CN109628409A - A kind of hybridoma cell strain that secreting anti-ractopamine monoclonal antibody and its application - Google Patents
A kind of hybridoma cell strain that secreting anti-ractopamine monoclonal antibody and its application Download PDFInfo
- Publication number
- CN109628409A CN109628409A CN201811578773.2A CN201811578773A CN109628409A CN 109628409 A CN109628409 A CN 109628409A CN 201811578773 A CN201811578773 A CN 201811578773A CN 109628409 A CN109628409 A CN 109628409A
- Authority
- CN
- China
- Prior art keywords
- ractopamine
- cell strain
- hybridoma cell
- monoclonal antibody
- detection
- 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.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N33/9406—Neurotransmitters
- G01N33/9413—Dopamine
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a kind of hybridoma cell strain for secreting anti-ractopamine monoclonal antibody and its applications.The hybridoma cell strain is named as hybridoma cell strain G-1-5, and deposit number is CGMCC No.16689.Mouse is immunized using the ractopamine artificial antigen of designed, designed in the present invention, hybridoma cell strain is prepared using immune mouse boosting cell, the anti-ractopamine monoclonal antibody of hybridoma cell strain secretion, potency is high, high specificity can be used for carrying out fast and accurately immune detection and immunoassay to Ractopamine.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of hybridoma for secreting anti-ractopamine monoclonal antibody
Cell strain and its application, it is residual particularly suitable for Ractopamine in animal tissue, animals urine, animal blood serum, feed equal samples
The detection of allowance.
Background technique
Ractopamine (Clebuterol) belongs to beta-receptor agonist, has and redistributes body nutrition, inhibits fat
Deposition improves protein content, increases ketoboidies lean meat percentage, improves meat and promotes the effect of growth of animal.However, in economy
Under the driving of interests, a large amount of Ractopamines are abused in animal husbandry and aquaculture.When human body excess intake Ractopamine, meeting
Lead to a series of adverse reactions, mainly has muscular tremor, heartbeat and accelerated breathing etc., or even can life threatening.Therefore, many states
Family has forbidden using Ractopamine as feed and feed addictive, and the Ministry of Agriculture, China was also clearly arranged in 2002 years
Enter and " forbids the types of drugs catalogue used in feed and animal drinking water ".
Currently, the more classical method of detection Ractopamine have tablets by HPLC-MS (HPLC-MS),
Gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), these are all the traditional method of comparison and mesh
Preceding confirmation method.The application of laboratories mass detection sample is usually immunization method, therefore obtains one plant with spy
The anti-ractopamine monoclonal antibody hybridoma cell strain of anisotropic affinity is very important.The present invention prepares a kind of point
The hybridoma cell strain of ractopamine monoclonal antibody is secreted, and is applied to immunoassay product, measures animal tissue, animal urine
The residual quantity of Ractopamine drug in liquid, there is detection to limit, and low, high specificity, easy to operate, detection speed is fast, testing cost
It is low, it is very easy to the advantages that promoting.
Summary of the invention
It is an object of that present invention to provide one plant to have the monoclonal antibody hybridoma of specific affinity to Ractopamine
The preparation method of cell strain.
The present invention provides one plant of ractopamine monoclonal antibody hybridoma cell strain, the antibody pair prepared by the cell strain
Ractopamine has preferable affinity and detection sensitivity, can be used to establish the immunology detection of Ractopamine total amount
Method, including enzyme linked immunological kit, colloidal gold colloidal gold detection test paper strip, immune affinity column etc..
Technical solution of the present invention: one plant of ractopamine monoclonal antibody hybridoma cell strain is named as G-1-5,
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.16689.
Ractopamine monoclonal antibody, the Ractopamine Dan Ke that it is CGMCC No.16689 by the deposit number
Grand antibody hybridoma cell strain G-1-5 is secreted to be generated.
The application of the ractopamine monoclonal antibody, the detection for Ractopamine content in food safety.
