CN109696548A - A kind of kit and its preparation method and application detecting Fipronil - Google Patents
A kind of kit and its preparation method and application detecting Fipronil Download PDFInfo
- Publication number
- CN109696548A CN109696548A CN201710990416.6A CN201710990416A CN109696548A CN 109696548 A CN109696548 A CN 109696548A CN 201710990416 A CN201710990416 A CN 201710990416A CN 109696548 A CN109696548 A CN 109696548A
- Authority
- CN
- China
- Prior art keywords
- fipronil
- solution
- kit
- liquid
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/10—Insecticides
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of kits and the preparation method and application thereof for detecting Fipronil.Fipronil detection kit provided by the present invention includes Fipronil ELISA Plate, Fipronil standard items working solution, Fipronil antibody working solution, enzyme marker working solution, substrate solution, terminate liquid, further includes fipronil hapten shown in Formulas I and the resulting antigen of carrier protein couplet.Fipronil detection kit provided by the present invention, easy to operate, quick, high sensitivity, high specificity, accuracy are strong, and for Fipronil residual in animal products, quickly detection has substantial worth.Formulas I.
Description
Technical field
The invention belongs to medicament residue field of fast detection, are related to a kind of kit and preparation method thereof for detecting Fipronil
With application.
Background technique
Fipronil is that a kind of Phenylpyrazole insecticides, insecticidal spectrum are wide, for preventing and treating ground and subterranean pest-insect, cauline leaf process
With soil treatment, seed treatment etc..Its mechanism of action is that the chloride for hindering the control of insect γ-aminobutyric acid is metabolized, therefore
There is very high insecticidal activity to important pests such as aphid, leafhopper, plant hopper, lepidopterous larvae, flies and coleopteras, is by numerous agricultures
Medicine expert is recommended as one of the preferred kind instead of high-toxic organic phosphorus pesticide, and in recent years, Fipronil is also widely used in hygienic biocide
Agent is mainly used for preventing killing the harmful organisms such as cockroach, ant.
Although Fipronil pest control effect is fine, it is extremely unfriendly to environment, can be to the butterfly around crops
Butterfly, dragonfly etc. impact, to fish, shrimp, honeybee, silkworm high poison.And existing animal experiment study shows to absorb in short term a large amount of
Fipronil can cause adverse effect to nervous system, and taken long-term Fipronil may damage liver, thyroid gland and kidney.Therefore
China provides disabling Fipronil from October 1st, 2009, can only be used to domestic hygiene pest at present.European legislation also provides, fluorine
Worm nitrile must not be used for the livestock and poultry cultivation process of human food's industrial chain.
However, on July 20th, 2017, Belgium is notified to detect Fipronil in egg by RASFF system.Problem egg is
Sold to 12 countries.It is reported that problem egg originates from Holland, Fipronil is by inappropriate cleaning for chicken farm
In article, egg is caused to be detected residue.This time Event Description, pollution by pesticides run through product cultivating, producing, processing and selling
Links, the supervision subsequent for government such as circulation propose more acid test.It also mentions in " 13 " planning from farmland
To the full chain supervision of dining table, edible agricultural product overall process tracing management is realized.
And influence government regulation or the most important factor of management effect is the detection means to Fipronil, Fipronil at present
Detection is also detected primarily directed to the insecticide Fipronil in vegetables, environment, and method mainly has high-efficient liquid phase technique and liquid chromatogram
Matter combination method, these method high specificities, high sensitivity, but cumbersome, expensive equipment, it is not suitable for batch samples
Selective mechanisms.Immunodetection has unique advantage, quick, cost easy to operate in terms of the qualitative, quantitative of antigen-antibody
It is low, analysis sample size is big, but the sample of existing immune detection fails to cover cultivation processing link, and sensitivity cannot reach
To the requirement monitored for Fipronil.
Summary of the invention
The object of the present invention is to provide a kind of kits and the preparation method and application thereof for detecting Fipronil, have sensitivity
It is high, accuracy is strong, high sensitivity, advantage easy to operate, the Edible tissues, interior especially suitable for animals such as ox, sheep, pig, chickens
Dirty, egg, milk, Fipronil residual quickly detection in animal product.
It is an object of the present invention to provide a kind of fipronil hapten compounds, and structural formula is as shown in formula I:
(formula I).
Fipronil hapten is that Fipronil and mercaptopropionic acid react to obtain, which contains pendant carboxylic group, Ke Yiyu
Carrier protein couplet forms coating antigen and immunogene.
The conjugate of compound shown in above-mentioned formula I and carrier protein also belongs to protection scope of the present invention.Wherein, the load
Body protein be bovine serum albumin(BSA) (BSA), human serum albumins (HSA), mouse haemocyanin (MSA), thyroprotein (BCG),
Rabbit serum proteins (RSA), hemocyanin (KLH) or oralbumin (OVA).
The conjugate of the fipronil hapten and carrier protein refers to that fipronil hapten and carrier protein pass through activity
The product that ester process obtains.
The conjugate of compound shown in compound shown in above-mentioned formula I and/or above-mentioned formula I and carrier protein is preparing Fipronil
Application in monoclonal antibody also belongs to protection scope of the present invention.
The conjugate and/or above-mentioned Fipronil of compound shown in above-mentioned formula I, compound and carrier protein shown in above-mentioned formula I
Application of the monoclonal antibody in the enzyme linked immunological kit of preparation detection Fipronil also belongs to protection scope of the present invention.
It is a further object to provide a kind of method for preparing compound shown in above-mentioned formula I and it is a kind of prepare it is above-mentioned
The method of the conjugate of compound shown in formula I and carrier protein.
The method provided by the present invention for preparing compound shown in above-mentioned formula I includes the following steps: Fipronil, 3- mercapto
Base propionic acid is dissolved in methanol with the weight ratio of 1000:365, and purifying is spin-dried for, and is terminated reaction, is obtained the compound.
