CN107389958A - The detection gel column of lincomycin and the detection method of lincomycin - Google Patents
The detection gel column of lincomycin and the detection method of lincomycin Download PDFInfo
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Abstract
The present invention relates to a kind of detection gel column of lincomycin and the detection method of lincomycin, the detection gel column of lincomycin includes open tubular column and the detection layers and Quality Control layer that are filled in open tubular column.During detection, the lincomycin enzyme-labelled antigen that testing sample and chemiluminescent labels mark is added in the detection gel column of the lincomycin cocurrent layer and Quality Control layer after testing.Lincomycin monoclonal antibody in the common competition detection layers of both lincomycin enzyme-labelled antigens of testing sample and chemiluminescent labels mark, judges whether contain lincomycin in testing sample according to the color of the color of detection layers and Quality Control layer.The detection gel column of above-mentioned lincomycin is easy to carry, whether contains lincomycin in quick, sensitive detection testing sample, is adapted to this large amount of quick detections of scene.
Description
Technical field
The present invention relates to detection technique field, detection gel column and lincomycin more particularly to a kind of lincomycin
Detection method.
Background technology
Lincomycin (Lincomycin, LIN), also known as lincomycinum, it is the elite stand gram obtained from strepto- bacteria culture fluid
Amide-type narrow-spectrum antibiotic.On veterinary clinic, lincomycin is mainly used in treating gram-positive bacteria particularly penicillin resistant
Gram-positive bacteria caused by various infection, poultry chronic respiratory tract disease, mycoplasma pneumonia of swine caused by Mycoplasma, anaerobic bacteria sense
The necrotic enteritis of such as chicken is contaminated, is also used for treating pig treponema dysentery, toxoplasmosis and dog, the lumpy jawl clams of cat.Lin Ke
Mycin has the effect of outstanding to the mastitis for milk cows as caused by gram-positive bacteria and anaerobic bacteria, can be used for whole body or part is controlled
Treat.It is mainly used in the preventing and treating of bee larva bacterial disease, especially gram-positive bacillus larvae in the production of honeybee industry
Cause the preventing and treating of American foul brood.During Animal husbandry production, generally addition is in feed or drinking-water for veterinary drug, for controlling
Treat and prevention Animal diseases, also there are some veterinary drugs (such as beta-stimulants class) to be used as growth accelerator, increase feed conversion rate and thin
Meat rate, residue of veterinary drug thus may be caused in animal derived food, so as to which serious threat human health is (such as allergic reaction, straight
Connect poisoning, diffusion of drug resistance pathogen etc.).If consumer takes in the animal sources food of the medicament residue of class containing lincomycin for a long time
Product, renal damage can be caused, destroy the hemopoietic system of people, death even can be caused to the people of allergic constitution.It is in addition, animal derived
Food nitrite dosage lincomycin class medicine may Induction of bacterial drug resistance, cause potential public health security.
Many countries have all formulated MRL (MRLs) standard of lincomycin.The Ministry of Agriculture of the People's Republic of China, MOA
In issue in 2002《Animal food herbal medicine MRL》Define lincomycin in various animal target tissues
Residue limits：100 μ g/kg in ox/sheep/pig/fowl muscle, 100 μ g/kg in fat, 500 μ g/kg in liver, 1500 μ g/kg in kidney,
150 μ g/kg in ox/goat milk, 50 μ g/kg in egg.It is 0~30 μ g/kg that day, which is permitted amount (ADI),.Above-mentioned regulation and the regulation of European Union
Compare, in addition to being 50 μ g/kg in fat, other are identical.It is 200 μ g/kg that the regulation of Codex Committee on Food, which is removed in muscle, other
It is identical.The residue limits standard to lincomycin of the U.S. and Japan is also basically identical.
The detection method majority of lincomycin residual is instrumental method and ELISA detection method at present
(Enzyme-linked immunosorbent assay, ELISA).Wherein instrumental method such as chromatograph mass spectrum analysis method is for big
Most chemical pollutant retention analysis has enough sensitivity, but detects except needing expensive analytical instrument, also requires
The horizontal and substantial amounts of pre-treatment reagent of the operation of technical staff.And detection process it is previous as will to analyte carry out complexity spread out
Biochemical treatment, pretreatment process is complicated, takes height, efficiency is low, is not suitable for the selective mechanisms of great amount of samples.High-efficient liquid phase color
Spectrometry and Liquid Chromatography-Tandem Mass Spectrometry, instrument and equipment and cumbersome process due to costliness, are not suitable for live great amount of samples
Quick detection.Compare, ELISA detection method sensitivity is higher, but traditional EUSA (ELISA
Method) operation it is complex, human error is bigger, is also not suitable for field quick detection.
To sum up, the detection method of traditional lincomycin is not suitable for this large amount of quick detections of scene.
The content of the invention
Based on this, it is necessary to provide a kind of lincomycin that can carry out this live a large amount of quick detection lincomycins
Detect the detection method of gel column and lincomycin.
A kind of detection gel column of lincomycin, including open tubular column and the detection layers and matter that are arranged in the open tubular column
Layer is controlled, the open tubular column is provided with inlet and outlet, and the detection layers go out close to the import, the Quality Control layer described in
Mouthful；
The detection layers include gel carrier, the first polyclonal secondary antibody being coupled on the gel carrier and are incorporated in
Lincomycin monoclonal antibody on the first polyclonal secondary antibody, wherein the first polyclonal secondary antibody is the lincomycin
The antibody of monoclonal antibody；
The Quality Control layer includes gel carrier, the second polyclonal secondary antibody being coupled on the gel carrier and is incorporated in
Anti- chemiluminescent labels antibody on the second polyclonal secondary antibody, wherein the second polyclonal secondary antibody is the anti-chemistry
The antibody of luminous marker antibody.
In one embodiment, the described first polyclonal secondary antibody is sheep anti mouse secondary antibody, and the lincomycin monoclonal resists
Body is the lincomycin monoclonal antibody in mouse source.
In one embodiment, the described second polyclonal secondary antibody is goat-anti rabbit secondary antibody, the anti-chemiluminescent labels
Antibody is the anti-HRP polyclonal antibodies in rabbit source.
In one embodiment, there is gap between the detection layers and the Quality Control layer.
In one embodiment, the gel carrier is the Ago-Gel through cyanogen bromide-activated.
