CN107991487A - A kind of fluorescent microsphere immune test paper card, preparation and detection method for detecting carbostyril antibiotic - Google Patents
A kind of fluorescent microsphere immune test paper card, preparation and detection method for detecting carbostyril antibiotic Download PDFInfo
- Publication number
- CN107991487A CN107991487A CN201711220850.2A CN201711220850A CN107991487A CN 107991487 A CN107991487 A CN 107991487A CN 201711220850 A CN201711220850 A CN 201711220850A CN 107991487 A CN107991487 A CN 107991487A
- Authority
- CN
- China
- Prior art keywords
- pad
- sample
- detecting
- card
- fluorescent microsphere
- 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
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The present invention relates to a kind of fluorescent microsphere immune test paper card, preparation and detection method for detecting carbostyril antibiotic, belong to field of immunological detection, test card includes getting stuck and test strips, and test strips include bottom plate and overlap joint is pasted onto water absorption pad, detecting pad, bonding pad and sample pad on bottom plate successively；Detecting pad is the nitrocellulose filter equipped with nature controlling line C, detection line T, and nature controlling line C is coated with goat-anti mouse monoclonal antibody, detection line T coating Enrofloxacin bovine serum albumin(BSA) conjugates；The glass fibre element film for the enrofloxacin monoclonal antibody that bonding pad marks for embedding time-resolved fluorescence microballoon；Sample pad is dry glass fibre element film after sample treatment liquid immersion treatment；Bottom plate is the PVC board not absorbed water, and water absorption pad is absorbent filter.Test card stability prepared by the present invention is good, high sensitivity, high specificity, can detect that 16 kinds of carbostyril antibiotics, and can achieve the purpose that fast quantification and batch detection to Enrofloxacin by fluorescence signal.
Description
Technical field
The invention belongs to field of immunological detection, relates generally to a kind of fluorescent microsphere for detecting carbostyril antibiotic and is immunized
Test card, preparation and detection method.
Background technology
Carbostyril antibiotic is the general medicine of a kind of people and animals, it be the DNA supercoils enzyme using bacterium as target, hinder this
Enzyme plays supercoil effect, causes the irreversible lesion of DNA of bacteria, bacterial cell is no longer divided.Because it is with broad-spectrum antiseptic
Property, antibacterial activity it is strong, with other antibacterials without cross resistance and toxic side effect it is small the features such as, be widely used in herding,
In the Animal diseases prevention and treatment of the aquacultures such as aquatic products.Continuous with animal-breeding scale expands, in breeding process blindly,
Antibiotic, which is excessively used, to be caused to remain quinolone drugs in animal derived food, people eat after by the serious anti-medicine of the medicine
Property, moment threaten human health.
The current detection method in relation to carbostyril antibiotic is high performance liquid chromatography, although the method high sensitivity,
Stablize, but because of high cost, cumbersome, detection cycle is grown, be only applicable to testing agency and grass-roots unit can not be applied to.In addition
The methods of fluorescence spectrum, Capillary Electrophoresis, is also had been reported that applied to detection carbostyril antibiotic, but sensitivity is not so good as immunology
Detection method.It is that in the market is most widely used with the simple and efficient immunoassay technology for advantage, wherein, fluorescent microsphere is made
Immunolabelling technique for tracer is the current research hotspot in this area.Fluorescent microsphere refers to that fluorescent material passes through and embeds, altogether
The modes such as valence link connection are introduced into organic or inorganic nano-particle, and the parcel of nano material makes it have good stability.With
Widely used Enzyme-linked Immunosorbent Assay (ELISA) kit compares, and fluorescent micro-ball immune chromatography technology need not be equipped with microplate reader, behaviour
Make simplicity, more fast.Compared to the colloidal gold immunochromatographimethod technology of same type, embed a large amount of fluorescence molecules and make it have higher
Sensitivity；Fluorescence signal can reach the effect of fast quantification by fluorescence analyzer detection at the same time.But how using glimmering
Light micro-ball immune chromatography technology for detection or preparation detection product testing carbostyril antibiotic residual have no document and clearly remember so far
Carry, it is even more blank to detect a variety of quinolone antibiotics at the same time using one kind detection product.
The content of the invention
To be immunized in view of the above-mentioned problems, it is an object of the invention to provide a kind of fluorescent microsphere for detecting carbostyril antibiotic
Test card, preparation and detection method.The kit can be detected simultaneously by 16 kinds of carbostyril antibiotics, and sensitivity is good, special
Property it is high, stability is strong, Laboratory Instruments and condition can not depended on directly outdoors or basic unit's Rapid identification.
A kind of preparation method for the fluorescent microsphere immune test paper card for detecting carbostyril antibiotic, comprises the following steps：
Step 1: the preparation of detecting pad：The goat-anti mouse monoclonal antibody coating buffer that concentration is 0.8mg/mL is prepared, concentration is
Two kinds of coating buffers are each individually drawn to Film-cutting machine pipe by the Enrofloxacin of 0.4mg/mL-bovine serum albumin(BSA) conjugate coating buffer
In line, nature controlling line C and detection line T are marked successively on nitrocellulose filter according to 0.8 μ L/cm, wherein, goat-anti murine monoclonal
Antibody coating buffer marks nature controlling line C, and Enrofloxacin-bovine serum albumin(BSA) conjugate coating buffer marks detection line T, by scribed line
Nitrocellulose filter is placed in 37 DEG C of air dry ovens dry 3-4h, and detecting pad is made.