The method of the conjugate provided by the present invention for preparing compound shown in above-mentioned formula I and carrier protein, including it is as follows
Step:
1) fipronil hapten (Formulas I) is dissolved in dimethylformamide (DMF), 1- (3- dimethylamino is then added
Propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and n-hydroxysuccinimide (NHS), 20-25 DEG C of magnetic agitation react 2-
3h obtains solution I；
Wherein, the fipronil hapten (Formulas I), the dimethylformamide (DMF), the 1- (3- dimethylamino-propyl)-
3- ethyl-carbodiimide hydrochloride (EDC), the n-hydroxysuccinimide (NHS) proportion be 15.93mg:1.5mL:
21.5mg:13mg.
2) carrier protein is placed in 0.1M sodium bicarbonate buffer liquid, 200rpm stirs 10min, sufficiently dissolves, obtains
To solution II；The proportion of the carrier protein and the 0.1M sodium bicarbonate buffer liquid is 33.6-50mg:3.5mL；
Wherein, if the carrier protein is bovine serum albumin(BSA) (BSA), the bovine serum albumin(BSA) (BSA) and the 0.1M
The proportion of sodium bicarbonate buffer liquid is 50mg:3.5mL；If the carrier protein is ovalbumin (OVA), the ovalbumin
It (OVA) is 33.6mg:3.5mL with the proportion of the 0.1M carbonic acid buffer；
3) solution I and the solution II are mixed, specially under the conditions of 0-4 DEG C, under 1000rpm stirring, by solution I by
It is added dropwise in the solution II, 500rpm is stirred to react for 24 hours, obtains solution III；
4) phosphate buffer (0.01M PBS, pH7.2) is used, in 4 DEG C to the solution III stirring dialysis 3 days, obtained described
Fipronil antigen.
The application of the fipronil hapten (Formulas I) or the Fipronil antigen in qualitatively or quantitatively detection Fipronil
It belongs to the scope of protection of the present invention.
Sample after weighing 1 ± 0.05 g homogeneous is in 50mL centrifuge tube；Sequentially add 1 g purified reagent, 3 mL
Acetonitrile (analysis is pure), abundant 5 min of whirling motion；4000 g or more are centrifuged 5 min；Take 1 mL supernatant in clean centrifuge tube；
50-60 DEG C of water-bath is dried with nitrogen；1 mL sample diluting liquid, abundant 1 min of whirling motion is added；Up to detection solution.
Of the invention additionally provides a kind of enzyme linked immunological kit for detecting Fipronil.
The enzyme linked immunological kit of detection Fipronil provided by the present invention, for it is following 1), 2), 3) or 4) in it is any described
Enzyme linked immunological kit:
1) conjugate, the above-mentioned fluorine including compound and carrier protein shown in any of the above-described formula I in enzyme linked immunological kit
Worm nitrile monoclonal antibody and enzyme mark antiantibody；Wherein, the conjugate is as coating antigen；
2) include in enzyme linked immunological kit the enzyme marker of compound shown in above-mentioned formula I, above-mentioned Fipronil monoclonal antibody and
Antiantibody；Wherein, the antiantibody is as coating antigen；
3) enzyme marker, the above-mentioned Fipronil monoclonal antibody including compound shown in above-mentioned formula I in enzyme linked immunological kit；Its
In, the Fipronil monoclonal antibody is as coating antigen；
4) conjugate, the above-mentioned fluorine including compound and carrier protein shown in any of the above-described formula I in enzyme linked immunological kit
The enzyme marker of worm nitrile monoclonal antibody；Wherein, the conjugate is as coating antigen.
The testing principle of above-mentioned 4 kinds of kits is as follows:
When the conjugate of fipronil hapten pre-coated on micropore of enzyme marker plate item and carrier protein, sample solution or mark is added
After quasi- product solution, Fipronil monoclonal antibody solution is added, remaining Fipronil or Fipronil standard items and enzyme mark in sample
On plate the conjugate of coated fipronil hapten and carrier protein compete Fipronil monoclonal antibody, be added enzyme mark secondary antibody into
Row amplification, is developed the color with developing solution, and sample light absorption value and the content of Fipronil in sample are negatively correlated, compared with standard curve
The content of Fipronil in you can get it sample.It simultaneously can also be according to the depth of color on ELISA Plate, with series of concentrations Fipronil mark
The comparison of quasi- product solution colour can in rough judgement sample Fipronil content concentration range.
When the pre-coated secondary antibody on micropore of enzyme marker plate item, after the incubation of Fipronil monoclonal antibody is added, it is molten that sample is added
Liquid or standard solution add enzyme label fipronil hapten solution, Fipronil or Fipronil standard items and enzyme in sample
It marks fipronil hapten to compete Fipronil specific antibody, is developed the color with developing solution, Fipronil in sample absorbance value and sample
Content be negatively correlated, the content of Fipronil in you can get it compared with standard curve sample.It simultaneously can also be according on ELISA Plate
Shade, compared with series of concentrations Fipronil standard solution color can in rough judgement sample Fipronil content it is dense
Spend range.
When Fipronil monoclonal antibody pre-coated on micropore of enzyme marker plate item, sample solution or standard solution is added
Afterwards, enzyme label fipronil hapten solution, remaining Fipronil or Fipronil standard items and enzyme-labelled antigen in sample are added
Compete the Fipronil monoclonal antibody that is coated on ELISA Plate, developed the color with developing solution, the content of sample light absorption value and Fipronil at
Negative correlation, the content of Fipronil in you can get it compared with standard curve sample.It simultaneously can also be deep according to the color on ELISA Plate
Shallowly, compared with the Fipronil standard solution color of series of concentrations can in rough judgement sample Fipronil content concentration model
It encloses.
