CN109212200A - A kind of Nicarbazin haptens, artificial antigen and the preparation method and application thereof - Google Patents

A kind of Nicarbazin haptens, artificial antigen and the preparation method and application thereof Download PDF

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CN109212200A
CN109212200A CN201710520105.3A CN201710520105A CN109212200A CN 109212200 A CN109212200 A CN 109212200A CN 201710520105 A CN201710520105 A CN 201710520105A CN 109212200 A CN109212200 A CN 109212200A
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nicarbazin
antigen
solution
haptens
added
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聂靖东
秦誉
邢维维
贾良曦
刘薇
王照鹏
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BEIJING WDWK BIOTECHNOLOGY Co Ltd
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BEIJING WDWK BIOTECHNOLOGY Co Ltd
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    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
    • G01N2333/455—Eimeria
    • 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

Abstract

The invention discloses a kind of Nicarbazin haptens, artificial antigen and the preparation method and application thereof.Nicarbazin artificial antigen provided by the present invention is by Nicarbazin haptens shown in Formulas I and the resulting antigen of carrier protein couplet.Nicarbazin synthesizing artificial antigen provided by the present invention is simple, and purity is high and yield are high, and the preparation and the detection of Nicarbazin medicament residue for Nicarbazin antibody have substantial worth.

Description

A kind of Nicarbazin haptens, artificial antigen and the preparation method and application thereof
Technical field
The invention belongs to medicament residue field of fast detection, be related to a kind of Nicarbazin artificial antigen and preparation method thereof with Using.
Background technique
Nicarbazin (Nicarbazin) also known as nitre urea pyrimidine, Nicarbazin, be mainly used for preventing chicken caecum coccidia and heap-type, Huge, murder by poisoning, E.brunetti.Codex Committee on Food (Codex Aliment Commission, CAC) in 1999 Determine that maximum residue limit (MRL) of the Nicarbazin in chicken tissues is 0.2 mg/kg, it is 0-400 μ g/ that (ADI) is measured about day kg·bw.2002, FAO/WHO announcement was forbidden in import animal derived food using Nicarbazin, Japan disclose in succession into The maximum residue limit of Nicarbazin is 0.2 mg/kg in mouth poultry.No. 235 bulletin " animals that the Ministry of Agriculture of China 2002 issues Property food herbal medicine maximum residue limit " to define MRL of the Nicarbazin in chicken tissues be 0.2 mg/kg.Current US mark Standard is defined as 4 ppm (in chicken tissues).
The content of Nicarbazin in ultraviolet spectrometry measurement feed and poultry is mostly used before both at home and abroad.It is big both at home and abroad at present High effective liquid chromatography for measuring is mostly used, these method high specificities, high sensitivity, still sample pre-treatments operating procedure is numerous Trivial, higher cost is not suitable for the selective mechanisms of batch samples yet.Immunochemistry is analyzed due in the qualitative fixed of antigen-antibody Amount aspect unique advantage and advantage quick, at low cost, that sensitivity is higher, analysis sample size is big easy to operate compensate for physics and chemistry The deficiency of analysis.Immunochemistry analysis due to the unique advantage in terms of the qualitative, quantitative of antigen-antibody and it is easy to operate quickly, At low cost, sensitivity is higher, analysis sample size is big advantage compensates for the deficiency of physico-chemical analysis, the system of Nicarbazin artificial antigen It is standby to have established important foundation for the implementation of this method.
The fundamental factor for influencing immunochemistry analysis quality is the specificity and compatibility of antibody, these properties are decided by again The structure of immune hapten molecule, therefore the MOLECULE DESIGN of immune haptens and synthesis are to generate specific antibody and establish small point The step of sub- the most basic of residue of veterinary drug Fast Detection Technique, most critical.
Summary of the invention
The object of the present invention is to provide a kind of Nicarbazin artificial antigens and the preparation method and application thereof.