A kind of detection method of lincomycin, comprises the following steps：
Testing sample is added in the detection gel column described in any of the above-described；
The lincomycin enzyme-labelled antigen of chemiluminescent labels mark is added into the detection gel column, so that describedization
Learn luminescent label lincomycin enzyme-labelled antigen with it is described in detection layers described in the testing sample competitive binding
Lincomycin monoclonal antibody；
Nitrite ion is added into the detection gel column, wherein, the nitrite ion can react with chemiluminescent labels
Produce color change；And
Whether judged according to the color of the color of the detection layers and the Quality Control layer can containing woods in the testing sample
Mycin.
In one embodiment, in addition to：
By the color of the detection layers compared with the standard established by lincomycin standard items, test sample is treated described in acquisition
The content of lincomycin described in product.
In one embodiment, the woods that chemiluminescent labels mark is added into the detection gel column can be mould
In the step of plain enzyme-labelled antigen, the concentration of the lincomycin enzyme-labelled antigen of chemiluminescent labels mark for 400ng/mL~
600ng/mL, the addition of the lincomycin enzyme-labelled antigen of the chemiluminescent labels mark is the volume of the detection layers
1 times~3 times.
In one embodiment, the lincomycin enzyme-labelled antigen that the chemiluminescent labels mark is by the following method
It is prepared：
Lincomycin is dissolved in DMF, then adds NaIO4, A liquid is obtained after reaction；
Chemiluminescent labels are dissolved in NaHCO3Solution obtains B liquid；
The B drops are added in the A liquid, obtain intermediate product；And
Use NaBH4The intermediate product is reduced, obtains the lincomycin enzyme-labelled antigen of the chemiluminescent labels mark.
In one embodiment, the lincomycin enzyme-labelled antigen of the chemiluminescent labels mark is HRP marks
Lincomycin enzyme-labelled antigen, the nitrite ion are TMB nitrite ions.
The detection gel column of above-mentioned lincomycin includes open tubular column and the detection layers and Quality Control layer that are arranged in open tubular column.
Detection layers are close to the import of open tubular column, and Quality Control layer is close to the outlet of open tubular column.Detection layers include gel carrier, are coupled at gel load
The first polyclonal secondary antibody on body and the lincomycin monoclonal antibody being incorporated on the first polyclonal secondary antibody.Quality Control layer includes
Gel carrier, the second polyclonal secondary antibody being coupled on gel carrier and the anti-chemistry hair being incorporated on the second polyclonal secondary antibody
Signal thing antibody.During detection, the lincomycin enzyme-labelled antigen that testing sample and chemiluminescent labels mark is added to this
Flow through in the detection gel column of lincomycin and successively detection layers and Quality Control layer.What testing sample and chemiluminescent labels marked
Both lincomycin enzyme-labelled antigens compete the lincomycin monoclonal antibody in detection layers jointly.If can without woods in testing sample
Mycin, lincomycin enzyme-labelled antigen and the lincomycin monoclonal antibody knot whole in detection layers of chemiluminescent labels mark
Close, after adding nitrite ion, detection layers and Quality Control layer can develop the color.If containing lincomycin in testing sample, test sample is treated
Lincomycin in product can be combined with all or part of lincomycin monoclonal antibody in detection layers, add nitrite ion
Afterwards, the lighter of detection layers or do not develop the color.And because the lincomycin enzyme-labelled antigen of chemiluminescent labels mark can be with
Anti- HRP antibody bindings in Quality Control layer, therefore under normal circumstances, no matter whether contain lincomycin in testing sample, add aobvious
Quality Control layer should all develop the color after color liquid, as Quality Control layer can not develop the color, illustrate that testing result is invalid, avoid false negative or false positive
Testing result.The detection gel column of above-mentioned lincomycin is easy to carry, can take scene detection sample specific at any time to
In content of lincomycin situation.The woods of detection method application testing sample and the chemiluminescent labels mark of the lincomycin
Lincomycin monoclonal antibody that can be in the common competition detection layers of both mycin enzyme-labelled antigens, further according to detection layers and Quality Control layer
Color can quickly, sensitively detect in testing sample whether contain lincomycin, be adapted to this large amount of quick detections of scene.
Brief description of the drawings
Fig. 1 is the structural representation of the detection gel column of the lincomycin of an embodiment；
Fig. 2 is the flow chart of the detection method of the lincomycin of an embodiment；
Fig. 3 is the different use state figures of the detection gel column of lincomycin as shown in Figure 1；
Fig. 4 is the lincomycin standard for detecting 6 concentration in embodiment 3 respectively with the detection gel column of 6 lincomycins
Result figure after product.
Embodiment
In order to facilitate the understanding of the purposes, features and advantages of the present invention, with reference to specific embodiment and
Accompanying drawing is described in detail to the embodiment of the present invention.Elaborate in the following description many details in order to
Fully understand the present invention.But the invention can be embodied in many other ways as described herein, art technology
Personnel can do similar improvement in the case of without prejudice to intension of the present invention, therefore the present invention is not by following public specific implementation
Limitation.
Referring to Fig. 1, the detection gel column 10 of the lincomycin of an embodiment, including open tubular column 100 and it is arranged on
Detection layers 200 and Quality Control layer 300 in open tubular column 100.
Open tubular column 100 is provided with import 101 and outlet 103, and detection layers 200 are close to import 101, and Quality Control layer 300 is close to going out
Mouth 103.Detection layers 200 include gel carrier, the first polyclonal secondary antibody being coupled on gel carrier and are incorporated in more than first
Clone the lincomycin monoclonal antibody on secondary antibody.First polyclonal secondary antibody is the antibody of lincomycin monoclonal antibody.Quality Control
Layer 300 includes gel carrier, the second polyclonal secondary antibody being coupled on gel carrier and is incorporated on the second polyclonal secondary antibody
Anti- chemiluminescent labels antibody.Second polyclonal secondary antibody is the antibody of anti-chemiluminescent labels antibody.
Specifically, after detection layers 200 are filled, the outer wall of detection layers 200 abuts with the inwall of open tubular column 100, Quality Control layer 300
The outer wall of Quality Control layer 300 abuts with the inwall of open tubular column 100 after filling.
In one embodiment, there is gap between detection layers 200 and Quality Control layer 300.Testing sample is from open tubular column 100
Import 101 add, flow through detection layers 200 and Quality Control layer 300 successively, testing sample first with the association reaction of detection layers 200, it is ensured that
Detection liquid energy is enough first combined with lincomycin monoclonal antibody.