The preparation step of cell strain G-1-5 provided by the invention are as follows:
(1) preparation with identification of artificial antigen: chapter amine and 4- Mehtoxy-ethoxy-benzo methyl valerate react, and obtain Lay
Gram dopamine haptens, haptens and protein are coupled, and prepare artificial antigen, i.e. immunogene, coating antigen；
(2) preparation of monoclonal antibody: by the way that mouse is immunized, monoclonal antibody is obtained；
(3) by cell fusion and cloning, it is miscellaneous the preparation of cell strain G-1-5: to obtain ractopamine monoclonal antibody
Hand over tumor cell strain G-1-5.
(4) application of cell strain G-1-5: it is applied to enzyme linked immunological kit.
(5) application of cell strain G-1-5: it is applied to colloidal gold immuno-chromatography test paper strip.
(6) application of cell strain G-1-5: it is applied to chemiluminescence immune detection reagent kit, immuno magnetic cell separation enrichment examination
Agent box, magnetic granule chemiluminescence kit, time-resolved fluoroimmunoassay chromatograph test strip, fluorescent micro-ball immune chromatography test paper strip,
Other immune detection products such as immune affinity column.
Detailed description of the invention
Fig. 1: Ractopamine hapten synthesis route map
Specific embodiment
Below with reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this
Invention, and be not intended to limit the scope of the invention.
The preparation of 1 Ractopamine reagent constituents of embodiment
1, the preparation of Ractopamine haptens
Chapter amine 1.0g is taken, adds methanol 50mL to dissolve, adds 4- Mehtoxy-ethoxy-benzo methyl valerate 1.73g, add cyano
7h is stirred in sodium borohydride 0.44g, 60 DEG C of oil baths, stops reaction, and revolving removes methanol, adds ethyl acetate 50ml, add water 30mL,
Extraction removes water phase, and organic phase is evaporated, and ethyl alcohol/n-hexane (v/v, 1/2) 70mL recrystallization obtains methoxyl group Ractopamine
Methyl acetate 2.0g, yield 83%.
Methoxyl group Ractopamine methyl acetate 2.0g is taken, the KOH aqueous solution 60ml of 1.0mol/L is added, is heated, 90 DEG C anti-
3h is answered, stops reaction, is cooled to room temperature, adds 6mol/L hydrochloric acid, pH value is adjusted to 6, adds ethyl acetate 100ml to extract, be evaporated, on
Silicagel column, chloroform/methanol (v/v, 5/1) elution separation, obtains acetic acid methoxyl group Ractopamine 1.6g, yield
84.21%.
2, the preparation of antigen
Immunogene preparation --- Ractopamine haptens and bovine serum albumin(BSA) (BSA) coupling obtain immunogene.
Acetic acid methoxyl group Ractopamine haptens 23mg is taken, adds tetrahydrofuran 3ml to dissolve, adds 120 μ L of triethylamine, chlorination
57 μ L of iso-butyl formate, is stirred at room temperature 5h, obtains haptens activating solution A liquid；Bovine serum albumin(BSA) (BSA) 50mg is taken, 3ml is added
0.05mol/L PB buffer solution, obtains B liquid, and A drop is added in B liquid, stirs for 24 hours, 0.02mol/L PBS dialysis three
It, obtains Ractopamine-BSA immunogene, and -20 DEG C of preservations are spare.
Coating antigen preparation --- Ractopamine haptens and ovalbumin (OVA) coupling obtain immunogene.
Acetic acid methoxyl group Ractopamine haptens 17mg is taken, adds DMSO 4ml to dissolve, adds 79 μ L of triethylamine, chlorination formic acid
46 μ L of isobutyl ester, is stirred at room temperature 5h, obtains haptens activating solution A liquid；Oralbumin (OVA) 50mg is taken, 3ml is added
0.05mol/L PB buffer solution, obtains B liquid, and A drop is added in B liquid, and for 24 hours, 0.02M PBS dialyses three days for stirring, obtains
To Ractopamine-OVA coating antigen, -20 degree are saved, spare.
3, the preparation of ractopamine monoclonal antibody
Animal immune: the immunogene that above-mentioned steps are obtained is injected into Balb/c Mice Body, and immunizing dose is 150 μ g/
Only, it is made to generate antiserum.
