CN104383889B - A kind of Dihydrocapsaicin polyclonal antibody adsorbent, immune affinity column and its preparation method and application - Google Patents
A kind of Dihydrocapsaicin polyclonal antibody adsorbent, immune affinity column and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to Dihydrocapsaicin polyclonal antibody adsorbent, immune affinity column and its preparation method and application.This immunosorbent includes the Dihydrocapsaicin polyclonal antibody of coupling on solid phase carrier and this solid phase carrier, described Dihydrocapsaicin polyclonal antibody is the Dihydrocapsaicin artificial antigen immune animal that Dihydrocapsaicin artificial semiantigen (E) 4 [2 methoxyl group 4 (pelargonamide methyl) phenoxy group] 4 oxygen 2 butenoic acids described in employing formula I obtain with bovine serum albumin(BSA) (BSA) coupling, and the acquisition of isolated and purified postlyophilization.The immune affinity column of the present invention can be specific binding with Dihydrocapsaicin, and maximum column capacity is 220 250ng；Using methyl alcohol to elute, subscript the rate of recovery up to 83.1%, RSD is respectively less than 6%.Can be used for the sample pre-treatments of Dihydrocapsaicin content, simple to operate, low cost, efficiency high, organic reagent consumption is few.
Description
Technical field:
The present invention relates to Dihydrocapsaicin polyclonal antibody adsorbent, immune affinity column and preparation method thereof and answer
With.
Background technology
In recent years, improper edible oil (being commonly called as waste oil) mix in qualified edible oil with obtain interests behavior prohibit repeatly
Incessantly, the health of consumer in serious threat, becomes the problem that current China field of food safety need solve.In China's tradition
The flavouring that in eating habit, capsicum is that usage amount is relatively big, has wide range of applications, Dihydrocapsaicin is mainization causing pungent
Study point, be primarily present in fruit and the seed of capsicum plants.Dihydrocapsaicin have fat-soluble, stiff stability good, boiling
The high character of point, and the swill oil of catering industry is one of main source of waste oil, current waste oil processing method has been difficult to
Full removal has the compound of above character.Normal edible oil is substantially free of Dihydrocapsaicin, and the kitchen contacting capsicum is given up
Abandon grease be difficult to avoid that containing this constituents, therefore Dihydrocapsaicin can refer to as the feature differentiating kitchen waste grease
Mark.
The detection method of existing Dihydrocapsaicin includes TLC and precision instrument analytic approach, and TLC is relatively
Early for detecting the common detection methods of Dihydrocapsaicin, this method need not special instrument and equipment, and common laboratory all can be entered
OK, but organic reagent consumption is big, complex steps, other component serious interference, poor accuracy, it is impossible to accurate quantitative analysis, and to experiment
Personnel and ambient contamination harm are relatively big, excitant is strong, is unsuitable for field quick detection.Precision instrument analytic approach mainly includes
High performance liquid chromatography (HPLC) and liquid chromatogram matter combination method (LC-MS/MS), these methods are highly sensitive, and accuracy is good, so
And requiring that Dihydrocapsaicin Sample Purification on Single degree is high, traditional Sample Pretreatment Technique, such as liquid-liquid extraction, SPE, solid phase
Micro-extractions etc., pretreatment process is loaded down with trivial details, the strongest.Therefore, set up Sample Pretreatment Technique efficiently and effectively and become two
The detection of hydrogen capsicim needs the primary and bottleneck problem solved in analyzing.Immune affinity chromatographic column (Immunoaffinity
Chromatography) be a kind of new and effective Sample Pretreatment Technique, utilize high degree of specificity that antigen-antibody combines and
Affinity, is attached to specific antibody on chromosorb with chemical coupling bonding method, based on specific between antigen-antibody
Reversible binding realize in complex sample target substance enrichment purify.At present, there is not yet Dihydrocapsaicin polyclone
The relevant report of antibody mediated immunity affinity post.
Summary of the invention
It is an object of the present invention to provide a kind of Dihydrocapsaicin polyclonal antibody adsorbent, its preparation method and at sample
Application in product examine survey.
For solving to realize the object of the invention, the technical solution used in the present invention is as follows:
A kind of Dihydrocapsaicin polyclonal antibody adsorbent, it is characterised in that: this immunosorbent includes that solid phase carries
The Dihydrocapsaicin polyclonal antibody of coupling on body and this solid phase carrier, described Dihydrocapsaicin polyclonal antibody is employing formula I
Described Dihydrocapsaicin artificial semiantigen (E)-4-[2-methoxyl group-4-(pelargonamide methyl) phenoxy group]-4-oxygen-2-butenoic acid
The Dihydrocapsaicin artificial antigen immune animal obtained with bovine serum albumin(BSA) (BSA) coupling, and isolated and purified postlyophilization
The Dihydrocapsaicin polyclonal antibody obtained,
By such scheme, described immunity is to use Freund's complete adjuvant or Freund the completeest Dihydrocapsaicin artificial antigen
After full adjuvant emulsion, immune animal is repeated at interval, wherein；Initial immunity Freund's complete adjuvant, follow-up immunization Freund is incomplete
Adjuvant, reaches more than 4000 to antiserum titre, i.e. puts to death Balb/c mouse, takes whole antiserum；Described purifying is octanoic acid
Ammonium sulfate method purifies.