When the conjugate of fipronil hapten pre-coated on micropore of enzyme marker plate item and carrier protein, sample solution is added
Or after standard solution, enzyme label Fipronil monoclonal antibody solution, Fipronil or Fipronil standard items in sample are added
With Fipronil antigenic competition Fipronil monoclonal antibody coated on ELISA Plate, developed the color with developing solution, sample light absorption value and sample
The content of middle Fipronil is negatively correlated, the content of Fipronil in you can get it compared with standard curve sample.It simultaneously can also basis
Shade on ELISA Plate, can Fipronil in rough judgement sample compared with series of concentrations Fipronil standard solution color
The concentration range of content.
It is dilute to may also include Fipronil standard solution, developing solution, cleaning solution and/or sample in any of the above-described kit
Release liquid.
In any of the above-described kit, the antiantibody is sheep anti mouse antiantibody or goat-anti rabbit-anti antibody.
In any of the above-described kit, the concentration of the Fipronil standard solution be respectively 0 μ g/L, 0.3 μ g/L,
0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L and 24.3 μ g/L.
In any of the above-described kit, the substrate developing solution is made of A liquid and B liquid, and A liquid is final concentration of
The aqueous solution of the urea peroxide of 1.5%-2.5%, concentration of the final concentration of urea peroxide in the A liquid；B liquid is dense eventually
Degree is the aqueous solution of the tetramethyl benzidine of 0.5%-1.5%, and the final concentration of tetramethyl benzidine is dense in the B liquid
Degree.
In any of the above-described kit, polysorbas20, final concentration of the cleaning solution by final concentration of 0.8%~1.2%
For the composition of 0.5% sodium azide, solvent is 0.01 M phosphate buffer；Final concentration of each substance is in cleaning solution
Concentration；The pH value of the cleaning solution is 7.4.
In any of the above-described kit, the sample diluting liquid is the phosphorus of 20 × (0.03 mol/L-0.05 mol/L)
Phthalate buffer.
Marker enzyme in the enzyme label is horseradish peroxidase or alkaline phosphatase, wherein it is preferred that horseradish peroxidating
Object enzyme；The sheep anti mouse secondary antibody or goat-anti rabbit secondary antibody of enzyme label be using glutaraldehyde method or Over-voltage protection by marker enzyme and secondary antibody into
Row coupling obtains, and horseradish peroxidase can be used a variety of methods in the prior art and be coupled it with secondary antibody, and such as penta
Dialdehyde method, Over-voltage protection etc., the present invention are improved Over-voltage protection by long-term labor and creation, make its is time saving,
The concentration rate for reducing horseradish peroxidase (HRP) and secondary antibody, saves raw material.
Of the invention additionally provides the detection method of a Fipronil kit:
When coating antigen is Fipronil coupled antigen, into micropore of enzyme marker plate, addition standard solution or sample solution are added anti-
Body is washed after incubation and is patted dry, adds ELIAS secondary antibody, wash and pat dry after incubation, develop the color, terminate, and measures absorbance with microplate reader
Value；
When coating antigen is Fipronil coupled antigen, standard solution is added into micropore of enzyme marker plate or sample solution adds enzyme
Labelled antibody is washed after incubation and is patted dry, develops the color, terminates, and measures absorbance value with microplate reader；
When coating antigen is Fipronil specific antibody, standard solution is added into micropore of enzyme marker plate or sample solution adds
Enzyme marks fipronil hapten, washs and pats dry after incubation, develops the color, terminates, and measures absorbance value with microplate reader；
When coating antigen is secondary antibody, Fipronil antibody is added into micropore of enzyme marker plate, washs and pats dry after incubation, add standard items
Enzyme mark fipronil hapten is added after solution or sample solution, washs and pats dry after incubation, develop the color, terminate, is measured and is inhaled with microplate reader
Shading value.
Analysis of test results process provided by the invention are as follows:
With the absorbance values (B) of the standard solution of each concentration obtained divided by first standard solution (0 standard)
Absorbance value (B0) multiplied by 100%, i.e. percentage absorbance value.Calculation formula are as follows: percentage absorbance value (%)=(B/B0)
× 100%
Using the semilog value of the concentration (μ g/L) of Fipronil standard solution as X-axis, percentage absorbance value is Y-axis, draws standard
Curve graph.The percentage absorbance value of sample solution is calculated with same method, the concentration of each corresponding sample then can be from mark
The content of Fipronil in sample is read on directrix curve.
The analysis of testing result can also use regression equation method in the present invention, calculate sample solution concentration.
The analysis of testing result can also utilize computer professional software in the present invention, this method be more convenient for the fast of a large amount of samples
Speed analysis, entire detection process only need the short period, to complete in 1.5 h.
Fipronil detection kit provided by the present invention, easy to operate, quick, high sensitivity, high specificity, accuracy
By force, for Fipronil residual in animal products, quickly detection has substantial worth.
Detailed description of the invention
Fig. 1 Fipronil canonical plotting.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The preparation of embodiment 1, Fipronil detection kit component
One, the preparation of fipronil hapten
50ml round-bottomed flask is rinsed well, is dried up with ethyl alcohol, is fixed on blender, stirrer is added.Weigh raw material
1g is dissolved with 10ml methanol, is in yellow, and 365mg 3- mercaptopropionic acid, stirring, contact plate detection reaction, purifying, column is added in stirring
Chromatographic purifying, solution where collecting product point, is spin-dried for get fipronil hapten.
Reaction equation is as follows:
Mass Spectrometer Method is carried out to gained fipronil hapten, its chemical structural formula is (MW=427) shown in formula I as the result is shown, as
Fipronil hapten.
Formulas I
Two, the preparation of fipronil artificial antigen
1, the synthesis of Fipronil immunogene (Fipronil-BSA)
(1) 15.93mg fipronil hapten 1.5mL DMF is dissolved, 200rpm stirs 10min, and it is molten that EDC 21.5mg is added
NHS 13mg is added after solution, and (500rpm) activation 2-3h is stirred at room temperature.
