CN106771273A - 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 base plate, sample pad, colloidal gold pad, coated film and adsorptive pads, and test strips two ends are provided with diaphragm, and the sample pad, colloidal gold pad, coated film, adsorptive pads are pasted on base plate successively；The monoclonal antibody that can recognize 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 features such as colloidal gold chromatographic test strip of the invention has convenient, quick, directly perceived, ageing strong, applied widely, low cost is easy to utilize.
Description
Technical field
The present invention relates to a kind of colloidal gold immuno-chromatography test paper strip for detecting Formoterol and preparation method and application, its
It is particularly suitable for the detection of Formoterol residual in animal tissue, belongs to immunological technique field and wild animal resources technology neck
Domain.
Background technology
Formoterol (Formoterol) is the third generation β developed by center research institute of Japanese Yamanouchi Pharmaceutical Co., Ltd
2- adrenoceptor agonists suppressing panting calming medicines, with bronchiectatic activity, the also effect of 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 of China, the supervision to violated use veterinary drug gradually adds
Greatly, the phenomenon for abusing violated veterinary drug has been controlled, but is occurred in that and supervised instead of emphasis using the similar substitute of bioactivity
The new phenomenon of veterinary drug.Conventional " clenbuterol hydrochloride " materials of three kinds of new " clenbuterol hydrochloride " Formoterol and Clenbuterol etc. have same rush
Enter growth, increase the effect of 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 used as feed addictive.Formoterol detection means is delayed, and especially quick detection product lacks, and country and each province and city are
Step up to set up the confirmation method of new " clenbuterol hydrochloride " material, while being also badly in need of being adapted to batch, the detection product of quick screening, meet
The need for government and enterprise.
The detection technique of relevant Formoterol residual mainly has high performance liquid chromatography (HPLC), liquid chromatogram-matter at present
Spectrum combination method (LC-MS), liquid chromatography tandem mass spectrometry (LC-MS/MS) and immuno analytical method, first three methods are sensitive, standard
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), with sensitivity it is high, it is specific it is good, into
The features such as this is low, easy to operate, is suitable to batch samples screening.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, the characteristics of be not required to 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
Full level.
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 has simple to operate, quickly, is adapted to batch samples primary dcreening operations
Advantage.Additionally, the present invention further provides the detection Formoterol colloidal gold immuno-chromatography test paper strip preparation method with should
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 base plate, sample pad, colloid
Gold pad, coated film and adsorptive pads composition, test paper two ends are provided with diaphragm, the sample pad, colloidal gold pad, coated film and adsorptive pads
It is pasted on base plate successively；Wherein colloidal gold pad is the Formoterol monoclonal antibody for being adsorbed with 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:The hybridoma cell strain Formoterol-3E6 of C2016182 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
It is CCTCC NO:C2016182.
Above-mentioned colloidal gold immuno-chromatography test paper strip, the Formoterol monoclonal antibody is produced by Formoterol antigen, its
It is conjugate synthesize using carbodlimide method by Formoterol and carrier protein, 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 the former preferred vector of detection is ovalbumin；
Above-mentioned colloidal gold immuno-chromatography test paper strip, the base plate is 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 fine for nitric acid
The plain film of dimension or CAM；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 () activates the preparation of p-aminobenzoic acid：6mg natrium nitrosums are weighed, is dissolved in 0.35mL distilled water, then weighed
10mg p-aminobenzoic acid, dissolves in the hydrochloric acid of 1.1mL 1mol/L, and be added dropwise over for above-mentioned sodium nitrite solution by ice bath stirring
To in p-aminobenzoic acid solution, lucifuge reaction 1h obtains activating p-aminobenzoic acid；
The preparation of the formoterol active intermediate of (b) with carboxyl：Weigh Formoterol 30mg be dissolved in 5mL,
In 0.05mol/L ice-cold borax buffer solution (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；
The synthesis of (c) 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 on a small quantity in solution and is stirred at room temperature
3h.BSA 200mg are weighed to be dissolved in the PBS of 1mL 0.01mol/L, in adding above-mentioned solution while stirring, after reaction overnight,
Conjugate solution is transferred to bag filter, then 4 DEG C of dialysis 72h in PBS use 4 DEG C of dialysis 48h of distilled water instead, often