Specifically, the gap between detection layers 200 and Quality Control layer 300 is 0.5cm~1.0cm, detection layers when avoiding developing the color
200 and Quality Control layer 300 influence each other.
Further, it is air layer 400 among detection layers 200 and Quality Control layer 300.
Specifically, the first polyclonal secondary antibody is the antibody of lincomycin monoclonal antibody, and the first polyclonal secondary antibody can with woods
Two kinds of antibody of mycin monoclonal antibody have homology, so that what lincomycin monoclonal antibody can be specifically is incorporated in
On one polyclonal secondary antibody.
Specifically, the second polyclonal secondary antibody is the antibody of anti-chemiluminescent labels antibody, and the second polyclonal secondary antibody is with resisting
Two kinds of antibody of chemiluminescent labels antibody have homology, so that anti-chemiluminescent labels antibody can be combined specifically
On the second polyclonal secondary antibody.
In one embodiment, the first polyclonal secondary antibody is sheep anti mouse secondary antibody, and lincomycin monoclonal antibody is mouse source
Lincomycin monoclonal antibody.
Specifically, chemiluminescent labels for it is a kind of developer or other it is specific under the conditions of can produce color change
Material.Further, chemiluminescent labels are different luminol, tris (bipyridine) ruthenium, acridinium ester, alkaline phosphatase or horseradish mistake
Oxide enzyme (HRP).
In present embodiment, chemiluminescent labels are horseradish peroxidase (HRP), i.e., anti-chemiluminescent labels resist
Body is anti-HRP antibody.
In one embodiment, the second polyclonal secondary antibody is goat-anti rabbit secondary antibody, and anti-chemiluminescent labels antibody is rabbit
The anti-HRP polyclonal antibodies in source.
In one embodiment, gel carrier is the Ago-Gel through cyanogen bromide-activated, the fine jade after cyanogen bromide-activated
The efficiency high of the coupling of sepharose and the first polyclonal secondary antibody and the second polyclonal secondary antibody so that the first polyclonal secondary antibody and
Second polyclonal secondary antibody is firmly incorporated on Ago-Gel.
In one embodiment, detection layers are clamped between two pieces of sieve plates, and Quality Control layer is clamped between two pieces of sieve plates.
The detection gel column 10 of above-mentioned lincomycin is easy to carry, can take in live detection sample specific at any time
Content of lincomycin situation.The detection gel column 10 of the lincomycin also at least has the advantages that：(1) in detection layers
The first polyclonal secondary antibody is coupled on gel carrier, lincomycin monoclonal antibody is incorporated on the first polyclonal secondary antibody, i.e., woods can
Mycin monoclonal antibody is by the first polyclonal secondary antibody absorption on gel carrier.Compared to lincomycin monoclonal antibody directly with
The mode that gel carrier combines, this detection gel column 10 are coupled the first polyclonal secondary antibody by elder generation on gel carrier, in conjunction with
Lincomycin monoclonal antibody.Due to the antibody that the first polyclonal secondary antibody is lincomycin monoclonal antibody, and first is polyclonal
Secondary antibody is polyclonal antibody, and the first polyclonal secondary antibody can be combined with a variety of epitopes of lincomycin monoclonal antibody.
Lincomycin monoclonal antibody can be incorporated in above the first polyclonal secondary antibody with different azimuth and angle, increase the stabilization of combination
Property.And when adding testing sample, the reserved epitope binding site of lincomycin monoclonal antibody has diversity so as to be measured
Lincomycin in sample smoothly can be combined and rested in detection layers with corresponding epitope binding site.Avoid lincomycin
Monoclonal antibody bound site of lincomycin monoclonal antibody and lincomycin directly in conjunction with caused by directly with gel carrier
The phenomenon that point is shielded completely by gel carrier.Lincomycin monoclonal antibody and it is incorporated in lincomycin monoclonal antibody
Lincomycin can firmly be adsorbed and rested on gel carrier in detection layers, be not easy to be lost in nitrite ion or eluent etc.,
Improve the accuracy of detection.(2) similarly, in Quality Control layer on gel carrier be coupled the second polyclonal secondary antibody, second polyclonal two
Resist for the antibody of anti-chemiluminescent labels antibody, and the second polyclonal secondary antibody is polyclonal antibody.Second polyclonal secondary antibody
It can be combined with a variety of epitopes of anti-chemiluminescent labels antibody.Anti- chemiluminescent labels antibody with different azimuth and
Angle can be incorporated in above the second polyclonal secondary antibody, avoid anti-chemiluminescent labels antibody from being flowed with nitrite ion or eluent etc.
Lose, improve the accuracy of detection.(3) it is chemical when adding when containing anti-chemiluminescent labels antibody in Quality Control layer, therefore detecting
After the lincomycin enzyme-labelled antigen of luminescent label, due to the lincomycin enzyme-labelled antigen energy of chemiluminescent labels mark
Anti- chemiluminescent labels antibody binding in enough and Quality Control layer, under normal circumstances, no matter whether contain woods in testing sample
Can mycin, Quality Control layer should all develop the color after adding nitrite ion, as Quality Control layer can not develop the color, illustrate that testing result is invalid, avoid vacation
Negative or false positive testing result.(4) the detection gel column can specifically detect lincomycin, and qualitative or quantitative sentences
The content of lincomycin in disconnected testing sample.(5) easy to operate, rapid reaction is easy to carry, and it is special at any time can to take scene to
The detection sample of property, has a good application prospect and higher learning value.
Referring to Fig. 2, the detection method of the lincomycin of an embodiment comprises the following steps S110~S140.
S110, testing sample is added and detected in gel column.
Wherein, the structure of detection gel column 10 refers to described above, and therefore not to repeat here.
Specifically, the import 101 of testing sample from detection gel column 10 is slowly added to, to enable testing sample to fill
Divide and contacted with the lincomycin monoclonal antibody in detection layers 200.
Specifically, testing sample be thing to be checked in itself, such as milk.Or testing sample configures what is formed by thing to be checked
Solution, the solution formed after being dissolved such as veterinary drug.
In one embodiment, testing sample is added in the step in detection gel column 10, the volume of testing sample
For 1 times~2 times of detection gel open tubular column product, so that testing sample can fully soak detection layers 200 and Quality Control layer 300.Treat
The addition speed of test sample product is 1 drop second~5 drop/sec, and unnecessary testing sample flows out from outlet 103.
If containing lincomycin in testing sample, the lincomycin in testing sample can be with being incorporated in detection layers
In 200 lincomycin monoclonal antibody.