(2) it weighs BSA 50mg to be dissolved in 3.5mL 0.1M sodium bicarbonate solution, 200rpm stirs 10min, makes it sufficiently
Dissolution, ice bath cool down 0-4 DEG C, and under 1000rpm stirring, step 1 reaction solution is added dropwise (1mL/min), and 500rpm stirring is anti-
Answer for 24 hours
(3) reaction product is packed into the clean bag filter of distilled water flushing (10cm), 1L0.01M PBS(1 ×, pH7.2) 4 DEG C of stirrings
(100rpm) dialysis 3d changes liquid 3 times (each primary in the morning, afternoon and evening) daily, total to change liquid 9 times, will dialysis product 5000rpm centrifugation
The packing of 6min, 1.5mL/ pipe, antigen is numbered, -20 DEG C save backup.
2, the synthesis of Fipronil coating antigen (Fipronil-OVA)
(1) 15.93mg fipronil hapten 1.5mL DMF is dissolved, 200rpm stirs 10min, and it is molten that EDC 21.5mg is added
NHS 13mg is added after solution, and (500rpm) activation 2-3h is stirred at room temperature.
(2) it weighs OVA 33.6mg to be dissolved in 3.5mL 0.1M sodium bicarbonate solution, 200rpm stirs 10min, fills it
Divide dissolution, ice bath cools down 0-4 DEG C, under 1000rpm stirring, step 1 reaction solution is added dropwise (1mL/min), 500rpm stirring
Reaction is for 24 hours.
(3) reaction product is packed into the clean bag filter of distilled water flushing (10cm), 1L0.01M PBS(1 ×, pH7.2) 4 DEG C
Stir (100rpm) to dialyse 3d, change liquid daily 3 times (in the morning, afternoon and evening each primary), it is total to change liquid 9 times, will dialysis product 5000rpm from
The packing of heart 6min, 1.5mL/ pipe, antigen is numbered, -20 DEG C save backup.
Fipronil artificial antigen is identified with MALDI-TOF-MS.
Three, the preparation of Fipronil monoclonal antibody
1, animal immune
With the immunogene (Fipronil-BSA) prepared by 100 μ g/, with physiological saline solution immunogene and Freund's complete adjuvant
Isometric to mix, immune 6 ~ 8 week old Balb/c female mices are subcutaneously injected in the nape of the neck, after initial immunity the 7th, 14,28 day with immunogene
It is mixed in equal volume with incomplete Freund's adjuvant, each supplementary immunization is primary, merges first 3 days with 100 μ of immune complex g/, is not added
Supplementary immunization is primary again for Freund's adjuvant.
2, cell fusion and clone
It carries out according to a conventional method, takes the splenocyte of immune mouse and the murine myeloma cell (SP2/0) for being in logarithmic growth phase
Mixing, the fusion agent (PEG4000) that preheating is then slowly added in 45s are merged, and are suspended uniformly with HAT culture medium, then
Suitable feeder cells are added, are incubated at 96 well culture plates, in 37 DEG C, 5%CO2It is cultivated in incubator, HT culture medium is used after 5 days
Partly change liquid, 9 days whens carry out changing liquid entirely.
After cell fusion, when cell grows to the 1/4 of culture hole area, hybridoma is screened using substep screening method.
Primary election uses indirect ELISA method, (uses its best peridium concentration of square matrix method conventional titration and positive serum in advance with envelope antigen
Dilution) coated elisa plate, measured hole culture supernatant is added, is incubated for, sheep anti-mouse igg-HRP and IgM-HRP is added after cleaning,
OPD carries out chromogenic reaction.The positive Kong Zaiyong indirect competitive ELISA method screening filtered out, first by cell conditioned medium and 100 μ g/
The Fipronil of mL mixes in equal volume, and 37 DEG C of water-baths act on 30min, is then added in the ELISA Plate being coated with.Replaced simultaneously with PBS
Fipronil compares, remaining step is same as above.If the OD after Fipronil blocks450Nm value drops to the 50% of control wells hereinafter, then
It is judged to the positive, is all positive hole through 2 ~ 3 detections, carries out subcloning with limiting dilution assay immediately.
3, the preparation and purifying of monoclonal antibody
The hybridoma that 2 ~ 3 subclones are built after strain is expanded into culture, supernatant is collected with indirect ELISA and measures potency, freeze
It deposits；And take 8 ~ 10 week old Balb/c mouse peritoneal injecting fluid paraffin 0.5mL/ only, hybridoma 1 is injected intraperitoneally after 7 ~ 10 days
Mouse ascites are extracted after ~ 2 × 105/, 7 ~ 10 days.Cell conditioned medium or ascites are collected, its potency is measured using indirect ELISA method
(being indicated when measurement potency with the cell conditioned medium of P/N > 2.1 or ascites maximum dilution multiple), the results showed that the potency of cell conditioned medium
For 1:10000, the potency of ascites is 1:50000.Then, it is purified with octanoic acid-saturated ammonium sulfate method, is put after purification
Enter -20 DEG C of environment to save.
Four, the sero-fast preparation of Fipronil
1, animal immune
Use fipronil artificial antigen " Fipronil-BSA " as immunogen immune new zealand white rabbit.Each immunizing dose is 100 ~
200 μ g, immunization ways be double shoulder and the subcutaneous multi-point injection of rear thigh, each region with about 1/4 immunogene.It will when head exempts from
Then immunogene normal saline dilution carries out 1:1(volume ratio with incomplete Freund's adjuvant) it is mixed and made into emulsifier, at interval of
Same dose immunogene is taken within 2 weeks to add booster immunization after isometric incomplete Freund's adjuvant mixing and emulsifying primary, it is total using this mode
Add after exempting from 3 times, interval takes same dose immunogene Jia Fushi Freund's incomplete adjuvant to carry out final immunization for 3~4 weeks again, and arteria auricularis takes blood
Detect antibody titer.Arteria carotis bloodletting is used after final immunization 7-10 days, every rabbit can obtain blood 100-120mL or so, take
Blood placed 3 ~ 4 hours in 4 DEG C of refrigerators, serum is isolated in centrifugation.
