CN106771273B - One kind detection Formoterol colloidal gold immuno-chromatography test paper strip and preparation method and application - Google Patents
One kind detection Formoterol colloidal gold immuno-chromatography test paper strip and preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of colloid gold chromatographic test paper strip for detecting Formoterol and preparation method and application, belong to immunological technique field and wild animal resources technical field.The test strips structure includes bottom plate, sample pad, colloidal gold pad, coated film and adsorptive pads, and test strips both ends are provided with diaphragm, and the sample pad, colloidal gold pad, coated film, adsorptive pads are pasted on bottom plate successively；The monoclonal antibody that can identify Formoterol of colloid gold label is coated with colloidal gold pad；Coated film is provided with the stealthy detection line of the carrier protein solution printing of coupling Formoterol, and the stealthy nature controlling line printed with goat anti-mouse igg solution.The colloidal gold chromatographic test strip of the present invention has the characteristics that convenient, quick, directly perceived, ageing strong, and applied widely, cost is low, easy to utilize.
Description
Technical field
The present invention relates to a kind of colloidal gold immuno-chromatography test paper strip and preparation method and application for detecting Formoterol, its
The detection remained particularly suitable for Formoterol in animal tissue, belong to immunological technique field and wild animal resources technology neck
Domain.
Background technology
Formoterol (Formoterol) is the third generation β developed by Japanese Yamanouchi Pharmaceutical Co., Ltd center research institute
2- adrenoceptor agonists suppressing panting calming medicines, there is the effect of bronchiectatic activity, also antiallergy and suppression oedema, can
For treating chronic bronchial asthma, Nocturnal, exercise-induced asthma, chronic obstructive pulmonary disease and the non-asthmatic breathing of children
Tract disease.In recent years, with the reinforcement of the food-safe supervision in China, the violated supervision using veterinary drug is gradually added
Greatly, the phenomenon for abusing violated veterinary drug has controlled, but occurs supervising instead of emphasis using the similar substitute of bioactivity
The new phenomenon of veterinary drug.Conventional " clenbuterol hydrochloride " material of three kinds of new " clenbuterol hydrochloride " Formoterol and Clenbuterol etc. has same rush
Enter growth, the effect of increase lean meat percentage, No. 1519 regulations of rules food animals of the Ministry of Agriculture in 2002 prohibit the use of beta-agonists class medicine
Thing is as feed addictive.Formoterol detection means lags, and especially quick detection product lacks, and country and each province and city are
Step up to establish the confirmation method of new " clenbuterol hydrochloride " material, while be also badly in need of the detection product for being adapted to batch, quickly screening, meet
Government and the needs of enterprise.
The detection technique of relevant Formoterol residual mainly has high performance liquid chromatography (HPLC), liquid chromatogram-matter at present
It is sensitive, accurate to compose combination method (LC-MS), liquid chromatography tandem mass spectrometry (LC-MS/MS) and immuno analytical method, first three methods
Really, it is but cumbersome, it is higher to experimental facilities and technical requirements, be not suitable for the quick detection of batch samples.Immunoassay side
Method includes enzyme linked immunosorbent assay (ELISA) and colloidal gold immunity chromatography (GICA), have high sensitivity, specificity it is good, into
The features such as this is low, easy to operate, screened suitable for batch samples.GICA is existing to exempt from as a kind of new immunological detection method
The specificity of epidemiology method, have again it is easy quick, be not required to the characteristics of instrument, it is more in medical science, the animal and plant quarantine, food inspection etc.
Field has been widely applied.Exploitation Formoterol colloidal gold strip contributes to Formoterol residual fast and accurately to examine
Survey, effectively contain its Misuse and residual excessive problem in aquatic products, improve people's aquatic products dining table quality peace
It is complete horizontal.
The content of the invention
The technical problems to be solved by the invention are to overcome prior art defect to provide a kind of colloid for detecting Formoterol
Golden immuno-chromatographic test paper strip, the colloidal gold immuno-chromatography test paper strip have simple to operate, quickly, are adapted to batch samples primary dcreening operations
Advantage.In addition, the present invention further provides the preparation method of the colloidal gold immuno-chromatography test paper strip of the detection Formoterol with answering
With.
Technical problem of the present invention is realized by following technical scheme.
A kind of colloidal gold immuno-chromatography test paper strip for detecting Formoterol, the test strip is by bottom plate, sample pad, colloid
Gold pad, coated film and adsorptive pads composition, test paper both ends are provided with diaphragm, the sample pad, colloidal gold pad, coated film and adsorptive pads
It is pasted on successively on bottom plate；Wherein colloidal gold pad is to be adsorbed with the Formoterol monoclonal antibody of colloid gold label, on coated film
Pre-coated detection line (T lines) containing useful Formoterol coupling carrier albumen, it is pre-coated with goat-anti or rabbit anti-mouse IgG solution
Nature controlling line (C lines), two lines are arranged in parallel vertically.