Cell fusion and cloning: after mice serum measurement result is higher, taking its splenocyte, compares by 8:1 (quantitative proportion)
Example is merged with SP2/0 myeloma cell, measures cell supernatant using indirect competitive ELISA, screens positive hole.Using limited dilute
Interpretation of the law carries out cloning to positive hole, until obtaining the hybridoma cell strain of secretion ractopamine monoclonal antibody.
Biological material specimens preservation: one plant of ractopamine monoclonal antibody hybridoma cell strain G-1-5 has been preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, abbreviation CGMCC, address are as follows: Chaoyang District, Beijing City North Star west
The institute 3 of road 1, Institute of Microorganism, Academia Sinica, deposit number are CGMCC No.16689.The deposit date is 2018 10
The moon 29.
Cell cryopreservation and recovery: monoclonal hybridoma strain is made 1 × 10 with frozen stock solution6The cell suspension of a/ml,
It is saved for a long time in liquid nitrogen.Cryopreservation tube is taken out when recovery, 37 DEG C of water-bath middling speeds is immediately placed in and melts, and after centrifugation removal frozen stock solution, is moved
Enter to cultivate culture in glassware.
The production and purifying of monoclonal antibody: Balb/c mouse peritoneal is injected into sterilizing paraffin oil 0.5ml/ only, abdomen after 7 days
Chamber injects stable monoclonal hybridoma strain 5 × 105A/only, ascites is acquired after 7 days.With octanoic acid-saturated ammonium sulfate method into
The purifying of row ascites, -20 DEG C of preservations.
4, the preparation of sheep anti mouse antiantibody
It is that immune animal obtains sheep anti mouse antiantibody using source of mouse antibody as immunogen immune pathogen-free domestic sheep with sheep.
5, the preparation of enzyme label antiantibody
Sheep anti mouse antiantibody is coupled with horseradish peroxidase (HRP) using the Over-voltage protection after improvement.It passes
It is 4:1 that the Over-voltage protection of system, which requires the molar concentration rate of enzyme and antibody in reaction system, since horseradish peroxidase is strong
Many sites in conjunction with antibody are generated under oxidation, the horseradish peroxidase molecule activated in this way acts as each point of connection
The bridge of son, reduces the enzymatic activity of enzyme marker, makes to be mixed with many condensates in the conjugate of preparation.It is asked to solve this
Topic, we are improved traditional method, it may be assumed that
(1) closed process of amino is eliminated, because the amino that can generate the connection of itself amino is actually seldom；
(2) molar concentration ratio of horseradish peroxidase and antibody is reduced to 2:1, and the method after improvement is than traditional side
Method is easy, reduces to the loss of enzymatic activity.
6, the preparation of ELISA Plate
Coating antigen is diluted to 20 μ g/ml with coating buffer, 100 μ l are added in every hole, and 37 DEG C are protected from light incubation 2h, hole of inclining
Middle liquid is washed 2 times with cleaning solution, and each 30s is patted dry, and 200 μ l confining liquids is then added in every hole, 37 DEG C are protected from light incubation
2h, liquid pats dry in hole of inclining, and is saved after dry with aluminium film vacuum sealing.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of Ractopamine
The enzyme linked immunological kit for setting up detection Ractopamine, makes that it includes following components:
(1) it is coated with the ELISA Plate of Ractopamine coupled antigen；
(2) 6 bottles of Ractopamine standard solution, concentration be respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L,
2.7μg/L,8.1μg/L；
(3) ractopamine monoclonal antibody working solution；
(4) the sheep anti mouse antiantibody of horseradish peroxidase-labeled is used；
(5) substrate developing solution is made of A liquid and B liquid, and A liquid is urea peroxide, and B liquid is tetramethyl benzidine；
(6) terminate liquid is 2mol/L sulfuric acid；
(7) it is 7.4 that cleaning solution, which is pH value, anti-containing 0.5%~1.0% Tween-20,0.01 ‰~0.03 ‰ sodium azide
Rotten agent, 0.1~0.3mol/L phosphate buffer, the percentage be percent weight in volume；
(8) redissolve liquid be pH value be 7.0, the phosphate buffer of 0.02mol/L, the percentage be weight volume basis
Than.The detection of Ractopamine in 3 animal tissue of embodiment, animals urine sample
1, sample pre-treatments
(1) animal tissue
Sample after weighing 2.0 ± 0.05g homogeneous is into 50ml polystyrene centrifuge tube；Addition 1ml redissolution liquid working solution,
1ml ethyl alcohol and 1ml 0.25M dilute sulfuric acid vibrate 5min, (20-25 DEG C) centrifugation 5min of 3000g room temperature with oscillator；It pipettes
It is mixed that 0.5ml redissolution working solution is added into 2ml polystyrene centrifuge tube in 0.5ml supernatant (if upper layer has suspended matter that should avoid)
It is even；Take 20 μ l for analyzing.
