CN108530373A - A kind of furans metabolite hapten, artificial antigen and its application in fluorescent quantitation immunochromatography - Google Patents
A kind of furans metabolite hapten, artificial antigen and its application in fluorescent quantitation immunochromatography Download PDFInfo
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Abstract
The invention discloses a kind of furans metabolite hapten, artificial antigen and its applications in fluorescent quantitation immunochromatography, there are two the feature structures of determinand in haptens structure, so that the spatial position of haptens in carrier proteins is more prominent, increase recognition site, the immunogenicity for effectively raising small haptens also improves the potency of antigen.Artificial antigen in the present invention has very high sensitivity for ELISA detection furans metabolites.Coordinate the detection method in the present invention using fluorescent quantitation immuno-chromatographic test paper strip prepared by the artificial antigen in the present invention, can quickly and conveniently realize the detection of furans metabolite, and the test strips high sensitivity, precision are high, stability is good.
Description
Technical field
The invention belongs to immunochemical technique fields, and in particular to a kind of furans metabolite hapten, artificial antigen and
Its application in fluorescent quantitation immunochromatography.
Background technology
Furans are the antibiotic that artificial synthesized one kind shares 5- nitrofuran ring structures, to gram-positive bacteria, leather
Lan Shi negative bacteriums, some fungi all have antibacterial effect.Because cheap, it is widely used in livestock and poultry and fish culture,
For treating the enteritis caused by Salmonella, Escherichia coli, globidiosis etc., and frequently as farming animals and aquatic products fish
The growth promoter of shrimps is inserted in feed.Itrofurans includes mainly：Furaltadone, nitrofurazone, furantoin and
Furazolidone.Itrofurans prototype medicine is metabolized rapidly in vivo, and metabolite is respectively AOZ, AMOZ, AHD, SEM,
Therefore often the residual situation of Nitrofuran metabolites is reacted using the detection of metabolin.
In food security, nitrofurans a large amount of or continuous use not only to the toxic effect of cultivated animals, but also
With carcinogenic, teratogenesis, mutagenesis, metabolin also has malicious influences to human health.Therefore, many national explicit orders
Forbid using nitrofurans during raising animal derived food, and defines the inspection and quarantine in animal derived food
Standard.
Method currently used for detecting furans metabolite mainly has traditional detection method, such as spectrophotometry, efficient liquid
Phase chromatography and its joint technology etc., although above method can be with accurate quantification, due to equipment and instrument costliness, detection time
It is long, and professional is needed to operate, therefore cannot achieve field quick detection truly；Another detection method is immunology
Detection technique, the method have the characteristics that high specific and highly selective, are highly suitable for the separation of complex matrices trace components
Or detection, it being interpenetrated with interdisciplinary, detection method emerges one after another, and immunoassay detection technique is also evolving,
From ELISA ELISAs technology and colloidal gold immunochromatographimethod technology to biosensor technology (such as fluorescence immune chromatography skill
Art).For ELISA and colloidal gold technique, fluorescence immune chromatography technology has as new detection method than the former huge
Advantage.Although existing enzyme-linked immune analytic method high sensitivity, flux are big, also professional is needed to operate, and detection time
It is longer, it equally also cannot be satisfied the demand of on-site quick screening；And although colloidal gold method has high-throughput, easy to operate, detection
The advantages that time is short and is operated without professional technician, but its poor sensitivity, but also colloidal gold method cannot be satisfied state
The requirement of family's examination criteria.And fluorescence immune chromatography technology is as a kind of new quantitative approach quickly detected, is based on immune
What principle and colloidal gold immunochromatographimethod technology grew up, which combines the high sensitivity and colloidal gold method of ELISA method
Quick feature, detection time only needs 10~15min, but detection sensitivity is 3~5 times of colloidal gold method, have operation letter
Just, highly sensitive, high-throughput and quick feature.
Invention content
It is immunized the purpose of the present invention is to provide a kind of furans metabolite hapten, artificial antigen and its in fluorescent quantitation
Application in chromatography.
The technical solution used in the present invention is：
A kind of furans metabolite hapten, which is characterized in that the general structure of furans metabolite hapten is：
The structural formula of R group is any one in following four：
The preparation method of furans metabolite hapten includes the following steps：
1) chemical compounds I and compound ii are dissolved in acetone, acid binding agent is added, 2~5h is reacted at 80~90 DEG C, obtain chemical combination
Object III；
2) compound III is dissolved in absolute ethyl alcohol, it is 10~12 that 6M lithium hydroxide solutions to pH value of reaction system, which is added,
15~26h is reacted at room temperature, purified water is added, the pH value that 1M dilute hydrochloric acid is added to reaction system is 4~5, and filtering obtains compound
Ⅳ；
3) compounds Ⅳ is dissolved in methanol, X, 60~70 DEG C of 1.5~2.5h of reaction is added, reaction finishes, filters, obtain
Haptens V, any one in X AMOZ, AOZ, AHD, SEM；
Formula I~V is as follows：
Further, in step 1), acid binding agent K2CO3, the molar ratio of acid binding agent and compound ii is (1.5~2):1,
The molar ratio of chemical compounds I and compound ii is (2~2.5):1.
Further, the molar ratio of the X and compounds Ⅳ that are added in step 3) are (2.0~3.0):1.
A kind of furans metabolite artificial antigen obtains for above-mentioned furans metabolite hapten with carrier protein couplet,
The general structure of furans metabolite artificial antigen is：
Carrier protein is any one in bovine serum albumin(BSA), ovalbumin, human serum albumins or hemocyanin.
Furans metabolite monoclonal antibody prepared by above-mentioned furans metabolite artificial antigen, furans metabolite are artificial
The carrier protein of antigen is hemocyanin.