Above-mentioned colloidal gold immuno-chromatography test paper strip, Formoterol monoclonal antibody is mouse resource monoclonal antibody, by CCTCC
NO:C2016182 hybridoma cell strain Formoterol-3E6 is produced.The hybridoma cell strain is named as hybridoma
Strain Formoterol-3E6, China typical culture collection center (CCTCC) preservation, preserving number are delivered on October 26th, 2016
For CCTCC NO:C2016182.
Above-mentioned colloidal gold immuno-chromatography test paper strip, the Formoterol monoclonal antibody are produced by Formoterol antigen, its
For the conjugate synthesized by Formoterol with carrier protein using carbodlimide method, including immunogene and detection original；The carrier
Albumen is bovine serum albumin(BSA), mouse serum albumin, gpv protein, thyroprotein, ovalbumin, hemocyanin or people
Seralbumin；Immunogene preferred vector is bovine serum albumin(BSA), and it is ovalbumin to detect former preferred vector；
Above-mentioned colloidal gold immuno-chromatography test paper strip, the bottom plate are PVC bars or other hard materials not absorbed water；The sample
Product pad and colloidal gold pad are made up of glass fibre cotton；The adsorptive pads are made up of absorbent filter；The coated film can be that nitric acid is fine
Tie up plain film or cellulose acetate film；The diaphragm is opaque glued membrane.
Colloid gold chromatographic test paper strip preparation method of the present invention, comprises the following steps：
(1) Formoterol antigen working solution is prepared：
(a) preparation of p-aminobenzoic acid is activated：6mg natrium nitrosums are weighed, is dissolved in 0.35mL distilled water, then weighs
10mg p-aminobenzoic acid, dissolve in 1.1mL 1mol/L hydrochloric acid, ice bath stirring, above-mentioned sodium nitrite solution is added dropwise
Into p-aminobenzoic acid solution, lucifuge reaction 1h, obtain activating p-aminobenzoic acid；
(b) preparation of the formoterol active intermediate with carboxyl：Weigh Formoterol 30mg be dissolved in 5mL,
In borax buffer solution ice-cold 0.05mol/L (pH=9, the sodium chloride containing 0.15mol/L), ice bath stirring, by this solution by
It is added dropwise in activation p-aminobenzoic acid solution, 4 DEG C of lucifuges react 3h, obtain the formoterol active intermediate with carboxyl
Solution；
(c) synthesis of immunogene：0.1mol/ is added in the above-mentioned formoterol active midbody solution with carboxyl
LNaOH adjusts pH to 7.4, and EDC 140mg, NHS 60mg are dissolved in 2mL PBS, is repeatedly added in solution is stirred at room temperature on a small quantity
3h.Weigh BSA 200mg to be dissolved in 1mL 0.01mol/L PBS, add while stirring in above-mentioned solution, after reaction overnight,
Conjugate solution is transferred to bag filter, 4 DEG C of dialysis 72h in PBS, then uses 4 DEG C of dialysis 48h of distilled water instead, often
It changes liquid 2 times.Solution after dialysis centrifuges 30min in 6000rmp, takes -20 DEG C of preservations of supernatant.Labeled as Formoterol-
BSA；
(d) the former synthesis of detection：0.1mol/ is added in the above-mentioned formoterol active midbody solution with carboxyl
LNaOH adjusts pH to 7.4, and EDC 140mg, NHS 60mg are dissolved in 2mL PBS, is repeatedly added in solution is stirred at room temperature on a small quantity
3h.Weigh OVA 150mg to be dissolved in 1mL 0.01mol/L PBS, add while stirring in above-mentioned solution, after reaction overnight,
Conjugate solution is transferred to bag filter, 4 DEG C of dialysis 72h in PBS, then uses 4 DEG C of dialysis 48h of distilled water instead, often
It changes liquid 2 times.Solution after dialysis centrifuges 30min in 6000rmp, takes -20 DEG C of preservations of supernatant.Labeled as Formoterol-
OVA。
(2) Formoterol monoclonal antibody working solution is prepared：
(a) animal immune：Immunogene of the carrier protein for bovine serum albumin(BSA) is selected, the female Balb/ of 6-8 week old is immunized
C mouse, are spaced 2 weeks immune 1 time, 3 times it is immune after docking take hematometry potency and inhibiting rate, the optimal mouse preparation of selection result
Fusion；
(b) cell fusion：The splenocyte and mouse myeloma SP2/0 cells for taking the selected mouse of step (a) are merged,
Indirect elisa method measure supernatant chooses positive high hole, and positive hole is subcloned by limiting dilution assay, until establishing
Produce the hybridoma cell strain of the monoclonal antibody of single anti-Formoterol；
(c) a large amount of preparations of monoclonal antibody：The larger female Balb/c mouse of individual are chosen, using inducing ascites in vivo
Method, ascites is largely prepared, and ascites is purified by octanoic acid-ammonium sulfate precipitation, be divided into tubule, -20 DEG C of preservations, obtain Formoterol list
Clonal antibody；
(3) the Formoterol monoclonal antibody of colloid gold label is prepared：
Formoterol monoclonal antibody solution 10000r/min to be marked is centrifuged into 30min, takes colloidal gold solution 10mL,
With 0.1mol/L K2CO3The pH value of solution regulation colloidal gold solution is to 8.2；Take equivalent Formoterol monoclonal antibody solution, magnetic