It changes liquid 2 times.Solution after dialysis is centrifuged 30min in 6000rmp, takes -20 DEG C of preservations of supernatant.Labeled as Formoterol-
BSA；
The former synthesis of (d) 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 on a small quantity in solution and is stirred at room temperature
3h.OVA 150mg are weighed to be dissolved in the PBS of 1mL 0.01mol/L, in adding above-mentioned solution while stirring, after reaction overnight,
Conjugate solution is transferred to bag filter, then 4 DEG C of dialysis 72h in PBS use 4 DEG C of dialysis 48h of distilled water instead, often
It changes liquid 2 times.Solution after dialysis is centrifuged 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：Selection carrier protein is the immunogene of bovine serum albumin(BSA), and 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 determines supernatant and chooses positive hole high, and positive hole is subcloned by limiting dilution assay, until setting up
Produce the hybridoma cell strain of the monoclonal antibody of single anti-Formoterol；
A large amount of preparations of (c) monoclonal antibody：Individual larger female Balb/c mouse are chosen, using inducing ascites in vivo
Method, largely prepares ascites, and purifies ascites by octanoic acid-ammonium sulfate precipitation, is divided into tubule, and -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 30min, colloidal gold solution 10mL is taken,
Use 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, abandons supernatant, and gained is precipitated
With every liter of BSA containing 10g, 0.5g Sodium azides, the Na that concentration is 0.02mol/L2B4O7Solution dilutes, that is, obtain 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 with gold spraying instrument, 37 DEG C dry 1h, is sealed, 4 DEG C saves 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 is molten
Liquid is put in storage pool B, and fixed fire, in film center, forms the linear stealthy detection line of spacing 0.5cm and stealthy Quality Control respectively for start
Line, spontaneously dries, sealing, and 4 DEG C save backup；
(6) test strips assembling：Sample pad, colloidal gold pad, coated film and adsorptive pads are pasted on base plate successively in order,
And in the surface mount diaphragm at test strips two ends, test strips are cut on slot-cutting machine.
Above-mentioned colloid gold chromatographic test paper strip is for detecting animal tissue, urine and the application in feed in Formoterol.
The above-mentioned application for detecting Formoterol, comprises the following steps：
(1) sample pre-treatments
(a) urine sample：Urine sample room temperature 5000rpm centrifugation 5min are taken, the μ L sample diluting liquids of supernatant 100 is taken and is diluted 4 times, taken
Detected clearly；
(b) (meat, kidney, liver etc.) tissue：Meat sample tissue 2.0g ± 0.05g are weighed, 5mL ethyl acetate is added, shaken
Swing 1min, 5000rpm centrifugations 10min；Upper strata ethyl acetate layer solution is pipetted in another centrifuge tube, water mutually uses 5mL acetic acid again
Ethyl ester repeats to extract, and merges the drying of organic phase nitrogen；Add 1mL to redissolve working solution dissolving, take 50 μ L for analyzing；
(c) feed：Feed 2.0g ± 0.05g are weighed, 5mL ethyl acetate is added, 1min, 5000rpm centrifugations is vibrated
10min；Upper strata ethyl acetate layer solution is pipetted in another centrifuge tube, water mutually repeats extraction with 5mL ethyl acetate again, is associated with
Machine phase nitrogen is dried up；Add 1mL to redissolve working solution dissolving, take 50 μ L for analyzing.
(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, and horizontal positioned, observation in 3-5 minutes is sentenced
Result is invalid after determining testing result, 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 is the positive, illustrate to contain Formoterol in testing sample；
If b () feminine gender T, location of C on coated film show two days reddish brown colo(u)r streaks, represent that result is feminine gender, illustrate
Formoterol is free of in testing sample；
C () failure does not show brownish red band as quality control region C, then no matter whether detection zone T shows brownish red band
It is invalid that the test paper is judged to.
The detection of Formoterol colloid gold chromatographic test paper strip of the invention is limited to 4 μ g/L, with other several beta-agonists class medicines
Thing (phenolethanolamine A, salbutamol, Tulobuterol, clenobuterol hydrochloride, Ractopamine, Cimaterol) is in the absence of intersection
Reaction, the detection to sample is consistent with high performance liquid chromatography result.Formoterol is detected with colloid gold chromatographic test paper strip of the present invention
Method is easy, quick, directly perceived, accurate, applied widely, low cost, easily promote the use of.
Brief description of the drawings
Fig. 1 Formoterol monoclonal antibody affinity of the present invention determines figure
Fig. 2 Formoterol monoclonal antibody hypotypes of the present invention determine figure
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 is protected
Tibetan center, postcode：430072) preservation, deposit number CCTCC NO:C2016182.