Step 2: the preparation of bonding pad：Fluorescent microsphere dispersion liquid is placed in centrifuge tube, MES buffer solutions are added, in ultrasound
Under the conditions of mix, centrifugation abandon supernatant collect microballoon precipitation, repeat this step to clean microballoon, microballoon is precipitated and dissolved in MES and is delayed
In fliud flushing, mixing, add EDC solution, 22-25 DEG C of lucifuge activation, centrifugation abandons supernatant, adds MES buffer solutions, and ultrasound breaks up microballoon,
Supernatant is abandoned in centrifugation, adds borate buffer, and ultrasound is broken up, and adds enrofloxacin monoclonal antibody, lucifuge reaction, adds glycine
Solution, lucifuge reaction 30min, adds BSA solution, lucifuge reaction 30min, completes closing, centrifugation is redissolved, and it is micro- to form fluorescence
Ball-antibody complex solution, fluorescent microsphere-antibody complex solution is sprayed onto on glass fibre element film, dry, is made and is combined
Pad.
Step 3: the preparation of sample pad：The tiling of glass fibre element film is put into the sample pad treatment fluid configured, is soaked
0.5h, is put into 37 DEG C of air dry ovens, dry 8-10h, and sample pad is made, and the sample pad treatment fluid is the PBS of pH 7.4,
The component and its concentration wherein contained be respectively：Mass fraction is 0.5% sucrose, and mass fraction is 0.5% PVP-K30,
Mass fraction is 0.5% PEG-20000, and mass fraction is 0.1% PEG-6000, and mass fraction is 0.3% casein,
Volume fraction is 0.5% Tween 20, and mass fraction is 0.1% S9, and mass fraction is 0.1% S17 and mass fraction
For 0.1% S21.
Step 4: the assembling of test card：Using PVC board as bottom plate, by sample pad, bonding pad, detecting pad and water absorption pad successively
Overlap joint is pasted onto on bottom plate, there is when each adjacent overlap joint that 1-2mm is overlapping, and makes the detection line T on detecting pad close to sample pad
The PVC board posted is cut into strip with cutting machine, test strips is formed, by test strips by side, nature controlling line C close to water absorption pad side
Combined with getting stuck, form test card, be sealed.
Further, the particle diameter of fluorescent microsphere described in step 2 is 200nm.
Further, in fluorescent microsphere described in step 2-antibody complex solution, the concentration of enrofloxacin monoclonal antibody
For 3 μ g/mL.
The present invention also protects a kind of fluorescent microsphere of the detection carbostyril antibiotic prepared using the above method that examination is immunized
Paper card, the test card include the interior test strips that get stuck that get stuck and be located at, and test strips include bottom plate and overlap joint is pasted onto successively
Water absorption pad, detecting pad, bonding pad and sample pad on bottom plate；The detecting pad is that the nitric acid equipped with nature controlling line C, detection line T is fine
The plain film of dimension, nature controlling line C coating goat-anti mouse monoclonal antibodies, detection line T coating Enrofloxacin-bovine serum albumin(BSA) conjugates；Institute
State glass fibre element film of the bonding pad for the enrofloxacin monoclonal antibody of embedding time-resolved fluorescence microballoon mark；The bottom plate
For the PVC board not absorbed water, water absorption pad is absorbent filter.
It is further, described to get stuck including the Ka Gai and deck mutually fastened, the interior card slot for being equipped with fixed test strips of deck,
The region that card is covered corresponding to detecting pad is equipped with form, blocks the region covered corresponding to sample pad and is equipped with well.
The method of the above-mentioned fluorescent microsphere immune test paper card detection carbostyril antibiotic of further Sustainable use of the invention, tool
Gymnastics is made as follows：
Sample treatment：Weigh the solid sample through homogeneous to be placed in centrifuge tube, add 70% ethyl acetate solution, oscillator is acute
Violent shock swings at least 5min, and centrifuging and taking supernatant, is dried up with nitrogen evaporator, forms coagulation, adds sample diluting liquid and dissolves coagulation
And dilute, obtain sample detection liquid；Or directly dilute liquid sample through sample diluting liquid, obtain sample detection liquid；
Measure：Sample detection drop is added in the well of test card, reacts 5min, test card insertion Portable fluorescence is exempted from
In epidemic disease analyzer, T/C values are read, according to concentration standard curve, is calculated in counter sample and examines carbostyril antibiotic
Concentration.
Further, the sample diluting liquid is the PBS solution of concentration 0.02mol/L, wherein being containing volume fraction
0.5% methanol.