Power stirring is lower to be mixed with colloidal gold solution, is incubated at room temperature 15min, 10000r/min centrifugation 30min, is abandoned supernatant, gained is precipitated
The Na for being 0.02mol/L with every liter of BSA containing 10g, 0.5g Sodium azides, concentration2B4O7Solution dilutes, that is, obtains colloid gold label
Formoterol monoclonal antibody, 4 DEG C save backup；
(4) prepared by colloidal gold pad：Glass fibre cotton is cut by specification, by the Formoterol monoclonal antibody of colloid gold label
Uniformly it is sprayed on glass fibre cotton, 37 DEG C of dry 1h, is sealed, 4 DEG C save backup with gold spraying instrument；
(5) preparation of detection line and nature controlling line：Formoterol antigen is put in gold spraying instrument storage pool A, goat anti-mouse igg is molten
Liquid is put in storage pool B, and fixed fire in film center, forms spacing 0.5cm linear stealthy detection line and stealthy Quality Control respectively for start
Line, spontaneously dry, sealing, 4 DEG C save backup；
(6) test strips assemble：Sample pad, colloidal gold pad, coated film and adsorptive pads are pasted on bottom plate successively in order,
And in the surface mount diaphragm at test strips both ends, test strips are cut on slot-cutting machine.
Above-mentioned colloid gold chromatographic test paper strip is for detecting the application in animal tissue, urine and feed in Formoterol.
The above-mentioned application for detecting Formoterol, comprise the following steps：
(1) sample pre-treatments
(a) urine sample：Take urine sample room temperature 5000rpm to centrifuge 5min, take the μ L of supernatant 100 to dilute 4 times with sample diluting liquid, take
Detected clearly；
(b) (meat, kidney, liver etc.) tissue：Meat sample tissue 2.0g ± 0.05g is weighed, 5mL ethyl acetate is added, shakes
Swing 1min, 5000rpm centrifugations 10min；Upper strata ethyl acetate layer solution is pipetted in another centrifuge tube, aqueous phase uses 5mL acetic acid again
Ethyl ester repeats to extract, and merges the drying of organic phase nitrogen；Add 1mL and redissolve working solution dissolving, take 50 μ L to be used to analyze；
(c) feed：Feed 2.0g ± 0.05g is weighed, adds 5mL ethyl acetate, vibrates 1min, 5000rpm centrifugations
10min；Upper strata ethyl acetate layer solution is pipetted in another centrifuge tube, aqueous phase repeats to extract with 5mL ethyl acetate again, is associated with
Machine phase nitrogen dries up；Add 1mL and redissolve working solution dissolving, take 50 μ L to be used to analyze.
(2) detected with colloid gold chromatographic test paper strip
The colloidal gold immuno-chromatography test paper strip of the detection Formoterol is taken out from the package, and test sample is treated into sample end insertion
In product liquid, insertion depth is no more than mark line, takes out test strip within about 10-20 seconds, horizontal positioned, 3-5 minutes, which are observed, to be sentenced
Determine testing result, result is invalid after 10 minutes；
(3) result judgement
If (a) corresponding quality control region location of C shows a reddish brown colo(u)r streak on positive coated film, detection zone T location does not show
Show reddish brown colo(u)r streak, represent that testing result for the positive, illustrates to contain Formoterol in testing sample；
If (b) feminine gender T, location of C on coated film shows two days reddish brown colo(u)r streaks, represent that result for feminine gender, illustrates
Formoterol is free of in testing sample；
(c) fail when quality control region C does not show brownish red band, then no matter whether detection zone T shows brownish red band
It is invalid that the test paper is judged to.
The detection of the Formoterol colloid gold chromatographic test paper strip of the present invention is limited to 4 μ g/L, with other several beta-agonists class medicines
Thing (phenolethanolamine A, salbutamol, Tulobuterol, clenobuterol hydrochloride, Ractopamine, Cimaterol), which is not present, to intersect
Reaction, the detection to sample are consistent with high performance liquid chromatography result.Formoterol is detected with colloid gold chromatographic test paper strip of the present invention
Method it is easy, quick, directly perceived, accurate, applied widely, cost is low, easily promotes the use of.
Brief description of the drawings
Fig. 1 Formoterol monoclonal antibody affinity measure figures of the present invention
Fig. 2 Formoterol monoclonal antibody hypotype measure figures of the present invention
Fig. 3 Formoterol colloid gold chromatographic test paper strip test paper cross-sectional views of the present invention
Fig. 4 Formoterol colloid gold chromatographic test paper strip test paper overlooking the structure diagrams of the present invention
Fig. 5 Formoterol colloid gold chromatographic test paper strip test paper result process decision charts of the present invention
The hybridoma cell strain Formoterol-3E6 of anti-Formoterol monoclonal antibody of the present invention, in 2016
On October 26, in delivers China typical culture collection center (abbreviation CCTCC, address：Wuhan University, Chinese Typical Representative culture are protected
Tibetan center, postcode：430072) preservation, deposit number CCTCC NO:C2016182.