(2) animals urine
Take the 20 limpid urine samples of μ l directly measure (such as urine sample muddiness must by filtering or 3000g room temperature be centrifuged 5min until
It is limpid), the sample that wouldn't be used answers freezen protective.
2, it is detected with kit
Add 20 μ l of standard items/sample into corresponding micropore, 50 hole μ l/ of ELIAS secondary antibody is then added, adds antibody work
Make 80 hole μ l/ of liquid, gently oscillation mixes, and reacts 45min in cover board membrane cover plate 25 DEG C of light protected environments of postposition.Carefully open cover board
Film dries liquid in hole, and 250 hole μ l/ of wash operating solution, sufficiently washing 4-5 times, every minor tick 10s is added, sprinkles in board falling hole
Cleaning solution is patted dry with blotting paper.50 hole μ l/ of substrate solution A liquid is added, adds 50 hole μ l/ of substrate solution B liquid, gently oscillation mixes,
15min is reacted in cover board membrane cover plate 25 DEG C of light protected environments of postposition.50 hole μ l/ of terminate liquid is added, gently oscillation mixes, and sets enzyme
Instrument is marked at 450nm, measures every hole OD value.
3, Analysis of test results
The percentage absorptance of standard items or sample be equal to the average value (diplopore) of the absorbance value of standard items or sample divided by
The average value of the absorbance value of first standard items (0 standard) obtains the percentage extinction of standard items or sample multiplied by 100%
Angle value.Using standard items percentage absorptance as ordinate, using the logarithm of Ractopamine standard concentration (μ g/L) as abscissa, draw
Canonical plotting processed.The percentage absorptance of sample is substituted into standard curve, is read from standard curve dense corresponding to sample
Degree, is the actual concentrations of Ractopamine in sample multiplied by its corresponding extension rate.
4 Ractopamine technical parameter of embodiment determines test
1, kit sensitivity and detection limit
Conventionally assay kit sensitivity, the range of standard curve are 0.1~8.1 μ g/L, IC50(50% suppression
Concentration processed) floating range be 0.37~0.75 μ g/L；20 parts of samples are detected, are found from standard curve corresponding to each hundred
Divide the concentration of absorbance value, detection limit is indicated plus 3 times of standard deviations with the average value of 20 parts of concentration of specimens, the results show that the party
Method is respectively 0.5 μ g/kg, 0.3 μ g/kg to the detection limit of animal tissue, animals urine.
2, sample preci-sion and accuracy is tested
Using the rate of recovery as accuracy estimating index, the testing result relative standard deviation of a certain concentration samples of replication
(RSD%) it is used as precision evaluation index.Calculation formula are as follows: the rate of recovery (%)=actual measured value/theoretical value × 100%,
Middle theoretical value is the addition concentration of sample；Relative standard deviation RSD%=SD/X × 100%, wherein SD is standard deviation, and X is
The average value of determination data.
Recycling measurement is added to pork sample respectively by the Ractopamine of 1 μ g/kg, 2 two concentration of μ g/kg, is pressed
0.6 μ g/kg, 1.2 two concentration of μ g/kg Ractopamine respectively to pig urine samples be added recycling measurement, each sample
It does 4 in parallel, is measured with three batches of different reagents, the average recovery rate and precision result for calculating sample see the table below.
1 precision of table and accuracy test
Recycling measurement is added to pork sample respectively by the Ractopamine of 1 μ g/kg, 2 two concentration of μ g/kg, is pressed
0.6 μ g/kg, 1.2 two concentration of μ g/kg Ractopamine recycling measurement is added to pig urine samples respectively, it is average to recycle
Rate is between 81.2%~90.7%；Relative standard deviation is respectively less than 10% in batch, between criticizing.