The lincomycin enzyme mark that chemiluminescent labels mark is added in S120, the detection gel column obtained into S110 resists
Original, so that the lincomycin enzyme-labelled antigen of chemiluminescent labels mark can with the woods in testing sample competitive binding detection layers
Mycin monoclonal antibody.
The quantity of lincomycin monoclonal antibody in detection layers is certain, and the woods of chemiluminescent labels mark can be mould
Plain enzyme-labelled antigen competes limited lincomycin monoclonal antibody jointly with testing sample.After adding nitrite ion, according to detection layers
Shade be the content situation of lincomycin in testing sample of can determine whether.
In one embodiment, the lincomycin enzyme mark that chemiluminescent labels mark is added into detection gel column resists
In former step, the concentration of the lincomycin enzyme-labelled antigen of chemiluminescent labels mark is 400ng/mL~600ng/mL, is changed
The addition for learning the lincomycin enzyme-labelled antigen of luminescent label is 1 times~3 times of the volume of detection layers.Chemiluminescence mark
The lincomycin enzyme-labelled antigen of note substance markers can soak detection layers 200, compete limited lincomycin jointly with testing sample
Monoclonal antibody.
In one embodiment, the lincomycin enzyme-labelled antigen of chemiluminescent labels mark is prepared via a method which
Obtain, this method specifically comprises the following steps S121~S124.
S121, lincomycin is dissolved in DMF, then adds NaIO4, A liquid is obtained after reaction.
Specifically, NaIO4DMF (DMF) is first dissolved in in the mixed liquor of water, forming NaIO4Solution,
Then by NaIO4Solution, which is added drop-wise in lincomycin solution, to react.Wherein DMF in the mixed liquor of DMF and water
∶H2O=2: 3.
S122, chemiluminescent labels are dissolved in NaHCO3Solution obtains B liquid.
Specifically, chemiluminescent labels for it is a kind of developer or other it is specific under the conditions of can produce color change
Material.Further, chemiluminescent labels are different luminol, tris (bipyridine) ruthenium, acridinium ester, alkaline phosphatase or horseradish mistake
Oxide enzyme (HRP).Specifically, react and carried out under the conditions of lucifuge
In present embodiment, chemiluminescent labels are horseradish peroxidase (HRP).
Specifically, in B liquid, NaHCO3The concentration of solution is 0.1mol/L~0.2mol/L, HRP (horseradish peroxidase)
Mass fraction be 5%~15%.
S123, the B drops obtained in S122 are added in the A liquid obtained in S121, obtain intermediate product.
Specifically, react and carried out under the conditions of lucifuge.
S124, use NaBH4The intermediate product obtained in reduction S123, obtains the lincomycin of chemiluminescent labels mark
Enzyme-labelled antigen.
Specifically, the reaction equation for preparing the lincomycin enzyme-labelled antigen of HRP marks is as follows：
Wherein, Lincomycin represents lincomycin, and LIN-HRP represents the lincomycin enzyme-labelled antigen of HRP marks.
Being marked on the lincomycin enzyme-labelled antigen (LIN-HR) of above-mentioned HRP marks has, and can show after adding nitrite ion
Color.And LIN-HR properties are stable, can compete limited lincomycin monoclonal antibody jointly with testing sample, but do not influence
The property of testing sample and lincomycin monoclonal antibody in itself.
Nitrite ion is added in S130, the detection gel column obtained into S120, wherein, nitrite ion can be with chemiluminescence mark
Remember that thing reaction produces color change.
In one embodiment, before adding nitrite ion into detection gel column, in addition to add into detection gel column
Enter cleaning buffer solution to elute the lincomycin enzyme-labelled antigen of uncombined detection liquid and chemiluminescent labels mark.
Specifically, cleaning buffer solution is the PBS containing 1%~5% Tween-100.
Detection gel column has been incorporated into the lincomycin enzyme-labelled antigen of testing sample and chemiluminescent labels mark.
After adding nitrite ion, nitrite ion produces color change with chemiluminescent labels therein reaction.
In one embodiment, the lincomycin enzyme-labelled antigen of chemiluminescent labels mark can for the woods of HRP marks
Mycin enzyme-labelled antigen, nitrite ion are TMB nitrite ions.TMB nitrite ions can show blueness with HRP reactions, and color change is rapid, spirit
It is quick, and aberration is larger, is easy to observe.
S140, according to the color of detection layers and the color of Quality Control layer judge whether contain lincomycin in testing sample.
Detection gel column has been incorporated into the lincomycin enzyme-labelled antigen of testing sample and chemiluminescent labels mark.
If without lincomycin, the lincomycin enzyme-labelled antigen of chemiluminescent labels mark and the woods of detection layers whole in testing sample
Can mycin monoclonal antibody combine, add nitrite ion after, detection layers and Quality Control layer can develop the color.If contain in testing sample
Lincomycin, then the lincomycin in testing sample can be with all or part of lincomycin monoclonal antibody in detection layers
With reference to after adding nitrite ion, the lighter of detection layers or not developing the color.And due to the lincomycin of chemiluminescent labels mark
Enzyme-labelled antigen can with the anti-chemiluminescent labels antibody binding in Quality Control layer, therefore under normal circumstances, no matter testing sample
In whether contain lincomycin, Quality Control layer should all develop the color after adding nitrite ion, the explanation detection knot if Quality Control layer can not develop the color
Fruit is invalid, avoids the testing result of false negative or false positive.
Specifically, refer to shown in Fig. 3, if detection layers 200 and Quality Control layer 300 develop the color, illustrate that testing sample is free of
There is lincomycin, this is negative sample；If detection layers 200 do not develop the color, and Quality Control layer 300 develops the color, then illustrates that testing sample contains
Lincomycin, this is positive.And if Quality Control layer 300 does not develop the color, no matter detection layers 200 develop the color or not developed the color, detection knot
Fruit is invalid.
In one embodiment, in addition to：The standard that the color of detection layers and lincomycin standard items are established is carried out
Compare, obtain the content of lincomycin in testing sample.
In one embodiment, by the standard that lincomycin standard items are established by operating to obtain as follows：Can be mould by woods
Plain standard items carry out gradient dilution, the lincomycin standard items of gradient concentration are obtained, by the lincomycin standard of each concentration
Product are separately added into detection gel column, then add the lincomycin enzyme-labelled antigen of chemiluminescent labels mark with can be mould with woods
Lincomycin monoclonal antibody in plain standard items competitive binding detection layers, nitrite ion is added into detection gel column, wherein,
Nitrite ion can react with chemiluminescent labels produces color change, and the lincomycin standard items for obtaining each concentration are shown
The color of layer.