Embodiment
The present invention is described in further detail with reference to embodiment, cited embodiment is not to the present invention
Content and protection domain form any restrictions.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
The preparation of the Formoterol antigen of the present invention of embodiment one
(1) preparation of p-aminobenzoic acid is activated：6mg natrium nitrosums are weighed, is dissolved in 0.35mL distilled water, then weighs
10mg p-aminobenzoic acid, dissolve in 1.1mL 1mol/L hydrochloric acid, ice bath stirring, above-mentioned sodium nitrite solution is added dropwise
Into p-aminobenzoic acid solution, lucifuge reaction 1h, obtain activating p-aminobenzoic acid；
(2) preparation of the formoterol active intermediate with carboxyl：Weigh Formoterol 30mg be dissolved in 5mL,
In borax buffer solution ice-cold 0.05mol/L (pH=9, the sodium chloride containing 0.15mol/L), ice bath stirring, by this solution by
It is added dropwise in activation p-aminobenzoic acid solution, 4 DEG C of lucifuges react 3h, obtain the formoterol active intermediate with carboxyl
Solution；
(3) synthesis of immunogene：0.1mol/ is added in the above-mentioned formoterol active midbody solution with carboxyl
LNaOH adjusts pH to 7.4, and EDC 140mg, NHS 60mg are dissolved in 2mL PBS, is repeatedly added in solution is stirred at room temperature on a small quantity
3h.Weigh BSA 200mg to be dissolved in 1mL 0.01mol/L PBS, add while stirring in above-mentioned solution, after reaction overnight,
Conjugate solution is transferred to bag filter, 4 DEG C of dialysis 72h in PBS, then uses 4 DEG C of dialysis 48h of distilled water instead, often
It changes liquid 2 times.Solution after dialysis centrifuges 30min in 6000rmp, takes -20 DEG C of preservations of supernatant.Labeled as Formoterol-
BSA；
(4) the former synthesis of detection：0.1mol/ is added in the above-mentioned formoterol active midbody solution with carboxyl
LNaOH adjusts pH to 7.4, and EDC 140mg, NHS 60mg are dissolved in 2mL PBS, is repeatedly added in solution is stirred at room temperature on a small quantity
3h.Weigh OVA 150mg to be dissolved in 1mL 0.01mol/L PBS, add while stirring in above-mentioned solution, after reaction overnight,
Conjugate solution is transferred to bag filter, 4 DEG C of dialysis 72h in PBS, then uses 4 DEG C of dialysis 48h of distilled water instead, often
It changes liquid 2 times.Solution after dialysis centrifuges 30min in 6000rmp, takes -20 DEG C of preservations of supernatant.Labeled as Formoterol-
OVA。
The preparation of the Formoterol monoclonal antibody of the present invention of embodiment two
(1) animal immune：Carrier protein is small for the female Balb/c of the immunogen immune 6-8 week old of bovine serum albumin(BSA)
Mouse, is spaced 2 weeks immune 1 time, 3 times it is immune after docking take hematometry potency and inhibiting rate, the optimal mouse preparation of selection result is melted
Close；
(2) cell fusion：The splenocyte and mouse myeloma SP2/O cells of mouse are taken, is merged, indirect elisa method
Determine supernatant and choose positive high hole, positive hole is subcloned by limiting dilution assay, single anti-good fortune is produced until establishing
The hybridoma cell strain of Mo Teluo monoclonal antibody；
(3) a large amount of preparations of monoclonal antibody：The larger female Blab/c mouse of individual are chosen, using inducing ascites in vivo
Method, ascites is largely prepared, and ascites is purified by octanoic acid-ammonium sulfate precipitation, be divided into tubule, -20 DEG C of preservations, obtain Formoterol list
Clonal antibody.
The Formoterol monoclonal antibody CHARACTERISTICS IDENTIFICATION of the present invention of embodiment three
1st, affinity determines
Using the affinity costant of non-competing ELISA measure Formoterol monoclonal antibody, as a result as shown in figure 1, parent
With constant Ka=7.24 × 108L/mol。
2nd, hypotype determines
Hypotype measure is carried out using purchased from the mouse source monoclonal antibody hypotype identification kit of Sigma companies, as a result as shown in Figure 2,
Formoterol monoclonal antibody hypotype is IgG1.