The principle of test card of the present invention detection carbostyril antibiotic is：It will be added dropwise containing the sample of antibiotic to be measured
Small molecule antibiotic and the fluorescent microsphere in bonding pad-enrofloxacin monoclonal antibody compound in sample application zone, sample is special
The opposite sex is combined and chromatographed through capillary action to water absorption pad side, after the detection line of fixed antigen is reached, in detection line
Antigen and the binding site on the antibiotic competition antibody in sample, and unnecessary Fluorescent microsphere marker continues to chromatograph forward,
Combined with the sheep anti mouse monoclonal antibody for being fixed on nature controlling line.After reaction, detecting pad is scanned with ultraviolet source, detection line T and matter
The fluorescent microsphere of control line C sends high intensity fluorescence.In addition the fluorescence decay time of fluorescent microsphere rare earth elements is glimmering compared to common
Light blob is grown, and is its 103~106 times.By delaying time of measuring, treat that luminous short life fluorescence all declines naturally in sample substrate
After change, then the specificity fluorescent of rare earth element is measured, reach the effect that background interference is completely eliminated, realize high s/n ratio.
Beneficial effects of the present invention：
1st, test card of the present invention is prepared based on fluorescent microsphere, utilizes antibiotic molecule and the antibody of fluorescent microsphere mark
Compound combines, and then the antigen in detection line and the binding site on carbostyril antibiotic molecule competition antibody, fluorescence are micro-
Ball sends high intensity fluorescence, and the quantitative detection to carbostyril antibiotic is realized by the power and standard curve of fluorescence signal,
Compared to ELISA kit, colloid gold immune test paper bar or other common fluorescent immunochromatography techniques, test card of the present invention
Sensitivity to be higher by the 2-3 order of magnitude, secondly, the fluorescent microsphere with carboxyl group is connected with antibody with covalent bond, stability
Higher.In addition, test card of the present invention, in addition to having the advantages that general ELISA test strip convenience etc., it can detect 16 kinds of quinoline promises
Ketone antibiotic, compared to ELISA kit detection time, whole process only needs 10min, substantially increase detection efficiency, save into
This, is highly suitable for on-site quick screening, and a kind of effective technology is provided for the remaining detection of carbostyril antibiotic
Means.
2nd, of the present invention in the preparation process of test card, the preparation method of each component determines its detection sensitivity
Or the characteristic such as stability, wherein, in bonding pad preparation, EDC processing promotes itself and antibody to activate microsphere surface carboxylic group
Combination, then be sequentially added glycine and BSA and carry out closed for second time, nonspecific reaction can be avoided to do testing result
Disturb, in sample pad preparation, Multiple components are included in sample pad treatment fluid, acts synergistically between the component of various concentrations, makes leaching
Bubble processing after sample pad while mobility is ensured, homogeneity and stability are preferable, improve immune effect, for accurately,
The content for delicately detecting carbostyril antibiotic in sample is laid a good foundation.
Brief description of the drawings
Fig. 1 is test strips of the present invention and stuck structure schematic diagram, wherein：1st, sample pad, 2, bonding pad, 3, detecting pad, 4,
Water absorption pad；
Fig. 2 is Enrofloxacin canonical plotting.
Embodiment
Below by specific embodiment the present invention will be further explained explanation.
It is prepared by embodiment one, test strips
Preparation process includes：The preparation of fluorescent microsphere-antibody complex, the preparation of bonding pad, sample pad preparation, the system of detecting pad
Standby and test strips assemblings.
1st, the preparation of fluorescent microsphere-antibody complex：
(1) clean：50 μ L fluorescent microspheres are taken into 1.5mL centrifuge tubes, are added in 1mL 0.01M MES buffer solutions, concussion mixes,
15000r/min centrifuges 15min, abandons supernatant, adds 1mL 0.01M MES buffer solutions, and ultrasound breaks up microballoon, repeats this step 3
It is secondary, achieve the purpose that to clean microballoon,
(2) activate：Into the 1mL microballoon liquid after cleaning, 250 μ L EDC solutions, lucifuge activation 2h, 15000r/min centrifugation are added
15min, abandons supernatant, adds 1mL 0.01M MES buffer solutions, and ultrasound breaks up microballoon, and 15000r/min centrifugation 15min, abandon supernatant,
(3) mark：1mL 0.05M borate buffer solutions are added, ultrasound is broken up, and adds 10 μ g enrofloxacin monoclonal antibodies, is mixed
It is even, lucifuge reaction 2h,
(4) 1 is closed：100 μ L1M glycine solutions are added, lucifuge reacts 30min,
(5) 2 are closed：100 μ L10%BSA solution are added, lucifuge reacts 30min,
(6) centrifugation is redissolved：15000r/min centrifuges 15min, abandons supernatant, adds 100 μ L dilutions, and ultrasound breaks up microballoon, prepares
Into fluorescent microsphere-antibody complex solution.
Wherein, the particle diameter of fluorescent microsphere used is 200nm；Absorbing wavelength is 365nm, and Detection wavelength is 610nm.
2nd, the preparation of bonding pad：
The fluorescent microsphere of above-mentioned preparation-antibody complex solution is sprayed onto on glass fibre element film according to 3 μ L/cm, 37 DEG C of dryings
3-4h is the bonding pad, and dried bonding pad is put into spare in drier.