Above-mentioned furans metabolite artificial antigen and above-mentioned furans metabolite monoclonal antibody are in ELISA detection method
Application.
Above-mentioned furans metabolite artificial antigen and above-mentioned furans metabolite monoclonal antibody are immune in fluorescent quantitation
Application in chromatography.
A kind of furans metabolite fluorescent quantitation immuno-chromatographic test paper strip, test strips include being coated with above-mentioned furans generation
Thank to the reaction film of object artificial antigen, the carrier protein of furans metabolite artificial antigen is bovine serum albumin, preparation method packet
Include following steps：
1) reaction film for being coated with artificial antigen and rabbit IGg is prepared：By the furans generation that carrier protein is bovine serum albumin
It thanks to object artificial antigen and is added to the uniformly mixed detection zone for being sprayed onto reaction film in coating buffer solution, rabbit IGg is added to coating buffer solution
In be uniformly mixed and be sprayed onto the control zone of reaction film, detection zone and control zone are separated from each other, are saved backup after drying process；
2) preparation of sample pad：The blank sample pad cut is immersed in sample pad treatment fluid and is impregnated, sample is taken out
Pad saves backup after being dried；
3) assembling of fluorescent quantitation immuno-chromatographic test paper strip：Reaction film is stacked in the middle part of backing, stacked sample is distinguished at both ends
Pad and water absorption pad, reaction film are mutually connected with water absorption pad, sample pad, and detection zone is obtained close to sample pad, control zone close to water absorption pad
Test strips are loaded into test card by test paper plate, obtain fluorescent quantitation immuno-chromatographic test paper strip.
The method for detecting furans metabolite using above-mentioned furans metabolite fluorescent quantitation immuno-chromatographic test paper strip：
1) preparation of the furans metabolite monoclonal antibody of fluorescent microsphere label：After fluorescent microsphere is activated with above-mentioned furan
Metabolite monoclonal antibody of muttering is coupled, and centrifugation removal of impurities is added fluorescence buffer solution and fluorescent microsphere is resuspended, and obtains fluorescent microsphere label
Furans metabolite monoclonal antibody；
2) preparation of the furans metabolite monoclonal antibody detection liquid of fluorescent microsphere label：With fluorescence buffer solution by fluorescence
The furans metabolite monoclonal antibody dilution of microballoon label, obtains the furans metabolite monoclonal antibody of fluorescent microsphere label
Detect liquid；
3) the furans metabolite standard solution of series concentration, the furans for taking fluorescent microsphere to mark are equipped with PBS buffer solution
The sample area of fluorescent quantitation immuno-chromatographic test paper strip is added drop-wise to after metabolite monoclonal antibody detection liquid and standard solution mixing,
T/C values are read with fluorescent quantitative detector, standard curve is established by the corresponding T/C values of concentration of standard solution, are surveyed
Determine the T/C values of sample to be tested, the Quantitative detection of furans metabolite can be realized in combined standard curve.
The beneficial effects of the invention are as follows：The common method for preparing the haptens of furans metabolite in the prior art is by it
It transform the haptens with 4-CP or 3-CP derivant structures as, then coupling protein prepares artificial antigen, and such method is common
Point does not have to retain the dominant group nitro end of object to be measured object when being haptens structure of modification, and the antibody prepared is caused to exist
The low deficiency of poor specificity, affinity.The haptens for the furans metabolite that the present invention designs spreads out with traditional 4-CP or 3-CP
Biochemical haptens is compared, and is had a clear superiority.The arm not only active group that haptens introduces, also completely remains to be measured
The nitro end of object so that haptens is consistent with the cloud density of object to be measured object, while having in a haptens structure
The feature structure of two determinands so that the spatial position of haptens in carrier proteins is more prominent, increases recognition site,
The immunogenicity for effectively raising small haptens also improves the potency of antigen.
Furans metabolite artificial antigen is detected for ELISA, to furaltadone, furazolidone, furantoin, furans
The minimum detection limit in XiLin is respectively 0.06 μ g/L, 0.04 μ g/L, 0.02 μ g/L, 0.08 μ g/L.
Furans metabolite artificial antigen is used for fluorescent quantitation immunochromatography technique, can quickly and conveniently realize furans
The detection of metabolite, the fluorescent quantitation immuno-chromatographic test paper strip prepared in of the invention are appropriate to furaltadone, furazolidone, furans
Because the detection sensitivity of, nitrofurazone is respectively 0.2 μ g/L, 0.1 μ g/L, 0.1 μ g/L, 0.2 μ g/L, in addition the fluorescent quantitation is exempted from
Epidemic disease chromatograph test strip precision is high, and can stablize at room temperature and preserve 1 year or more, can meet market in storage and transport mistake
Requirement in journey.
Description of the drawings
Fig. 1 is the mass spectrogram of V-AMOZ；
Fig. 2 is the mass spectrogram of V-AMOZ；
Fig. 3 is the mass spectrogram of V-AMOZ；
Fig. 4 is the mass spectrogram of V-AMOZ；
Fig. 5 is ELISA examination criteria curves；
Fig. 6 is fluorescent quantitation immunochromatography quantitative measurement standard curve.
Specific implementation mode
Below in conjunction with embodiment, the present invention is further explained, it should be appreciated that following embodiment be merely to illustrate the present invention and
It is not used in and limits the scope of the invention.
Hapten synthesis
The synthetic line figure of furans metabolite hapten is as follows in Examples 1 to 4：
Wherein X is any one in AMOZ, AOZ, AHD, SEM；
The structural formula of R group is any one in following four：
Embodiment 1
A kind of furans metabolite hapten, preparation method are as follows：
(1) it takes 4.8g (28.9mmol) chemical compounds Is in 50ml round-bottomed flasks, sequentially adds 20ml acetone, 3.0g
(11.5mmol) compound ii and 3.2g (23.0mmol) potassium carbonate.5h or more is reacted in 80 DEG C.Reaction finishes, and decompression is gone molten
Agent is added 40ml purified waters, is extracted with ethyl acetate twice, then depressurize solvent, recrystallizes to obtain 3.2g compound IIIs (acetic acid second
Ester/petroleum ether, 1/5, v/v).