3rd, titration
With 1：40000 dilution coating primordial covering detection plates, 1 is carried out by monoclonal antibody after purification:2000,1:4000,
1:8000 ... ... 1:1024000 dilutions, are added in ELISA Plate hole, the sheep anti mouse secondary antibody of HRP marks are added after reaction, finally
Developed the color with TMB, the potency when Formoterol MAb concentration for as a result showing after purification is 1mg/ml is up to 1：512000.It is real
Apply the colloid gold chromatographic test paper strip of the detection Formoterol of the present invention of example four
1st, prepared by collaurum liquid：Take 100mL distilled water, boil 5min, add gold chloride 1mL that mass concentration is 1% and
Mass concentration is 1% citric acid three sodium solution 1.5mL, continues agitating and heating, and solution eventually passes through grey, claret in saturating
Bright red, room temperature is cooled to, Quality Identification is carried out by ocular estimate, ultraviolet spectrophotometry respectively, qualified collaurum is molten
4 DEG C of liquid is kept in dark place；
2nd, the preparation of the Formoterol monoclonal antibody of colloid gold label：Formoterol monoclonal antibody to be marked is molten
Liquid 10000r/min centrifuges 30min, colloidal gold solution 10mL is taken, with 0.1mol/L K2CO3Solution adjusts the pH of colloidal gold solution
It is worth to 8.2；Equivalent Formoterol monoclonal antibody solution is taken, is mixed under magnetic agitation with colloidal gold solution, is incubated at room temperature
15min, 10000r/min centrifuge 30min, abandon supernatant, are with every liter of BSA containing 10g, 0.5g Sodium azides, concentration by gained precipitation
0.02mol/L Na2B4O7Solution dilutes, that is, obtains the Formoterol monoclonal antibody of colloid gold label, 4 DEG C save backup；
3rd, prepared by colloidal gold pad：Glass fibre cotton is cut by specification, gold labeling antibody is uniformly sprayed on glass with gold spraying instrument
On cellucotton, 37 DEG C of dry 1h, sealing, 4 DEG C save backup；
4th, the preparation of detection line and nature controlling line：Formoterol antigen is put in gold spraying instrument storage pool A, goat anti-mouse igg is molten
Liquid is put in storage pool B, and fixed fire in film center, forms spacing 0.5cm linear stealthy detection line T lines and stealthy matter respectively for start
Line C lines are controlled, are spontaneously dried, sealing, 4 DEG C save backup；
5th, test strips assemble：Sample pad, colloidal gold pad, coated film and adsorptive pads are pasted on bottom plate successively in order,
And in the surface mount diaphragm at test strips both ends, test strips are cut on slot-cutting machine.
6th, the structure of Formoterol colloid gold chromatographic test paper strip, referring to Fig. 3, Fig. 4.Figure bottom plate 1 is made of PVC board, sample
Product pad 2 is made of glass fibre cotton, and anti-Formoterol monoclonal antibody is adsorbed with colloidal gold pad 3, and coated film 4 is fine using nitric acid
Plain film is tieed up, adsorptive pads 7 are made of absorbent filter, and numbering 2,3,4,7 each layers are pasted and fixed on bottom plate 1 successively from right to left,
Intersection fiber crosses one another infiltration each other.Stealthy detection line 5 and stealthy nature controlling line 6, stealthy detection line are provided with coated film 4
To be printed with chicken ovalbumin (OVA) solution of coupling Formoterol, stealthy nature controlling line is printed with goat anti-mouse igg antibody solution,
Two traces are arranged in parallel into " ︱ | ".8 is are covered in the sample end diaphragm above sample pad 2 and colloidal gold pad 3, and 9 be covering
Handle end diaphragm on adsorptive pads 7, arrow and MAX printed words are printed on the diaphragm on the right side of sample mark line.
Formoterol in five colloid gold chromatographic test paper strip of the present invention of embodiment detection sample
(1) sample pre-treatments
(a) urine sample：Take urine sample room temperature 5000rpm to centrifuge 5min, take the μ L of supernatant 100 to dilute 4 times with sample diluting liquid, take
Detected clearly；
(b) (meat, kidney, liver etc.) tissue：Meat sample tissue 2.0g ± 0.05g is weighed, 5mL ethyl acetate is added, shakes
Swing 1min, 5000rpm centrifugations 10min；Upper strata ethyl acetate layer solution is pipetted in another centrifuge tube, aqueous phase uses 5mL acetic acid again
Ethyl ester repeats to extract, and merges the drying of organic phase nitrogen；Add 1mL and redissolve working solution dissolving, take 50 μ L to be used to analyze；
(c) feed：Feed 2.0g ± 0.05g is weighed, adds 5mL ethyl acetate, vibrates 1min, 5000rpm centrifugations
10min；Upper strata ethyl acetate layer solution is pipetted in another centrifuge tube, aqueous phase repeats to extract with 5mL ethyl acetate again, is associated with
Machine phase nitrogen dries up；Add 1mL and redissolve working solution dissolving, take 50 μ L to be used to analyze.
(2) detected with colloid gold chromatographic test paper strip
The colloidal gold immuno-chromatography test paper strip of the detection Formoterol is taken out from the package, and test sample is treated into sample end insertion
In product liquid, insertion depth is no more than mark line, takes out test strip within about 10-20 seconds, horizontal positioned, 3-5 minutes, which are observed, to be sentenced
Determine testing result, result is invalid after 10 minutes.