Specific embodiment
The present invention is described in further detail with reference to specific embodiment, cited embodiment is not to the present invention
Content and protection domain constitute any limitation.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
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 weighed
10mg p-aminobenzoic acid, dissolves in the hydrochloric acid of 1.1mL 1mol/L, and be added dropwise over for above-mentioned sodium nitrite solution by ice bath stirring
To in p-aminobenzoic acid solution, lucifuge reaction 1h obtains activating p-aminobenzoic acid；
(2) preparation of the formoterol active intermediate with carboxyl：Weigh Formoterol 30mg be dissolved in 5mL,
In 0.05mol/L ice-cold borax buffer solution (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 on a small quantity in solution and is stirred at room temperature
3h.BSA 200mg are weighed to be dissolved in the PBS of 1mL 0.01mol/L, in adding above-mentioned solution while stirring, after reaction overnight,
Conjugate solution is transferred to bag filter, then 4 DEG C of dialysis 72h in PBS use 4 DEG C of dialysis 48h of distilled water instead, often
It changes liquid 2 times.Solution after dialysis is centrifuged 30min in 6000rmp, takes -20 DEG C of preservations of supernatant.Labeled as Formoterol-
BSA；
(4) former synthesis is detected：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 on a small quantity in solution and is stirred at room temperature
3h.OVA 150mg are weighed to be dissolved in the PBS of 1mL 0.01mol/L, in adding above-mentioned solution while stirring, after reaction overnight,
Conjugate solution is transferred to bag filter, then 4 DEG C of dialysis 72h in PBS use 4 DEG C of dialysis 48h of distilled water instead, often
It changes liquid 2 times.Solution after dialysis is centrifuged 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 hole high, positive hole is subcloned by limiting dilution assay, until set up producing single anti-good fortune
The hybridoma cell strain of the monoclonal antibody of Mo Teluo；
(3) a large amount of preparations of monoclonal antibody：Individual larger female Blab/c mouse are chosen, using inducing ascites in vivo
Method, largely prepares ascites, and purifies ascites by octanoic acid-ammonium sulfate precipitation, is divided into tubule, and -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 is determined
The affinity costant of Formoterol monoclonal antibody is determined using non-competing ELISA, as a result as shown in figure 1, parent
With constant Ka=7.24 × 108L/mol。
2nd, hypotype is determined
Hypotype measure is carried out using the mouse source monoclonal antibody hypotype identification kit purchased from 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, potency when as a result showing that Formoterol MAb concentration 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 heating, and solution eventually passes through grey, claret in saturating
Bright red, is cooled to room temperature, carries out Quality Identification by ocular estimate, ultraviolet spectrophotometry respectively, and qualified collaurum is molten
4 DEG C of liquid keeps 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 is centrifuged 30min, takes colloidal gold solution 10mL, uses 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 with colloidal gold solution under magnetic agitation, be incubated at room temperature
15min, 10000r/min are centrifuged 30min, abandon supernatant, are with every liter of BSA containing 10g, 0.5g Sodium azides, concentration by gained precipitation
The Na of 0.02mol/L2B4O7Solution dilutes, that is, obtain the Formoterol monoclonal antibody of colloid gold label, and 4 DEG C save backup；
3rd, prepared by colloidal gold pad：Glass fibre cotton is cut by specification, gold labeling antibody gold spraying instrument is uniformly sprayed on glass
On cellucotton, 37 DEG C dry 1h, sealing, and 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 the linear stealthy detection line T lines and stealthy matter of spacing 0.5cm respectively for start
Control line C lines, spontaneously dry, sealing, and 4 DEG C save backup；
5th, test strips assembling：Sample pad, colloidal gold pad, coated film and adsorptive pads are pasted on base plate successively in order,
And in the surface mount diaphragm at test strips two 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.Base plate 1 is made of PVC board in figure, 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
The plain film of dimension, adsorptive pads 7 are made of absorbent filter, numbering 2,3,4,7 each layers are pasted and fixed on base 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
Printed with chicken ovalbumin (OVA) solution of coupling Formoterol, stealthy nature controlling line is printed with goat anti-mouse igg antibody solution,
Two trace is arranged in parallel into " ︱ | ".8 to be covered in the sample end diaphragm above sample pad 2 and colloidal gold pad 3, and 9 is covering
Handle end diaphragm on adsorptive pads 7, is printed on arrow and MAX printed words on the diaphragm on the right side of sample mark line.
Formoterol in the colloid gold chromatographic test paper strip of the present invention of embodiment five detection sample
(1) sample pre-treatments
(a) urine sample：Urine sample room temperature 5000rpm centrifugation 5min are taken, the μ L sample diluting liquids of supernatant 100 is taken and is diluted 4 times, taken
Detected clearly；
(b) (meat, kidney, liver etc.) tissue：Meat sample tissue 2.0g ± 0.05g are weighed, 5mL ethyl acetate is added, shaken
Swing 1min, 5000rpm centrifugations 10min；Upper strata ethyl acetate layer solution is pipetted in another centrifuge tube, water mutually uses 5mL acetic acid again
Ethyl ester repeats to extract, and merges the drying of organic phase nitrogen；Add 1mL to redissolve working solution dissolving, take 50 μ L for analyzing；
(c) feed：Feed 2.0g ± 0.05g are weighed, 5mL ethyl acetate is added, 1min, 5000rpm centrifugations is vibrated
10min；Upper strata ethyl acetate layer solution is pipetted in another centrifuge tube, water mutually repeats extraction with 5mL ethyl acetate again, is associated with
Machine phase nitrogen is dried up；Add 1mL to redissolve working solution dissolving, take 50 μ L for analyzing.
(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, and horizontal positioned, observation in 3-5 minutes is sentenced
Result is invalid after determining testing result, 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 on coated film
Put and do not show reddish brown colo(u)r streak, represent that testing result is the positive, illustrate to contain Formoterol in testing sample；B is feminine gender, in bag
T, location of C show two days reddish brown colo(u)r streaks on envelope, represent that result is feminine gender, illustrate to be free of Formoterol in testing sample；
C, d for 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 methyl alcohol) are diluted to 0,1,2,3,4,5,10 with PBS respectively
The series of standards solution of μ g/L, then take 90 μ L above-mentioned standard solution respectively and drop in the sample pad of test strips, observed after 3min
The colour developing situation of test strips.
When Formoterol addition concentration is 3 μ g/L, the lighter of T lines when T lines are relative to 0 μ g/L, 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, specificity
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 the PBS of 0.01mol/L, PH7.4, the sample well of test strips is added drop-wise to, testing result shows, preparation
Formoterol colloidal gold strip does not exist cross reaction with other several beta receptor activators.
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, 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
The detection of Mo Teluo colloidal gold strips, as a result complies fully with HPLC methods, is shown in Table 1.
The Formoterol colloidal gold strip of table 1 compares with high performance liquid chromatography testing result
Note："+" represents positive, and "-" represents negative.
Above-described embodiment technology design and advantage only to illustrate the invention, the present invention can also become with other forms
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 (8)
1. it is a kind of to detect Formoterol colloidal gold immuno-chromatography test paper strip, it is characterised in that the test strip is by base plate, sample
Pad, colloidal gold pad, coated film and adsorptive pads composition, test paper two ends are provided with diaphragm；The sample pad, colloidal gold pad, coated film
It is pasted on base plate successively with adsorptive pads；Wherein colloidal gold pad is the Formoterol monoclonal antibody for being adsorbed with 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.
2. colloid gold chromatographic test paper strip according to claim 1, it is characterised in that the base plate be PVC bars or other not
The hard material of water suction；The sample 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 nitrocellulose filter or CAM；The diaphragm is opaque glued membrane.
3. colloid gold chromatographic test paper strip according to claim 1, it is characterised in that the Formoterol monoclonal antibody is
Mouse resource monoclonal antibody, by CCTCC NO:The hybridoma cell strain Formoterol-3E6 of C2016182 is produced；The hybridoma is thin
Born of the same parents' strain is named as hybridoma cell strain Formoterol-3E6, and China typical culture collection is delivered on October 26th, 2016
Center preservation, preserving number is CCTCC NO:C2016182.
4. colloid gold chromatographic test paper strip according to claim 1, it is characterised in that the Formoterol monoclonal antibody by
Formoterol antigen is produced, and the Formoterol antigen is the idol synthesized using carbodiimide method by Formoterol and carrier protein
Connection thing, including immunogene is former with detection；The carrier protein be bovine serum albumin(BSA), mouse serum albumin, gpv protein,
Thyroprotein, ovalbumin, hemocyanin or human serum albumins.
5. colloid gold chromatographic test paper strip according to claim 4, it is characterised in that the Formoterol immunogene uses ox
Seralbumin is carrier；It is carrier using ovalbumin that the Formoterol detection is former.