(2) 3.2g (7.4mmol) compound III is dissolved in 10ml ethyl alcohol, the lithium hydroxide solution of 6M is added by compound
The pH value of III ethanol solution is adjusted to 10, reacts at room temperature 15h or more, and 40ml purified waters are then added, and acquired solution passes through 1M's
PH value is adjusted to 4 by dilute hydrochloric acid, and filtering, drying obtains 2.3g compounds Ⅳs.
(3) compounds Ⅳ of 500mg (1.2mmol) is dissolved in 10ml methanol, the furan of 750mg (3.7mmol) is added
Mutter its bupropion metabolite object AMOZ, reacts 1.5h or more in 60 DEG C, and reaction is finished, is cooled to room temperature, and is filtered, is obtained furanone metabolites
The mass spectrogram of haptens V-AMOZ 350mg, V-AMOZ are as shown in Figure 1, ESI-MS：771[M+1].
Embodiment 2
A kind of furans metabolite hapten, preparation method are as follows：
(1) it takes 3.8g (23.0mmol) chemical compounds Is in 50ml round-bottomed flasks, sequentially adds 20ml acetone, 3.0g
(11.5mmol) compound ii and 2.4g (17.3mmol) potassium carbonate.2h is reacted in 90 DEG C.Reaction finishes, and solvent is removed in decompression, adds
Enter 40ml purified waters, be extracted with ethyl acetate twice, then depressurize solvent, column chromatography purifies to obtain 2.4g compound IIIs (acetic acid second
Ester/petroleum ether, 1/5, v/v).
(2) compound III of 2.4g (5.6mmol) is dissolved in 10ml ethyl alcohol, the lithium hydroxide solution of 6M is added by chemical combination
The pH value of the ethanol solution of object III is adjusted to 12, reacts at room temperature 26h, and 40ml purified waters are then added, and acquired solution passes through the dilute of 1M
PH value is adjusted to 5 by hydrochloric acid, and filtering, drying obtains 1.7g compounds Ⅳs.
(3) compounds Ⅳ of 500mg (1.2mmol) is dissolved in 10ml methanol, the furan of 247mg (2.4mmol) is added
Mutter oxazolone metabolin AOZ, reacts 1.5h or more in 60 DEG C, and reaction is finished, is cooled to room temperature, and is filtered, is obtained Furaxone metabolite
The mass spectrogram of haptens V-AOZ 475mg, V-AOZ are as shown in Fig. 2, ESI-MS：573[M+1].
Embodiment 3
A kind of furans metabolite hapten, preparation method are as follows：
(1) it takes 3.8g (23.0mmol) chemical compounds Is in 50ml round-bottomed flasks, sequentially adds 20ml acetone, 3.0g
(11.5mmol) compound ii and 2.4g (17.3mmol) potassium carbonate.2h is reacted in 90 DEG C.Reaction finishes, and solvent is removed in decompression, adds
Enter 40ml purified waters, be extracted with ethyl acetate twice, then depressurize solvent, column chromatography purifies to obtain 2.6g compound IIIs (acetic acid second
Ester/petroleum ether, 1/5, v/v).
(2) compound III of 2.6g (6.1mmol) is dissolved in 10ml ethyl alcohol, the lithium hydroxide solution of 6M is added by chemical combination
The pH value of the ethanol solution of object III is adjusted to 11, reacts at room temperature 20h, and 40ml purified waters are then added, and acquired solution passes through the dilute of 1M
PH value is adjusted to 4.5 by hydrochloric acid, and filtering, drying obtains 1.9g compounds Ⅳs.
(3) compounds Ⅳ of 500mg (1.2mmol) is dissolved in 10ml methanol, the furan of 287mg (2.4mmol) is added
It mutters appropriate because of metabolin AHD, reacts 1.5h or more in 60 DEG C, reaction is finished, is cooled to room temperature, and is filtered, is obtained Cistofuran metabolite
The mass spectrogram of haptens V-AHD 300mg, V-AHD are as shown in figure 3, ESI-MS：599[M+1].
Embodiment 4
A kind of furans metabolite hapten, preparation method are as follows：
(1) it takes 3.8g (23.0mmol) chemical compounds Is in 50ml round-bottomed flasks, sequentially adds 20ml acetone, 3.0g
(11.5mmol) compound ii and 3.2g (23.0mmol) potassium carbonate.Reaction finishes, and solvent is removed in decompression, and 40ml purified waters are added,
Be extracted with ethyl acetate twice, then depressurize solvent, column chromatography purify 2.6g compound IIIs (ethyl acetate/petroleum ether, 1/5,
v/v)。
(2) compound III of 2.6g (6.1mmol) is dissolved in 10ml ethyl alcohol, the lithium hydroxide solution of 6M is added by chemical combination
The pH value of the ethanol solution of object III is adjusted to 12, reacts at room temperature 26h, and 40ml purified waters are then added, and acquired solution passes through the dilute of 1M
PH value is adjusted to 5 by hydrochloric acid, and filtering, drying obtains 1.7g compounds Ⅳs.
(3) compounds Ⅳ of 500mg (1.24mmol) is dissolved in 10ml methanol, is added 280mg's (3.72mmol)
Furacilin metabolite SEM reacts 1.5h or more in 60 DEG C, and reaction is finished, is cooled to room temperature, and is filtered, and obtains nitrofurazone metabolism
The mass spectrogram of object haptens V-SEM 640mg, V-SEM are as shown in figure 4, ESI-MS：519[M+1].