(3) result judgement
As shown in figure 5, a is the positive, corresponding quality control region location of C shows a reddish brown colo(u)r streak, detection zone T positions on coated film
Put and do not show reddish brown colo(u)r streak, represent that testing result for the positive, illustrates to contain Formoterol in testing sample；B is feminine gender, is being wrapped
T, location of C show two days reddish brown colo(u)r streaks on envelope, represent that result for feminine gender, illustrates to be free of Formoterol in testing sample；
C, d is failure, when quality control region C does not show brownish red band, then no matter detection zone T show brownish red band whether the test paper
It is invalid to be judged to.
The Formoterol colloid gold chromatographic test paper strip performance detection of the present invention of embodiment six
1st, sensitivity
1mg/mL Formoterols (1mg Formoterols are dissolved in 1mL methanol) are diluted to 0,1,2,3,4,5,10 with PBS respectively
μ g/L series of standards solution, then take 90 μ L above-mentioned standard solution to drop in the sample pad of test strips respectively, observed after 3min
The colour developing situation of test strips.
When Formoterol addition concentration be 3 μ g/L when, T lines relative to T lines during 0 μ g/L lighter, and work as good fortune not
When the addition concentration of special sieve is 4 μ g/L, T lines disappear line, illustrate that the detection of colloidal gold strip is limited to 4 μ g/L.
2nd, it is specific
Choose 5 kinds of beta receptor activators (phenolethanolamine A, salbutamol, Tulobuterol, clenobuterol hydrochloride, Rec DOPA
Amine, Cimaterol) test, observe test strips color developing effect.
By phenolethanolamine A, salbutamol, Tulobuterol, clenobuterol hydrochloride, Ractopamine, Cimaterol mother liquor point
50 μ g/L are not diluted to 0.01mol/L, PH7.4 PBS, are added drop-wise to the sample well of test strips, testing result is shown, preparation
Cross reaction is not present with other several beta receptor activators in Formoterol colloidal gold strip.
3rd, accuracy
8 parts of pork samples are randomly selected, as sample of having a try.The good fortune that every part of sample is prepared with HPLC methods and this research respectively
Mo Teluo colloidal gold colloidal gold detection test paper strips are detected, and compare testing result.
8 parts of pork samples are through high performance liquid chromatography detection, wherein 6 parts of samples do not detect Formoterol residual, No. 4 sample inspections
The μ g/kg of formoterol content 2.14 are measured, No. 5 sample detections go out the μ g/kg of formoterol content 1.53.These samples use good fortune simultaneously
Mo Teluo colloidal gold strips detect, and are as a result complied fully with HPLC methods, are shown in Table 1.
The Formoterol colloidal gold strip of table 1 is compared with high performance liquid chromatography testing result
Note："+" represents positive, and "-" represents negative.
The technical concept and advantage of above-described embodiment only to illustrate the invention, the present invention can also have other forms to become
Change, as well known to the skilled person, above-described embodiment is functioned only as to the exemplary role in foregoing invention protection domain, right
For those of ordinary skill in the art, there are many conventional deformations and other implementations in the protection domain that the present invention is limited
Example, these deformations and embodiment are all by within the pending protection domain of the present invention.
Claims (4)
1. one kind detection Formoterol colloidal gold immuno-chromatography test paper strip, it is characterised in that the test strip is by bottom plate, sample
Pad, colloidal gold pad, coated film and adsorptive pads composition, test paper both ends are provided with diaphragm；The sample pad, colloidal gold pad, coated film
It is pasted on successively on bottom plate with adsorptive pads；Wherein colloidal gold pad is to be adsorbed with the Formoterol monoclonal antibody of colloid gold label,
The pre-coated detection line T lines containing useful Formoterol coupling carrier albumen, pre- with goat-anti or rabbit anti-mouse IgG solution on coated film
Coated nature controlling line C line, two lines are arranged in parallel vertically；The bottom plate is PVC bars；The sample pad and colloidal gold pad are by glass
Cellucotton is made；The adsorptive pads are made up of absorbent filter；The coated film can be nitrocellulose filter or cellulose acetate film；
The diaphragm is opaque glued membrane；The Formoterol monoclonal antibody is mouse resource monoclonal antibody, by CCTCC NO:
C2016182 hybridoma cell strain Formoterol-3E6 is produced；The hybridoma cell strain is named as hybridoma cell strain
Formoterol-3E6, China typical culture collection center preservation, preserving number CCTCC are delivered on October 26th, 2016
NO:C2016182；The Formoterol monoclonal antibody is produced by Formoterol antigen, Formoterol antigen Wei You Fu Mote
The conjugate that sieve is synthesized with carrier protein using carbodiimide method, including immunogene and detection original；The carrier protein is ox
Seralbumin, mouse serum albumin, gpv protein, thyroprotein, ovalbumin, hemocyanin or human seralbumin egg
In vain；The Formoterol immunogene is carrier using bovine serum albumin(BSA)；The Formoterol detection is former to be using ovalbumin
Carrier.