2, antiserum titre measures
It is specific as follows using the antibody titer of one gained serum of indirect elisa method determination step:
1) it is coated with: " Fipronil-OVA " solution that 100 μ L concentration are 2 μ g/mL being added in 96 hole elisa Plates and (uses coating buffer
It is diluted), while the control of not envelope antigen is set, 4 DEG C of coatings overnight, are washed 3 times with PBS buffer solution.
Be coated with buffer: (solvent is water to the sodium carbonate-bicarbonate buffer of pH9.6,0.05M, and solute and its concentration are such as
Under: Na2CO31.59g/L and NaHCO32.93g/L).
2) it closes: the confining liquid in 150 holes μ L/ is added, in 37 DEG C of incubation 2h, abandon confining liquid, wash 3 times, pat dry.It is placed in 4
DEG C refrigerator saves backup.
Confining liquid: contain 0.5%(volumn concentration) calf serum, 3%(3g/100mL) casein phosphate it is slow
Fliud flushing, pH7.4.
3) add sample to be tested: drawing the 100 μ l of test serum of different dilutions, be added in corresponding ELISA Plate, 37 DEG C incubate
30min is educated, board-washing 4 times, is patted dry.
The control of non-immunized rabbit anteserum is set simultaneously；The control (negative control hole) of sample to be tested is replaced with PBS.
4) add ELIAS secondary antibody: the goat anti-rabbit igg antibody for taking horseradish peroxidase to mark dilutes for 1:5000 times by volume
Afterwards, 100 hole μ l/, 37 DEG C are incubated for 20 to 30min, wash 4 times, pat dry.
5) it develops the color: 20 × TMB is diluted to 1 × TMB, be added by 100 holes μ l/, 37 DEG C of colour developing 15-30min.
6) it terminates: terminate liquid (2MH is added2SO4) 50 holes μ l/.
7) it reads: each hole OD value is measured with 450nm Single wavelength, (to replace pair of sample to be tested with PBS with negative control hole
According to) ratio (P/N) of OD value is greater than 2.1 and is limited, as the critical point for being judged as serum titer.
ELISA result judgement method: it is indicated with the serum maximum dilution multiple of P/N > 2.1.
The result shows that the antibody titer in serum is 1:16000.
The preparation and application of embodiment 2, Fipronil kit
One, Fipronil kit is made of following substances:
1, Fipronil standard items working solution: 6 bottles, 1.5mL/ bottles, concentration 0ng/mL, 0.3ng/mL, 0.9ng/mL, 2.7ng/
mL,8.1ng/mL,24.3ng/mL；
2, Fipronil ELISA Plate: 1 piece (8 hole × 12), for the enzyme mark for being coated with " Fipronil-OVA " that embodiment 1 is prepared
Plate.
3, Fipronil antibody working solution: 1 bottle (10mL) is antibody diluent by antibody progress 1:8000 dilution, and antibody is dilute
Releasing liquid is volume fraction containing 6%() 0.2MPBS of lowlenthal serum, the Fipronil antibody is the antiserum that is prepared of embodiment 3
Purify resulting antibody.
4, enzyme marker working solution: the antibody of the sheep anti mouse through horseradish peroxidase label.
5, sample diluting liquid: the PBS of 0.01M pH7.4.
6, cleaning solution: the PBST solution of 0.01M pH7.4.
7, substrate A liquid, substrate B liquid are each 1 bottle (7mL).Wherein, substrate A is 2% urea peroxide aqueous solution.Substrate B is 1% 4
Methyl biphenyl amine aqueous solution.
8, terminate liquid: 1 bottle (7mL), be 2 M H2SO4Solution.
9, cover board film；
10, valve bag.
Two, the application of Fipronil kit
(1) detection method
1, sample pre-treatments
Sample (animal flesh and internal organ, egg, milk, animal product) after weighing 1 ± 0.05 g homogeneous is in 50mL centrifuge tube
In；Sequentially add 1 g sodium chloride, 3 mL acetonitriles (analysis is pure), abundant 5 min of whirling motion；4000 g or more are centrifuged 5 min；
Take 1 mL supernatant in clean centrifuge tube；50-60 DEG C of water-bath is dried with nitrogen；1 mL sample diluting liquid, abundant whirling motion 1 is added
min。
2, it is detected with kit
(1) lath is inserted on ELISA Plate frame, and records the position of each standard items and sample, it is proposed that it is parallel to do diplopore, not
After the lath used is sealed with valve bag, it is stored in 2-8 DEG C of environment immediately；
(2) the Fipronil standard items working solution (or testing sample solution) of 50 each concentration of μ L is separately added into corresponding standard items
In (or sample to be tested hole)；
(3) 50 μ L enzyme marker working solutions are added in every hole, then 80 μ l antibody working solutions are added in every hole；
(4) cover board film is covered, ELISA Plate 10s is gently vibrated, mixes well, at room temperature (25 ± 2 DEG C), be protected from light 30min；
(5) cover board film is opened；
(6) liquid in plate hole is outwelled, 260 μ L wash operating solutions, sufficiently washing 4 times are added in every hole, impregnate 15-30s every time；
(7) liquid in plate hole is outwelled, ELISA Plate is inverted on blotting paper, is patted dry；
(8) 100 μ LA, B mixed liquor are added in every hole immediately；
(9) cover board film is covered, ELISA Plate 10s is gently vibrated, mixes well, at room temperature (25 ± 2 DEG C), be protected from light 15-
20min；
(10) cover board film is opened, 50 μ L terminate liquids are added in every hole, ELISA Plate 10s is gently vibrated, mixes well；
(11) the interior microplate reader of 5min reads ELISA Plate absorbance value at dual wavelength 450nm, 630nm after terminating.
3, result calculates or determines
(1) mean absorbance values of each standard items (or sample to be tested), divided by zero standard (standard items that concentration is 0ng/mL) extinction
Angle value, multiplied by 100, the percentage of the corresponding absorbance of available each standard items, i.e. percentage absorbance value.