3rd, prepared by sample pad：
(1) PBS of pH 7.4 is configured：Weigh 8g NaCl, 5.8g Na2HPO4, 0.2g KCl, 0.2g KH2PO4It is dissolved in 1L purifying
In water, pH to 7.4 is adjusted,
(2) sample pad treatment fluid is configured：Accurately weigh 5g sucrose, 5g PVP-K30,5g PEG-20000,1g PEG-6000,3g
Casein, 1g S9,1g S17,1g S21,5mL Tween-20, into the PBS of 1L pH 7.4, mixes on magnetic stirring apparatus,
Sample pad treatment fluid is obtained, it is spare.Wherein, the S9 is a kind of amphoteric surfactant for Tetronic 1307, and the S17 is
Ohodasurf ON-870, are a kind of emulsifying agents, and the S21 is BRIJ 35, is a kind of nonionic surfactant.S9、S17
After the sample pad treatment fluid being added to S21, the good immune effect of more general processing method,
(3) processing of sample pad：Glass fibre element film is loaded in valve bag, by the 80mL/ sample pads for adding above-mentioned preparation
Treatment fluid, soaks 30min,
(4) drying of sample pad：The glass fibre element film fully soaked is put into 37 DEG C of air dry ovens, dry 8-10h,
Sample pad is obtained, sample pad is put into stand-by in drier.
4th, the preparation of detecting pad：
(1) preparation of nature controlling line coating buffer：Sheep anti mouse monoclonal antibody is diluted to 0.8mg/mL with coating working solution,
(2) preparation of detection line coating buffer：Enrofloxacin-bovine serum albumin(BSA) conjugate is diluted to coating working solution
0.4mg/mL,
(3) coated film：Above two coating buffer is distinguished into suck-back into Film-cutting machine pipeline, according to 0.8 μ L/cm, is marked successively
Nature controlling line C, detection line T, the spacing between two lines is 4-5mm, is then placed in 37 DEG C of air dry ovens, dry 3-4h, i.e.,
Detecting pad is prepared into, is put into stand-by in drier.The detecting pad is active ingredient and the active matter being fixed on film in sample
The region that matter is specifically bound.
5th, the assembling of test strips：
Using the PVC adhesive sheets not absorbed water as bottom plate, sample pad, sample bonding pad, detecting pad and water absorption pad are overlapped into stickup successively
On bottom plate, each component has that 1-2mm is overlapping in adjacent, and the PVC board posted is cut into the test strips of 3mm wide with cutting machine, leads to
The card slot crossed in deck is combined with deck, and deck closes to form test card with card cover buckle.Wherein, card covers the region of corresponding detecting pad
Equipped with form, the T lines on detecting pad have " T " mark, nature controlling line on the form side that sample pad side, corresponding card cover
C lines have " C " mark on the form side that water absorption pad side, corresponding card cover, and card covers the region corresponding to sample pad
Equipped with well, well has " S " mark on side, as shown in Figure 1.The water absorption pad is absorbent filter, and absorbent filter is cut
Grow up 30cm, wide 1.9-2.1cm.The water absorption pad has siphon effect.
The specification of the Ka Gai is 70mm*20mm*5mm, and the form size at Ka Gai centers is 16mm*3mm.
The drafting of embodiment two, standard curve
1st, the configuration of standard items：
With the PBS of 0.1M pH 7.4 by Enrofloxacin standard items gradient dilution into 5ppb, 10ppb, 20ppb, 40ppb, 80ppb,
2nd, reading：
50 μ L above-mentioned standards product point cards are taken respectively, after reacting 5min, are corresponded to card slot in Portable fluorescence immunity analysis instrument and are read
Number, the result is shown in shown in table 1 below for reading：
3rd, the verification of computational methods：
(1) four parametric method, after Enrofloxacin curve relevant parameter is inputted Portable fluorescence immunity analysis instrument, in standard curve model
Enclose interior selection 5ppb and 20ppb configuration standard product, point card, reacts 5min, read two concentration be respectively 2.454ppb and
0.885ppb,
(2) piecewise linearity, it is bent in standard after Enrofloxacin standard curve relevant parameter is inputted Portable fluorescence immunity analysis instrument
In the range of line select 5ppb and 20ppb configuration standard product, point card, react 5min, read two concentration be respectively 4.891ppb and
19.990ppb,
By two groups of data comparisons, it can be concluded that, the computational methods of sample concentration should select piecewise linearity.
(4) verification and drafting of standard curve：Reading value in step 2 is inputted into recurrence/the Fitting Calculation program, draws curve
Figure, as shown in Figure 2.
Embodiment three, to detect in meat products exemplified by carbostyril antibiotic, compare fluorescent microsphere test strips and colloidal gold
The sensitivity of test strips
1st, with homogenizer homogeneous fresh pork sample；
2nd, the sample of two parts of 5.0 ± 0.05g grindings is weighed into two 50mL polystyrene centrifuge tubes, is separately added into 25ml
70% ethyl acetate, its middle pipe 2 add 10ng Enrofloxacins, acutely vibrate 5min with oscillator；
3rd, the sample after shaken well is placed in centrifuge, 6000r/min centrifugations 5min；
4th, take 1mL centrifuged supernatants to be dried up with nitrogen evaporator, 1mL sample diluting liquids are accurately added after drying coagulation is fully molten
Solve up to sample extracting solution；The sample diluting liquid is the PBS of pH7.4,0.05mol/L；
5th, 1 is pressed with sample diluting liquid:9 dilution proportion (examples：+ 900 μ L samples dilution of 100 μ L samples extracting solution)；
6th, take 50 μ L of liquid after being diluted after being diluted in pipe 1 in 50 μ L of liquid and pipe 2 that card 1 is added dropwise respectively and blocks 2 respectively, clock reaction
5min；
7th, card 1 is inserted into reading in Portable fluorescence immunity analysis instrument card slot, display density value is 0, card 2 is inserted into portable glimmering
Reading in light immunity analysis instrument card slot, display Enrofloxacin concentration value is 1.964ng/mL；
8th, with the comparison of colloidal-gold detecting-card：The dilution handled well in pipe 1, pipe 2 is separately added into carbostyril antibiotic glue
Body gold detection card 1 and detection card 2, react 10min.As a result：1 outlet two lines of colloid gold card, are presented negative findings, colloid gold card
2 there are two lines, and negative findings is presented.