3, stabilization of kit is tested
Kit preservation condition is 2~8 DEG C, by measurement in 12 months, the maximum absorbance value (zero standard) of kit,
50% inhibition concentration, Ractopamine addition actual measured value are within normal range (NR).Consider in transport and use process,
It has improper preservation condition to occur, kit is placed 7 days under 37 DEG C of preservation conditions, carry out accelerated aging tests, as a result
Show that the kit indices comply fully with requirement.In view of kit freezing happens, kit is put into -20 DEG C of ice
Case freezes 7 days, and measurement result also indicates that kit indices are completely normal.It can show that kit can be 2 from result above
~8 DEG C at least save 12 months or more.
The preparation of 5 colloidal gold immuno-chromatography test paper strip of embodiment
The preparation method of the test strips mainly comprises the steps that
1) preparation is coated with ractopamine monoclonal antibody-colloid gold label object conjugate release pad；
2) preparation has the detection line for being coated with Ractopamine hapten-carrier protein conjugate and is coated with sheep anti mouse
The reaction film of the nature controlling line of antiantibody；
And 2) 3) 1) conjugate release pad, reaction film and the sample absorption pad, water absorption pad and PVC bottom plate that prepare are assembled
At test strips.
Substep narration in detail below:
1, ractopamine monoclonal antibody-colloid gold label object preparation
(1) preparation of colloidal gold
1% gold chloride is diluted to 0.01% (mass fraction) with double distilled deionized water, 100ml is taken to be placed in conical flask,
It is heated to boiling with thermostatic electromagnetic blender, in continuous high temperature, is persistently added with stirring 1% trisodium citrate of 2.5ml, continue
Solution is at the uniform velocity heated with stirring in stopping when bright red, is restored to original volume, 4 DEG C of guarantors with deionized water after being cooled to room temperature
It deposits.The colloidal gold appearance prepared is pure, it is bright, without precipitating and floating material.
(2) ractopamine monoclonal antibody-colloid gold label object preparation
Under magnetic stirring, molten by every milliliter of colloidal gold with the pH value of 0.2mol/L solution of potassium carbonate tune colloidal gold to 7.0
Ractopamine monoclonal antibody is added into colloidal gold solution for the standard that 20~50 μ g are added in liquid, continues to stir and evenly mix
10%BSA is added in 30min, makes its final concentration of 1% (volume fraction) in colloidal gold solution, stands 10min.12000r/
Min, 4 DEG C of centrifugation 40min abandon supernatant, and it is initial colloid gold volume 1/10 with volume that precipitating is washed twice with redissolution buffer
Redissolution buffer will precipitating be resuspended, set 4 DEG C it is spare.
Redissolve buffer: casein containing protein 0.02%~0.1% (mass fraction), 0.05%~0.2% (quality of Tween-80
Score), the 0.02mol/L phosphate buffer of pH7.2.
2, the preparation of conjugate release pad
Conjugate release pad is soaked in (concentration of the bovine serum albumin(BSA) in buffer is containing bovine serum albumin(BSA)
0.5%), in the phosphate buffer of pH 7.2,0.5mol/L, 1h is uniformly soaked, 37 DEG C of baking 3h are spare.With Isoflow point film
By the ractopamine monoclonal antibody prepared-colloid gold label object even application in conjugate release pad, every 1cm is tied instrument
After closing object release pad spraying 0.01ml ractopamine monoclonal antibody-colloid gold label object, it is placed in (humidity < in 37 DEG C of environment
20%) it is taken out after 60min, is placed in dry environment (humidity < 20%) and saves backup.
3, the preparation of reaction film
Detection line will be constituted in Ractopamine haptens-ovalbumin conjugate coating to reaction film, sheep anti mouse is resisted
Antibody, which is coated on reaction film, constitutes nature controlling line.