(2) it using the percentage absorbance value of each standard items as ordinate, is drawn and is marked as abscissa using corresponding Fipronil concentration
Directrix curve.Canonical plotting is as shown in Figure 1.
(3) the percentage absorbance value of sample to be tested is substituted into calibration curve equation, can obtains the corresponding concentration of sample to be tested,
Multiplied by the extension rate of respective sample, side obtains the actual content of Fipronil in raw sample to be measured.
Three, Fipronil kit detects Fipronil
1, accuracy and precision test
Tissue, internal organ, meat products, milk, egg blank sample in add Fipronil, make the final concentration of Fipronil in the sample
Respectively 1.0 μ g/L, 2.0 μ g/L；Place before sample after addition is carried out according to method described in the present embodiment step 2 respectively
Reason, obtains test sample solution.
It is detected from the kit of 3 different batches, each experiment is repeated 5 times, and calculates separately the coefficient of variation.As a result divide
It is not shown in Table 1- table 5.
The calculation method of variation within batch coefficient: variation within batch coefficient=with the variation lines of each parallel samples in primary measurement
Number.
The calculation method of interassay coefficient of variation: change of the interassay coefficient of variation=same sample in different batches measurement result
Different coefficient takes its average value.
1 accuracy of table and precision (tissue)
2 accuracy of table and precision (internal organ)
3 accuracy of table and precision (meat products)
4 accuracy of table and precision (milk)
5 accuracy of table and precision (egg)
The result shows that all samples respectively add the rate of recovery of concentration between 80.5~120%.Criticizing for each addition concentration is interior, criticizes
Between the coefficient of variation be below 15%.
2, sample recovery test
To tissue, internal organ, meat products, milk, egg blank sample in add the Fipronil standard items of various concentration respectively, make
The concentration of Fipronil in the sample is respectively 1 μ g/L；By the sample after addition respectively according to method described in embodiment step two into
Row pre-treatment obtains sample solution, then is detected, and each sample does 20 in parallel, the calculating rate of recovery (rate of recovery=measured value/
Add value).It the results are shown in Table 6.
6 blank sample measurement result statistical form (μ g/L) of table
The result shows that tissue minimum detectability is 0.97 μ g/L, internal organ minimum detectability is 0.94 μ g/L, the minimum detection of meat products
It is limited to 0.98 μ g/L, milk minimum detectability is 0.94 μ g/L, and egg minimum detectability is 0.98 μ g/L, to ensure present invention examination
The stability of agent box, it is believed that Fipronil detection kit detects in tissue, internal organ, meat products, milk, egg and is limited to 1.0 μ
g/L。
4, cross reacting rate is tested:
The specificity of Fipronil enzyme linked immunological kit is determined by carrying out cross reaction test with corresponding substance.It hands over
Fork reaction is smaller, and specificity is better.
Fipronil and other analogs (fluorine formonitrile HCN, Fipronil sulfone, Fipronil sulfoxide, Fipronil thioether) are done into series respectively
Dilution, is operated according to step 22 as above respectively, is substituted with the serial dilutions of Fipronil and other analogs therein
" Fipronil standard items working solution " makes standard curve, and finds out respective 50% inhibition concentration (IC on curve50), specific method
It is as follows: to obtain Y value equal to 50% corresponding Fipronil concentration (ng/mL), i.e. IC50Value.Kit pair is calculated with following formula
The cross reacting rate of Fipronil and each analog:
Cross reacting rate (%)=(the Fipronil concentration for causing 50% inhibition/cause the Fipronil analog concentration of 50% inhibition) ×
100%.
The results are shown in Table 7.
The specificity of 7 kit of table
| Title | Cross reacting rate (%) |
| Fipronil | 100.0 |
| Fipronil sulfone | 102.0 |
| Fipronil sulfoxide | 40.0 |
| Fluorine formonitrile HCN | 24.0 |
| Fipronil thioether | 20.0 |
Experiment shows that kit of the present invention is good to the specificity of Fipronil, and can detecte fipronil substance, i.e. present invention examination
Agent box can detecte Fipronil and the like.
5, kit storage life is tested
Kit preservation condition is 2-8 DEG C, by measurement in 12 months, the maximum absorbance value (zero standard) of kit, 50% suppression
Concentration processed, Fipronil addition actual measured value are within normal range (NR).In view of having improper in transport and use process
Preservation condition occurs, and kit is placed 8 days under conditions of 37 DEG C of preservations, carries out accelerated aging tests, the results showed that the examination
The indices of agent box comply fully with requirement.In view of kit freezing happens, kit is put into -20 DEG C of refrigerator freezings
8 days, measurement result also indicated that kit indices are completely normal.From result above can obtain kit can 2-8 DEG C to
It can save 12 months or more less.
Claims (7)
1. a kind of detection kit of Fipronil, comprising: Fipronil ELISA Plate, Fipronil standard items working solution, Fipronil antibody
Working solution, enzyme marker working solution, substrate solution, terminate liquid, it is characterised in that:
The Fipronil ELISA Plate is made of Fipronil antigen coat, and the Fipronil antigen is by fipronil hapten and carrier
The resulting antigen of albumen coupling, fipronil hapten are compound shown in Formulas I:
Formulas I.
2. a kind of detection kit of Fipronil according to claim 1, which is characterized in that the compound is Fipronil
It is reacted with mercaptopropionic acid, which contains pendant carboxylic group, can form coating antigen with carrier protein couplet and be immunized
It is former.
3. a kind of detection kit of Fipronil according to claim 1, which is characterized in that the preparation side of the compound
Method includes the following steps: to rinse well 50ml round-bottomed flask, is dried up, is fixed on blender with ethyl alcohol；Weigh raw material
1g is dissolved with 10ml methanol, is in yellow, and 365mg 3- mercaptopropionic acid, stirring, contact plate detection reaction, purifying, column is added in stirring
Chromatographic purifying, solution where collecting product point, is spin-dried for get fipronil hapten.