Nicarbazin artificial antigen provided by the present invention is that resulting resist is constructed on the basis of Nicarbazin haptens It is former.
The Nicarbazin haptens belongs to the scope of protection of the present invention, and structure is shown in formula I.
Formulas I
The method provided by the present invention for preparing the Nicarbazin haptens, specifically may include following steps:
It dimethylformamide (DMF) is added in aniline, adds succinic anhydride after stirring, the aniline, DMF, succinic anhydride Proportion is 1000mg:25ml:1290mg, and required reaction temperature is 65-70 DEG C (such as 65 DEG C) reaction 6-7h(such as 6h), it is reacted Liquid；
Reaction solution is added drop-wise in 100ml ice water, 25ml ethyl acetate extracts 3 times, combined ethyl acetate phase, and anhydrous sodium sulfate is dry Dry 4 hours, it is filtered to remove desiccant, filtrate is spin-dried for, and obtains Nicarbazin haptens intermediate 1870mg；
It is sufficiently stirred in the 15ml concentrated sulfuric acid and is slowly added to 5ml concentrated nitric acid, stir 20min under ice bath, be slowly added to Nicarbazin half Antigen intermediate 1500mg, finishes and removes ice bath, is slowly increased to 20-24 DEG C of room temperature magnetic agitation 2-3 hours, reaction solution is delayed Slowly it is poured into ice water and yellow solid is precipitated, filter, filter cake is washed with 100ml, is collected forced air drying 5-6 hours of 50 DEG C of filter cake and is obtained 924.6 mg Nicarbazin haptens.
Resulting Nicarbazin antigen is constructed on the basis of Nicarbazin haptens also belongs to protection scope of the present invention.
The Nicarbazin antigen, for by the Nicarbazin haptens (Formulas I) and the resulting antigen of carrier protein couplet. In one embodiment of the invention, the carrier protein is specially bovine serum albumin(BSA) (BSA) or ovalbumin (OVA).
The preparation method of the Nicarbazin antigen also belongs to protection scope of the present invention.
The preparation method of the Nicarbazin antigen, specifically may include following steps: by the Nicarbazin haptens (formula I it) is coupled with carrier protein by amido bond, obtains the Nicarbazin antigen.
Wherein, the molar ratio of the Nicarbazin haptens (Formulas I) and the carrier protein couplet is 17.6:1.
In the present invention, the Nicarbazin antigen is specifically to prepare according to the method included the following steps:
(1) the Nicarbazin haptens (Formulas I) is dissolved in dimethylformamide (DMF), 1- (3- diformazan ammonia is then added Base propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and n-hydroxysuccinimide (NHS), 20-25 DEG C of magnetic agitation reaction 2-3h obtains solution I；
Wherein, the Nicarbazin haptens (Formulas I), the dimethylformamide (DMF), the 1- (3- dimethylamino third Base) -3- ethyl-carbodiimide hydrochloride (EDC), the n-hydroxysuccinimide (NHS) proportion be 10.66mg: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 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 Nicarbazin antigen.
The Nicarbazin haptens (Formulas I) or the Nicarbazin antigen are in qualitatively or quantitatively detection Nicarbazin Using also belonging to protection scope of the present invention.
Protection scope of the present invention is also belonged to using antibody prepared by the Nicarbazin antigen.The antibody can be more grams Grand antibody, monoclonal antibody or antiserum.
Nicarbazin haptens provided by the present invention and the Nicarbazin antigen, synthetic method is simple, purity High, yield height, preparation and the detection of Nicarbazin medicament residue for Nicarbazin antibody have substantial worth.
Detailed description of the invention
Fig. 1 is Nicarbazin haptens intermediate mass spectrogram.
Fig. 2 Nicarbazin haptens mass spectrogram.
The MALDI-TOF-MAS that Fig. 3 is BSA schemes.
Fig. 4 is that the MALDI-TOF-MAS of Nicarbazin BSA compound schemes.
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.