By such scheme, described Dihydrocapsaicin polyclonal antibody is 3.06 to 50% inhibition concentration of Dihydrocapsaicin
μ g/mL, measures through conventional cross reaction, this antibody and the chemical residual pollutant such as fenvalerate, decis and aspergillus flavus poison
The element equal no cross reaction of biotoxin pollutant.
By such scheme, described Dihydrocapsaicin artificial semiantigen is by N, N '-dicyclohexylcarbodiimide and N-hydroxyl
After base succinimide (NHS) activation, then carry out coupling with bovine serum albumin(BSA) and obtain.
By such scheme, described coupling concretely comprises the following steps:
(1) by Dihydrocapsaicin artificial semiantigen at N,N-dimethylformamide (DMF) and N-hydroxy-succinamide
Room temperature reaction 1-2 hour in mixed solution, the DMF being subsequently adding N, N '-dicyclohexylcarbodiimide is molten
Liquid, room temperature reaction 4-6 hour, stand overnight, take the Dihydrocapsaicin artificial semiantigen solution that supernatant i.e. activates；
(2) supernatant is added drop-wise to the phosphate buffer of the carrier protein concentration carrier protein more than or equal to 2mg/mL
In, room temperature reaction 12-14h, wherein: the Dihydrocapsaicin artificial semiantigen in described step (1) and the mol ratio of carrier protein
More than 5:1, then dialysis obtains Dihydrocapsaicin artificial antigen, i.e. compound II.
By such scheme, N-hydroxy-succinamide and N in described step (1), N '-dicyclohexylcarbodiimide mole
Ratio is 1:1-1.1.
By such scheme, the phosphate buffer of the bovine serum albumin(BSA) in described step (2) is by bovine serum albumin(BSA)
Dissolve with the phosphate buffer of 0.2MpH 8 and to obtain.
By such scheme, described solid phase carrier is Ago-Gel or silica gel microball.
The preparation method of above-mentioned Dihydrocapsaicin polyclonal antibody adsorbent, it is characterised in that:
When described solid phase carrier selects polyacrylamide silica gel microball, preparation method is: by polyacrylamide silica gel microball
Alternately rinse with the phosphate buffer of pure water and pH=6, with the phosphate buffer of pH=6 join polyacrylamide silica gel is micro-
Ball microsphere suspension liquid；Dissolve Dihydrocapsaicin polyclonal antibody with the phosphate buffer of pH=6, be then added dropwise to poly-third
In acrylamide silica gel microball aaerosol solution, add carbodiimide (EDC), after 4 DEG C of condition stirring reaction 20-22h, obtain with poly-
Acrylamide silica gel microball is the Dihydrocapsaicin polyclonal antibody adsorbent of solid phase carrier；
When described solid phase carrier selects Ago-Gel, preparation method is: repeatedly washed through hydrochloric acid by Ago-Gel
It is dissolved in coupling buffer after removal of impurity activation, adds above-mentioned Dihydrocapsaicin polyclonal antibody, stir at ambient temperature,
To Ago-Gel solution, then by after in Ago-Gel solution, antibody-solutions with agarose gel coupling does not filters, use
Coupling buffer rinses Ago-Gel, adds the Tris-HCl buffer solution of 0.1M, pH=8.0, room temperature reaction 1.5-2.5h,
Then agar is alternately rinsed with the Tris-HCl buffer solution of 0.1M, pH=8.0 and the Tris-HCl buffer solution of 0.1M, pH=4.0
Sugar gel, removes Dihydrocapsaicin polyclonal antibody and other impurity of non-coupling, obtains the Dihydrocapsaicin polyclone purified
Antibody mediated immunity adsorbent.
By such scheme, when described solid phase carrier selects polyacrylamide silica gel microball: polyacrylamide silica gel microball, two
Hydrogen capsicim polyclonal antibody, the mass ratio of carbodiimide are 0.4-1.2g:0.8-4.0mg:28-84mg；The phosphorus of described pH=6
The volume of phthalate buffer and the ratio of polyacrylamide silica gel microball quality are 1-5mL/g；
When described solid phase carrier selects Ago-Gel: described Ago-Gel and the matter of Dihydrocapsaicin polyclonal antibody
Amount ratio is 0.2-0.6g:0.4-2mg；The volume of the coupling buffer of described dissolving Ago-Gel and the quality of Ago-Gel
Ratio be 20-40mL/g.