Above-mentioned experimental result further verifies that the sensitiveness of fluorescent microsphere test strips is compared with colloidal gold strip height.
Example IV, compare fluorescent microsphere mark enrofloxacin monoclonal antibody compound and colloid gold label Enrofloxacin
The stability of monoclonal antibody complex
1st, by the bonding pad of the colloidal gold prepared-enrofloxacin monoclonal antibody compound and fluorescent microsphere-Enrofloxacin list
The bonding pad of clonal antibody compound is placed in 37 DEG C of air dry ovens,
2nd, on day 3, respectively to negative sample and positive sample at 5 days, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 20 weeks, 36 weeks, 48 weeks
Originally it is detected, observes its stability,
3rd, inspection result is as shown in table 2 below：
By upper table 2 it could be assumed that：The stability of fluorescent microsphere labelled antibody compound is compared with colloidal gold labeled monoclonal antibody compound
By force.
Embodiment five, cross reaction experiment
Norfloxacin, Enoxacin, Ciprofloxacin, Pefloxacin, Ofloxacin, Danofloxacin, Lomefloxacin, fiber crops are selected to protect husky
15 kinds of star, flumequine, oxolinic acid, Difloxacin, sarafloxacin, Nadifloxacin, Orbifloxacin and Lomefloxacin Du-6859as
Thing；7 kinds of selection sulphadiazine, ampicillin, streptomysin, erythromycin, tetracycline, chloramphenicol and nitrofurazone are not belonging to quinoline promise
The antibiotic of ketone, cross reacting rate is measured according to the test strips application method of embodiment three respectively.The results show that with En Nuosha
Asterisk directrix curve is above 50% for the cross reacting rate of other 15 kinds of quinolone drugs of curve determination built in fluorescence detector；
Other 7 kinds other equal no cross reactions of class medicine, high specificity.It follows that fluorescent microsphere immune test paper prepared by the present invention
Enrofloxacin can be detected outside by blocking, and can also determine Norfloxacin in sample to be tested, Enoxacin, Ciprofloxacin, training
Flucloxacillin, Ofloxacin, Danofloxacin, Lomefloxacin, marbofloxacin, flumequine, oxolinic acid, Difloxacin, sarafloxacin, that
15 kinds of Flucloxacillin, Orbifloxacin and Lomefloxacin carbostyril antibiotic residuals.
Claims (7)
- A kind of 1. preparation method for the fluorescent microsphere immune test paper card for detecting carbostyril antibiotic, it is characterised in that：Including with Lower step：Step 1: the preparation of detecting pad：Prepare the goat-anti mouse monoclonal antibody coating buffer that concentration is 0.8mg/mL, concentration 0.4 Two kinds of coating buffers are each individually drawn to Film-cutting machine pipeline by the Enrofloxacin of mg/mL-bovine serum albumin(BSA) conjugate coating buffer In, nature controlling line C and detection line T are marked successively on nitrocellulose filter according to 0.8 μ L/cm, wherein, goat-anti murine monoclonal resists Body coating buffer marks nature controlling line C, and Enrofloxacin-bovine serum albumin(BSA) conjugate coating buffer marks detection line T, by the nitre of scribed line Acid cellulose film is placed in 37 DEG C of air dry ovens dry 3-4 h, and detecting pad is made；Step 2: the preparation of bonding pad：Fluorescent microsphere dispersion liquid is placed in centrifuge tube, MES buffer solutions are added, in ultrasound condition Lower mixing, centrifugation abandon supernatant and collect microballoon precipitation, repeat this step to clean microballoon, microballoon is precipitated and dissolved in MES buffer solutions In, mix, add EDC solution, 22-25 DEG C of lucifuge activation, centrifugation abandons supernatant, adds MES buffer solutions, and ultrasound breaks up microballoon, centrifugation Supernatant is abandoned, adds borate buffer, ultrasound is broken up, and adds enrofloxacin monoclonal antibody, lucifuge reaction, it is molten to add glycine Liquid, lucifuge react 30 min, add BSA solution, and lucifuge reacts 30 min, complete closing, and centrifugation is redissolved, and it is micro- to form fluorescence Ball-antibody complex solution, fluorescent microsphere-antibody complex solution is sprayed onto on glass fibre element film, dry, is made and is combined Pad；Step 3: the preparation of sample pad：The tiling of glass fibre element film is put into the sample pad treatment fluid configured, immersion 0.5 H, is put into 37 DEG C of air dry ovens, dry 8-10 h, and sample pad is made, and the sample pad treatment fluid is the PBS of pH 7.4, its In the component that contains and its concentration be respectively：Mass fraction be 0.5% sucrose, mass fraction be 0.5% PVP-K30, matter Measure fraction be 0.5% PEG-20000, mass fraction be 0.1% PEG-6000, mass fraction be 0.3% casein, volume Fraction is 0.5% Tween 20, and mass fraction is 0.1% S9, and the S17 and mass fraction that mass fraction is 0.1% are 0.1% S21；Step 4: the assembling of test card：Using PVC board as bottom plate, sample pad, bonding pad, detecting pad and water absorption pad are overlapped successively It is pasted onto on bottom plate, there is when each adjacent overlap joint that 1-2 mm are overlapping, and makes the detection line T on detecting pad close to sample pad one The PVC board posted is cut into strip by side, nature controlling line C close to water absorption pad side, with cutting machine, forms test strips, by test strips with Get stuck combination, form test card, be sealed.