2. the preparation method of detection Formoterol colloidal gold immuno-chromatography test paper strip as claimed in claim 1, it is characterised in that bag
Include following steps：
(1) Formoterol antigen working solution is prepared：
(a) preparation of p-aminobenzoic acid is activated：6mg natrium nitrosums are weighed, is dissolved in 0.35mL distilled water, then weighs 10mg
P-aminobenzoic acid, dissolve in 1.1mL 1mol/L hydrochloric acid, ice bath stirring, above-mentioned sodium nitrite solution is added dropwise to pair
In aminobenzoic acid solution, lucifuge reaction 1h, obtain activating p-aminobenzoic acid；
(b) preparation of the formoterol active intermediate with carboxyl：Weigh Formoterol 30mg and be dissolved in 5mL, 0.05mol/L ice
In cold borax buffer solution, the borax buffer solution pH=9 and the sodium chloride containing 0.15mol/L, ice bath stirring, by this
Solution is added dropwise in activation p-aminobenzoic acid solution, and 4 DEG C of lucifuges react 3h, obtain the formoterol active with carboxyl
Midbody solution；
(c) synthesis of immunogene：0.1mol/LNaOH is added in the above-mentioned formoterol active midbody solution with carboxyl
PH to 7.4 is adjusted, EDC 140mg, NHS 60mg are dissolved in 2mL PBS, is repeatedly added in solution 3h is stirred at room temperature on a small quantity；Claim
Take BSA 200mg to be dissolved in 1mL 0.01mol/L PBS, add while stirring in above-mentioned solution, after reaction overnight, will be even
Connection thing solution is transferred to bag filter, 4 DEG C of dialysis 72h in PBS, then uses 4 DEG C of dialysis 48h of distilled water instead, changes daily
Liquid 2 times；Solution after dialysis centrifuges 30min in 6000rmp, takes -20 DEG C of preservations of supernatant；Labeled as Formoterol-BSA；
(d) the former synthesis of detection：0.1mol/LNaOH is added in the above-mentioned formoterol active midbody solution with carboxyl
PH to 7.4 is adjusted, EDC 140mg, NHS 60mg are dissolved in 2mL PBS, is repeatedly added in solution 3h is stirred at room temperature on a small quantity；Claim
Take OVA 150mg to be dissolved in 1mL 0.01mol/L PBS, add while stirring in above-mentioned solution, after reaction overnight, will be even
Connection thing solution is transferred to bag filter, 4 DEG C of dialysis 72h in PBS, then uses 4 DEG C of dialysis 48h of distilled water instead, changes daily
Liquid 2 times；Solution after dialysis centrifuges 30min in 6000rmp, takes -20 DEG C of preservations of supernatant；Labeled as Formoterol-OVA；
(2) Formoterol monoclonal antibody working solution is prepared：
(a) animal immune：Immunogene of the carrier protein for bovine serum albumin(BSA) is selected, the female Balb/c that 6-8 week old is immunized is small
Mouse, it is spaced 2 weeks immune 1 time, measure potency and inhibiting rates after being immunized three times, the optimal mouse preparation fusion of selection result；
(b) cell fusion：The splenocyte and mouse myeloma SP2/0 cells for taking the selected mouse of step (a) are merged, indirectly
ELISA method measure supernatant chooses positive high hole, and positive hole is subcloned by limiting dilution assay, produced until establishing
The hybridoma cell strain of the monoclonal antibody of single anti-Formoterol；
(c) a large amount of preparations of monoclonal antibody：The larger female Blab/c mouse of individual are chosen, using inducing ascites method in vivo,
It is a large amount of to prepare ascites, and ascites is purified by octanoic acid-ammonium sulfate precipitation, it is divided into tubule, -20 DEG C of preservations, obtains Formoterol Dan Ke
Grand antibody；
(3) the Formoterol monoclonal antibody of colloid gold label is prepared：
Formoterol monoclonal antibody solution 10000r/min to be marked is centrifuged into 30min, takes colloidal gold solution 10mL, is used
0.1mol/LK2CO3The pH value of solution regulation colloidal gold solution is to 8.2；Equivalent Formoterol monoclonal antibody solution is taken, magnetic force stirs
Mix down and mixed with colloidal gold solution, be incubated at room temperature 15min, 10000r/min centrifugation 30min, supernatant abandoned, by gained precipitation with often
Rise BSA containing 10g, 0.5g Sodium azides, the Na that concentration is 0.02mol/L2B4O7Solution dilutes, that is, obtains the good fortune of colloid gold label not
Special sieve monoclonal antibody, 4 DEG C save backup；
(4) prepared by colloidal gold pad：Glass fibre cotton is cut by specification, the Formoterol monoclonal antibody of colloid gold label is sprayed
Golden instrument is uniformly sprayed on glass fibre cotton, 37 DEG C of dry 1h, and sealing, 4 DEG C save backup；
(5) preparation of detection line and nature controlling line：Formoterol antigen is put in gold spraying instrument storage pool A, goat anti-mouse igg solution is put
In storage pool B, fixed fire in film center, forms spacing 0.5cm linear stealthy detection line and stealthy nature controlling line, certainly respectively for start
So dry, sealing, 4 DEG C save backup；
(6) test strips assemble：Sample pad, colloidal gold pad, coated film and adsorptive pads are pasted on bottom plate successively in order, and
The surface mount diaphragm at test strips both ends, cuts into test strips on slot-cutting machine.