Nicarbazin: Town in Shanghai spectrum experiment Science and Technology Co., Ltd.'s product, product number CDCT-C15508000.
The preparation of embodiment 1, Nicarbazin haptens
One, the preparation of Nicarbazin haptens
Aniline 1000mg is added in 50ml round-bottomed flask, the DMF of 25ml stirs 15min, 20-24 DEG C of addition succinic anhydride 1290mg, 65-70 DEG C of magnetic agitations are reacted 6 hours.Reaction solution is added drop-wise in 100ml ice water, 25ml ethyl acetate extraction 3 Secondary, combined ethyl acetate phase, anhydrous sodium sulfate is 4 hours dry, is filtered to remove desiccant, filtrate is spin-dried for, and obtains faint yellow oily Object 1870mg is Nicarbazin haptens intermediate.
Under ice bath, concentrated sulfuric acid 15ml is added into 50ml round-bottomed flask, is sufficiently stirred and is slowly added to concentrated nitric acid 5ml, ice bath Lower stirring 20min is slowly added to Nicarbazin haptens intermediate 1500mg, removes ice bath, is slowly increased to 20-24 DEG C of magnetic of room temperature Power stirs 2-3 hours, reaction solution is slowly poured into ice water, yellow solid is precipitated, and filters, and filter cake is washed with 100ml, collects filter Forced air drying 5-6 hours of 50 DEG C of cake obtains 924.6 mg Nicarbazin haptens.
Reaction equation is as follows:
Two, the Structural Identification of Nicarbazin haptens intermediate, Nicarbazin haptens
1, Mass Spectrometer Method (Fig. 1) is carried out to Nicarbazin haptens intermediate, as the result is shown its MW=193.2, as Nicarbazin Haptens intermediate.
2, Mass Spectrometer Method (Fig. 2) is carried out to 924.6 mg Nicarbazin haptens of gained, as the result is shown its chemical structural formula (MW=238.20) shown in formula I, as Nicarbazin haptens.
Formulas I
Embodiment 2, the preparation of Nicarbazin artificial antigen and Structural Identification
One, the preparation of Nicarbazin artificial antigen
1, the synthesis of immunogene
(1) 10.66mg Nicarbazin haptens 1.5mL DMF is dissolved, 200rpm stirs 10min, and EDC 21.5mg is added NHS 13mg is added after dissolution, 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 coating antigen
(1) 10.66mg Nicarbazin haptens 1.5mL DMF is dissolved, 200rpm stirs 10min, and EDC 21.5mg is added NHS 13mg is added after dissolution, 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.
Two, the identification of Nicarbazin artificial antigen
Immunogene MALDI-TOF-MS qualification result shows coupling ratio are as follows: R=(69034.671-64839.862)/238.2= 17.61(Fig. 3 and Fig. 4).I.e. in immunogene, the Nicarbazin haptens (Formulas I) is rubbed with what bovine serum albumin(BSA) (BSA) was coupled You are than being 17.6:1.
Embodiment 3, Nicarbazin artificial antigen are immunized animal and prepare monoclonal antibody
One, animal immune
The immunogene (Nicarbazin-BSA) prepared with embodiment 2 is by 100 μ g/, with physiological saline solution immunogene and not Family name's Freund's complete adjuvant mixes in equal volume, and immune 6 ~ 8 week old Balb/c female mices, the 7th, 14,28 after initial immunity is subcutaneously injected in the nape of the neck It is mixed in equal volume with immunogene and incomplete Freund's adjuvant, and each supplementary immunization is primary, merges first 3 days with 100 μ of immune complex Only, Freund's adjuvant is not added, and supplementary immunization is primary again by g/.
Two, 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 Nicarbazin 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.It is taken simultaneously with PBS It is compared for Nicarbazin, remaining step is same as above.If the OD after Nicarbazin blocks450Nm value drops to the 50% of control wells Hereinafter, being then judged to the positive, all it is positive hole through 2 ~ 3 detections, carries out subcloning with limiting dilution assay immediately.