4. a kind of detection kit of Fipronil according to claim 1, which is characterized in that the carrier protein is ox blood
Pure albumen, ovalbumin, human serum albumins, hemocyanin, mouse haemocyanin, thyroprotein or rabbit serum proteins.
5. a kind of detection kit of Fipronil according to claim 1, which is characterized in that the system of the Fipronil antigen
Preparation Method includes the following steps:
(1) fipronil hapten (Formulas I) is dissolved in dimethylformamide, 1- (3- dimethylamino third is then added
Base) -3- ethyl-carbodiimide hydrochloride and n-hydroxysuccinimide, 20-25 DEG C of magnetic agitation react 2-3h, obtain solution I；
Wherein, the fipronil hapten (Formulas I), the dimethylformamide, the 1- (3- dimethylamino-propyl) -3- ethyl
Carbodiimide hydrochloride, the n-hydroxysuccinimide proportion be 15.93mg:1.5mL:21.5mg:13mg；
(2) carrier protein is placed in 0.1M sodium bicarbonate buffer liquid, 200rpm stirs 10min, sufficiently dissolves, obtains molten
Liquid II；The proportion of the carrier protein and the 0.1M sodium bicarbonate buffer liquid is 33.6-50mg:3.5mL；
(3) solution I and the solution II are mixed, specially under the conditions of 0-4 DEG C, under 1000rpm stirring, by solution I
It is added dropwise in the solution II, 500rpm is stirred to react for 24 hours, obtains solution III；
(4) phosphate buffer (0.01M PBS, pH7.2) is used, in 4 DEG C to the solution III stirring dialysis 3 days, obtained described
Fipronil antigen.
6. a kind of preparation method of the detection kit of Fipronil, it is characterised in that:
(1) concentration of Fipronil standard solution described in kit is respectively 0 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ
G/L, 8.1 μ g/L and 24.3 μ g/L；
(2) Fipronil ELISA Plate described in kit；
(3) working solution of Fipronil antibody described in kit is antibody diluent by antibody progress 1:8000 dilution, and antibody dilutes
Liquid is the 0.2MPBS of lowlenthal serum containing 6%；
(4) enzyme marker working solution described in kit, for the antibody of the sheep anti mouse marked through horseradish peroxidase；
(5) sample diluting liquid described in kit is the phosphate buffer of 20 × (0.03 mol/L-0.05 mol/L)；
(6) cleaning solution described in the kit is folded by final concentration of 0.8%~1.2% polysorbas20, final concentration of 0.5%
The composition of sodium nitride, solvent are 0.01 M phosphate buffer；Concentration of the final concentration of each substance in cleaning solution；It is described
The pH value of cleaning solution is the PBST solution of 7.40.01M pH7.4；
(7) substrate solution described in kit includes being made of A liquid and B liquid, and A liquid is the peroxidating of final concentration of 1.5%-2.5%
The aqueous solution of urea, concentration of the final concentration of urea peroxide in the A liquid；B liquid is the four of final concentration of 0.5%-1.5%
The aqueous solution of methyl biphenyl amine, concentration of the final concentration of tetramethyl benzidine in the B liquid；
(8) terminate liquid: 1 bottle (7mL), be 2 M H2SO4Solution.
7. the application of Fipronil detection kit according to claim 1 or 6, it is characterised in that detection sample can be group
It knits, internal organ, meat products, milk, egg, the detection of Fipronil is limited to 1 μ g/L in the above-mentioned sample of detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710990416.6A CN109696548A (en) | 2017-10-23 | 2017-10-23 | A kind of kit and its preparation method and application detecting Fipronil |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710990416.6A CN109696548A (en) | 2017-10-23 | 2017-10-23 | A kind of kit and its preparation method and application detecting Fipronil |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109696548A true CN109696548A (en) | 2019-04-30 |
Family
ID=66225788
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710990416.6A Pending CN109696548A (en) | 2017-10-23 | 2017-10-23 | A kind of kit and its preparation method and application detecting Fipronil |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109696548A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110376372A (en) * | 2019-07-25 | 2019-10-25 | 苏州微测生物技术有限公司 | One-step method detects the remaining VHH-ELISA kit of Fipronil and its application |
| CN111187346A (en) * | 2020-01-15 | 2020-05-22 | 江苏大学 | Colloidal gold test strip for detecting fipronil and its metabolite and preparation method thereof |
| CN111273015A (en) * | 2020-04-13 | 2020-06-12 | 北京维德维康生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting Gymnodinium breve toxin and preparation and application thereof |
| CN113636954A (en) * | 2021-10-18 | 2021-11-12 | 天津国际生物医药联合研究院 | Fipronil artificial antigen, preparation method and application |
| CN114113617A (en) * | 2021-11-15 | 2022-03-01 | 湖北省食品质量安全监督检验研究院 | Fipronil rapid quantitative detection kit and detection method |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK200600092U1 (en) * | 2006-03-31 | 2006-04-28 | Cheminova As | Chemical compounds with useful properties |
| JP2006321766A (en) * | 2005-05-20 | 2006-11-30 | Horiba Ltd | Haptene compound of fipronyl, antibody, hybridoma and its measuring means, and measuring kit or measuring method |
| CN101100456A (en) * | 2007-07-26 | 2008-01-09 | 浙江大学 | Fipronil artificial hapten, synthetic method for the same, and its antigen, antibody and use |
| CN101165055A (en) * | 2006-10-19 | 2008-04-23 | 上海赫腾精细化工有限公司 | Method for preparing 5-amino-3-cyano-4-thiocyano-1-(2,6-dichloro-4-fluoromethylphenyl)pyrazole |
| US20100047927A1 (en) * | 2005-12-16 | 2010-02-25 | Bayer Cropscience K.K. | Kit for measurement of termite insecticide active ingredient by immunoassay method |