The synthesis of furans metabolite artificial antigen
Carrier protein is that the synthetic method of the furans metabolite artificial antigen of bovine serum albumin(BSA) is as follows：
(1) the furans metabolite hapten in the 10mg present invention is taken, is dissolved in 0.5ml dimethylformamides (DMF),
After stirring fully, 10mg EDC and 10mg n-hydroxysuccinimide (NHS) is added, stirs 4h at room temperature, you can it is anti-to obtain half
Former Acibenzolar；
(2) 30mg bovine serum albumins (BSA) are weighed, keep the PBS that it is substantially dissolved in a concentration of 0.01mol/L of 4ml molten
In liquid, carrier protein solution is formed, the haptens Acibenzolar is slowly added dropwise dropwise under stiring molten to the carrier protein
In liquid, and it is stirred at room temperature 16~for 24 hours；
(3) solution made from step (2) is dialysed 3 days with the PBS room temperatures of 0.01mol/L, 3 dialyzates is changed daily, to remove
Remove unreacted small-molecule substance；
(4) it dispenses, is saved backup in 4 DEG C.
Carrier protein is that the synthetic method of the furans metabolite artificial antigen of hemocyanin is as follows：
(1) the furans metabolite hapten in the 10mg present invention is taken, is dissolved in 0.5ml dimethylformamides (DMF),
After stirring fully, 10mg EDC and 10mg n-hydroxysuccinimide (NHS) is added, stirs 4h at room temperature, you can it is anti-to obtain half
Former Acibenzolar；
(2) 30mg hemocyanin (KLH) is weighed, it is made to be substantially dissolved in the PBS solution of a concentration of 0.01mol/L of 4ml
In, carrier protein solution is formed, the haptens Acibenzolar is slowly added dropwise dropwise to the carrier protein solution under stiring
In, and it is stirred at room temperature 16~for 24 hours；
(3) solution made from step (2) is dialysed 3 days with the PBS room temperatures of 0.01mol/L, 3 dialyzates is changed daily, to remove
Remove unreacted small-molecule substance；
(4) it dispenses, is saved backup in 4 DEG C.
The preparation of furans metabolite monoclonal antibody
It is immunogene by the furans metabolite artificial antigen of hemocyanin of above-mentioned carrier protein, is helped with isometric Freund
After agent emulsification, immune balb/c mice.Every mouse immunizing dose is 50~100ug, and immunization interval 3 weeks is adopted small after being immunized 3 times
Rat-tail portion venous blood detects serum titer.It is required as antibody titer does not reach, needs booster immunization, after antibody titer no longer increases,
Subcutaneous booster immunization is carried out with 100ug holoantigens, mouse boosting cell and SP20 cell fusions are taken after 5 days.Cell after fusion exists
It is screened in HAT culture mediums, being substituted for HAT culture mediums after 5 days with complete medium is cultivated.With ELISA to cell conditioned medium into
The hole inner cell that testing result is strong positive is carried out limiting dilution assay clone's culture, is detected through 3 time cloning cultures by row detection
Afterwards, the hole inner cell being positive is the hybridoma of secrete monoclonal antibody.After hybridoma amplification culture, connect
Kind generates the ascites containing antibody to mouse peritoneal.Purify ascites with octanoic acid-ammonium sulfate precipitation method, you can obtain high-purity, Gao Te
Anisotropic monoclonal antibody.
With containing 0.05% Sodium azide, monoclonal antibody is diluted to 0.05 μ g/ml by the phosphate buffer of pH7.4,4 DEG C
It preserves.
Using the haptens in above-mentioned preparation method and Examples 1 to 4, the furan that carrier protein is bovine serum albumin(BSA) is prepared
It mutters its ketone antigen, carrier protein is the furaltadone antigen of hemocyanin, and furaltadone monoclonal antibody, carrier protein is ox blood
The furazolidone antigen of pure albumen, carrier protein are the furazolidone antigen of hemocyanin, and furazolidone monoclonal antibody carries
Body protein is the furantoin antigen of bovine serum albumin(BSA), and carrier protein is the furantoin antigen of hemocyanin, furantoin
Monoclonal antibody, carrier protein are the nitrofurazone antigen of bovine serum albumin(BSA), and carrier protein is the nitrofurazone of hemocyanin
Antigen, nitrofurazone monoclonal antibody.
Application and effect assessment of the furans metabolite artificial antigen in ELISA
Make coating dilution with the carbonate buffer solution of pH9.6, by the furans generation that carrier protein is bovine serum albumin(BSA)
It thanks to object artificial antigen and is diluted to 0.5 μ g/ml, be added in polystyrene micropore plate by 100 holes μ L/, 4 DEG C of coatings overnight, press by drying
1%BSA is added in 250 holes μ L/, closes 1h for 37 DEG C in phosphate buffer, drying is vacuum-packed after dry and preserves.
It is molten to the addition furans range of metabolic object standard in the micropore ELISA Plate of furans metabolite artificial antigen that is coated with
50 holes μ L/ of liquid, then corresponding 50 holes μ L/ of addition furans metabolite monoclonal antibody solution, 37 DEG C of reaction 0.5h；After drying, add
Enter 300 holes μ L/ of washing lotion, is patted dry after washing 3 times；Add 100 holes μ L/ of ELIAS secondary antibody, 37 DEG C of reaction 0.5h；It washs 3 times again
After pat dry, be separately added into the developing solution A and developing solution B in 50 holes μ L/, 37 DEG C of reaction 15min；50 holes μ L/ of 2M sulfuric acid are added to terminate,
It sets microplate reader and measures the OD values per hole in 450nm wavelength.As a result such as following table.