6. the preparation method of detection Formoterol colloidal gold immuno-chromatography test paper strip as claimed in claim 1 is prepared, and its feature exists
In comprising the following steps：
(1) Formoterol antigen working solution is prepared：
A () activates the preparation of p-aminobenzoic acid：6mg natrium nitrosums are weighed, is dissolved in 0.35mL distilled water, then weigh 10mg
P-aminobenzoic acid, is dissolved in the hydrochloric acid of 1.1mL 1mol/L, and ice bath is stirred, and it is right that above-mentioned sodium nitrite solution is added dropwise to
In aminobenzoic acid solution, lucifuge reaction 1h obtains activating p-aminobenzoic acid；
The preparation of the formoterol active intermediate of (b) 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；
The synthesis of (c) 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, be repeatedly added on a small quantity in solution and 3h is stirred at room temperature；Claim
Take BSA 200mg to be dissolved in the PBS of 1mL 0.01mol/L, add while stirring in above-mentioned solution, after reaction overnight, incite somebody to action 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 is centrifuged 30min in 6000rmp, takes -20 DEG C of preservations of supernatant；Labeled as Formoterol-BSA；
The former synthesis of (d) 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, be repeatedly added on a small quantity in solution and 3h is stirred at room temperature；Claim
Take OVA 150mg to be dissolved in the PBS of 1mL 0.01mol/L, add while stirring in above-mentioned solution, after reaction overnight, incite somebody to action 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 is centrifuged 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：Selection carrier protein is the immunogene of bovine serum albumin(BSA), and the female Balb/c that 6-8 week old is immunized is small
Mouse, is spaced 2 weeks immune 1 time, three times it is immune after docking take hematometry potency and inhibiting rate, the optimal mouse preparation of selection result is melted
Close；
(b) cell fusion：The splenocyte and mouse myeloma SP2/0 cells for taking the selected mouse of step (a) are merged, indirectly
ELISA method determines supernatant and chooses positive hole high, and positive hole is subcloned by limiting dilution assay, until set up producing
The hybridoma cell strain of the monoclonal antibody of single anti-Formoterol；
A large amount of preparations of (c) monoclonal antibody：Individual larger female Blab/c mouse 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 obtain 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 30min, colloidal gold solution 10mL is taken, used
0.1mol/L K2CO3The pH value of solution regulation colloidal gold solution is to 8.2；Take equivalent Formoterol monoclonal antibody solution, magnetic force
Stirring is lower to be mixed with colloidal gold solution, is incubated at room temperature 15min, 10000r/min centrifugation 30min, abandons supernatant, gained is precipitated and is used
Every liter of BSA containing 10g, 0.5g Sodium azides, concentration are the Na of 0.02mol/L2B4O7Solution dilutes, that is, obtain the good fortune of colloid gold label
Mo Teluo monoclonal antibodies, 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, and 37 DEG C dry 1h, sealing, and 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 the linear stealthy detection line and stealthy nature controlling line of spacing 0.5cm respectively for start, from
So dry, sealing, 4 DEG C save backup；
(6) test strips assembling：Sample pad, colloidal gold pad, coated film and adsorptive pads are pasted on base plate successively in order, and
The surface mount diaphragm at test strips two ends, cuts into test strips on slot-cutting machine.
7. colloid gold chromatographic test paper strip as claimed in claim 1 is for detecting animal tissue, urine and Fu Mote in feed
The application of sieve.
8. application according to claim 7, it is characterised in that comprise the following steps：
(1) sample pre-treatments
(a) urine sample：Urine sample room temperature 5000rpm centrifugation 5min are taken, the μ L sample diluting liquids of supernatant 100 is taken and is diluted 4 times, taken supernatant and enter
Row detection；
(b) (meat, kidney, liver etc.) tissue：Meat sample tissue 2.0g ± 0.05g are weighed, 5mL ethyl acetate, vibration is added
1min, 5000rpm are centrifuged 10min；Upper strata ethyl acetate layer solution is pipetted in another centrifuge tube, water mutually uses 5mL acetic acid second again
Ester repeats to extract, and merges the drying of organic phase nitrogen；Add 1mL to redissolve working solution dissolving, take 50 μ L for analyzing；
(c) feed：Feed 2.0g ± 0.05g are weighed, 5mL ethyl acetate is added, 1min, 5000rpm centrifugations 10min is vibrated；Move
Upper strata ethyl acetate layer solution is taken in another centrifuge tube, water mutually repeats extraction with 5mL ethyl acetate again, merges organic phase nitrogen
Drying；Add 1mL to redissolve working solution dissolving, take 50 μ L for analyzing；
(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 about 10-20 seconds test strip, and horizontal positioned, observation in 3-5 minutes judges inspection
Result is invalid after surveying result, 10 minutes；
(3) result judgement
A () 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, represents that testing result is the positive, illustrates to contain Formoterol in testing sample；
B () is negative：If T, location of C show two days reddish brown colo(u)r streaks on coated film, represent that result is feminine gender, illustrate to be measured
Formoterol is free of in sample；
C () is failed：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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| CN109709339A (en) * | 2018-12-28 | 2019-05-03 | 河北省科学院生物研究所 | Detect colloidal gold immuno-chromatography test paper strip and the application of ox or ovine skeletal muscle Troponin I |
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