- 2. a kind of preparation method of fluorescent microsphere immune test paper card for detecting carbostyril antibiotic as claimed in claim 1, It is characterized in that：The particle diameter of fluorescent microsphere described in step 2 is 200 nm.
- 3. a kind of preparation method of fluorescent microsphere immune test paper card for detecting carbostyril antibiotic as claimed in claim 1, It is characterized in that：In fluorescent microsphere described in step 2-antibody complex solution, the concentration of enrofloxacin monoclonal antibody is 3 μ g/mL。
- 4. examination is immunized in the fluorescent microsphere such as detection carbostyril antibiotic prepared by methods of the claim 1-3 as described in any one Paper card, it is characterised in that：Test card includes the interior test strips that get stuck that get stuck and be located at, and test strips include bottom plate and overlap successively Water absorption pad, detecting pad, bonding pad and the sample pad being pasted onto on bottom plate；The detecting pad is equipped with nature controlling line C, detection line T Nitrocellulose filter, nature controlling line C coating goat-anti mouse monoclonal antibodies, detection line T coatings Enrofloxacin-bovine serum albumin(BSA) coupling Thing；The glass fibre element film for the enrofloxacin monoclonal antibody that the bonding pad marks for embedding time-resolved fluorescence microballoon；Institute It is the PVC board not absorbed water to state bottom plate, and water absorption pad is absorbent filter.
- A kind of 5. fluorescent microsphere immune test paper card for detecting carbostyril antibiotic as claimed in claim 4, it is characterised in that： Described get stuck covers to correspond to and detects including the Ka Gai and deck mutually fastened, the interior card slot for being equipped with fixed test strips of deck, card The region of pad is equipped with form, and the region that card covers corresponding to sample pad is equipped with well.
- 6. utilize the method for fluorescent microsphere immune test paper card as claimed in claim 5 detection carbostyril antibiotic, its feature It is：Concrete operations are as follows：Sample treatment：Weigh the solid sample through homogeneous to be placed in centrifuge tube, add 70% ethyl acetate solution, oscillator is acute Violent shock swings at least 5 min, and centrifuging and taking supernatant, is dried up with nitrogen evaporator, forms coagulation, adds sample diluting liquid and dissolves coagulation And dilute, obtain sample detection liquid；Or directly dilute liquid sample through sample diluting liquid, obtain sample detection liquid；Measure：Sample detection drop is added in the well of test card, 5 min is reacted, test card is inserted into Portable fluorescence In immunity analysis instrument, T/C values are read, according to concentration standard curve, is calculated in counter sample and examines carbostyril antibiotic Concentration.
- 7. the method for fluorescent microsphere immune test paper card detection carbostyril antibiotic, its feature are utilized as claimed in claim 6 It is：The sample diluting liquid is the PBS solution of 0.02 mol/L of concentration, wherein containing the methanol that volume fraction is 0.5%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711220850.2A CN107991487A (en) | 2017-11-29 | 2017-11-29 | A kind of fluorescent microsphere immune test paper card, preparation and detection method for detecting carbostyril antibiotic |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711220850.2A CN107991487A (en) | 2017-11-29 | 2017-11-29 | A kind of fluorescent microsphere immune test paper card, preparation and detection method for detecting carbostyril antibiotic |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107991487A true CN107991487A (en) | 2018-05-04 |
Family
ID=62033927
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711220850.2A Pending CN107991487A (en) | 2017-11-29 | 2017-11-29 | A kind of fluorescent microsphere immune test paper card, preparation and detection method for detecting carbostyril antibiotic |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107991487A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108680747A (en) * | 2018-06-26 | 2018-10-19 | 宁波奥丞生物科技有限公司 | A kind of placenta growth factor detection immunofluorescence technique kit |
| CN108918866A (en) * | 2018-06-21 | 2018-11-30 | 河南省生物工程技术研究中心有限公司 | A kind of marker of inflammation POCT combined detection kit suit |
| CN109781691A (en) * | 2019-02-28 | 2019-05-21 | 渤海大学 | A method of based on pipe/polyhenylethylene nano fluorescent microsphere probe in detecting chloramphenicol |
| CN110702896A (en) * | 2019-10-12 | 2020-01-17 | 青海省动物疫病预防控制中心 | Time-resolved fluorescence immunochromatographic assay quantitative detection method for quinolone drugs in yak meat |