| CN102060774A (en) * | 2009-11-11 | 2011-05-18 | 李家波 | Method for synthesizing fipronil |
| CN102079788A (en) * | 2009-11-27 | 2011-06-01 | 北京维德维康生物技术有限公司 | Method for detecting sulfanilamide medicine and special enzyme-linked immunoassay reagent kit thereof |
-
2017
- 2017-10-23 CN CN201710990416.6A patent/CN109696548A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006321766A (en) * | 2005-05-20 | 2006-11-30 | Horiba Ltd | Haptene compound of fipronyl, antibody, hybridoma and its measuring means, and measuring kit or measuring method |
| US20100047927A1 (en) * | 2005-12-16 | 2010-02-25 | Bayer Cropscience K.K. | Kit for measurement of termite insecticide active ingredient by immunoassay method |
| DK200600092U1 (en) * | 2006-03-31 | 2006-04-28 | Cheminova As | Chemical compounds with useful properties |
| CN101165055A (en) * | 2006-10-19 | 2008-04-23 | 上海赫腾精细化工有限公司 | Method for preparing 5-amino-3-cyano-4-thiocyano-1-(2,6-dichloro-4-fluoromethylphenyl)pyrazole |
| CN101100456A (en) * | 2007-07-26 | 2008-01-09 | 浙江大学 | Fipronil artificial hapten, synthetic method for the same, and its antigen, antibody and use |
| CN102060774A (en) * | 2009-11-11 | 2011-05-18 | 李家波 | Method for synthesizing fipronil |
| CN102079788A (en) * | 2009-11-27 | 2011-06-01 | 北京维德维康生物技术有限公司 | Method for detecting sulfanilamide medicine and special enzyme-linked immunoassay reagent kit thereof |
Non-Patent Citations (1)
| Title |
|---|
| 蒋富国等: "氟虫腈的催化氧化合成研究", 《上海化工》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110376372A (en) * | 2019-07-25 | 2019-10-25 | 苏州微测生物技术有限公司 | One-step method detects the remaining VHH-ELISA kit of Fipronil and its application |
| CN111187346A (en) * | 2020-01-15 | 2020-05-22 | 江苏大学 | Colloidal gold test strip for detecting fipronil and its metabolite and preparation method thereof |
| CN111187346B (en) * | 2020-01-15 | 2023-05-05 | 江苏大学 | Colloidal gold test strip for detecting fipronil and metabolites thereof and preparation method thereof |
| CN111273015A (en) * | 2020-04-13 | 2020-06-12 | 北京维德维康生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting Gymnodinium breve toxin and preparation and application thereof |
| CN111273015B (en) * | 2020-04-13 | 2023-04-11 | 北京维德维康生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting Gymnodinium breve toxin and preparation and application thereof |
| CN113636954A (en) * | 2021-10-18 | 2021-11-12 | 天津国际生物医药联合研究院 | Fipronil artificial antigen, preparation method and application |
| CN114113617A (en) * | 2021-11-15 | 2022-03-01 | 湖北省食品质量安全监督检验研究院 | Fipronil rapid quantitative detection kit and detection method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109696548A (en) | A kind of kit and its preparation method and application detecting Fipronil | |
| CZ303174B6 (en) | Immunoassay for neonicotinyl insecticides | |
| CN105044365B (en) | The preparation method of the time resolved fluoro-immunoassay test paper of detection enrofloxacin residual | |
| CN101100486A (en) | Fipronil artificial antigen, antibody and use thereof | |
| CN101100456A (en) | Fipronil artificial hapten, synthetic method for the same, and its antigen, antibody and use | |
| CN101799472A (en) | Diethylstilbestrol detection kit and detection method | |
| CN105675874A (en) | Colloidal gold test strip for detection of imidacloprid and application thereof | |
| CN106866568A (en) | Furaxone metabolite AOZ derivatizations haptens, the preparation method and applications of artificial antigen | |
| CN110286240A (en) | A kind of purposes of triclosan monoclonal antibody and/or triclocarban monoclonal antibody in detection triclosan and/or triclocarban | |
| CN101021535B (en) | Enzyme-linked immunalogical kit for detecting gentamicin medicine and method | |
| CN105277708A (en) | Enzyme linked immunosorbent assay kit for detecting aflatoxin B1 in chili | |
| CN107941785A (en) | A kind of surface-enhanced Raman gold label test strip for quickly detecting micro ethiprole and preparation method thereof | |
| CN109212200A (en) | A kind of Nicarbazin haptens, artificial antigen and the preparation method and application thereof | |
| CN106831498B (en) | Furacilin metabolite SEM derivatizations haptens, artificial antigen preparation method and applications | |
| CN101429241A (en) | Penicillin and carrier protein couplet product, method for producing beta-lactam penicillin antibody, and uses thereof | |
| CN107119022A (en) | One plant of iprodione monoclonal antibody hybridoma cell strain ZXL 2 and its application | |
| CN110117575A (en) | One plant of pyrimethanil monoclonal antibody hybridoma cell strain HFG and its application | |
| CN106367396A (en) | Carbofuran monoclonal antibody hybridoma cell strain YH1 and application thereof | |
| CN109824599A (en) | A kind of albendazole haptens and its preparation method and application | |
| CN108120832A (en) | Probenazole haptens, coupled antigen, antibody and colloidal gold quick detection device and application thereof | |
| CN109053477A (en) | A kind of preparation method and application of butralin haptens and antigen | |
| CN103288661A (en) | Preparation method and application of malachite green hapten | |
| CN109956848A (en) | A kind of triclosan haptens and antigen and its preparation method and application | |
| CN108558718A (en) | Florfenicol and florfenicol amine antigen, antibody and its enzyme-linked immune analytic method is detected simultaneously | |
| CN109752531A (en) | The kit and its detection method of Fipronil in a kind of detection egg |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190430 |
|
| RJ01 | Rejection of invention patent application after publication |