Three, 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.
Embodiment 4, Nicarbazin artificial antigen are immunized animal and prepare antiserum
One, animal immune
Use the Nicarbazin artificial antigen " Nicarbazin-BSA " of the acquisition of step embodiment 2 as immunogen immune New Zealand great Bai Rabbit.Each immunizing dose is 100 ~ 200 μ g, and immunization ways are double shoulder and the subcutaneous multi-point injection of rear thigh, and each region is about With 1/4 immunogene.By immunogene normal saline dilution when head exempts from, 1:1(volume then is carried out with incomplete Freund's adjuvant Than) it is mixed and made into emulsifier, add after taking same dose immunogene to add isometric incomplete Freund's adjuvant mixing and emulsifying at interval of 2 weeks It is strong immune primary, added altogether after exempting from 3 times using this mode, interval takes same dose immunogene Jia Fushi Freund's incomplete adjuvant in 3~4 weeks again Final immunization is carried out, arteria auricularis takes blood examination to survey antibody titer.Arteria carotis bloodletting is used after final immunization 7-10 days, every rabbit can Blood 100-120mL or so is obtained, the blood taken is placed 3 ~ 4 hours in 4 DEG C of refrigerators, and serum is isolated in centrifugation.
Two, 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: " Nicarbazin-OVA " solution that 100 μ L concentration are 2 μ g/mL being added in 96 hole elisa Plates and (is buffered with coating Liquid 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.
Embodiment 5, Nicarbazin enzyme linked immunological kit detect Nicarbazin
One, the assembling of Nicarbazin enzyme linked immunological kit
1, the composition of Nicarbazin enzyme linked immunological kit includes the following:
(1) Nicarbazin standard items working solution: 6 bottles, 1.5mL/ bottles, concentration 0ng/mL, 0.10 ng/mL, 0.3ng/mL, 0.9ng/mL,2.7ng/mL,8.1ng/mL；
(2) Nicarbazin ELISA Plate: 1 piece (8 hole × 12), to be coated with " Nicarbazin-OVA " that embodiment 2 is prepared ELISA Plate.
(3) 1 bottle (10mL), antibody Nicarbazin antibody working solution: is subjected to 1:8000 dilution, antibody for antibody diluent Dilution is volume fraction containing 6%() 0.2MPBS of lowlenthal serum, it is anti-that the Nicarbazin antibody is that embodiment 3 is prepared Serum purifies 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 MH2SO4Solution.
(9) cover board film；
(10) valve bag.
2, the equipment and material for needing and not providing
(1) equipment
Microplate reader (Detection wavelength 450nm, reference wavelength 630nm), balance (precision: 0.01g), vortex oscillator, centrifuge (4000g), shaking table (300rpm), nitrogen evaporator, micropipettor, timer.
(2) reagent
Sodium chloride, acetonitrile, n-hexane, acetic acid solution (take 1mL acetic acid, be added in 99 mL deionized waters, mix).
3, it stores
The kit is stored in 2-8 DEG C, is sure not to freeze, and validity period 1 year.
The ELIAS strip being not used should seal, 2-8 DEG C of preservation.
4, kit testing principle
Fixed antigen-specific sexual competition antibody on Nicarbazin and ELISA Plate in sample, is added enzyme marker, catalysis substrate Colour developing, according to the depth of colour developing come the content of Nicarbazin in judgement sample.Colour developing is deep, and content is few, and colour developing is shallow, and content is more.
Two, the application method of Nicarbazin enzyme linked immunological kit
1, sample pre-treatments
(1) animal flesh and the tissue (coefficient of dilution: 12)
A) take 1 ± 0.05g tissue sample in 50mL centrifuge tube；B) 0.1mL acetic acid solution, 3 mL n-hexanes and 6 mL are added Acetonitrile, immediately abundant 1 min of whirling motion；C) 4000g or more is centrifuged 5min；D) upper layer n-hexane is discarded；E) 0.5mL supernatant is taken In new 4mL centrifuge tube；F) it in 50-60 DEG C of water-bath, is dried with nitrogen；G) 1mL sample diluting liquid, abundant whirling motion 30s is added；H) 20 μ L are taken to be detected.