Coating process: being diluted to 10mg/ml for Ractopamine haptens-ovalbumin conjugate with phosphate buffer,
It is coated in the detection line (T line) on nitrocellulose filter with Isoflow point film instrument, package amount is 0.8 μ l/cm；With
Sheep anti mouse antiantibody is diluted to 200 μ g/ml by the phosphate buffer of 0.01mol/L, pH7.4, with Isoflow point film instrument by its
The nature controlling line (C line) being coated on nitrocellulose filter, package amount are 1.0 μ l/cm.The reaction film being coated with is placed in 37 DEG C of items
Dry 2h, spare under part.
4, the preparation of sample absorption pad
Sample absorption pad is placed in slow containing 0.5% bovine serum albumin(BSA) (volume fraction), pH7.2,0.1mol/L phosphate
2h is impregnated in fliud flushing, 37 DEG C of baking 2h are spare.
5, the assembling of test strips
Sample absorption pad, conjugate release pad, reaction film, water absorption pad are successively pasted on PVC bottom plate in order；In conjunction with
Object release pad has 1/3 region to be absorbed by the sample pad covering from starting point, and the end of conjugate release pad and the beginning of reaction film connect
It connects, the end of reaction film is connected with the beginning of water absorption pad, and the beginning of sample absorption pad is aligned with the beginning of PVC bottom plate, water absorption pad
End be aligned with the end of PVC bottom plate；There are detection line and nature controlling line, detection line (T line) and nature controlling line (C on the reaction film
Line) it is the strip tape perpendicular with the length of the test strips；Detection line is located at the side close to the end of conjugate release pad；
Nature controlling line is located remotely from the side of the end of conjugate release pad；Test strips are cut into the small item of 3mm wide with machine, mounted in special
Plastics fabrication in, under the conditions of 4~30 DEG C can be reserved for 12 months.
The detection of Rct opamine residue in 6 sample of embodiment
1, it is detected with test strips
Measuring samples solution, which to be drawn, with suction pipe 3 drops is vertically added dropwise in well, liquid starts timing when flowing, reaction 5~
10min determines result.
2, analysis detection result
Negative (-): T line and C line all develop the color, and indicate that Ractopamine concentration is lower than detection limit in sample.
Positive (+): T line indicates that Ractopamine concentration is equal to or higher than detection limit in sample without colour developing, the colour developing of C line.
It is invalid: not occur C line, show the deterioration failure of incorrect operating process or test strips.It in the case, should be again
It is secondary to read over specification, and retested with new test strips.
7 sample detection example of embodiment
1, detection limit test
Blank diaper, chicken, pork, the flesh of fish, shrimp, feed sample are taken, adds Ractopamine respectively to end in urine
Concentration is 1.5,3,6ng/ml, chicken, pork, the flesh of fish, shrimp add respectively Ractopamine to final concentration of 2.5,5,
10ng/ml adds Ractopamine to final concentration of 10,20,40ng/ml in feed respectively, takes test strips to be detected, each
Sample is repeated three times.
When detecting urine, chicken, pork, the flesh of fish, shrimp, feed sample with test strips, judgement inspection is shown according to test strips
Limit is surveyed, shows that this test strips is limited to 3ng/ml to the detection of Ractopamine in urine, to Lay in chicken, pork, the flesh of fish, shrimp
The detection of gram dopamine is limited to 5ng/ml, is limited to 20ng/ml to the detection of Determination of Ractopamine in Feeds.
2, false positive rate, false negative rate test
Known Ractopamine content is taken to be greater than urine, chicken, pork, the flesh of fish, shrimp, the feed positive of detection limit respectively
Each 20 parts of sample and content are less than each 20 parts of negative sample of detection limit, are detected with three batches of test strips, calculate its yin and yang attribute
Rate.As a result it see the table below.
2 false positive rate of table, false negative rate test result
The result shows that: positive urine, chicken, pork, the flesh of fish, shrimp, feeding are detected with the test strips of 3 batch productions respectively
When expecting sample, as a result it is all positive, it is known that positive sample coincidence rate is 100%, false negative rate 0；20 parts of feminine genders are detected respectively
When urine, chicken, pork, the flesh of fish, shrimp, feed sample, as a result it is all negative, it is known that negative match-rate 100%, false positive
Rate is 0.Illustrate that the test strips of detection Ractopamine of the invention can be in urine, chicken, pork, the flesh of fish, shrimp, feed
Rct opamine residue is used for quickly detecting.
