sample freeze-drying concentrates CAP: draw supernatant 20mL in glass dish, be placed on freeze-drying in the freeze dryer (available from U.S. LABCONCO company), with the CAP in the saline crystallization of freeze-drying formation in the ethyl acetate extracting plate, extract is put into 70 ℃ of water-baths and is volatilized, until no ethyl acetate smell.
Direct competitive ELISA detects freeze-drying concentrating sample CAP, and its step detects the operation of enrichment with magnetic bead sample CAP with direct competitive ELISA in [3].The data of reading are calculated by the typical curve formula, and it is as shown in table 3 to obtain data.
CAP detects data in the supernatant of table 3 magnetic bead absorption back
By the data of table 2 and table 3, the chloramphenicol immune detecting system that can calculate by marked by magnetic bead detects chloromycetin content total in the sample, and actual chloromycetin addition is 0.06ng, therefore can calculate the error amount of detection, as table 4.
Error analysis between table 4 CAP detection limit and addition
Can draw by table 4, the chloramphenicol immune detecting system of marked by magnetic bead has good detection effect to this small molecule antigen of chlorine detection mycin, detects good stability.
Six, experiment conclusion
In the present invention, the effect of magnetic bead is the CAP molecule in the adsorption sample, further isolate CAP molecule in the CAP sample by magnetic separation technique, the volatile organic solvent effect is that the CAP molecule is extracted from immunomagnetic beads, water-bath adds the organic solvent ethyl acetate in the heat abstraction extract then, micro-CAP in the extract does not vapor away with organic solvent at this moment, but be trapped in the centrifuge tube, in this centrifuge tube, add the 0.01M PBS dissolving CAP of 110uL, make the CAP sample to be checked after concentrating.CAP sample after concentrating by the direct competitive ELISA detection is at last extrapolated the CAP content in the CAP sample to be checked.This method has realized the CAP sample that exceeds conventional sense method detection limit is detected, and has effectively improved detection sensitivity, and through the front experimental data as can be known, the detection limit of the inventive method can reach 0.002ng/mL.And with respect to the MB-ELISA method, this method has been avoided carrying out detection reaction on immunomagnetic beads, has effectively got rid of its labile factor, good stability.
Claims (5)
1, a kind of chloramphenicol immune detecting system of marked by magnetic bead, it is characterized in that, after utilizing coupling to have the immunomagnetic beads of anti--CAP monoclonal antibody of high specificity to adsorb CAP molecule in the CAP sample to be checked, adopt magnetic separation technique to remove supernatant, go out the CAP molecule with volatile organic solvent extracting from the immunomagnetic beads that has adsorbed the CAP molecule then, to extract the CAP molecule afterwards again and be mixed with concentrated CAP sample, and adopt the direct competitive ELISA method that the CAP sample that concentrates is detected at last.
2, the chloramphenicol immune detecting system of a kind of marked by magnetic bead according to claim 1, it is levied and is, and its concrete steps comprise:
The first step prepares the immunomagnetic beads that coupling has the anti--CAP monoclonal antibody of high specificity through magnetic bead activation → antibody coupling → steps such as magnetic bead washing → magnetic bead sealing → magnetic bead preservation;
Second step joined the immunomagnetic beads for preparing in the first step in the CAP sample to be checked in proportion, adopted magnetic separation technique to remove supernatant behind the incubation;
The 3rd step, utilize volatile organic solvent that the immunomagnetic beads compound of removing in second step behind the supernatant is carried out the extracting of CAP molecule, then extract is put into water-bath and volatilize, till the organic solvent-free smell;
In the 4th step, dissolve the precipitate after the organic solvent volatilization in the 3rd step, the CAP sample to be checked that preparation is concentrated with the PBS solution of 110ul;
In the 5th step, detect the CAP sample that concentrates that obtains in the 4th step with the direct competitive ELISA detection method;
In the 6th step, calculate CAP content in the sample according to the CAP typical curve.
3, the chloramphenicol immune detecting system of a kind of marked by magnetic bead according to claim 2, it is levied and is, after the immunomagnetic beads of anti--CAP monoclonal antibody that coupling specificities is strong joins in the sample to be checked, incubation 40min in 37 ℃, the constant temperature shaking table of 180rpm is adsorbed in the immunomagnetic beads CAP in the sample.
4, the chloramphenicol immune detecting system of a kind of marked by magnetic bead according to claim 2, it is levied and is, and described volatility organic solution is ethyl acetate, and the number of times that the immunomagnetic beads compound is carried out the extracting of CAP molecule is 2 times, and be 5min each interval time.
5, the chloramphenicol immune detecting system of a kind of marked by magnetic bead according to claim 2, it is levied and is, described extract is placed in 70 ℃ of water-baths volatilizees, and removes organic solvent ethyl acetate.
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