ELISA tests the OD values of the furans metabolite standard solution of various concentration
By data in table, four parameter Logistic curve matchings are carried out using ELISA Calc softwares and draw standard curve
The linear equation of (such as Fig. 5), furaltadone is：Y=(A-D)/[1+ (x/C) ^B]+D, r2=0.997, A=2.56195, B=
1.02386, C=0.21452, D=-0.07147, x indicate that testing concentration, y indicate OD values, and IC50 values, which are obtained by calculation, is
0.29 μ g/L, it is in a linear relationship in 0.05~1.6 μ g/L.By measuring the titer of 20 0 μ g/L, by formula Z=average values-
3 standard deviations, it is 2.0368 to calculate Z values, and Z values can be calculated lowest detection as Y value substitution standard curve is limited to 0.06 μ g/
L。
Identical method can calculate the curvilinear line r of furazolidone2=0.998, IC50 value are 0.22 μ g/L, lowest detection
It is limited to 0.04 μ g/L.
Identical method can calculate the curvilinear line r of furantoin2=0.997, IC50 value are 0.25 μ g/L, lowest detection
It is limited to 0.02 μ g/L.
Identical method can calculate the curvilinear line r of nitrofurazone2=0.996, IC50 value are 0.28 μ g/L, lowest detection
It is limited to 0.08 μ g/L.
The preparation of fluorescent quantitation immuno-chromatographic test paper strip
The preparation method of fluorescent quantitation immuno-chromatographic test paper strip is as follows：
(1) reaction film for being coated with artificial antigen and rabbit IGg is prepared：
With nitrocellulose filter (NC films) for reaction film, the artificial antigen that carrier protein is bovine serum albumin(BSA) is coated with
Buffer solution adjusts concentration to 0.1~0.5mg/mL, and rabbit IGg is also adjusted concentration to 0.1~1mg/mL with coating buffer solution, presses
According to the film liquid amount of 0.8~1.2 μ L/cm, antigen and rabbit IGg are sprayed onto the corresponding detection zone of reaction film and control zone, detection zone and
Be divided into 5mm between control zone, place 40~45 DEG C of 3~35h of oven, be placed in it is spare in constant temperature and humidity safe, it is used
It is the 0.01M PBS bufferings containing 0.5%PEG20000,1% sucrose, 0.5%BSA, 0.05% sodium azide to be coated with buffer solution
Liquid；
(2) preparation of sample pad：
The blank sample pad cut is immersed in sample copy treatment fluid, after impregnating 5min, is taken out in 37 DEG C of dryings
16h, be placed in it is spare in constant temperature and humidity safe, used sample pad treatment fluid be contain 0.3% polysorbas20,1% sucrose, 0.5%
The 0.01M PBS buffer solution of BSA, 0.05% sodium azide；
(3) assembling of fluorescent quantitation immuno-chromatographic test paper strip：
Sample made from superimposition step (2) is distinguished at reaction film made from superimposition step (1), both ends in the middle part of the PVC board backing
Pad and water absorption pad, reaction film are mutually connected with water absorption pad, sample pad, and detection zone is obtained close to sample pad, control zone close to water absorption pad
Test paper plate obtains test paper plate, and test paper plate is cut into test strips, test strips are loaded into test card, and it is immune to obtain fluorescent quantitation
Chromatograph test strip.
The performance evaluation of fluorescent quantitation immuno-chromatographic test paper strip
(1) preparation of the furans metabolite monoclonal antibody of fluorescent microsphere label：Take 500 μ L fluorescent microsphere solution (glimmering
Light microballoon solid content 1% contains fluorescent microsphere 5mg), ultrasonication is used in combination；Be separately added under stirring 5mg NHS and
The temperature of 4mgDEC, control fluorescent microsphere solution are 4~10 DEG C, and activate 20min at this temperature；After having activated, 0.1M is used
Solution of potassium carbonate tune pH is 8~9, and the temperature of control fluorescent microsphere solution is 4~10 DEG C；0.25mg furans metabolite lists are added
Clonal antibody after stirring evenly, removes ice bath, it is allowed to be warmed to room temperature naturally, and 4~6h is stirred at room temperature；After the completion of coupling,
Reaction solution centrifuges 10min at 10000rpm, removes supernatant, and 1mL fluorescence buffer solutions are added, after ultrasound is resuspended, in centrifugation, removing
Clearly, which is repeated 3 times, and last time centrifuges, after removing supernatant, addition 0.5mL fluorescence buffer solution ultrasounds resuspension fluorescent microsphere, and 2
~8 DEG C of refrigerators save backup, and fluorescence buffer solution used is containing 0.5%PEG20000,2% sucrose, 0.1% polysorbas20 0.5%
The 0.01M PBS buffer solution of BSA, 0.05% sodium azide.
(2) preparation of the furans metabolite monoclonal antibody detection liquid of fluorescent microsphere label：By above-mentioned fluorescent microsphere mark
The furans metabolite monoclonal antibody of note, which is taken out, places to room temperature, micro- at fluorescence with 100~1000 times of fluorescence buffer solution dilution
Ball label furans metabolite monoclonal antibody detect liquid, fluorescence buffer solution be containing 0.5%PEG20000,2% sucrose,
0.1% polysorbas20,0.5%BSA, 0.05% sodium azide 0.01M PBS buffer solution, be subsequently placed in 2~8 DEG C of refrigerators preserve it is standby
With.