3, specific test
The beta-stimulants such as salbutamol, clenbuterol, Cimaterol with Ractopamine test strips detection 500ng/ml
Class drug.The results show that test strips nature controlling line and detection line develop the color, it is negative.Illustrate this test strips to the sand of 500ng/ml
The beta-stimulants no cross reaction such as butylamine alcohol, clenbuterol, Cimaterol.
Claims (8)
1. a kind of hybridoma cell strain for secreting anti-ractopamine monoclonal antibody, which is characterized in that be named as G-1-5, protect
Hiding number is CGMCC No.16689.
2. preparing hybridoma cell strain as described in claim 1, need first to prepare Ractopamine haptens, the system of haptens
Preparation Method is as follows:
Chapter amine 1.0g is taken, adds methanol 50mL to dissolve, adds 4- Mehtoxy-ethoxy-benzo methyl valerate 1.73g, add cyano boron hydrogen
Changing sodium 0.44g, 7h is stirred in 60 DEG C of oil baths, stops reaction, and revolving removes methanol, adds ethyl acetate 50ml, add water 30mL, it extracts,
Water phase is removed, organic phase is evaporated, and ethyl alcohol/n-hexane (v/v, 1/2) 70mL recrystallization obtains methoxyl group Ractopamine acetic acid first
Ester 2.0g, yield 83%；
Methoxyl group Ractopamine methyl acetate 2.0g is taken, the KOH aqueous solution 60ml of 1.0mol/L is added, is heated, 90 DEG C of reaction 3h,
Stop reaction, be cooled to room temperature, add 6mol/L hydrochloric acid, adjusts pH value to 6, add ethyl acetate 100ml to extract, be evaporated, upper silica gel
Column, chloroform/methanol (v/v, 5/1) elution separation, obtains acetic acid methoxyl group Ractopamine 1.6g, yield 84.21%.
3. hybridoma cell strain as described in claim 1 answering in the enzyme linked immunological kit of preparation detection Ractopamine
With.
4. hybridoma cell strain as described in claim 1 is in the colloidal gold immuno-chromatography test paper strip of preparation detection Ractopamine
In application.
5. hybridoma cell strain as described in claim 1 is in the chemiluminescence immunoassay detection reagent of preparation detection Ractopamine
It is box, immuno magnetic cell separation enrichment kit, magnetic granule chemiluminescence kit, time-resolved fluoroimmunoassay chromatograph test strip, glimmering
Application in other immune detection products such as light micro-ball immune chromatography test paper strip, immune affinity column.
6. a kind of anti-ractopamine monoclonal antibody, which is characterized in that by hybridoma cell strain described in claim 1 or its
Passage cell strain secretion generates.
7. application of the anti-ractopamine monoclonal antibody as claimed in claim 6 in detection Ractopamine.
8. a kind of detection kit of Ractopamine, which is characterized in that contain anti-Ractopamine as claimed in claim 6
Monoclonal antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811578773.2A CN109628409B (en) | 2018-12-24 | 2018-12-24 | Hybridoma cell strain secreting anti-ractopamine monoclonal antibody and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811578773.2A CN109628409B (en) | 2018-12-24 | 2018-12-24 | Hybridoma cell strain secreting anti-ractopamine monoclonal antibody and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109628409A true CN109628409A (en) | 2019-04-16 |
| CN109628409B CN109628409B (en) | 2022-08-19 |
Family
ID=66076634
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811578773.2A Active CN109628409B (en) | 2018-12-24 | 2018-12-24 | Hybridoma cell strain secreting anti-ractopamine monoclonal antibody and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109628409B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110950960A (en) * | 2019-11-26 | 2020-04-03 | 中国农业大学 | Preparation method of small molecule compound antibody based on high-throughput sequencing and hybrid hybridoma technology |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6274334B1 (en) * | 2000-07-13 | 2001-08-14 | The United States Of America As Represented By The Secretary Of Agriculture | Monoclonal antibody, cell line and immunoassay for ractopamine |
| CN101921730A (en) * | 2010-01-19 | 2010-12-22 | 泰州康正生物技术有限公司 | A kind of monoclonal antibody of Ractopamine hydrochloride, its preparation method and application |