| CN110780067A (en) * | 2019-11-04 | 2020-02-11 | 南京欧凯生物科技有限公司 | Fluorescence immunochromatographic test paper for detecting procalcitonin and preparation method thereof |
| CN111308067A (en) * | 2020-02-03 | 2020-06-19 | 南京农业大学 | Quantum dot microsphere immunochromatography test strip for quantitatively detecting fluoroquinolone drugs |
| CN111505271A (en) * | 2020-03-10 | 2020-08-07 | 玉林师范学院 | Preparation method of olaquindox rapid detection test strip |
| CN111624335A (en) * | 2020-06-10 | 2020-09-04 | 光景生物科技(苏州)有限公司 | Norovirus GI/GII antigen combined quantitative detection kit and preparation method thereof |
| CN111638327A (en) * | 2020-07-01 | 2020-09-08 | 成都赛普克生物科技股份有限公司 | Colloidal gold immunochromatographic test paper gold label pad, treatment solution for sample pad and treatment method |
| CN113295861A (en) * | 2021-05-20 | 2021-08-24 | 深圳无微华斯生物科技有限公司 | Rapid detection method of near-infrared fluorescent microsphere immunochromatographic test strip |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101487840A (en) * | 2009-01-14 | 2009-07-22 | 天津九鼎医学生物工程有限公司 | Reagent kit for quantitatively detecting enrofloxacin content in food and its use |
| CN102023210A (en) * | 2009-09-21 | 2011-04-20 | 北京中德大地食品安全技术开发有限责任公司 | Fluorescent microsphere immunochromatography detection card for detecting enrofloxacin and preparation method thereof |
| CN102618502A (en) * | 2012-01-09 | 2012-08-01 | 浙江大学 | Hybridoma cell strain capable of secreting monoclonal antibodies to quinolones and application of monoclonal antibodies thereof |
| CN103091495A (en) * | 2013-01-16 | 2013-05-08 | 河南知微生物工程有限公司 | Test paper card for quickly detecting residues of fluoroquinolones and preparation method of test paper card |
| CN104090248A (en) * | 2013-12-24 | 2014-10-08 | 上海容晖生物科技有限公司 | Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay |
| CN104530459A (en) * | 2014-12-10 | 2015-04-22 | 南京邮电大学 | Preparation method of polystyrene fluorescent microsphere coupled with antibody |
| CN105044365A (en) * | 2015-07-10 | 2015-11-11 | 天水师范学院 | Preparation method of time-resolved fluoroimmunoassay test paper for detection of Enrofloxacin residue |
| CN105785014A (en) * | 2016-04-25 | 2016-07-20 | 成都盛泰尔生物医药科技有限公司 | Colloidal-gold-based quantitative mycotoxin detection device and preparation method |
| CN105891468A (en) * | 2016-04-08 | 2016-08-24 | 四川新健康成生物股份有限公司 | Sample release pad treatment solution |
-
2017
- 2017-11-29 CN CN201711220850.2A patent/CN107991487A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101487840A (en) * | 2009-01-14 | 2009-07-22 | 天津九鼎医学生物工程有限公司 | Reagent kit for quantitatively detecting enrofloxacin content in food and its use |
| CN102023210A (en) * | 2009-09-21 | 2011-04-20 | 北京中德大地食品安全技术开发有限责任公司 | Fluorescent microsphere immunochromatography detection card for detecting enrofloxacin and preparation method thereof |
| CN102618502A (en) * | 2012-01-09 | 2012-08-01 | 浙江大学 | Hybridoma cell strain capable of secreting monoclonal antibodies to quinolones and application of monoclonal antibodies thereof |
| CN103091495A (en) * | 2013-01-16 | 2013-05-08 | 河南知微生物工程有限公司 | Test paper card for quickly detecting residues of fluoroquinolones and preparation method of test paper card |
| CN104090248A (en) * | 2013-12-24 | 2014-10-08 | 上海容晖生物科技有限公司 | Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay |
| CN104530459A (en) * | 2014-12-10 | 2015-04-22 | 南京邮电大学 | Preparation method of polystyrene fluorescent microsphere coupled with antibody |
| CN105044365A (en) * | 2015-07-10 | 2015-11-11 | 天水师范学院 | Preparation method of time-resolved fluoroimmunoassay test paper for detection of Enrofloxacin residue |
| CN105891468A (en) * | 2016-04-08 | 2016-08-24 | 四川新健康成生物股份有限公司 | Sample release pad treatment solution |
| CN105785014A (en) * | 2016-04-25 | 2016-07-20 | 成都盛泰尔生物医药科技有限公司 | Colloidal-gold-based quantitative mycotoxin detection device and preparation method |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108918866A (en) * | 2018-06-21 | 2018-11-30 | 河南省生物工程技术研究中心有限公司 | A kind of marker of inflammation POCT combined detection kit suit |
| CN108680747A (en) * | 2018-06-26 | 2018-10-19 | 宁波奥丞生物科技有限公司 | A kind of placenta growth factor detection immunofluorescence technique kit |
| CN109781691A (en) * | 2019-02-28 | 2019-05-21 | 渤海大学 | A method of based on pipe/polyhenylethylene nano fluorescent microsphere probe in detecting chloramphenicol |
| CN110702896A (en) * | 2019-10-12 | 2020-01-17 | 青海省动物疫病预防控制中心 | Time-resolved fluorescence immunochromatographic assay quantitative detection method for quinolone drugs in yak meat |
| CN110780067A (en) * | 2019-11-04 | 2020-02-11 | 南京欧凯生物科技有限公司 | Fluorescence immunochromatographic test paper for detecting procalcitonin and preparation method thereof |