(3) it is equipped with a series of standard solution of the furans metabolite of various concentrations with 0.01M PBS buffer solution, then takes
The prepared series mark of furans metabolite monoclonal antibody detection liquid 20 μ L and 100 μ L of the fluorescent microsphere label of above-mentioned preparation
After quasi- product solution mixing 1min, the sample area for the fluorescent quantitation immuno-chromatographic test paper strip that 80 μ L are added drop-wise in the present invention, 15min are taken
Afterwards, the fluorescence signal value ratio of T/C, detection experiment 3 groups of repetitions of setting are read with fluorescent quantitative detector.Pass through serial mark product again
The corresponding T/C values of concentration establish four parameter Logistic curves, four parameter value typing fluorescent quantitative detectors obtained by curve
Calibration software, you can realize fluorescent quantitation immuno-chromatographic test paper strip on fluorescence immune chromatography analyzer fast quantification inspection
It surveys.
Measurement result is as follows：
Use the measurement result of fluorescent quantitation immuno-chromatographic test paper strip and corresponding furans metabolite titer
It is determined by the T/C values to furans series standard liquid, and calculates the B/B0 (standard items of various concentration
The ratio for the standard items test card T/C values that test card T/C values are 0 with content), as a result as shown above.As can be seen from the table, when
When the standard liquid hold-up of furaltadone is 0.2 μ g/L, B/B0 values are 74.94%, illustrate the T/C values detected under this concentration and contain
The test card T/C values of the furaltadone titer of 0 μ g/L concentration have notable difference, therefore layer is immunized in fluorescent quantitation prepared by the present invention
It to furaltadone detection sensitivity is respectively 0.2 μ g/L to analyse test strips, similarly to furazolidone, furantoin, nitrofurazone
Detection sensitivity is respectively 0.1 μ g/L, 0.1 μ g/L, 0.2 μ g/L.
By upper table data, four parameter Logistic curve matchings are carried out using ELISA Calc softwares and draw fluorescent quantitation
The quantitation curves (as shown in Figure 6) of immuno-chromatographic test paper strip, the linear equation of curve obtained are respectively：
Furaltadone y=(A-D)/[1+ (x/C) ^B]+D, r2=0.998, A=4.32042, B=1.44536, C=
0.42066, D=-0.17617, furazolidone y=(A-D)/[1+ (x/C) ^B]+D, r2=0.999, A=3.47841, B=
1.59301, C=0.17753, D=0.05243, furantoin y=(A-D)/[1+ (x/C) ^B]+D, r2=0.999, A=
5.38069, B=1.75562, C=0.18283, D=0.25605, nitrofurazone y=(A-D)/[1+ (x/C) ^B]+D, r2=
0.998, A=3.84599, B=1.83320, C=0.32952, D=0.20005, x indicates testing concentration, y in linear equation
Indicate T/C values.In the quantitation curves setting interface of fluorescence immune chromatography analyzer, respectively by A, B, C, the value typing of D is glimmering
The calibration software of light immunochromatographiassays assays instrument, you can realize fluorescent quantitation immuno-chromatographic test paper strip in fluorescence immune chromatography analyzer
On Quantitative detection.
The stability study of fluorescent quantitation immuno-chromatographic test paper strip
The condition that fluorescent quantitation immuno-chromatographic test paper strip preserves is room temperature, in order to ensure the stability of test strips, to test paper
Item has carried out accelerated shelf life test, is continuously placed 60 days under the conditions of room temperature, 50 DEG C respectively, and on day 3, the 6th day, the 15th
It, detects fluorescence signal value, 3 groups of repetitions of experimental setup in the 30th day and the 60th day respectively；
As seen from the above table, the fluorescent quantitation immuno-chromatographic test paper strip being sealed at room temperature and 50 DEG C is after 60 days, four
The T/C values of the test strips of project have no significant change, and illustrate fluorescent quantitation immuno-chromatographic test paper strip at 50 DEG C of Acceleration study most
Preservation 60 days can be stablized less.Therefore, four kinds of furans metabolin fluorescent quantitation immuno-chromatographic test paper strips that prepared by the present invention can be in room
Temperature is lower to be stablized and preserves 1 year or more, can meet requirement of market during storage and transport completely.
The precision of fluorescent quantitation immuno-chromatographic test paper strip is tested
With the T/C values of test strips batch in error and batch between error indicate the precision of this method, and respectively with batch in
The coefficient of variation and interassay coefficient of variation come error in embodying batch and batch between error.The measurement of variation within batch coefficient is to same batch
10 fluorescent quantitation immuno-chromatographic test paper strips T/C values data, and to these data carry out dispersion degree analysis.Become between batch
The measurement of different coefficient is the data to the T/C values of 10 fluorescent quantitation immuno-chromatographic test paper strips of different batches, and to these numbers
According to progress dispersion degree analysis.By coefficient of variation formula CV%=[standard deviation S D values/average value] × 100% can respectively based on
Calculate the variation within batch coefficient and interassay coefficient of variation of fluorescent quantitation immuno-chromatographic test paper strip.
As seen from the above table, by the test of 10 fluorescent quantitation immuno-chromatographic test paper strips in same batch, test strips
T/C values change smaller.The variation within batch coefficient furans of the T/C values of this 10 test strips is obtained by coefficient of variation calculation formula
Its ketone is 2.64%, furazolidone 2.89%, furantoin 2.25%, and nitrofurazone 3.14% illustrates test strips
Variation within batch coefficient is small, and precision is high, can meet the quantitative requirement of test strips.
As seen from the above table, by the test of 10 fluorescent quantitation immuno-chromatographic test paper strips to different batches, test strips
T/C values change smaller.The interassay coefficient of variation furans of the T/C values of this 10 test strips is obtained by coefficient of variation calculation formula
Its ketone is 3.83%, furazolidone 4.07%, furantoin 4.10%, and nitrofurazone 4.12% illustrates test strips
Interassay coefficient of variation is small, and precision is high, can meet the quantitative requirement of test strips.