3. colloid gold chromatographic test paper strip as claimed in claim 1 is for detecting Fu Mote in animal tissue, urine and feed
The application of sieve.
4. application according to claim 3, it is characterised in that comprise the following steps：
(1) sample pre-treatments
(a) urine sample：Take urine sample room temperature 5000rpm to centrifuge 5min, take the μ L of supernatant 100 to dilute 4 times with sample diluting liquid, take supernatant to enter
Row detection；
(b) meat, kidney, liver organization：Meat sample tissue 2.0g ± 0.05g is weighed, adds 5mL ethyl acetate, vibrates 1min,
5000rpm centrifuges 10min；Upper strata ethyl acetate layer solution is pipetted in another centrifuge tube, aqueous phase is repeated with 5mL ethyl acetate again
Extraction, merge the drying of organic phase nitrogen；Add 1mL and redissolve working solution dissolving, take 50 μ L to be used to analyze；
(c) feed：Feed 2.0g ± 0.05g is weighed, adds 5mL ethyl acetate, vibrates 1min, 5000rpm centrifugations 10min；Move
Upper strata ethyl acetate layer solution is taken in another centrifuge tube, aqueous phase repeats to extract with 5mL ethyl acetate again, merges organic phase nitrogen
Drying；Add 1mL and redissolve working solution dissolving, take 50 μ L to be used to analyze；
(2) detected with colloid gold chromatographic test paper strip
The colloidal gold immuno-chromatography test paper strip of the detection Formoterol is taken out from the package, and sample end is inserted into analyte sample fluid
In, insertion depth is no more than mark line, takes out within 10-20 seconds test strip, and horizontal positioned, 3-5 minutes observe judgement detection
As a result, result is invalid after 10 minutes；
(3) result judgement
(a) it is positive：If corresponding quality control region location of C shows a reddish brown colo(u)r streak on coated film, detection zone T location does not show palm fibre
Red line, represent that testing result for the positive, illustrates to contain Formoterol in testing sample；
(b) it is negative：If T, location of C show two days reddish brown colo(u)r streaks on coated film, represent that result for feminine gender, illustrates to be measured
Formoterol is free of in sample；
(c) fail：When quality control region C does not show brownish red band, then no matter detection zone T show brownish red band whether the examination
It is invalid that paper is judged to.
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| CN109709339B (en) * | 2018-12-28 | 2022-03-15 | 河北省科学院生物研究所 | Colloidal gold immunochromatographic test strip for detecting skeletal muscle troponin I of cattle or sheep and application thereof |
| CN113588958B (en) * | 2021-08-17 | 2023-11-10 | 合肥工业大学智能制造技术研究院 | Lamotrigine lateral chromatography test strip with multiple concentration detection lines and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101446588A (en) * | 2008-12-29 | 2009-06-03 | 山东大学 | Chemiluminescent enzyme-linked immunosorbent assay (CELISA) kit for detecting ractopamine |
| CN101597330A (en) * | 2009-06-26 | 2009-12-09 | 青岛康地恩药业有限公司 | The synthetic method of a kind of Ractopamine hydrochloride artificial antigen |
| CN202196068U (en) * | 2011-08-10 | 2012-04-18 | 无锡安迪生物工程有限公司 | Phenylethanolamine A rapid detection card |
| CN102768278A (en) * | 2012-05-31 | 2012-11-07 | 华中农业大学 | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting beta-receptor stimulant |
| CN104569408A (en) * | 2014-12-30 | 2015-04-29 | 无锡中德伯尔生物技术有限公司 | Fenoterol colloidal gold detection card and preparation method thereof |
-
2016
- 2016-12-08 CN CN201611143061.9A patent/CN106771273B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101446588A (en) * | 2008-12-29 | 2009-06-03 | 山东大学 | Chemiluminescent enzyme-linked immunosorbent assay (CELISA) kit for detecting ractopamine |
| CN101597330A (en) * | 2009-06-26 | 2009-12-09 | 青岛康地恩药业有限公司 | The synthetic method of a kind of Ractopamine hydrochloride artificial antigen |
| CN202196068U (en) * | 2011-08-10 | 2012-04-18 | 无锡安迪生物工程有限公司 | Phenylethanolamine A rapid detection card |
| CN102768278A (en) * | 2012-05-31 | 2012-11-07 | 华中农业大学 | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting beta-receptor stimulant |
| CN104569408A (en) * | 2014-12-30 | 2015-04-29 | 无锡中德伯尔生物技术有限公司 | Fenoterol colloidal gold detection card and preparation method thereof |
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