The detection method of above-mentioned lincomycin, testing sample and the lincomycin monoclonal in detection layers in detection gel column
After antibody binding, add chemiluminescent labels mark lincomycin enzyme-labelled antigen with testing sample competitive binding examine
The lincomycin monoclonal antibody surveyed in layer.The detection method of the lincomycin also at least has the advantages that：(1) detect
The first polyclonal secondary antibody is coupled in layer on gel carrier, lincomycin monoclonal antibody is incorporated on the first polyclonal secondary antibody.Matter
Control and be coupled the second polyclonal secondary antibody in layer on gel carrier, anti-chemiluminescent labels antibody binding is in the second polyclonal secondary antibody
On.After the lincomycin enzyme-labelled antigen of testing sample or the chemiluminescent labels mark of addition, the woods in testing sample can be mould
The lincomycin enzyme-labelled antigen of element and chemiluminescent labels mark can be combined with lincomycin monoclonal antibody so as to stop
Stay in detection layers, the lincomycin enzyme-labelled antigen of chemiluminescent labels mark can be with anti-chemiluminescent labels antibody knot
Close so as to rest in Quality Control layer, the lincomycin enzyme-labelled antigen of lincomycin and chemiluminescent labels mark is not easy with aobvious
Color liquid or eluent etc. are lost in, and improve the accuracy of detection.(2) add after testing sample and add chemistry into detection gel column again
The lincomycin enzyme-labelled antigen of luminescent label with the lincomycin Dan Ke in testing sample competitive binding detection layers
Grand antibody.In the common competition detection layers of both lincomycin enzyme-labelled antigens marked using testing sample and chemiluminescent labels
Lincomycin monoclonal antibody, judge containing for lincomycin in testing sample further according to the color change of detection layers and Quality Control layer
Amount situation, accuracy and the sensitivity of detection are improved, realizes trace detection.(3) it specific can survey and contain for lincomycin
Amount, avoids the interference of other materials.(4) the lincomycin enzyme-labelled antigen for the chemiluminescent labels mark that detection adds can be with
No matter whether the anti-chemiluminescent labels antibody binding in Quality Control layer, under normal circumstances, can be mould containing woods in testing sample
Element, Quality Control layer should all develop the color after adding nitrite ion, as Quality Control layer can not develop the color, illustrate that testing result is invalid, avoid false negative
Or the testing result of false positive.(5) detection method is easy to operate, rapid reaction, rapid reaction, the more traditional ELISA side of sensitivity
Method is high more than 20 times, and does not need any expensive instrument, can realize this live a large amount of quick detections.
It is specific embodiment part below.
In embodiment using reagent and instrument if not otherwise indicated, it is this area conventional selection.It is unreceipted in embodiment
The experimental method of actual conditions, generally according to normal condition, such as the condition described in document, books or kit factory
The method that family is recommended is realized.
Immune affinity column and sieve plate empty 1mL is purchased from Agilent companies.The Ago-Gel of cyanogen bromide-activated
(Sepharose 4B) is purchased from GE companies, article No. 17-0430-01.Sheep anti-mouse antibody and goat anti-rabbit antibody are purchased from the U.S.
Jackson companies.Lincomycin standard items are purchased from Sigma-Aldrich.HRP and the lincomycin monoclonal in mouse source
Antibody is purchased from Abcam companies, and tmb substrate nitrite ion is Chinese Aladdin Products.
Embodiment 1
Prepare the detection gel column of lincomycin
First, detection gel is prepared
(1) gel carrier activates：The Ago-Gel (Sepharose 4B) for taking 1.0g cyanogen bromides (CN-Br) to activate is used
After 1mmol/L HCl swellings, fully washed with 200mL HCl solution, whole process is completed in 15min.What is be swelled is solidifying
Glue uses 0.1mol/L NaHCO again3Solution fully balances.The Sepharose 4B of general 1.0g cyanogen bromide-activateds can be swelled about
3.5mL gel.
(2) it is coupled：The first polyclonal secondary antibodies of 0.3mL (sheep anti-mouse antibody, secondary antibody, 2.3g/L) are taken to be dissolved in the coupling of 5mL gels
In solution, mixed with the gel carrier of above-mentioned activation, stir 20h.
(3) wash：Removing the first uncombined polyclonal secondary antibody using 50mL gel conjugate solution washing, (sheep anti mouse resists
Body).
(4) close：The gel lock solution for adding 20mL into gel solution closes the activation site on gel carrier.
(5) wash：The gel closed fully is washed using 100mL acetate buffer solutions (pH 4.0), after washing again
Fully washed with 100mL gels conjugate solution (pH 8.4), above-mentioned washing process replaces detergent gel 3 times.
(6) 0.1mL of above-mentioned preparation is coupled to the gel carrier of sheep anti mouse secondary antibody and the lincomycin Dan Ke in 0.1mL mouse source
Grand antibody mixes, the lincomycin monoclonal antibody in mouse source and the fixed specific combination of sheep anti mouse secondary antibody, excessive mouse source
Lincomycin monoclonal antibody flowed directly out by Action of Gravity Field, obtain mixture and be placed on shaking table, 400rpm reaction 6min, obtain
To detection gel.
(7) preserve：The detection gel prepared is transferred in 50mL sterile centrifugation tubes after adding 10mL PBS solution, is added
4 DEG C of refrigerators of placement save backup after entering 0.01mL Procline 300.
2nd, Quality Control gel is prepared
(1) gel carrier is prepared：The Ago-Gel (Sepharose 4B) of 1.0g cyanogen bromide-activateds is taken with 1mmol/L's
After HCl swellings, fully washed with 200mL HCl solution.Whole process is completed in 15min.The gel being swelled is used again
0.1mol/L NaHCO3Solution fully balances, and obtains gel carrier.The Sepharose 4B of general 1.0g cyanogen bromide-activateds can
To be swelled about 3.5mL gel.
(2) it is coupled：The second polyclonal secondary antibodies of 0.3mL (goat anti-rabbit antibody, secondary antibody, 2.3g/L) are taken to be dissolved in the coupling of 5mL gels
In solution, mixed with the gel carrier of above-mentioned activation, stir 20h.
(3) wash：The second uncombined polyclonal secondary antibody (goat-anti rabbit-anti is removed using 50mL gel conjugate solution washing
Body).
(4) close：The gel lock solution for adding 20mL into gel solution closes the activation site on gel carrier.
(5) wash：The gel closed fully is washed using 100mL acetate buffer solutions (pH 4.0), after washing again
Fully washed with 100mL gels conjugate solution (pH 8.4), above-mentioned washing process replaces detergent gel 3 times.
(6) 0.1mL of above-mentioned preparation is coupled the gel of goat-anti rabbit secondary antibody and the anti-HRP antibody in 0.1mL rabbits source mixes, obtained
It is placed in mixture on shaking table, 400rpm reaction 6min, obtains Quality Control gel.
(7) preserve：The Quality Control layer prepared is transferred in 50mL sterile centrifugation tube after adding 10mL PBS solution, is added
4 DEG C of refrigerators of placement save backup after entering Procline 300.
3rd, the detection gel column (visualization gel ELISA detections gel column) of lincomycin is prepared
(1) immune affinity column (specification 1mL) of sky is taken, sieve plate (i.e. the lower sieve plate of Quality Control layer) is added, then adds 200
μ L Quality Control gel, after natural sediment forms post bed, sieve plate (i.e. the upper sieve plate of Quality Control layer) is added, forms Quality Control layer.
(2) after completing step (1), immune affinity column is taken, adds sieve plate (i.e. the lower sieve plates of detection layers) and the sieve plate and matter
1.0cm interval is left between the upper sieve plate of control layer, that is, forms air layer.
(3) after completing step (2), immune affinity column is taken, adds 200 μ L detection gels, after natural sediment forms post bed,
Sieve plate (i.e. the upper sieve plates of detection layers) is added, that is, forms detection layers.
By above-mentioned steps, that is, the detection gel column of lincomycin it has been prepared into.The lincomycin prepared in the present embodiment
The structure of detection gel column refer to shown in Fig. 1.Visualization gel ELISA detection gel columns are divided into three parts, from up to
Under be followed successively by detection layers, air layer and Quality Control layer.The main function of air layer is the reaction for isolation detection layer and Quality Control layer.
Embodiment 2
Prepare the lincomycin enzyme-labelled antigen (LIN-HRP) of HRP marks
With 0.3mL ultra-pure waters (ddw) and 0.2mLN, dinethylformamide (DMF) mixed liquor, DMF: H in mixed liquor2O
=2: 3, by 2.7mg NaIO4It is dissolved in the mixed liquor, obtains NaIO4Solution.By NaIO4Solution is added dropwise with 0.2mL's
In the 5.8mg LIN of DMF (DMF) dissolving, 20min is stirred at room temperature, this is A liquid.Claim 3.35mg HRP, use
0.13mol/L NaHCO310%HRP activated solutions are made in solution (2.76mL), and this is B liquid.B liquid is added dropwise to A liquid
In, lucifuge stirring 2h, obtain intermediate product.Then toward the NaBH of addition 0.1mL in intermediate product4(2mg/mL) reductase 12 h.Close
Into lincomycin enzyme-labelled antigen at 4 DEG C with PBS stir dialysis 3d, change liquid daily 4 times, centrifugation go precipitation obtain purifying
LIN-HRP, reactional equation is such as：
Embodiment 3
Detection gel column (visualization gel ELISA detections gel column) detection of the lincomycin prepared using embodiment 1
Testing sample
Cleaning Principle：In detection layers, detection layers include the gel carrier (Sephrose activated through cyanogen bromide (CN-Br)
4B is as solid phase carrier), the first polyclonal secondary antibody (sheep anti mouse secondary antibody) for being coupled on gel carrier and be incorporated in more than first
Clone the mouse source lincomycin monoclonal antibody on secondary antibody.From the import of open tubular column sequentially add testing sample and HRP mark
Lincomycin enzyme-labelled antigen (LIN-HRP), the lincomycin in testing sample compete limited in detection layers jointly with LIN-HRP
The lincomycin monoclonal antibody in mouse source.Add the colour developing of TMB assay chromogenic substrate solutions.If blueness explanation testing sample is presented not in detection layers
Contain lincomycin.Detection layers, which are presented in colourless explanation testing sample, contains lincomycin.
In Quality Control layer, Quality Control layer includes gel carrier, and (the Sephrose 4B through cyanogen bromide (CN-Br) activation are as solid
Phase carrier), the second polyclonal secondary antibody (goat-anti rabbit secondary antibody) for being coupled on the gel carrier and to be incorporated in second polyclonal
The anti-HRP antibody in rabbit source on secondary antibody.The lincomycin enzyme mark that testing sample and HRP marks are sequentially added from the import of open tubular column resists
Former (LIN--HRP), LIN--HRP and the anti-HRP antibody bindings in rabbit source, it is eventually adding the colour developing of TMB assay chromogenic substrate solutions.Because HRP is marked
The lincomycin enzyme-labelled antigen of note can with the anti-HRP antibody bindings in Quality Control layer, therefore under normal circumstances, no matter testing sample
In whether contain lincomycin, Quality Control layer should all develop the color after adding nitrite ion, the explanation detection knot if Quality Control layer can not develop the color
Fruit is invalid, avoids the testing result of false negative or false positive.Therefore, blueness explanation testing result is presented in Quality Control layer in the present embodiment
Effectively, the colourless explanation testing result of Quality Control layer presentation is invalid, need to detect again.
First, the test limit of method
1st, lincomycin standard items are taken, respectively with PBS solution dilute to obtain lincomycin solution 1 that concentration is 0ng/mL,
Lincomycin solution 3 that lincomycin solution 2 that concentration is 0.05ng/mL, concentration are 0.1ng/mL, concentration 0.15ng/mL
Lincomycin solution 4 and concentration be 0.3ng/mL lincomycin solution 5.
2nd, after completing step 1,1mL lincomycins solution (lincomycin solution 1, lincomycin solution 2, lincomycin are taken
Solution 3, lincomycin solution 4 or lincomycin solution 5) it is separately added into the detection gel of lincomycin prepared by 5 embodiments 1
In post (flow velocity is 1 drop/sec, and purpose is that antigen-antibody fully reacts).
3rd, after completing step 2, the lincomycin enzyme-labelled antigen of the HRP marks of 300 μ L embodiments 2 preparation is continuously added
(LIN-HRP) dilution, dried up after being incubated 6min.Wherein LIN-HRP dilutions are to be buffered with pH 7.4,0.01mol/L PBS
Liquid dilutes LIN-HRP to 4000 times and obtained.
4th, after completing step 3, washed 7 times with the pH 7.4 containing Tween-100,0.01mol/L PBS.
5th, 150 μ L TMB assay chromogenic substrate solutions are added after completing step 4, drying, detection layers and Quality Control layer are observed after 5min
Color, according to detection layers and the color judged result of Quality Control layer.
(1 represents that adding lincomycin solution 1,2 represents that adding lincomycin solution 2,3 represents to experimental result as shown in Figure 4
Lincomycin solution 3,4 is added to represent to add the expression addition lincomycin of lincomycin solution 4,5 solution 5), the results showed that, matter
Control layer display blueness, it was demonstrated that this experimental result is effective.The degree of detection layers display blueness is with lincomycin solution concentration
Increase and gradually reduce, 0.05ng/mL i.e. low concentration can still detect.When lincomycin concentration reaches 0.3ng/mL,
Suppress complete.Therefore, the test limit that the lincomycin detection gel post detection lincomycin that embodiment 1 can be utilized to prepare remains
For 0.3ng/mL.
2nd, the specificity of method
1st, prepare for examination drug solution
Take for examination aflatoxin standard items, dissolved with sterilized water, obtain concentration and supply examination drug solution for 100 μ g/L.
It is lincomycin and Pirlimycin for reagent thing.
2nd, it will be separately added into the detection gel column of lincomycin prepared by 2 embodiments 1 that (flow velocity is for examination drug solution
1 drop/sec, purpose is that antigen-antibody fully reacts).
3rd, after completing step 2, the lincomycin enzyme-labelled antigen (LIN- of HRP marks prepared by 300 μ L embodiments 2 is added
HRP) dilution, dried up after being incubated 6min.Wherein LIN-HRP dilutions are dilute with pH 7.4,0.01mol/L PBS
LIN-HRP to 4000 times is released to obtain.
4th, after completing step 3, washed 7 times with the pH 7.4 containing Tween-100,0.01mol/L PBS.
5th, 150 μ L TMB assay chromogenic substrate solutions are added after completing step 4, drying, detection layers and Quality Control layer are observed after 5min
Color, according to detection layers and the color judged result of Quality Control layer.
As a result show, show blueness for the Quality Control layer for trying drug solution, it was demonstrated that this experimental result is effective.Lincomycin
The detection layers of solution show colourless, and the detection layers of Pirlimycin show blueness.Therefore, lincomycin provided by the present invention
Detect the detection lincomycin that gel column can be special.
3rd, milk sample is detected
The preparation of sample
Milk sample pretreatment process is as follows：By milk sample in 4 DEG C, 5000 × g centrifugal degreasings 10 minutes.Take 5mL's
Skim milk sample, it is separately added into 0.2mL sodium nitroprussides (0.36M) and 0.2mL zinc sulfate (1.04M) and is vortexed 1 point afterwards
Clock.Milk sample after vortex centrifuges 10 minutes under the conditions of 3000 × g, and after upper strata albumen is removed, it is (to be measured to obtain supernatant
Sample), it is measured according to following test procedures.
(1) (flow velocity is 1 drop/sec, mesh in the detection gel column for the lincomycin for preparing testing sample addition embodiment 1
Fully reacted for antigen-antibody).
(2) after completing step (1), the lincomycin enzyme-labelled antigen (LIN- of HRP marks prepared by 300 μ L embodiments 2 is added
HRP) dilution, void column pipe lower end liquid outlet is closed, is incubated at room temperature 5min.Wherein LIN-HRP dilutions be with pH 7.4,
0.01mol/L PBS dilutes LIN-HRP to 4000 times and obtained.
(3) after completing step (2), washed 7 times, washed with the pH 7.4 containing Tween-100,0.01mol/L PBS
Wash away uncombined LIN-HRP.
(4) after completing step (3), 150 μ L TMB assay chromogenic substrate solutions are added, are dried up, detection layers and Quality Control are observed after 5min
The color of layer.
As a result show：Quality Control layer display blueness, it was demonstrated that this experimental result is effective.Detection layers show colourless, show sample
In contain lincomycin.
4th, detection method is evaluated
The authenticity of immune diagnostic reagent is also known as accuracy, refers to the difference degree of measured value and actual value.Examination can be used
The indexs such as sensitivity, specificity, false positive rate and the false negative rate tested are evaluated, and Related Computational Methods are as follows.
The evaluation of test result standard of table 1.
(1) sensitivity is also known as True Positive Rate, refers to that confirming as lincomycin by instrumental method detection remains in exceeded sample
It is positive ability by the detection method detection screening of the lincomycin.Calculation formula is：
(2) specificity is also known as true negative rate, refer to by instrumental method detection confirm as be not present lincomycin remain it is exceeded
Ability in sample by the detection method detection screening of the lincomycin for feminine gender.Calculation formula is：
(3) false positive rate, refer to that instrumental method detection is confirmed as being not present in the exceeded sample of lincomycin residual by the woods
Can the detection method detection screening of mycin be positive ability.
(4) false negative rate, refer to instrumental method detection confirm as existing lincomycin remains can by the woods in exceeded sample
The detection method detection screening of mycin is negative ability.
In this experiment, we add 1/2MRLs (maximum residue limit) lincomycin in 100 portions of blank milk and 100 parts
Milk sample (positive sample) is detected, and calculates detection method (the visualization gel of the lincomycin of present embodiment respectively
ELISA method) accuracy.
Result of the test standard judges that result of the test can divide into milk sample the screening positive and screening is cloudy by naked eyes
Property sample.If the positive sample filtered out in above-mentioned experiment has lincomycin residual content through instrumental method confirmation exceedes rule
Calibration is accurate, then the testing result is true positives, is otherwise false positive.And if the negative sample filtered out in above-mentioned experiment is through instrument
Device method confirmation exceedes required standard in the absence of lincomycin residual content, then the testing result is true negative, is otherwise false cloudy
Property.Reference medical external diagnosis reagent evaluates goldstandard, using following table in visualization gel ELISA detection milk in this experiment
The accuracy of lincomycin residual is evaluated.
Visualization gel ELISA of the table 2. based on HRP observes detection lincomycin residual result
From the point of view of the result of table 2, detection method (visualization gel ELISA method) detection milk middle forest of the lincomycin
Can the false positive rate of mycin residual be respectively that 4.5%, false negative rate 0.0%, sensitivity 100.0%, specificity are
95.5%, meet the requirement of quick detection lincomycin.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that come for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. the detection gel column of a kind of lincomycin, it is characterised in that including open tubular column and be arranged in the open tubular column
Detection layers and Quality Control layer, the open tubular column are provided with inlet and outlet, and the detection layers are leaned on close to the import, the Quality Control layer
The nearly outlet；
The detection layers include gel carrier, the first polyclonal secondary antibody being coupled on the gel carrier and are incorporated in described
Lincomycin monoclonal antibody on first polyclonal secondary antibody, wherein the first polyclonal secondary antibody is the lincomycin Dan Ke
The antibody of grand antibody；
The Quality Control layer includes gel carrier, the second polyclonal secondary antibody being coupled on the gel carrier and is incorporated in described
Anti- chemiluminescent labels antibody on second polyclonal secondary antibody, wherein the second polyclonal secondary antibody is the anti-chemiluminescence
The antibody of marker antibody.
2. the detection gel column of lincomycin according to claim 1, it is characterised in that the first polyclonal secondary antibody is
Sheep anti mouse secondary antibody, the lincomycin monoclonal antibody are the lincomycin monoclonal antibody in mouse source.
3. the detection gel column of lincomycin according to claim 1, it is characterised in that the second polyclonal secondary antibody is
Goat-anti rabbit secondary antibody, the anti-chemiluminescent labels antibody are the anti-HRP polyclonal antibodies in rabbit source.
4. the detection gel column of lincomycin according to claim 1, it is characterised in that the detection layers and the Quality Control
There is gap between layer.
5. the detection gel column of lincomycin according to claim 1, it is characterised in that the gel carrier is through bromination
The Ago-Gel of cyanogen activation.
6. a kind of detection method of lincomycin, it is characterised in that comprise the following steps：
Testing sample is added in the detection gel column as described in any one of Claims 1 to 5；
The lincomycin enzyme-labelled antigen of chemiluminescent labels mark is added into the detection gel column, so that the chemistry hair
The lincomycin enzyme-labelled antigen of signal substance markers can with the woods in detection layers described in the testing sample competitive binding
Mycin monoclonal antibody；
Nitrite ion is added into the detection gel column, wherein, the nitrite ion can react with chemiluminescent labels to be produced
Color change；And
Judge whether contain lincomycin in the testing sample according to the color of the color of the detection layers and the Quality Control layer.
7. the detection method of lincomycin according to claim 6, it is characterised in that also include：
By the color of the detection layers compared with the standard established by lincomycin standard items, obtain in the testing sample
The content of the lincomycin.
8. the detection method of lincomycin according to claim 6, it is characterised in that described into the detection gel column
In the step of adding the lincomycin enzyme-labelled antigen of chemiluminescent labels mark, the woods of the chemiluminescent labels mark can
The concentration of mycin enzyme-labelled antigen is 400ng/mL~600ng/mL, and the lincomycin enzyme mark of the chemiluminescent labels mark resists
Former addition is 1 times~3 times of the volume of the detection layers.
9. the detection method of lincomycin according to claim 6, it is characterised in that the chemiluminescent labels mark
Lincomycin enzyme-labelled antigen be prepared via a method which to obtain：
Lincomycin is dissolved in DMF, then adds NaIO4, A liquid is obtained after reaction；
Chemiluminescent labels are dissolved in NaHCO3Solution obtains B liquid；
The B drops are added in the A liquid, obtain intermediate product；And
Use NaBH4The intermediate product is reduced, obtains the lincomycin enzyme-labelled antigen of the chemiluminescent labels mark.
10. the detection method of lincomycin according to claim 6, it is characterised in that the chemiluminescent labels mark
The lincomycin enzyme-labelled antigen of note is the lincomycin enzyme-labelled antigen of HRP marks, and the nitrite ion is TMB nitrite ions.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1811453A (en) * | 2006-01-26 | 2006-08-02 | 台州金芯生物工程有限公司 | Animal source food animal medicine residue detection reagent kit and detecting method thereof |
| CN101349696A (en) * | 2008-08-25 | 2009-01-21 | 深圳市绿诗源生物技术有限公司 | Enzyme-linked immunologic reagent and method for detecting alficetin |
| CN101813698A (en) * | 2010-04-02 | 2010-08-25 | 上海优你生物科技股份有限公司 | Kit for detecting beta-lactam antibiotic ligand in milk by receptor method and detection method thereof |
| CN103675256A (en) * | 2012-09-12 | 2014-03-26 | 天津科技大学 | Chloramphenicol immunoaffinity gel detection column |
| CN105388284A (en) * | 2015-11-20 | 2016-03-09 | 天津科技大学 | Immunoaffinity gel detection column for detecting enrofloxacin and preparation method thereof |
| CN106124265A (en) * | 2016-06-17 | 2016-11-16 | 中国海洋大学 | A kind of aquatic products remain extraction and the purification method of neomycin |
-
2017
- 2017-08-30 CN CN201710765091.1A patent/CN107389958A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1811453A (en) * | 2006-01-26 | 2006-08-02 | 台州金芯生物工程有限公司 | Animal source food animal medicine residue detection reagent kit and detecting method thereof |
| CN101349696A (en) * | 2008-08-25 | 2009-01-21 | 深圳市绿诗源生物技术有限公司 | Enzyme-linked immunologic reagent and method for detecting alficetin |
| CN101813698A (en) * | 2010-04-02 | 2010-08-25 | 上海优你生物科技股份有限公司 | Kit for detecting beta-lactam antibiotic ligand in milk by receptor method and detection method thereof |
| CN103675256A (en) * | 2012-09-12 | 2014-03-26 | 天津科技大学 | Chloramphenicol immunoaffinity gel detection column |
| CN105388284A (en) * | 2015-11-20 | 2016-03-09 | 天津科技大学 | Immunoaffinity gel detection column for detecting enrofloxacin and preparation method thereof |
| CN106124265A (en) * | 2016-06-17 | 2016-11-16 | 中国海洋大学 | A kind of aquatic products remain extraction and the purification method of neomycin |
Non-Patent Citations (2)
| Title |
|---|
| IRINA YU. GORYACHEVA ET AL.: "Immunoaffinity pre-concentration combined with on-column visual detection as a tool for rapid aflatoxin M1 screening in milk", 《FOOD CONTROL》 * |
| MAKSIM A. BURKIN ET AL.: "Development of a Competitive Indirect ELISA for the Determination of Lincomycin in Milk, Eggs, and Honey", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
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