(2) egg (coefficient of dilution: 12)
A) sample after accurately weighing 1 ± 0.05 g homogeneous is in 50 mL centrifuge tubes；B) sequentially add 1 g sodium chloride, 1.5mL acetic acid solution (see 4.2), 3 mL n-hexanes and 4.5 mL acetonitriles, immediately abundant whirling motion 1min；C) 4000 g with On, it is centrifuged 5 min；D) upper layer n-hexane is discarded；E) take 0.5 mL acetonitrile layer in 4 new mL centrifuge tubes；f) 50-60 In DEG C water-bath, it is dried with nitrogen；G) 1 mL sample diluting liquid, abundant 30 s of whirling motion is added；H) 20 μ L is taken to be detected.
2, detecting step
(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 Nicarbazin standard items working solution (or testing sample solution) of 20 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 by abscissa of corresponding Nicarbazin concentration Standard curve.
(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 Nicarbazin in raw sample to be measured.
Three, Nicarbazin enzyme linked immunological kit detects Nicarbazin
1, specific detection
The specificity of Nicarbazin enzyme linked immunological kit is determined by carrying out cross reaction test with corresponding substance. Cross reaction is smaller, and specificity is better.
Nicarbazin and other analogs (4.4 '-dinitro acardite, 2- hydroxyl -4.6- dimethyl pyrimidine) are done respectively It is serially diluted, is operated respectively according to step 22 as above, substitute it with the serial dilutions of Nicarbazin and other analogs In " Nicarbazin standard items working solution ", make standard curve, and find out respective 50% inhibition concentration (IC on curve50), tool Body method is as follows: obtaining Y value equal to 50% corresponding Nicarbazin concentration (ng/mL), i.e. IC50Value.It is calculated with following formula Cross reacting rate of the kit to Nicarbazin and each analog.
Cross reacting rate (%)=(the Nicarbazin concentration for causing 50% inhibition/cause the Nicarbazin analog of 50% inhibition Concentration) × 100%
The results are shown in Table 1, from table 1 it follows that Nicarbazin enzyme linked immunological kit is anti-to the intersection of various analogs Should rate be respectively less than 0.1%.This illustrates that Nicarbazin enzyme linked immunological kit has high specificity to Nicarbazin, can be effective The other analogs of exclusion interference, can be dedicated for the detection of Nicarbazin.
The specificity of 1 Nicarbazin enzyme linked immunological kit of table
Medicine name Cross reacting rate (%)
Nicarbazin 100
4.4 '-dinitro acardite < 0.1
2- hydroxyl -4.6- dimethyl pyrimidine < 0.1
2, the minimum detection limit measurement of different samples
When measurement detects Nicarbazin in animal flesh, tissue and egg using Nicarbazin enzyme linked immunological kit respectively Minimum detection limit.Specific method such as step 2.
The results show that being measured using sample-pretreating method processing animal flesh, tissue and egg in step 2 1(1) Minimum detection limit is up to 0.5 μ g/kg.
3, between Nicarbazin enzyme linked immunological kit plate inner panel error measurement
Error between error and plate in the plate of Nicarbazin enzyme linked immunological kit is measured respectively.Specific method such as step 2.
The results show that error is less than 5% in the plate of kit absorbance, error is less than 10% between plate.
4, the determination of recovery rates of Nicarbazin enzyme linked immunological kit detection Nicarbazin
Measurement detects the rate of recovery of Nicarbazin using Nicarbazin enzyme linked immunological kit.Specific method such as step 2.