| CN111308067A (en) * | 2020-02-03 | 2020-06-19 | 南京农业大学 | Quantum dot microsphere immunochromatography test strip for quantitatively detecting fluoroquinolone drugs |
| CN111505271A (en) * | 2020-03-10 | 2020-08-07 | 玉林师范学院 | Preparation method of olaquindox rapid detection test strip |
| CN111624335A (en) * | 2020-06-10 | 2020-09-04 | 光景生物科技(苏州)有限公司 | Norovirus GI/GII antigen combined quantitative detection kit and preparation method thereof |
| CN111638327A (en) * | 2020-07-01 | 2020-09-08 | 成都赛普克生物科技股份有限公司 | Colloidal gold immunochromatographic test paper gold label pad, treatment solution for sample pad and treatment method |
| CN111638327B (en) * | 2020-07-01 | 2023-10-13 | 成都赛普克生物科技股份有限公司 | Treatment fluid and treatment method for colloidal gold immunochromatographic test paper gold-labeled pad and sample pad |
| CN113295861A (en) * | 2021-05-20 | 2021-08-24 | 深圳无微华斯生物科技有限公司 | Rapid detection method of near-infrared fluorescent microsphere immunochromatographic test strip |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107991487A (en) | A kind of fluorescent microsphere immune test paper card, preparation and detection method for detecting carbostyril antibiotic | |
| CN107976541A (en) | Test card, preparation and the detection method of synchronous detection zearalenone and aflatoxin B1 | |
| CN107942062A (en) | Test card, preparation and the detection method of synchronous detection 2 toxin of ochracin, vomitoxin and T | |
| CN101470114B (en) | Sensitization detection method of colloidal gold immunity chromatography and use thereof | |
| CN104090248A (en) | Reagent for quantitative detection of Beta-receptor stimulant through Europium chelate latex time-resolved immunochromatographic assay | |
| CN107942061A (en) | A kind of test card, preparation and its detection method for detecting transmissible gastro-enteritis virus antibody | |
| CN201965131U (en) | Quick test paper of hepatitis A virus (HAV) IgM antibody | |
| CN205910196U (en) | Colloidal gold test paper card of short -term test dog coronavirus antibody | |
| JP2002539425A (en) | Sample collection and test system | |
| CA2324096A1 (en) | Integrated assay device and methods of production and use | |
| CN101576563A (en) | Test paper for rapidly detecting immunochromatography of cadmium ion colloidal gold and preparation method and application thereof | |
| CN101553729A (en) | Saturation assay | |
| CN102890152A (en) | Test strip and method for fast quantitative detection of drug in blood | |
| CN101881769A (en) | Colloidal gold immunochromatographic test strip for detecting shrimp allergen and preparation method thereof | |
| CN101441210A (en) | Nano magnetic particle chromatography test paper detection method | |
| CN205861689U (en) | A kind of detection device | |
| CN107621539A (en) | A kind of method of analyte in detection means and detection liquid sample | |
| CN106370861A (en) | C reactive protein saliva test paper strip and preparation method thereof | |
| CN107748252A (en) | Immune test paper card, preparation and the detection method of detection goats contagious pleuropneumonia antibody based on fluorescent microsphere | |
| CN109406778A (en) | The time-resolved fluorescence quantitative test paper item and its preparation method and application of ralstonia solanacearum in a kind of detection tobacco leaf | |
| CA2513579A1 (en) | Immunoassay and method of use | |
| CN104614511A (en) | Immune chromatography analysis method, immune chromatography analysis device, and immune chromatography analysis reagent box | |
| CN107328938A (en) | Propepsin and helicobacter pylori antibody detection method and its kit | |
| CN103175965A (en) | Fecal occult blood detection kit and preparation method | |
| CN105785041A (en) | Test strip for quantitatively detecting calprotectin, preparation method thereof and determining method for calprotectin concentration |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Li Xiumei Inventor after: Yang Zhi Inventor after: Xiao Aibo Inventor after: Xu Guorong Inventor after: Wang Hu Inventor after: Geng Yujing Inventor after: Zhang Xiaofei Inventor after: Yang Erxia Inventor after: Ji Yingbin Inventor before: Li Xiumei Inventor before: Yang Zhi Inventor before: Geng Yujing Inventor before: Zhang Xiaofei Inventor before: Yang Erxia Inventor before: Ji Yingbin |
|
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180420 Address after: 471000 West S8, Kaiyuan Avenue, Luolong District Science Park, Luoyang City, Henan Applicant after: Luoyang Modern Biotechnology Research Institute Co., Ltd. Applicant after: Liaoning Provincial Animal Products Safety Supervision Institute Address before: 471000 West S8, Kaiyuan Avenue, Luolong District Science Park, Luoyang City, Henan Applicant before: Luoyang Modern Biotechnology Research Institute Co., Ltd. |
|
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180504 |