Claims (10)
1. a kind of furans metabolite hapten, which is characterized in that the general structure of furans metabolite hapten is：
The structural formula of R group is any one in following four：
2. furans metabolite hapten according to claim 1, it is characterised in that：Preparation method includes following step
Suddenly：
1) chemical compounds I and compound ii are dissolved in acetone, acid binding agent is added, 2~5h is reacted at 80~90 DEG C, obtain compound
Ⅲ；
2) compound III is dissolved in absolute ethyl alcohol, it is 10~12 that 6M lithium hydroxide solutions to pH value of reaction system, which is added, in room temperature
15~26h is reacted, purified water is added, the pH value that 1M dilute hydrochloric acid is added to reaction system is 4~5, and filtering obtains compounds Ⅳ；
3) compounds Ⅳ is dissolved in methanol, X, 60~70 DEG C of 1.5~2.5h of reaction is added, reaction is finished, filtered, and it is anti-to obtain half
Any one in original V, X AMOZ, AOZ, AHD, SEM；
Formula I~V is as follows：
3. furans metabolite hapten according to claim 2, it is characterised in that：In step 1), acid binding agent K2CO3,
The molar ratio of acid binding agent and compound ii is (1.5~2):1, the molar ratio of chemical compounds I and compound ii is (2~2.5):1.
4. furans metabolite hapten according to claim 2, it is characterised in that：The X and chemical combination being added in step 3)
The molar ratio of object IV is (2.0~3.0):1.
5. a kind of furans metabolite artificial antigen, be Claims 1 to 4 any one of them furans metabolite hapten with
Carrier protein couplet obtains, and the general structure of furans metabolite artificial antigen is：
Carrier protein is any one in bovine serum albumin(BSA), ovalbumin, human serum albumins or hemocyanin.
6. furans metabolite monoclonal antibody prepared by furans metabolite artificial antigen according to claim 5, furan
Mutter metabolite artificial antigen carrier protein be hemocyanin.
7. the furans metabolite monoclonal described in furans metabolite artificial antigen and claim 6 described in claim 5
Application of the antibody in ELISA detection method.
8. the furans metabolite monoclonal described in furans metabolite artificial antigen and claim 6 described in claim 5
Application of the antibody in fluorescent quantitation immunochromatography.
9. a kind of furans metabolite fluorescent quantitation immuno-chromatographic test paper strip, test strips include being coated with described in claim 5
Furans metabolite artificial antigen reaction film, the carrier protein of furans metabolite artificial antigen is bovine serum albumin,
Preparation method includes the following steps：
1) reaction film for being coated with artificial antigen and rabbit IGg is prepared：By the furans metabolite that carrier protein is bovine serum albumin
Artificial antigen is added in coating buffer solution and is uniformly mixed the detection zone for being sprayed onto reaction film, and rabbit IGg is added in coating buffer solution and is mixed
The control zone for being uniformly sprayed onto reaction film is closed, detection zone and control zone are separated from each other, are saved backup after drying process；
2) preparation of sample pad：The blank sample pad cut is immersed in sample pad treatment fluid and is impregnated, it is dry to take out sample pad
It is saved backup after dry processing；
3) assembling of fluorescent quantitation immuno-chromatographic test paper strip：In the middle part of backing be stacked reaction film, both ends distinguish stacked sample pad and
Water absorption pad, reaction film and water absorption pad, sample pad mutually connect, and detection zone obtains test paper close to sample pad, control zone close to water absorption pad
Test strips are loaded into test card by plate, obtain fluorescent quantitation immuno-chromatographic test paper strip.
10. a kind of furans metabolite fluorescent quantitation immuno-chromatographic test paper strip using described in claim 9 detects furans generation
The method for thanking to object：
1) preparation of the furans metabolite monoclonal antibody of fluorescent microsphere label：After fluorescent microsphere is activated with claim 6
The furans metabolite monoclonal antibody coupling, centrifugation removal of impurities are added fluorescence buffer solution and fluorescent microsphere are resuspended, obtain fluorescence
The furans metabolite monoclonal antibody of microballoon label；
2) preparation of the furans metabolite monoclonal antibody detection liquid of fluorescent microsphere label：With fluorescence buffer solution by fluorescent microsphere
The furans metabolite monoclonal antibody of label dilutes, and obtains the furans metabolite monoclonal antibody detection of fluorescent microsphere label
Liquid；
3) the furans metabolite standard solution of series concentration, the furans generation for taking fluorescent microsphere to mark are equipped with PBS buffer solution
It is added drop-wise to the sample area of fluorescent quantitation immuno-chromatographic test paper strip after thanking object monoclonal antibody detection liquid and standard solution mixing, use is glimmering
Light instrument reads T/C values, establishes standard curve by the corresponding T/C values of concentration of standard solution, measures sample to be tested
The Quantitative detection of furans metabolite can be realized in T/C values, combined standard curve.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109239328A (en) * | 2018-09-26 | 2019-01-18 | 南京财经大学 | A kind of immunochromatographic method of four kinds of metabolites of nitrofuran of quick detection |
| CN110456041A (en) * | 2019-08-06 | 2019-11-15 | 珠海市医友生物科技有限公司 | A kind of norovirus GI type GII type combined detection reagent and preparation method thereof |
| CN110642743A (en) * | 2019-10-18 | 2020-01-03 | 中国农业大学 | Nifuroxazide hapten and artificial antigen as well as preparation methods and application thereof |
| CN110818599A (en) * | 2019-10-09 | 2020-02-21 | 广东达元绿洲食品安全科技股份有限公司 | Sulfonamide hapten, artificial antigen and application thereof in immunodetection |
| CN111007257A (en) * | 2019-12-13 | 2020-04-14 | 暨南大学 | Porcine pseudorabies virus gE protein antibody detection immune blocking chromatography kit and application thereof |
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101435823A (en) * | 2007-11-12 | 2009-05-20 | 无锡中德伯尔生物技术有限公司 | Fluorescent micro-ball immune chromatography test paper strip for detecting residual animal medicine and preparing method thereof |
| CN202599957U (en) * | 2011-12-28 | 2012-12-12 | 中国科学院苏州纳米技术与纳米仿生研究所 | Fluorescence immunochromatography test strip |
| CN203191384U (en) * | 2012-12-19 | 2013-09-11 | 广州万孚生物技术股份有限公司 | Furazolidone metabolite fluorescent quantitative detection system |
| KR20160050658A (en) * | 2014-10-30 | 2016-05-11 | 주식회사 엘지화학 | Detection method of persistent organic pollutants |
| CN106053787A (en) * | 2016-06-06 | 2016-10-26 | 上海师范大学 | Fluorescent immunochromatography test strip for detecting furazolidone metabolites as well as preparation and application |
| CN106483300A (en) * | 2016-10-21 | 2017-03-08 | 河北省科学院生物研究所 | A kind of colloidal gold immuno-chromatography test paper strip of detection Furaxone metabolite and preparation method and application |
| CN106588805A (en) * | 2017-01-12 | 2017-04-26 | 广州润坤生物科技有限公司 | Furaltadone metabolite (AMOZ) derivatization hapten, preparation method of artificial antigen and application of artificial antigen |
| CN106831498A (en) * | 2017-01-12 | 2017-06-13 | 广州润坤生物科技有限公司 | Furacilin metabolite SEM derivatizations haptens, the preparation method and applications of artificial antigen |
| CN106866568A (en) * | 2017-01-12 | 2017-06-20 | 广州润坤生物科技有限公司 | Furaxone metabolite AOZ derivatizations haptens, the preparation method and applications of artificial antigen |
| CN107118159A (en) * | 2017-01-12 | 2017-09-01 | 广州润坤生物科技有限公司 | Cistofuran metabolite AHD derivatizations haptens, the preparation method and applications of artificial antigen |
-
2018
- 2018-05-14 CN CN201810456832.2A patent/CN108530373A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101435823A (en) * | 2007-11-12 | 2009-05-20 | 无锡中德伯尔生物技术有限公司 | Fluorescent micro-ball immune chromatography test paper strip for detecting residual animal medicine and preparing method thereof |
| CN202599957U (en) * | 2011-12-28 | 2012-12-12 | 中国科学院苏州纳米技术与纳米仿生研究所 | Fluorescence immunochromatography test strip |
| CN203191384U (en) * | 2012-12-19 | 2013-09-11 | 广州万孚生物技术股份有限公司 | Furazolidone metabolite fluorescent quantitative detection system |
| KR20160050658A (en) * | 2014-10-30 | 2016-05-11 | 주식회사 엘지화학 | Detection method of persistent organic pollutants |
| CN106053787A (en) * | 2016-06-06 | 2016-10-26 | 上海师范大学 | Fluorescent immunochromatography test strip for detecting furazolidone metabolites as well as preparation and application |
| CN106483300A (en) * | 2016-10-21 | 2017-03-08 | 河北省科学院生物研究所 | A kind of colloidal gold immuno-chromatography test paper strip of detection Furaxone metabolite and preparation method and application |
| CN106588805A (en) * | 2017-01-12 | 2017-04-26 | 广州润坤生物科技有限公司 | Furaltadone metabolite (AMOZ) derivatization hapten, preparation method of artificial antigen and application of artificial antigen |
| CN106831498A (en) * | 2017-01-12 | 2017-06-13 | 广州润坤生物科技有限公司 | Furacilin metabolite SEM derivatizations haptens, the preparation method and applications of artificial antigen |
| CN106866568A (en) * | 2017-01-12 | 2017-06-20 | 广州润坤生物科技有限公司 | Furaxone metabolite AOZ derivatizations haptens, the preparation method and applications of artificial antigen |
| CN107118159A (en) * | 2017-01-12 | 2017-09-01 | 广州润坤生物科技有限公司 | Cistofuran metabolite AHD derivatizations haptens, the preparation method and applications of artificial antigen |
Non-Patent Citations (1)
| Title |
|---|
| RONALD L. WLLDER，ET AL.: "BIVALENT HAPTEN-ANTIBODY INTERACTIONS--I.A COMPARISON OF WATER SOLUBLE AND WATER INSOLUBLE BIVALENT HAPTENS", 《LMMUNOCHEMISTRY》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109239328A (en) * | 2018-09-26 | 2019-01-18 | 南京财经大学 | A kind of immunochromatographic method of four kinds of metabolites of nitrofuran of quick detection |
| CN110456041A (en) * | 2019-08-06 | 2019-11-15 | 珠海市医友生物科技有限公司 | A kind of norovirus GI type GII type combined detection reagent and preparation method thereof |
| CN110818599A (en) * | 2019-10-09 | 2020-02-21 | 广东达元绿洲食品安全科技股份有限公司 | Sulfonamide hapten, artificial antigen and application thereof in immunodetection |
| CN110818599B (en) * | 2019-10-09 | 2022-08-26 | 广东达元绿洲食品安全科技股份有限公司 | Sulfonamide hapten, artificial antigen and application thereof in immunodetection |
| CN110642743A (en) * | 2019-10-18 | 2020-01-03 | 中国农业大学 | Nifuroxazide hapten and artificial antigen as well as preparation methods and application thereof |
| CN111007257A (en) * | 2019-12-13 | 2020-04-14 | 暨南大学 | Porcine pseudorabies virus gE protein antibody detection immune blocking chromatography kit and application thereof |
| CN112557661A (en) * | 2020-10-31 | 2021-03-26 | 上海海洋大学 | Magnetic immunochromatographic test strip and method for rapidly detecting pre-S2 antigen of hepatitis B virus |
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