The results show that use the rate of recovery range of Nicarbazin enzyme linked immunological kit detection Nicarbazin for 90% ~ 110%。
5, the sensitivity determination of Nicarbazin enzyme linked immunological kit
Measure the sensitivity of Nicarbazin enzyme linked immunological kit.Specific method such as step 2.
The results show that the sensitivity of Nicarbazin enzyme linked immunological kit is 0.1 μ g/kg, standard curve range 0.1-8.1 μg/kg。
Embodiment 6, Nicarbazin colloidal gold strip detect Nicarbazin
One, the composition of Nicarbazin colloidal gold strip
Nicarbazin colloidal gold strip is made of sample absorption pad, colloidal gold pad, reaction film and water absorption pad；
Along the axial direction of test strips, sample absorption pad, colloidal gold pad, reaction film and water absorption pad are successively linked in sequence, and sample absorbs The end of pad is connected with the beginning of colloidal gold pad, and the end of colloidal gold pad is connected with the beginning of reaction film, the end of reaction film with The beginning of water absorption pad is connected；
The Nicarbazin monoclonal antibody that the embodiment 3 of colloid gold label obtains is coated in the colloidal gold pad；
There are detection zone and quality control region on the reaction film, detection zone (T line) and quality control region (C line) are axially vertical with test strips Ribbon；Detection zone is located at close to the side of colloidal gold pad end；Quality control region is located remotely from the side of colloidal gold pad end；Inspection Nicarbazin-OVA prepared by area's coating embodiment 2 is surveyed, quality control region is coated with sheep anti mouse secondary antibody.
Sample well is located at one end in sample absorbent far from colloidal gold pad end.
Two, the preparation of test strips
1, colloidal gold labeled monoclonal antibody
(1) preparation of colloidal gold solution
It takes 0.01% aqueous solution of chloraurate 100mL to be heated to boiling with thermostatic electromagnetic blender, is added in the case where lasting stirring 1% trisodium citrate aqueous solution 2.5mL, continues agitating and heating 20min, and solution is in bright red.Room temperature is cooling, uses deionization Water is restored to original volume, 2-8 DEG C of preservation.
(2) preparation of gold labeling antibody solution
With 0.1mol/L K2CO3Aqueous solution adjusts the pH to 8.2 of colloidal gold solution, then takes 10mL that 50mL beaker is added In, magnetic stirrer 250r/min stirring is added dropwise monoclonal antibody solution, 3mL 5g/100mL BSA water is added dropwise Solution persistently stirs 10min.
(3) 20-24 DEG C of low speed of gold labeling antibody solution (1500r/min) is centrifuged 20min, discards the gold particle shape by agglomerating At precipitating, take red supernatant solution.
(4) 4 DEG C of the solution of step (3), 11000r/min are centrifuged 40min, solution is divided into three layers of (transparent supernatant, pipe The gold particle layer of black densification in the flowable dark red precipitate in bottom and tube bottom wall), flowable dark red precipitate is transferred to In another centrifuge tube, it is molten that former gold labeling antibody is suspended to the 0.01mol/L phosphate buffer of the BSA containing 1g/100mL The volume of liquid, overnight, 4 DEG C, 11000r/min centrifugation 40min collect precipitating.
(5) with the 0.01mol/L phosphate buffer of BSA containing 1g/100mL and 0.02g/100mL NaN3 by step (4) precipitating is suspended to 1/40,2-8 DEG C of preservation of the volume of former gold labeling antibody solution.
2, metal spraying: the suspension that step (1) obtains is sprayed on glass fibre membrane, colloidal gold pad is made.
3, spray film: the T line position on reaction film sprays the Nicarbazin-OVA of the preparation of embodiment 2, the spray of C line position Upper sheep anti-mouse antibody.
4, it assembles: sample absorption pad (cellulose filter membrane), colloidal gold pad, nitrocellulose filter, water absorption pad is routinely square Method is assembled, and is then cut, and can be detected.Test strips can also be fitted into plastics fabrication, form test card for detecting.