| CN102778564A (en) * | 2012-05-31 | 2012-11-14 | 华中农业大学 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting ractopamine |
-
2018
- 2018-12-24 CN CN201811578773.2A patent/CN109628409B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6274334B1 (en) * | 2000-07-13 | 2001-08-14 | The United States Of America As Represented By The Secretary Of Agriculture | Monoclonal antibody, cell line and immunoassay for ractopamine |
| CN101921730A (en) * | 2010-01-19 | 2010-12-22 | 泰州康正生物技术有限公司 | A kind of monoclonal antibody of Ractopamine hydrochloride, its preparation method and application |
| CN102778564A (en) * | 2012-05-31 | 2012-11-14 | 华中农业大学 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting ractopamine |
Non-Patent Citations (1)
| Title |
|---|
| 王俊等: "莱克多巴胺单克隆抗体的制备及特性鉴定", 《广东农业科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110950960A (en) * | 2019-11-26 | 2020-04-03 | 中国农业大学 | Preparation method of small molecule compound antibody based on high-throughput sequencing and hybrid hybridoma technology |
| CN110950960B (en) * | 2019-11-26 | 2021-05-14 | 中国农业大学 | Preparation method of small molecule compound antibody based on high-throughput sequencing and hybrid hybridoma technology |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109628409B (en) | 2022-08-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100403030C (en) | ELISA kit for detecting Sudan red medicines and detection method thereof | |
| CN101571541B (en) | Enzyme-linked immunosorbent inspect kit for inspecting sulfa drugs and method thereof | |
| CN101013129B (en) | Enzyme linked immunosorbent reagent casing for detecting furacilin metabolite and uses thereof | |
| CN101256188B (en) | ELISA kit for detecting lincomycin medicine as well as usage thereof | |
| CN100445746C (en) | Method for detecting 19-nortestosterone and special enzyme-linked immune reagent kit thereof | |
| CN102967709B (en) | Detect enzyme linked immunological kit and the application thereof of zearalenone medicine | |
| CN100501409C (en) | ELISA kit for detecting chloramphenicols in animal derived food | |
| CN101021536B (en) | Enzyme-linked immunological kit for detecting neomycin drug and method | |
| CN103012592A (en) | Preparation and applications of ractopamine monoclonal antibody | |
| CN101046474A (en) | Enzyme-linked immunological kit for detecting quinoxaline medicine residue | |
| CN101021535B (en) | Enzyme-linked immunalogical kit for detecting gentamicin medicine and method | |
| CN101358967B (en) | Method for detecting chlorpromazine and special ELISA kit thereof | |
| CN101776685A (en) | Enzyme linked immunosorbent assay kit for detecting trimethoprim medicament and application thereof | |
| CN109917126A (en) | A method of test strips, the preparation method of imidacloprid haptens and the detection Determination of Imidacloprid Residue of detection imidacloprid | |
| CN109239336A (en) | A kind of test strips and its application detecting Mobucin | |
| CN100489530C (en) | Method for assaying sulfaquinoxaline and special enzyme-linked immune reagent kit | |
| CN101013131B (en) | Furantoin metabolite enzyme linked immunosorbent analytical reagent casing and uses thereof | |
| CN107271665A (en) | A kind of test strips for detecting salbutamol and its application | |
| CN100533148C (en) | Method for detecting tylosin and special enzyme-linked immune reagent kit thereof | |
| CN101782579B (en) | Enzyme-linked immunoassay kit for detecting tiamulin medicaments, and application thereof | |
| CN103364553A (en) | Enzyme linked immunosorbent assay kit for detecting nitroimidazole drugs and application thereof | |
| CN109628409A (en) | A kind of hybridoma cell strain that secreting anti-ractopamine monoclonal antibody and its application | |
| CN100492009C (en) | ELISA kit for detecting fluoromethylquinoline and detection method thereof | |
| CN109628408A (en) | A kind of hybridoma cell strain that secreting anti-clenbuterol monoclonal antibody and its application | |
| CN100489532C (en) | Method for detecting salinomycin and spcial enzyme-linked immune reagent kit thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |