CN106084059A - The general specific antibody of anti-Capsaicinoids, test strips and kitchen waste grease immunochromatography method for quick identification - Google Patents
The general specific antibody of anti-Capsaicinoids, test strips and kitchen waste grease immunochromatography method for quick identification Download PDFInfo
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- CN106084059A CN106084059A CN201610361935.1A CN201610361935A CN106084059A CN 106084059 A CN106084059 A CN 106084059A CN 201610361935 A CN201610361935 A CN 201610361935A CN 106084059 A CN106084059 A CN 106084059A
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- capsaicinoids
- kitchen waste
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- waste grease
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Classifications
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- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- C—CHEMISTRY; METALLURGY
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- 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
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Abstract
The present invention relates to the general specific antibody of anti-Capsaicinoids, test strips and kitchen waste grease immunochromatography method for quick identification.The general specific antibody of anti-Capsaicinoids, it is characterised in that: it is by the general artificial immunity antigen compound immune animal of the Capsaicinoids described in formula I, and the general specific antibody of anti-Capsaicinoids that isolated and purified postlyophilization obtains.Kitchen waste grease immunochromatography immunity test strip prepared therefrom can be used for kitchen waste grease and may be used for quickly detecting the total amount of Capsaicinoids in kitchen waste grease；Detection sensitivity is high, pH wide accommodation；Single step operates, simple and convenient.Need not Capsaicinoids standard solution as positive control.
Description
Technical field
The present invention relates to the general specific antibody of anti-Capsaicinoids, test strips and kitchen waste grease immunochromatography fast
Speed discrimination method, belongs to immunochemical technique field.
Background technology
Waste oil is one and refers to concept, is the general designation to all kinds of poor oils, generally comprises swill oil, frying waste oil, food
The waste grease that product and relevant enterprise produce, and one of from the swill oil of catering trade it is the primary raw material source of waste oil.?
In China's cooking culture, the Fructus Capsici containing capsaicin is one of requisite condiment.Capsaicinoid compounds is present in peppery
The dominant chemical making it have pungent stimulation in green pepper fruit, that has verified structure has kind more than 20.Capsaicin
Class material nutrition is worth height, is widely used and food processing field.Synthetic capsaicin also has and natural capsicum element class material phase
As structure, also have pungent feature, be a member in Capsaicinoids, be mainly used in industrial circle, but because of its preparation cost
Low, some lawless persons are substituted natural capsicum element, are applied to field of food.Capsaicin, Dihydrocapsaicin and synthesize peppery
Green pepper element is the Major Members in Capsaicinoids.Capsaicinoids is of high nutritive value, and is widely used and food processing is led
Territory.Capsaicin element class material has fat-soluble strong, good stability, boiling point high.Current waste oil processing technique is difficult to
Remove the material with such feature completely.Multiple studies have shown that, normal edible oil is substantially free of capsaicin, and contacts Fructus Capsici
Kitchen waste grease be difficult to avoid that containing this constituents.Therefore capsaicin constituents can be as differentiating kitchen waste grease
Characteristic index, by detecting the content of Capsaicinoids, can differentiate and whether be impregnated in kitchen abandoned oil in edible vegetable oil
Fat.At present, in detection edible vegetable oil, the method for Capsaicinoids mainly has high performance liquid chromatography, chromatograph-mass spectrometer coupling
Deng modern instrument detection method, it is highly sensitive, accuracy good, but instrument and equipment is expensive, experimental situation requires height, and wants
Ask professional operator, it is difficult to realize quickly detection.The immune analysis method developed rapidly in recent years owing to overcoming on
The shortcoming stating method, and have high specificity, highly sensitive, analyze that capacity is big, convenient and swift, with low cost, be suitable to on-the-spot batch
The advantages such as amount detection, are widely used in the every field such as medical science, agronomy.
Immunochromatography technique based on colloidal gold labeled monoclonal antibody and antigenic specificity association reaction is due to its testing result meat
Eye is visible, it is not necessary to large-scale instrument and equipment, testing cost is low, and analysis time is short, micro-in mycotoxin, pesticide residues etc. in recent years
It is widely applied in the detection qualitative, online, quick of amount residue.But, the most also not having should by the method
In the detection of kitchen waste grease Capsaicinoids.The core parts of colloidal gold immunochromatographimethod technology are specific anti-
Body, and kitchen waste grease matrix components is complicated, the pH of sample extracting solution is difficult to determine, it will usually make specific antibody activity drop
Low, detection sensitivity reduces.Therefore, in the urgent need to developing, affinity is high, Idiotype is good and organic solvent-resistant, pH subject range
Wide anti-Capsaicinoids specific antibody, and utilize this antibody to set up colloidal gold immuno-chromatography test paper strip detection method, it is used for
Differentiate kitchen waste grease.
Summary of the invention
Problem to be solved by this invention is to provide a kind of anti-Capsaicinoids specific antibody, kitchen waste grease glue
Body gold immuno-chromatographic test paper strip, kitchen waste grease immunochromatography method for quick identification.The anti-capsaicin class thing that the present invention provides
Matter specific antibody affinity is high, specificity is good and organic solvent-resistant, pH wide accommodation.The immunity-chromatography test made of it
Paper slip is the detection of Capsaicinoids in the kitchen waste grease, has simple to operate, with low cost, highly sensitive, pH and fits
Answer the feature that scope is wide.
For solving above-mentioned technical problem, the technical solution used in the present invention is:
Thering is provided Capsaicinoids general artificial immunity antigen compound, its structural formula is as described in formula I:
There is provided the preparation method of above-mentioned Capsaicinoids general artificial immunity antigen compound, including walking in detail below
Rapid: with the hydrochloric acid in sodium hydroxide and in Vanillylamine hydrochlorate, to obtain formula III compound, then anti-with maleic anhydride
Should, obtain artificial semiantigen compound, by Capsaicinoids artificial semiantigen by N, N '-dicyclohexylcarbodiimide DCC
Activate with N-hydroxy-succinamide NHS, or activated by tri-n-butylamine and isobutyl chlorocarbonate, then enter with carrier protein BSA
Row coupling obtains.
The synthetic route of artificial semiantigen compound is as follows:
The preparation method of above-mentioned Capsaicinoids general artificial immunity antigen compound specifically includes following steps:
(1) be 1:(0.7～1.1 by mol ratio) Vanillylamine hydrochlorate and sodium hydroxide soluble in water, be stirred at room temperature
10-30min, obtains white precipitate Vanillylamine (formula IV compound) after filtration；
The Vanillylamine being thoroughly dried and maleic anhydride are dissolved in chloroform with mol ratio for 1:0.8-1.2,
Room temperature reaction 1-2h, i.e. obtains Capsaicinoids artificial semiantigen after filtration.
(2) by general for Capsaicinoids artificial semiantigen in N,N-dimethylformamide with N-hydroxy-succinamide
(NHS) room temperature reaction 30min-2h, is subsequently adding the DMF solution of dicyclohexylcarbodiimide, room temperature reaction
4-6 hour, stand overnight, take capsaicin, Dihydrocapsaicin and synthetic capsaicin artificial semiantigen solution that supernatant i.e. activates,
The mol ratio of described N-hydroxy-succinamide (NHS) and dicyclohexylcarbodiimide is 1:1-1.2；
Or general for Capsaicinoids artificial semiantigen is dissolved in N,N-dimethylformamide order addition three positive fourths
Amine and isobutyl chlorocarbonate, room temperature reaction 0.5-3h hour, the Capsaicinoids artificial semiantigen that gained reactant liquor i.e. activates
Solution, the mol ratio of described tri-n-butylamine and isobutyl chlorocarbonate is 1:1-1.2.
(3) the Capsaicinoids general artificial semiantigen supernatant that above-mentioned (1) gained activates is added drop-wise to carrier protein
Concentration is more than or equal in the phosphate buffer of the carrier protein of 2mg/mL, and room temperature condition reacts 4-8h, wherein: described step
(1) the general artificial semiantigen of Capsaicinoids in is more than 10:1 with the mol ratio of carrier protein, and then dialysis obtains Fructus Capsici
Element artificial antigen.
By such scheme, the phosphate buffer of described carrier protein is by the phosphoric acid of carrier protein 0.2M pH8.0
Salt buffer dissolving obtains.
By such scheme, described dialysis is for using 0.01M PBS dialysis 72h, period, and 4-8h changes and once dialyses
Liquid.
The general specific antibody of anti-Capsaicinoids, it is for by the general artificial immunity of the Capsaicinoids described in formula I
Antigen compound immune animal, and the general specific antibody of anti-Capsaicinoids that isolated and purified postlyophilization obtains；
By such scheme, described immunity repeats for interval after using Freund's complete adjuvant or incomplete Freund's adjuvant emulsifying
Immune animal, wherein: initial immunity Freund's complete adjuvant, follow-up immunization incomplete Freund's adjuvant, reach to antiserum titre
To more than 10000, carotid artery blood sampling is dead to it, after the centrifugal blood adopted, antiserum purification is obtained Capsaicinoids
Antibody.The antiserum titre that obtains of antigen-immunized animal provided by the present invention up to 320000, the antibody obtained to capsaicin,
The 503nhibiting concentration IC of Dihydrocapsaicin and synthetic capsaicin50Under conditions of pH value is 5.0-8.0, for 0.27-0.41 μ g/
mL.Show that this antibody can react with capsaicin, Dihydrocapsaicin and synthetic capsaicin with specificity, it is possible to as capsaicin,
Dihydrocapsaicin and synthetic capsaicin universal antibody, affinity is good, highly sensitive, good stability, including tolerance organic solvent and
Salt ionic concentration ability is strong, pH is applied widely.
By such scheme, the immune consumption of the Capsaicinoids general artificial immunity antigen compound described in described formula I
It is calculated as 0.2-1.0mg with OVA albumen therein；The immunity number of plies is 4-6 time；First immunisation uses isopyknic Freund's complete adjuvant
Emulsified immunogen, multi-point injection under rabbit；After first immunisation interval carry out in 2-4 week second time immunity, second time and later
Immunization interval 2-3 week, by isopyknic incomplete Freund's adjuvant emulsified immunogen, in the subcutaneous multi-point injection of rabbit back.From the 4th
Secondary beginning, after the most immune one week in 6-10 days, the non-competing indirect ELISA method of rabbit ear edge vein exploitating blood measures serum titer,
After titer reaches 10000, i.e. rabbit carotid artery blood sampling is dead to it, after the centrifugal blood adopted, antiserum utilizes octanoic acid-sulphuric acid
Ammonium method is purified, and obtains Capsaicinoids antibody.
Kitchen waste grease immuno-chromatographic test paper strip (see Fig. 3), including cardboard, the one side of cardboard is pasted the most successively
Adsorptive pads, detecting pad, gold mark pad and sample pad, adjacent each pad is overlapping in junction to be connected, and described detecting pad is with celluloid
Film is base wad, and nitrocellulose filter arranges horizontal nature controlling line and detection line from top to bottom, and described nature controlling line is coated with rabbit and resists
Mus polyclonal antibody, described detection line is coated Capsaicinoids envelope antigen；Described gold mark pad transverse jet scribbles nanometer
The general specific antibody of above-mentioned anti-Capsaicinoids of gold labelling.
By such scheme, described adsorptive pads length 10～15mm, wide 3～4mm, detecting pad length 25～30mm, wide 3～4mm；
Gold mark pad long 6～9mm, wide 3～4mm；Sample pad length 12～15mm, wide 3～4mm, the overlapping length of adjacent each pad is 1～3mm.
By such scheme, described adsorptive pads is absorbent paper.
By such scheme, the detection line on described detecting pad is 8～20mm with the spacing on edge on nitrocellulose filter, described
Detection line is 5～10mm with the spacing of nature controlling line.
The molecular structural formula of described Capsaicinoids envelope antigen is as shown in formula II:
By such scheme, on described detecting pad detection line, the package amount of every centimetre of required envelope antigen is 0.1～0.6
μg；On nature controlling line, the package amount of every centimetre of required rabbit anti-Mus polyclonal antibody is 0.1～0.6 μ g.
By such scheme, the particle diameter of nanometer gold used in described gold mark pad is 15～20nm.
Anti-Capsaicinoids by the nano gold mark needed for such scheme, the upper every centimetre of spraying length of described gold mark pad
The consumption of general specific antibody is 0.40～0.98 μ g.
The most quickly preparation method of the colloidal gold immuno-chromatography test paper strip of detection Capsaicinoids, including following
Step:
(1) preparation of adsorptive pads
Absorbent paper is cut out and i.e. obtains adsorptive pads；
(2) preparation of detecting pad
Detection being coated of line: it is 0.17～1.0mg/mL be coated that Capsaicinoids envelope antigen is configured to concentration
Liquid, in position along 8～20mm on nitrocellulose filter, is laterally coated in it on nitrocellulose filter by line spray mode,
Obtaining detecting the package amount of the general artificial complete antigen-envelope antigen of Capsaicinoids needed for line, every centimetre of detection line is 0.1
～0.6 μ g, then it is dried 30～60 minutes under the conditions of 37 DEG C；
Being coated of nature controlling line: anti-for rabbit Mus polyclonal antibody is made into concentration be 0.17～1.0mg/mL be coated liquid, in away from
The position of detection line 5～10mm, is laterally coated in it on nitrocellulose filter by line spray mode, obtains nature controlling line, every centimetre of matter
The package amount of the rabbit anti-Mus polyclonal antibody needed for control line is 0.1～0.6 μ g, is then dried 30～60 points under the conditions of 37 DEG C
Clock；
(3) preparation of sample pad
Glass fibre membrane is put in confining liquid and soaks, take out, be dried 6～12 hours under the conditions of 37～40 DEG C, obtain sample
Product pad, then puts room temperature preservation in exsiccator；
(4) preparation of gold mark pad
Glass fibre membrane is put in confining liquid and soaks, take out, be dried 6～12 hours under the conditions of 37～40 DEG C, in
On the glass fibre membrane being dried, on the glass fibre membrane being dried, laterally spray the anti-Fructus Capsici of nano gold mark by a spray mode
Element class material general Anti-TNF-α liquid solution, the anti-Capsaicinoids of nano gold mark needed for every centimetre of spraying length is general
The consumption of polyclonal antibody is 0.4～0.98ng, then vacuum lyophilization 2～6 hours, puts room temperature preservation in exsiccator；
(5) assembling of test strips
One side at cardboard pastes adsorptive pads, detecting pad, gold mark pad and sample pad the most successively, and adjacent each pad is even
Connecing the overlapping connection in place, overlapping length is 1～3mm, obtains kitchen waste grease immune chromatography test paper.
By such scheme, Capsaicinoids envelope antigen ((2Z)-4-[(4-hydroxy-3-methoxy) benzyl amino]-4-carbonyl
Base-2-olefin(e) acid-OVA) it is coated the buffer that is coated used in liquid and is: containing ovalbumin OVA 0.1g, nitrine in every 10mL
Change sodium 0.002g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g；
Described rabbit anti-Mus polyclonal antibody is coated the buffer that is coated used in liquid: containing bovine serum albumin in every 10mL
0.1g, Hydrazoic acid,sodium salt 0.002g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, biphosphate
Potassium 0.002g；
Contain by the every 100mL of confining liquid in such scheme, described step (3) and step (4): oralbumin 1～
2g, sucrose 2～5g, Hydrazoic acid,sodium salt 0.02～0.05g, sodium chloride 0.8g, disodium hydrogen phosphate 0.29g, potassium chloride
0.02g, potassium dihydrogen phosphate 0.02g.
By such scheme, the anti-Capsaicinoids general Anti-TNF-α liquid solution of described nano gold mark is to use insatiable hunger
Prepare with labelling method, its method particularly includes: taking 50.0mL commercial available quality concentration is the nano-Au solution of 0.01%, use 0.5mL
0.1mol/L wet chemical regulation pH value, be slowly added to the anti-Fructus Capsici of 0.1mg/mL of 2.0mL when stirring
Element class material general polyclonal antibody aqueous solution, continues stirring 30min；Adding mass concentration is 10% oralbumin (OVA)
Aqueous solution is 1% to OVA mass concentration at end, continues stirring 30min；After placing 2h in 4 DEG C, 3000r/min is centrifuged 15min, takes
Supernatant, abandons precipitation；Supernatant 12000r/min is centrifuged 30min, abandoning supernatant, adds the washing of 40.0mL labelling and preserve
Liquid；It is centrifuged 30min, abandoning supernatant with 12000r/min again, the washing of precipitation labelling is preserved liquid resuspended, obtain 5.0mL dense
Contracting thing, puts 4 DEG C of refrigerators standby.
By such scheme, described 0.1mol/L wet chemical is: 13.8g potassium carbonate is dissolved in pure water and is settled to
1000mL, 0.22 μm membrane filtration gained；Described labelling washing preserves liquid and is: 2.0g PEG-400,0.2g nitrine
Sodium, 0.1235g boric acid, pure water is settled to 1000mL, 0.22 μm membrane filtration gained.
Kitchen waste grease immunochromatography method for quick identification, step is as follows: pick-up kitchen waste grease testing sample, extracts
Capsaicinoids therein, redissolves dried for extracting solution solubilizer, and the ratio of described testing sample and redissolution solvent is
20g:1ml, obtains testing sample solution, then this testing sample solution (preferably 80-200 μ L) is added dropwise to meal as detection liquid
Detecting in the sample pad of kitchen waste grease immune chromatography test paper, it is as test strip, separately takes isopyknic methanol dense
Spend consistent methanol aqueous solution as negative controls, be added dropwise over the sample pad of another kitchen waste grease immune chromatography test paper
On, it is as control stripes bar, carries out test strip and control stripes bar developing the color right after a period of time (10-20 minute)
According to:
Testing result:
(1) negative: when nature controlling line colour developing in test strip, and detect line color and detection line on control stripes bar
Color close to time, show that Capsaicinoids capsaicin in testing sample solution, Dihydrocapsaicin or synthetic capsaicin content are low
In 3ng/mL, determine whether this sample is kitchen waste grease or the oil mixed with kitchen waste grease in conjunction with authenticity detection method
Fat sample；Content is less than 3ng/mL, can determine whether that testing sample is that the probability of kitchen waste grease is little, need to be further by confirmation
Property detection method is determined；
(2) positive: when nature controlling line colour developing in test strip, and detect line color than detection line in comparison test strips
Time of light color, show that capsaicin in testing sample solution, Dihydrocapsaicin or synthetic capsaicin content equal to or higher than 3ng/mL,
Then can determine that this sample is kitchen waste grease or the oil sample mixed with kitchen waste grease；
(3) invalid: when nature controlling line does not develops the color, no matter whether the detection line of test strip develops the color, and this test strips is judged to
Invalid；
This immuno-chromatographic test paper strip work in kitchen waste grease capsaicin, Dihydrocapsaicin or synthetic capsaicin detect
Making principle: when in the sample pad that testing sample solution joins test strips lower end, testing sample solution passes through capillarity edge
Test strips moves to adsorptive pads direction, when it moves to gold mark pad, and anti-capsaicin, Dihydrocapsaicin or the conjunction of nano gold mark
The general polyclonal antibody of capsaicin is become to be dissolved.When in sample containing capsaicin, Dihydrocapsaicin or synthetic capsaicin, Fructus Capsici
Element class material will combine with the general polyclonal antibody of anti-Capsaicinoids of the nano gold mark on gold mark pad and the most upwards
Swimming, its arrive fixed Capsaicinoids envelope antigen detection line time, antigen will be with capsaicin, Dihydrocapsaicin or conjunction
Become antigen binding site limited on the general polyclonal antibody of anti-Capsaicinoids of capsaicin competition binding nano gold mark,
In sample, capsaicin, Dihydrocapsaicin or synthetic capsaicin content are the highest, detection line on antigen can in conjunction with nanometer gold mark
The general polyclonal antibody of anti-capsaicin, Dihydrocapsaicin or synthetic capsaicin of note will be the fewest, the colour developing formed on detection line
Band color is the most shallow.The general polyclonal antibody of anti-Capsaicinoids combined when the antigen on detection line is less than certain quantity
Time, red lines will not be had to occur at detection line.No matter whether sample contains Capsaicinoids, on the most tested survey line
The anti-Capsaicinoids antibody of the nano gold mark that antigen is intercepted and captured or the anti-Capsaicinoids antibody of nano gold mark are with peppery
The conjugate of green pepper element, Dihydrocapsaicin or synthetic capsaicin will move on nature controlling line many grams of Mus anti-with the rabbit on nature controlling line
Grand antibodies is also developed the color by enrichment.Accordingly, respectively by the detection line of Capsaicinoids envelope antigen in test strip with
On control stripes bar, corresponding detection line color carries out colour developing comparison, can obtain capsaicin in sample, Dihydrocapsaicin or synthesize peppery
Green pepper cellulose content situation, thus judge whether oil sample is kitchen waste grease.
By such scheme, the described addition volumetric concentration that is extracted as in testing sample is the ethanol water of 95%, mixed
Even, reflux 1 hour at 60～90 DEG C, cooling obtains extracting solution；Described redissolution solvent is 10% methanol-PBS.
Beneficial effects of the present invention:
(1) present invention is the most maximum by the artificial semiantigen that the amide groups of vanillyl amine in capsaicin carries out modifying offer
Degree remains the feature structure (Rhizoma et radix valerianae in capsaicin essential molecular structure of capsaicin, Dihydrocapsaicin and synthetic capsaicin
Base amine and partial fat chain group), and at hapten linking arm, form a big conjugated structure, this structure can promote half to resist
In original structure, electronics flows, it addition, it has certain rigidity, thus may all be conducive to avtive spot in hapten
Expose, and then and OVA carry out the antigen compound that coupling obtains and thus can be used as antigenic determinant, having again can be with carrier
The active group of albumen generation coupling, it is thus achieved that antigen compound make immunizing antigen can immunity to obtain affinity high, highly sensitive,
High specificity and organic solvent-resistant and salt ionic concentration, the capsaicin antibody of pH wide accommodation.
(2) the kitchen waste grease immunochromatography method for quick identification that the present invention provides can detect in kitchen waste grease
The total amount of Capsaicinoids；Detection sensitivity is high, pH wide accommodation.
(3) the kitchen waste grease immunochromatography method for quick identification that the present invention provides is simple to operate: only need to kitchen be given up
Abandoning the detection solution after oil sample processes to be added dropwise in the sample pad of test strips, single step operates, simple and convenient.Inspection
Capsaicinoids standard solution is need not as positive control, it is only necessary to blank sample as negative control during test sample product.
And the testing result naked eyes of the method get final product interpretation.
Accompanying drawing explanation
Fig. 1 is the anti-Capsaicinoids general artificial complete antigen ultraviolet spectrogram that the present invention synthesizes.
Fig. 2 is the general artificial complete antigen of anti-Capsaicinoids-envelope antigen ultraviolet spectrogram that the present invention synthesizes.
The structural representation of the kitchen waste grease immuno-chromatographic test paper strip that Fig. 3 provides for the present invention.1 cardboard；2 water suctions
Pad；5 detecting pads；6 gold medal mark pads；7 sample pad；3 nature controlling lines；4 detection lines.
Detailed description of the invention
Test method used in following example, if no special instructions, is conventional method.
Material used in following example, reagent etc., if no special instructions, the most commercially obtain.
The preparation of embodiment 1 Capsaicinoids artificial semiantigen
(1) weigh Vanillylamine hydrochlorate 1g and be dissolved in 11.77ml ultra-pure water, be stirred vigorously down and dropwise drip 2M's
NaOH2.12ml, room temperature reaction 10min.Filter to obtain white precipitate, white precipitate is carried out the most after drying the next step.
(2) weigh step product 60mg to be dissolved in 30ml chloroform (after anhydrous sodium sulfate dehydration processes), room
Temperature stirs 40min, adds 40mg maleic anhydride, room temperature reaction 1h.After filtration, to be Capsaicinoids artificial for gained precipitation
Hapten (2Z)-4-[(4-hydroxy-3-methoxy) benzyl amino]-4-carbonyl-2-olefin(e) acid ((2Z)-4-[(4-hydroxy-3-
methoxybenzyl)amino]-4-oxobut-2-enoic acid).Molecular formula is C12H13NO5。
The preparation of embodiment 2 Capsaicinoids artificial complete antigen-immunizing antigen
Weigh above-mentioned Capsaicinoids artificial semiantigen 20.8mg (about 0.08mmol) and 11.6mg (about 0.1mmol)
NHS, in reaction bulb, adds 400ulDMF and is dissolved in reaction bulb, 30min is stirred at room temperature, weighs 20.6mg (about 0.1mmol)
DCC is dissolved in 50ulDMF, DCC/DMF solution is dropwise dropped to above-mentioned reaction bulb, 4h is stirred at room temperature, and 4 DEG C stand overnight.
8000rpm/5min, takes supernatant active ester liquid.
By active for supernatant ester liquid, being dropwise added drop-wise in the BSA solution of 6ml 7mg/ml reaction, reaction buffer is 0.2M
The phosphate buffer of pH8.0.React indoor temperature under magnetic stirring and carry out 4h.Reactant liquor is put in bag filter, use 0.01M
4 DEG C of stirring dialysis in the PBS of pH7.4, every 4h changes a dialysis solution, and dialyse 72h altogether.I.e. obtain capsaicin artificial antigen-exempt from
Epidemic disease antigen.The continuous scanning spectra of ultraviolet-visible spectrum is shown in that Fig. 1, qualification result show artificial antigen's coupling success.
The preparation of embodiment 3 Capsaicinoids artificial complete antigen-envelope antigen
Deserve to be called and state Capsaicinoids artificial semiantigen 4.52mg and be dissolved in 200uL dry DMF, be then sequentially added into
4.27 μ L tri-n-butylamines, 2.34 μ L isobutyl chlorocarbonates, stir lucifuge reaction 1h, obtain the capsaicin of activation, dihydro peppery under room temperature
Green pepper element and synthetic capsaicin hapten solution.
The capsaicin of activation, Dihydrocapsaicin and synthetic capsaicin hapten solution are dropwise added drop-wise to 10ml 4.5mg/
Reacting in the OVA solution of ml, reaction buffer is the phosphate buffer of 0.2M pH8.0.React Indoor Temperature under magnetic stirring
Degree carries out 4h.Being put by reactant liquor in bag filter, with 4 DEG C of stirring dialysis in the PBS of 0.01M pH7.4, every 4h changes and once dialyses
Liquid, dialyse 72h altogether.I.e. obtain capsaicin artificial antigen-envelope antigen.The continuous scanning spectra of ultraviolet-visible spectrum is shown in Fig. 2, mirror
Determine result and show artificial antigen's coupling success.
Embodiment 4: the preparation of Capsaicinoids specific antibody
The above-mentioned anti-Capsaicinoids artificial complete antigen (immunizing antigen) prepared is used for the day of immunity more than 3
This big ear rabbit.Average antigen consumption about 0.84mg (with the gauge of protein) every time.First immunisation uses by isopyknic Freund
The immunogen of Freund's complete adjuvant emulsifying, multi-point injection under rabbit；Head be spaced after exempting from 3 weeks, later every 2 weeks with by isopyknic not
The immunogen of family name's Freund's incomplete adjuvant emulsifying, in the subcutaneous multi-point injection of rabbit back.From the beginning of the 4th time, immunity one week after, rabbit every time
The non-competing indirect ELISA method of ear edge vein exploitating blood measures serum titer.After five exempt from, the blood sampling of rabbit carotid artery is dead to it.Adopt
Blood place about 1h in 37 DEG C, 4 DEG C are overnight, centrifugal, take supernatant, and a small amount of mixed equal-volume glycerol-20 DEG C temporarily stores for future use.Its
Remaining antiserum utilizes caprylic acid-ammonium to be purified, and finally obtains lyophilized antibodies, stores for future use in-20 DEG C.
Embodiment 5: the mensuration of Capsaicinoids specific antibody
1) indirect non-competing ELISA method is used to detect Capsaicinoids specific antibody titres
It is coated: Capsaicinoids artificial complete antigen-envelope antigen 0.1mol/L pH9.6 carbonate buffer solution is dilute
Releasing 1 μ g/mL, 100 μ l/ holes, 4 DEG C of reactions are overnight.Inclining and be coated liquid, PBST washs 3 times in full hole, and button is dry.
Closing: 1%OVA PBST solution, 200 μ L/ holes, 37 DEG C of incubation 1h, PBST wash 3 times in full hole, and button is dry.
Antibody antigen specific reaction: by the Capsaicinoids specific antibody phosphate buffer of 0.01M PH7.4
It is configured to the solution of 1mg/mL, then antibody-solutions is started doubling dilution from 1:10000, and join each dilution be coated
Kong Zhong, addition is 100 μ L/ holes, arranges feminine gender and blank control wells, and negative control sera dilutes 2 000 times, and blank only adds
PBST, 37 DEG C of incubation 1h, PBST wash 3 times in full hole, detain dry；
It is anti-to add two: with PBST solution by sheep anti mouse ELIAS secondary antibody 5000 times dilution, mixing, addition is 100 μ L/ holes, and 37
DEG C incubation 1h, PBST wash 6 times in full hole, and button is dry.
Colour developing: nitrite ion is (containing 1mg/mL tetramethyl benzidine 0.5mL, citrate buffer solution 9.5mL, the hydrogen peroxide of 1%
Urea 32 μ L) now with the current, 100 μ L/ holes, 37 DEG C of incubation 15min of lucifuge.
Terminate: the H2SO4 of 2mol/L, 50 μ L/ holes, measure light absorption value i.e. OD450 value at 450nm by microplate reader immediately.
Result interpretation: with OD450 value more than or equal to 1 time corresponding antibody-solutions highly diluted multiple as antibody
ELISA titer, is 320000.
2) indirect competitive ELISA method detection Capsaicinoids specific antibody 503nhibiting concentration is used
(1) working concentration of antibody is determined with above-mentioned indirect ELISA method, with OD450Corresponding resisting when being about 1
Bulk concentration is the suitableeest working concentration.
(2) it is coated, washs and closes: method operates with indirect non-competing ELISA method.
(3) preparation capsaicin standard solution: capsaicin, Dihydrocapsaicin and synthetic capsaicin methanol solution are configured to
The mother solution (wherein the ratio of Capsaicinoids is 1:1:1) of 10mg/ml.Before sample-adding, use the methanol/PBS of 10%
(0.01mol/L pH7.4) solution starts 5 times of dilutions, altogether 5 concentration of dilution from 20 μ g/mL.Every hole adds 50 μ L doubling dilutions
Capsaicin, Dihydrocapsaicin and synthetic capsaicin standard solution, then every hole adds 50 μ L extension rates is 160000
Antiserum, 37 DEG C of reaction 1h.
(4) add two and resist, develop the color, terminate and reading: method operates with indirect non-competing ELISA method.
(5) data process: with the logarithm of each concentration of capsaicin, Dihydrocapsaicin and synthetic capsaicin as abscissa, Fructus Capsici
Element, Dihydrocapsaicin and OD value corresponding to each concentration of synthetic capsaicin be vertical coordinate, draw standard curve, and calculating 50% suppresses dense
Degree is 0.3 μ g/mL.
Change methanol concentration in standard dilutions, as stated above after detection, draw standard curve, calculate this antibody and exist
Methanol concentration is 10%, 20%, under the conditions of 40%, and 50% inhibition concentration 0.31 μ g/mL, 0.36 μ g/mL, 0.34 μ g/mL respectively.
Show that this antibody tolerance organic solvent ability is strong.
Change the salt ionic concentration value of antibody diluent, as stated above after detection, draw standard curve, calculate this antibody
Be 0.01M, 0.16M, 0.32M at sodium chloride concentration, under the conditions of, 50% inhibition concentration respectively 0.28 μ g/mL, 0.33 μ g/mL,
0.37μg/mL.Show that this antibody can be resistance to higher by salt ionic concentration.
Change the pH value of standard dilutions, as stated above after detection, draw standard curve, calculate this antibody and exist
Under the conditions of pH5.0/6.0/7.0/7.4/8.0,50% inhibition concentration respectively 0.41 μ g/mL, 0.33 μ g/mL, 0.29 μ g/mL,
0.27μg/mL、0.28μg/mL.Show that this antibody pH tolerant scope is wide.
Embodiment 6: the preparation method of kitchen waste grease immune chromatography test paper, step is as follows:
(1) preparation of adsorptive pads
Absorbent paper is cut out growth 15mm, the specification of wide 4mm, obtains adsorptive pads；
(2) preparation of detecting pad
Detection being coated of line: by Capsaicinoids envelope antigen ((2Z)-4-[(4-hydroxy-3-methoxy) benzyl amino]-
4-carbonyl-2-olefin(e) acid-OVA) be configured to concentration be 0.4mg/mL be coated liquid, in position along 9mm on nitrocellulose filter,
By line spray mode, it is laterally coated on nitrocellulose filter, obtains detecting capsaicin class thing needed for line, every centimetre of detection line
The package amount of matter envelope antigen is 0.24 μ g, is then dried 30 minutes under the conditions of 37 DEG C；
Described envelope antigen ((2Z)-4-[(4-hydroxy-3-methoxy) benzyl amino]-4-carbonyl-2-olefin(e) acid-OVA) bag
By the buffer that is coated used in liquid it is: containing ovalbumin OVA 0.1g, Hydrazoic acid,sodium salt 0.002g, sodium chloride in every 10mL
0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g；
Being coated of nature controlling line: anti-for rabbit Mus polyclonal antibody is made into concentration be 0.5mg/mL be coated liquid, in away from detection line
The position of 10mm, is laterally coated in it on nitrocellulose filter, needed for obtaining nature controlling line, every centimetre of nature controlling line by line spray mode
The package amount of rabbit anti-Mus polyclonal antibody is 0.3 μ g, is then dried 30 minutes under the conditions of 37 DEG C；
Described rabbit anti-Mus polyclonal antibody is coated the buffer that is coated used in liquid: containing Ox blood serum in every 10mL
Albumin 0.1g, Hydrazoic acid,sodium salt 0.002g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, phosphorus
Acid dihydride potassium 0.002g；
The long 28mm of described nitrocellulose filter, wide 4mm
(3) preparation of sample pad
Glass fibre membrane is cut out growth 15mm, the specification of wide 4mm, puts in confining liquid and soak, take out, in 37 DEG C of conditions
Under be dried 8 hours, obtain sample pad, then put room temperature preservation in exsiccator.
The every 100mL of described confining liquid contains: oralbumin 1g, sucrose 2g, Hydrazoic acid,sodium salt 0.02g, sodium chloride
0.8g, disodium hydrogen phosphate 0.29g, potassium chloride 0.02g, potassium dihydrogen phosphate 0.02g.
(4) preparation of gold mark pad
Glass fibre membrane is cut out the specification of growth 8mm width 4mm, puts in confining liquid and soak, take out, under the conditions of 37 DEG C
It is dried 8 hours, on the glass fibre membrane being dried, on the glass fibre membrane being dried, laterally sprays nanometer by a spray mode
The anti-Capsaicinoids general Anti-TNF-α liquid solution of gold labelling, nano gold mark anti-peppery needed for every centimetre of spraying length
The consumption of the green pepper element general polyclonal antibody of class material is 0.5 μ g, then vacuum lyophilization 6 hours, puts room temperature in exsiccator and protects
Deposit；
The every 100mL of described confining liquid contains: oralbumin 1g, sucrose 2g, Hydrazoic acid,sodium salt 0.02g, sodium chloride
0.8g, disodium hydrogen phosphate 0.29g, potassium chloride 0.02g, potassium dihydrogen phosphate 0.02g.
The anti-Capsaicinoids general Anti-TNF-α liquid solution of described nano gold mark is to use unsaturated labelling legal system
Standby, its method particularly includes: taking 50.0mL commercial available quality concentration is the nano-Au solution of 0.01%, uses 0.5mL0.1mol/L carbon
Acid aqueous solutions of potassium regulation pH value, the anti-Capsaicinoids being slowly added to 2.0mL0.1mg/mL when stirring is general many
Clonal antibody aqueous solution, continues stirring 30min；Adding mass concentration is that 10% oralbumin (OVA) aqueous solution is to OVA matter at end
Amount concentration is 1%, continues stirring 30min；After placing 2h in 4 DEG C, 3000r/min is centrifuged 15min, takes supernatant, abandons precipitation；Will
Supernatant 12000r/min is centrifuged 30min, abandoning supernatant, adds the washing of 40.0mL labelling and preserves liquid；Again with 12000r/min
Centrifugal 30min, abandoning supernatant, the washing of precipitation labelling is preserved liquid resuspended, obtain 5.0mL concentrate, put 4 DEG C of refrigerators standby
With.
In described nano-Au solution, the particle diameter of nanometer gold is 15nm；
Described 0.1mol/L wet chemical is: 13.8g potassium carbonate is dissolved in pure water and is settled to 1000mL, 0.22 μm filter
Membrane filtration gained；Described labelling washing preserves liquid and is: 2.0g PEG-400,0.2g sodium azide, 0.1235g boric acid,
Pure water is settled to 1000mL, 0.22 μm membrane filtration gained.
(5) assembling of test strips
One side at cardboard pastes adsorptive pads, detecting pad, gold mark pad and sample pad the most successively, and adjacent each pad is even
Connecing the overlapping connection in place, overlapping length is 1mm, obtains kitchen waste grease immune chromatography test paper.See Fig. 1.
Above-mentioned colloidal gold immuno-chromatography test paper strip is in the application quickly differentiated in kitchen waste grease
Weighing 3 edible vegetable oil blind sample 20g respectively, pH value is respectively 5.3, and 5.8,6.4, numbered 1,2,3.Add
50ml volumetric concentration is the ethanol water of 95%, mixing, refluxes 1 hour, after cooling, by extracting solution vacuum rotating at 90 DEG C
It is dried, adds 1ml 10% methanol-PBS solution and redissolve, obtain testing sample solution, then take 200 these testing sample solutions of μ L works
It is added dropwise to quickly differentiate to detect in the sample pad of kitchen waste grease colloidal gold immuno-chromatography test paper strip for detection liquid,
It is as test strip, separately takes the consistent methanol-PBS solution of isopyknic methanol concentration as negative controls, dropwise adds
Entering in another sample pad quickly differentiating kitchen waste grease colloidal gold immuno-chromatography test paper strip, it is as control stripes bar, and 10
After minute, test strip and control stripes bar are carried out colour developing comparison, reading result:
Testing result: the nature controlling line of three sample detection test strips demonstrates red stripes, and detects line and do not develop the color, and can sentence
For positive findings, showing that the content of Capsaicinoids is above 3ng/ml, 3 samples are kitchen waste grease sample.Warp
After the detection of capsaicin immune affinity column-tablets by HPLC-MS, result show 1, in 2, No. 3 samples capsaicin and
Dihydrocapsaicin total amount is respectively 36,86,1213ng/mL, can determine that as kitchen waste grease.This result provides with the present invention
Kitchen waste grease colloidal gold immuno-chromatography test paper strip method for quick identification result of determination is consistent.
In the present invention, the standard of perfection of kitchen waste grease sample is mainly based upon what following reason was made: logical to in a large number
The capsaicin content of actual sample (including multiple Oleum Arachidis hypogaeae semen, soybean oil, Oleum Helianthi, the sample such as Oleum Brassicae campestris and waste grease) enters
Row detection, detection method: capsaicin immune affinity column liquid matter method, result is as shown in table 1.Result shows: the food in addition to Oleum Arachidis hypogaeae semen
With having no the detection of Capsaicinoids in vegetable oil, in Oleum Arachidis hypogaeae semen, Capsaicinoids is less than 1.53ug/kg (1.53ng/
Ml), and in abandoned oil (waste oil), the content of Capsaicinoids is higher than 12.27ug/kg (12.7ng/ml), thus by Fructus Capsici
Element class content of material equals to or more than 3ng/ml as differentiating that sample is kitchen waste grease or the oil mixed with kitchen waste grease
The examination standard of fat sample.
Capsaicinoid compounds content in table 1 vegetable oil
Note: '-' do not detect.
Embodiment 7: the preparation method of kitchen waste grease immune chromatography test paper, step is as follows:
(1) preparation of adsorptive pads
Absorbent paper is cut out growth 16mm, the specification of wide 3.8mm, obtains adsorptive pads；
(2) preparation of detecting pad
Detection being coated of line: by Capsaicinoids envelope antigen ((2Z)-4-[(4-hydroxy-3-methoxy) benzyl amino]-
4-carbonyl-2-olefin(e) acid-OVA) be configured to concentration be 0.2mg/mL be coated liquid, in position along 10mm on nitrocellulose filter
Put, by line spray mode, it is laterally coated on nitrocellulose filter, obtain detecting capsaicin class needed for line, every centimetre of detection line
The package amount of material envelope antigen is 0.12 μ g, is then dried 30 minutes under the conditions of 37 DEG C；
Described envelope antigen ((2Z)-4-[(4-hydroxy-3-methoxy) benzyl amino]-4-carbonyl-2-olefin(e) acid-OVA) bag
By the buffer that is coated used in liquid it is: containing ovalbumin OVA 0.1g, Hydrazoic acid,sodium salt 0.002g, sodium chloride in every 10mL
0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g；
Being coated of nature controlling line: anti-for rabbit Mus polyclonal antibody is made into concentration be 0.2mg/mL be coated liquid, in away from detection line
The position of 8mm, is laterally coated in it on nitrocellulose filter, needed for obtaining nature controlling line, every centimetre of nature controlling line by line spray mode
The package amount of rabbit anti-Mus polyclonal antibody is 0.12 μ g, is then dried 30 minutes under the conditions of 37 DEG C；
Described rabbit anti-Mus polyclonal antibody is coated the buffer that is coated used in liquid: containing Ox blood serum in every 10mL
Albumin 0.1g, Hydrazoic acid,sodium salt 0.002g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, phosphorus
Acid dihydride potassium 0.002g；
The long 25mm of described nitrocellulose filter, wide 3.8mm
(3) preparation of sample pad
Glass fibre membrane is cut out growth 13mm, the specification of wide 3.8mm, puts in confining liquid and soak, take out, in 37 DEG C of bars
It is dried 8 hours under part, obtains sample pad, then put room temperature preservation in exsiccator.
The every 100mL of described confining liquid contains: oralbumin 1g, sucrose 2g, Hydrazoic acid,sodium salt 0.02g, sodium chloride
0.8g, disodium hydrogen phosphate 0.29g, potassium chloride 0.02g, potassium dihydrogen phosphate 0.02g.