Three, it is detected with test card
1, sample pre-treatments and detection
The step of sample-pretreating method such as embodiment five 21.
Test card is taken out, desktop is lain against behind Kaifeng, sample to be tested solution is drawn and 3 ~ 5 drops is added dropwise in sample well； 5-10min judging result, the judging result after 15min are invalid.
As a result judgment criteria:
Negative: the colour developing of C line, T line naked eyes are as it can be seen that no matter shade is judged to feminine gender；
Positive: the colour developing of C line, T line do not develop the color, and are judged to the positive；
Invalid: C line does not develop the color, and no matter whether T line develops the color, which is judged in vain.

Claims (10)

1. a kind of compound, structure are shown in formula I:
Formulas I.
2. the method for preparing compound described in claim 1, including step are as follows:
(1) dimethylformamide (DMF) is added in aniline, succinic anhydride, the aniline, DMF, succinic anhydride is added after stirring Proportion be 1000mg:25ml:1290mg, the reaction temperature be 65-70 DEG C, obtain reaction solution；
(2) reaction solution is added drop-wise in 100ml ice water, the extraction of 25ml ethyl acetate, combined ethyl acetate phase, anhydrous sodium sulfate is dry Dry 4 hours, it is filtered to remove desiccant, filtrate is spin-dried for, and obtains Nicarbazin haptens intermediate 1870mg；
(3) 5ml concentrated nitric acid is slowly added under ice bath in the 15ml concentrated sulfuric acid, stirs, is slow added among Nicarbazin haptens Body 1500mg, magnetic agitation 2-3h, reaction solution is poured into ice water, yellow solid is precipitated at room temperature, and filtering, collects filter at washing Cake, 50 DEG C forced air drying 5-6 hours, obtain 924.6 mg of Nicarbazin haptens.
3. Nicarbazin antigen, for by compound described in claim 1 and the resulting antigen of carrier protein couplet.
4. Nicarbazin antigen according to claim 3, it is characterised in that: the carrier protein can be bovine serum albumin White, ovalbumin, human serum albumins, hemocyanin, mouse haemocyanin, thyroprotein or rabbit serum proteins.
5. the preparation method of Nicarbazin antigen described in claim 3 or 4 includes the following steps: change described in claim 1 It closes object and carrier protein to be coupled by amido bond, obtains the Nicarbazin antigen；Compound described in claim 1 and the load The molar ratio of body protein coupling is 17.6:1.
6. the preparation method of Nicarbazin antigen according to claim 5, it is characterised in that: the method includes walking as follows It is rapid:
(1) the Nicarbazin haptens (Formulas I) is dissolved in dimethylformamide (DMF), 1- (3- diformazan ammonia is then added Base propyl) -3- ethyl-carbodiimide hydrochloride (EDC) and n-hydroxysuccinimide (NHS), 20-25 DEG C of magnetic agitation reaction 2-3h obtains solution I；
Wherein, the Nicarbazin haptens (Formulas I), the dimethylformamide (DMF), the 1- (3- dimethylamino third Base) -3- ethyl-carbodiimide hydrochloride (EDC), the n-hydroxysuccinimide (NHS) proportion be 24.8810.66mg: 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 Nicarbazin antigen.
7. the application of Nicarbazin antigen described in compound or claim 3 described in claim 1, it is characterised in that qualitative or fixed Amount detection Nicarbazin；Prepare Nicarbazin antibody.
8. utilizing the antibody of the preparation of Nicarbazin antigen described in claim 3.
9. the application of Nicarbazin antigen according to claim 7, it is characterised in that can be to prepare ELISA reagent Box and colloidal-gold detecting-card.
10. the application of Nicarbazin antigen according to claim 7 or 9, it is characterised in that detection sample can be animal Meat, tissue and egg, the detection for detecting Nicarbazin in above-mentioned sample are limited to 0.5 μ g/kg.
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