(4) preparation of gold mark pad
Glass fibre membrane is cut out the specification of growth 9mm width 3.8mm, puts in confining liquid and soak, take out, in 37 DEG C of conditions
Under be dried 8 hours, on the glass fibre membrane being dried, with a spray mode on the glass fibre membrane being dried laterally spraying receive
The anti-Capsaicinoids general Anti-TNF-α liquid solution of rice gold labelling, resisting of every centimetre of nano gold mark sprayed needed for length
The consumption of the general polyclonal antibody of Capsaicinoids is 0.6 μ g, then vacuum lyophilization 6 hours, puts room temperature in exsiccator
Preserve；
The every 100mL of described confining liquid contains: oralbumin 1g, sucrose 2g, Hydrazoic acid,sodium salt 0.02g, sodium chloride
0.8g, disodium hydrogen phosphate 0.29g, potassium chloride 0.02g, potassium dihydrogen phosphate 0.02g.
The anti-Capsaicinoids general Anti-TNF-α liquid solution of described nano gold mark is to use unsaturated labelling legal system
Standby, its method particularly includes: taking 50.0mL commercial available quality concentration is the nano-Au solution of 0.01%, uses 0.5mL0.1mol/L carbon
Acid aqueous solutions of potassium regulation pH value, the anti-Capsaicinoids being slowly added to 2.0mL0.1mg/mL when stirring is general many
Clonal antibody aqueous solution, continues stirring 30min；Adding mass concentration is that 10% oralbumin (OVA) aqueous solution is to OVA matter at end
Amount concentration is 1%, continues stirring 30min；After placing 2h in 4 DEG C, 3000r/min is centrifuged 15min, takes supernatant, abandons precipitation；Will
Supernatant 12000r/min is centrifuged 30min, abandoning supernatant, adds the washing of 40.0mL labelling and preserves liquid；Again with 12000r/min
Centrifugal 30min, abandoning supernatant, the washing of precipitation labelling is preserved liquid resuspended, obtain 5.0mL concentrate, put 4 DEG C of refrigerators standby
With.
In described nano-Au solution, the particle diameter of nanometer gold is 15nm；
Described 0.1mol/L wet chemical is: 13.8g potassium carbonate is dissolved in pure water and is settled to 1000mL, 0.22 μm filter
Membrane filtration gained；Described labelling washing preserves liquid and is: 2.0g PEG-400,0.2g sodium azide, 0.1235g boric acid,
Pure water is settled to 1000mL, 0.22 μm membrane filtration gained.
(5) assembling of test strips
One side at cardboard pastes adsorptive pads, detecting pad, gold mark pad and sample pad the most successively, and adjacent each pad is even
Connecing the overlapping connection in place, overlapping length is 1mm, obtains kitchen waste grease immune chromatography test paper.See Fig. 1.
Above-mentioned colloidal gold immuno-chromatography test paper strip is in the application quickly differentiated in kitchen waste grease
Weighing the kitchen waste grease sample of collection and the original each 20g of edible vegetable blend oil sample, pH value divides
It is not 5.4 and 7.2, is separately added into the ethanol water that 50ml volumetric concentration is 95%, mixing, reflux 1 hour at 90 DEG C, cold
But after, extracting solution vacuum rotating is dried, adds 1ml 10% methanol-PBS solution and redissolve, obtain testing sample solution, then take
180 these testing sample solutions of μ L are added dropwise to quickly detect the colloidal gold immunochromatographimethod examination of Capsaicinoids as detection liquid
Detecting in the sample pad of paper slip, it is as test strip, and the methanol-PBS separately taking isopyknic methanol concentration consistent is molten
Liquid, as negative controls, is added dropwise in the sample pad of another kitchen waste grease immune chromatography test paper, and it is as comparison examination
Paper slip, carries out colour developing comparison, reading result by test strip and control stripes bar after 10 minutes:
Testing result: the nature controlling line of original edible vegetable blend oil sample detection test strips demonstrates red bar
Band, detection line is all close with the detection of blank test strips limit color, then be judged to negative findings, thus judge: do not used
Edible vegetable blend oil sample in Capsaicinoids content less than 3ng/mL, show this mixed oil not mixed with or mixed with seldom
The kitchen waste grease sample of amount.The nature controlling line of the kitchen waste grease sample detection test strips collected demonstrates red stripes,
And detect line and do not develop the color, then it is judged to positive findings, shows the content of Capsaicinoids in the kitchen waste grease sample collected
More than 3ng/mL.
Claims (10)
- The general specific antibody of the most anti-Capsaicinoids, it is characterised in that: it is general by the Capsaicinoids described in formula I Artificial immunity antigen compound immune animal, and the general specificity of anti-Capsaicinoids that isolated and purified postlyophilization obtains Antibody；
- The general specific antibody of anti-Capsaicinoids the most according to claim 1, it is characterised in that: described immunity is After using Freund's complete adjuvant or incomplete Freund's adjuvant emulsifying, immune animal is repeated at interval, wherein: initial immunity Freund is complete Full adjuvant, follow-up immunization incomplete Freund's adjuvant, reach more than 10000 to antiserum titre, carotid artery blood sampling is dead to it, After the centrifugal blood adopted, antiserum purification is obtained Capsaicinoids antibody.
- The general specific antibody of anti-Capsaicinoids the most according to claim 1, it is characterised in that: described in described formula I The immune consumption of Capsaicinoids general artificial immunity antigen compound be calculated as 0.2-1.0mg with OVA albumen therein；Exempt from The epidemic disease number of plies is 4-6 time；First immunisation uses isopyknic Freund's complete adjuvant emulsified immunogen, multi-point injection under rabbit；First After secondary immunity, interval is carried out second time immunity, second time and later immunization interval 2-3 week in 2-4 week, by isopyknic Freund not Freund's complete adjuvant emulsified immunogen, in the subcutaneous multi-point injection of rabbit back.From the beginning of the 4th time, after the most immune one week in 6-10 days, The non-competing indirect ELISA method of rabbit ear edge vein exploitating blood measures serum titer, after titer reaches 10000, i.e. and rabbit carotid artery blood sampling Dead to it, after the centrifugal blood adopted, utilize caprylic acid-ammonium to be purified antiserum, obtain Capsaicinoids Antibody.
- 4. kitchen waste grease immuno-chromatographic test paper strip, it is characterised in that: including cardboard, the one side of cardboard is glued the most successively Patch adsorptive pads, detecting pad, gold mark pad and sample pad, adjacent each pad is overlapping in junction to be connected, and described detecting pad is with cellulose nitrate Element film is base wad, and nitrocellulose filter arranges horizontal nature controlling line and detection line from top to bottom, and described nature controlling line is coated with rabbit Anti-Mus polyclonal antibody, described detection line is coated Capsaicinoids envelope antigen；Described gold mark pad transverse jet scribbles to be received The general specific antibody of anti-Capsaicinoids described in claim 1 of rice gold labelling.
- Kitchen waste grease immuno-chromatographic test paper strip the most according to claim 4, it is characterised in that: described adsorptive pads is long 10～15mm, wide 3～4mm, detecting pad length 25～30mm, wide 3～4mm；Gold mark pad long 6～9mm, wide 3～4mm；Sample pad is long 12～15mm, wide 3～4mm, the overlapping length of adjacent each pad is 1～3mm；Described adsorptive pads is absorbent paper.
- Kitchen waste grease immuno-chromatographic test paper strip the most according to claim 4, it is characterised in that: on described detecting pad Detection line is 8～20mm with the spacing on edge on nitrocellulose filter, and described detection line is 5～10mm with the spacing of nature controlling line；Institute The particle diameter stating nanometer gold used in gold mark pad is 15～20nm.
- Kitchen waste grease immuno-chromatographic test paper strip the most according to claim 4, it is characterised in that: described capsaicin class The molecular structural formula of material envelope antigen is as shown in formula II:On described detecting pad detection line, the package amount of every centimetre of required envelope antigen is 0.1～0.6 μ g；On nature controlling line every li The package amount of the rabbit anti-Mus polyclonal antibody required for meter is 0.1～0.6 μ g；The general specific antibody of anti-Capsaicinoids of the nano gold mark needed for the upper every centimetre of spraying length of described gold mark pad Consumption be 0.40～0.98 μ g.
- 8. the preparation method of the kitchen waste grease immuno-chromatographic test paper strip described in claim 4, it is characterised in that: include following Step:(1) preparation of adsorptive padsAbsorbent paper is cut out and i.e. obtains adsorptive pads；(2) preparation of detecting padDetection being coated of line: Capsaicinoids envelope antigen is configured to concentration be 0.17～1.0mg/mL be coated liquid, in On nitrocellulose filter along 8～20mm position, with line spray mode it is laterally coated on nitrocellulose filter, examined Survey line, the package amount of the general artificial complete antigen-envelope antigen of Capsaicinoids is 0.1～0.6 μ needed for every centimetre of detection line G, is then dried 30～60 minutes under the conditions of 37 DEG C；Being coated of nature controlling line: anti-for rabbit Mus polyclonal antibody is made into concentration be 0.17～1.0mg/mL be coated liquid, in away from detection The position of line 5～10mm, is laterally coated in it on nitrocellulose filter by line spray mode, obtains nature controlling line, every centimetre of nature controlling line The package amount of required rabbit anti-Mus polyclonal antibody is 0.1～0.6 μ g, is then dried 30～60 minutes under the conditions of 37 DEG C；(3) preparation of sample padGlass fibre membrane is put in confining liquid and soaks, take out, be dried 6～12 hours under the conditions of 37～40 DEG C, obtain sample pad, Then room temperature preservation in exsiccator is put；(4) preparation of gold mark padGlass fibre membrane is put in confining liquid and soaks, take out, be dried 6～12 hours under the conditions of 37～40 DEG C, in the driest Glass fibre membrane on, on the glass fibre membrane being dried, laterally spray the anti-capsaicin class of nano gold mark by a spray mode Material general Anti-TNF-α liquid solution, general many grams of the anti-Capsaicinoids of every centimetre of nano gold mark sprayed needed for length The consumption of grand antibody is 0.4～0.98ng, then vacuum lyophilization 2～6 hours, puts room temperature preservation in exsiccator；(5) assembling of test stripsOne side at cardboard pastes adsorptive pads, detecting pad, gold mark pad and sample pad the most successively, and adjacent each pad is in junction Overlapping connection, overlapping length is 1～3mm, obtains kitchen waste grease immune chromatography test paper.
- 9. kitchen waste grease immunochromatography method for quick identification, it is characterised in that: step is as follows: pick-up kitchen waste grease is to be measured Sample, extracts Capsaicinoids therein, is redissolved by dried for extracting solution solubilizer, described testing sample and redissolution solvent Ratio be 20g:1ml, obtain testing sample solution, then this testing sample solution be added dropwise to kitchen as detection liquid and discard Detecting in the sample pad of oils and fats immune chromatography test paper, it is as test strip, separately takes isopyknic methanol concentration consistent Methanol aqueous solution as negative controls, be added dropwise in the sample pad of another kitchen waste grease immune chromatography test paper, its As control stripes bar, after a period of time, test strip and control stripes bar are carried out colour developing and compare:Testing result:(1) negative: when nature controlling line colour developing in test strip, and to detect the color detecting line on line color and control stripes bar Close to time, show that Capsaicinoids capsaicin in testing sample solution, Dihydrocapsaicin or synthetic capsaicin content are less than In conjunction with authenticity detection method, 3ng/mL, determines whether this sample is kitchen waste grease or the oils and fats mixed with kitchen waste grease Sample；Content is less than 3ng/mL, can determine whether that testing sample is that the probability of kitchen waste grease is little, need to use authenticity further Detection method is determined；(2) positive: when nature controlling line colour developing in test strip, and to detect the color detecting line in line color ratio comparison test strips Time shallow, show that capsaicin in testing sample solution, Dihydrocapsaicin or synthetic capsaicin content equal to or higher than 3ng/mL, then may be used Judge that this sample is as kitchen waste grease or mixed with the oil sample of kitchen waste grease；(3) invalid: when nature controlling line does not develops the color, no matter whether the detection line of test strip develops the color, and it is invalid that this test strips is judged to.
- Kitchen waste grease immunochromatography method for quick identification the most according to claim 9, it is characterised in that: step is such as Under:Described extracting method is that addition volumetric concentration is the ethanol water of 95% in testing sample, and mixing, 60～90 Refluxing 1 hour at DEG C, cooling obtains extracting solution；Described redissolution solvent is 10% methanol-PBS.
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| KR1020170062643A KR101963023B1 (en) | 2016-05-26 | 2017-05-22 | General complete antigen hapten, specific antibody, test strip, and rapid immunochromatographic method for identification of a recovered oil from kitchen |
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| CN108982862A (en) * | 2018-08-01 | 2018-12-11 | 山西公大智检科技有限公司 | It is a kind of for detecting the immuno-chromatographic test paper strip and preparation method thereof of capsaicine in gutter oil |
| CN109991412A (en) * | 2018-12-10 | 2019-07-09 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | A kind of method of capsicum alkali composition in detection grease |
| WO2020118526A1 (en) * | 2018-12-10 | 2020-06-18 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Method for detecting capsaicin in oil |
| CN109856318A (en) * | 2019-01-30 | 2019-06-07 | 扬州工业职业技术学院 | A kind of test paper of quick detection gutter oil |
| CN110045108A (en) * | 2019-05-28 | 2019-07-23 | 江南大学 | A kind of gutter oil enzyme-linked immunologic detecting kit and its detection method based on capsaicine index |
| CN110045108B (en) * | 2019-05-28 | 2021-10-29 | 江南大学 | Capsaicin index-based illegal cooking oil enzyme-linked immunosorbent assay kit and detection method thereof |
| CN110981744A (en) * | 2019-11-08 | 2020-04-10 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Capsaicin hapten and artificial antigen for illegal cooking oil detection and preparation method and application thereof |
| WO2021088209A1 (en) * | 2019-11-08 | 2021-05-14 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Capsaicin hapten and artificial antigen for detecting illegal cooking oil, preparation method therefor and application thereof |
| CN112415186A (en) * | 2020-11-19 | 2021-02-26 | 北京科技大学 | Reverse phase transfer extraction detection process and application of capsaicin compounds in grease |
| WO2022188302A1 (en) * | 2021-03-11 | 2022-09-15 | 杭州同舟生物技术有限公司 | Pregabalin artificial hapten and pregabalin artificial antigen, preparation methods therefor, and applications thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| KR101963023B1 (en) | 2019-03-27 |
| CN106084059B (en) | 2019-10-25 |
| KR20170134217A (en) | 2017-12-06 |
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