By such scheme, described coupling buffer is 0.1M Na2CO3, 0.5M NaCl, pH=8.3.
It is mounted with the Dihydrocapsaicin polyclonal antibody parent of above-mentioned Dihydrocapsaicin polyclonal antibody adsorbent
And post.
The preparation method of above-mentioned Dihydrocapsaicin polyclonal antibody affinity column, specifically comprises the following steps that first by dihydro
Capsicim polyclonal antibody adsorbent is filled in solid-phase extraction column, after adding the phosphate buffer of 0.01M, pH=6
Natural sedimentation, then with the phosphate buffer washing of 0.01M, pH=6, is stored in the pH 6 containing 0.02wt% Sodium azide,
The phosphate buffer of 0.01M.
The application of above-mentioned Dihydrocapsaicin polyclonal antibody affinity column, it is characterised in that the dihydro described in utilization is peppery
Sample extracting solution before upper machine is enriched with and purifies by green pepper element polyclonal antibody affinity column.
By such scheme, described application process concrete operations are: first by Dihydrocapsaicin polyclonal antibody parent
With post pure water rinsing, add pending sample extracting solution, then use pure water drip washing, after dried liquid stream, by methanol-eluted fractions,
Collecting eluent, described eluent is the sample extracting solution purified and after enrichment.Sample extracting solution after purifying and being enriched with can
It is directly used in the detections such as LC-MS/MS.
Above-mentioned specific Dihydrocapsaicin artificial semiantigen can be with N-(4-hydroxyl-3-methoxybenzy) pelargonamide and suitable fourth
Enedioic acid acid anhydride is that raw material obtains Dihydrocapsaicin artificial semiantigen i.e. chemical compounds I through ester condensation reaction, and synthetic route is as follows:
Concretely comprise the following steps: be 1:(1.2～1.5 by mol ratio): N-(the 4-hydroxy-3-methoxy benzyl of (0.12～0.15)
Base) pelargonamide, maleic anhydride and dicyclohexylcarbodiimide (DCC) be stirred at room temperature reaction in dichloromethane, then put
In ice bath, under the conditions of 0-5 DEG C, the dichloromethane solution of DMAP is slowly dropped in above-mentioned solution, institute
N-(4-hydroxyl-3-methoxybenzy) pelargonamide stated and the mol ratio of DMAP are 1:(0.12～0.15), remove
Going water-bath, room temperature reaction 5-7h, post processing obtains Dihydrocapsaicin artificial semiantigen.
Wherein, described post processing is: by reacting liquid filtering, and decompression is distilled off solvent and obtains flaxen grease and slightly produce
Product, then by this thick product by silica gel column chromatography, column chromatography reagent: volume ratio is the petroleum ether of 1:1 and mixing of ethyl acetate
Close solution, decolour and i.e. obtain Dihydrocapsaicin artificial semiantigen I.
The beneficial effects of the present invention is:
Dihydrocapsaicin polyclonal antibody affinity column prepared by the present invention can be specific binding with Dihydrocapsaicin,
Its maximum column capacity is 220-250ng；Use eluant methanol to elute, the Dihydrocapsaicin of vegetable oil sample is subscripted back
Yield is up to more than 83.1, and RSD is respectively less than 6%.Can be used for the sample pre-treatments of Dihydrocapsaicin content, have simple to operate,
Low cost, efficiency are high, organic reagent consumption is few, it is possible to be applied to the front place of Dihydrocapsaicin compounds in complex lipids matrix
Reason.
Accompanying drawing explanation
The Dihydrocapsaicin artificial antigen ultraviolet spectrogram of Fig. 1 present invention synthesis.In figure: 1: Dihydrocapsaicin haptens,
2: carrier protein, 3: Dihydrocapsaicin artificial antigen.
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.
Embodiment 1: the preparation of Dihydrocapsaicin artificial antigen
Weigh N-(4-hydroxyl-3-methoxybenzy) pelargonamide 5.86g, maleic anhydride 2.36g and N, N '-two hexamethylene
Base carbodiimide 0.49g, 25mL dichloromethane room temperature reaction 0.5h in reaction bulb, be then placed in above-mentioned reactant liquor in ice bath
Make its temperature control in the range of 0～5 DEG C, take DMAP 0.30g and 4mL dichloromethane is placed in constant pressure funnel
In fully mix, slowly drip in reaction bulb, 0.5h dropping is complete, waits to drip complete, keeps 25 DEG C of conditions of room temperature to continue to stir
Mixing 6 hours, by reacting liquid filtering, decompression is distilled off solvent and obtains the thick product of flaxen grease, then led to by this thick product
Crossing silica gel column chromatography, column chromatography reagent: petroleum ether and ethyl acetate volume ratio are the mixed solution of 1:1, it is peppery that decolouring obtains dihydro
Green pepper element haptens (E)-4-[2-methoxyl group-4-(pelargonamide methyl) phenoxy group]-4-oxygen-2-butenoic acid, molecular formula is
C21NO6H29。
This compound is more than 90% with the homology of Dihydrocapsaicin compound.Through Mass Spectrometric Identification, its molecular ion peak is
ESI-MS 390(M-H)-, consistent with the theoretical value of its result, show that this hapten compound successfully synthesizes.
Weigh Dihydrocapsaicin haptens (E)-4-[2-methoxyl group-4-(pelargonamide methyl) phenoxy group]-4-oxygen-2-butylene
Acid 6mg, then weigh 10mgNHS, it is dissolved in reaction unit with 0.2mL DMF, reacts room temperature (25-27 DEG C) under magnetic stirring
Carry out 1h；Weigh DCC 20mg to be dissolved in 200uL DMF, be added dropwise in above-mentioned reaction unit, room temperature reaction 4h, white precipitate
Generating, stand overnight, 8000r/min, 5min are centrifugal, take supernatant；
In the phosphate buffer of the bovine serum albumin(BSA) that supernatant is added drop-wise to 10ml 2mg/mL the most respectively, room temperature is stirred
Mix reaction 12h；The phosphate buffer of described bovine serum albumin(BSA) is to be delayed by the phosphate of bovine serum albumin(BSA) 0.2M pH 8
Rush what liquid dissolving obtained.Then change a dislysate with 0.01M PBS dialysis 72h, 4-8h, i.e. obtain dihydro capsicum
Element artificial antigen.The continuous scanning spectra of ultraviolet-visible spectrum is shown in that Fig. 1, qualification result show artificial antigen coupling success.
Embodiment 2: the preparation of Dihydrocapsaicin polyclonal antibody
The present embodiment is with the prepared Dihydrocapsaicin artificial antigen of embodiment 1 as immunogene, and immunity obtains Dihydrocapsaicin
Antiserum, uses saturated ammonium sulfate two-step method deposition and purification to obtain antiserum i.e. Dihydrocapsaicin polyclonal antibody, concrete operations
As follows:
Dihydrocapsaicin artificial antigen uses Freund's complete adjuvant or incomplete Freund's adjuvant emulsification, and first immunisation uses
Freund's complete adjuvant, follow-up immunization all emulsifies with incomplete Freund's adjuvant, uses dorsal sc injection to note with leg muscle
Penetrate the method immunity Balb/c mouse combined, every immunity in 3 weeks once, and in third time immunity, after each immunity
After within one week, obtaining the blood sample centrifugal treating of Balb/c mouse, obtain antiserum, use indirect enzyme-linked immunosorbent method to measure anti-
Serum titer, treats that its antiserum titre reaches more than 4000, i.e. puts to death Balb/c mouse, takes whole antiserum, then with octanoic acid
The freeze-drying after purification of ammonium sulfate method obtains Dihydrocapsaicin polyclonal antibody, saves it in-20 DEG C of conditions stand-by.
After measured: described Dihydrocapsaicin polyclonal antibody is 3.06 μ g/ to 50% inhibition concentration of Dihydrocapsaicin
ML, shows successfully to prepare Dihydrocapsaicin antibody.Measure through conventional cross reaction, this antibody and fenvalerate, decis
Deng equal no cross reactions of biotoxin pollutant such as chemical residual pollutant and aflatoxin, show antibody can specifically with
Dihydrocapsaicin reacts.
Embodiment 3: Dihydrocapsaicin polyclonal antibody affinity column adsorbent and the preparation of immune affinity column
The present embodiment immune affinity column adsorbent includes solid phase carrier (polyacrylamide silica gel microball) and carries with this solid phase
The Dihydrocapsaicin polyclonal antibody of body coupling, concrete preparation method is as follows:
Weigh 1.0g polyacrylamide silica gel microball, put in Erlenmeyer flask, hand over the phosphate buffer of pure water and pH=6
For rinsing microballoon；The phosphate buffer measuring 5mL pH=6 dissolves polyacrylamide silica gel microball, and room temperature 25 DEG C stirs and evenly mixs
10min, is added dropwise over the Dihydrocapsaicin Anti-TNF-α liquid solution (concentration is 2mg/mL) with 1mL pH=6, adds
70mgEDC, after 4 DEG C of condition stirring reaction 20-22h, obtains the dihydro capsicum with polyacrylamide silica gel microball as solid phase carrier
Element polyclonal antibody adsorbent.
The preparation of Dihydrocapsaicin polyclonal antibody affinity column: by the immune affinity sorbent of above-mentioned preparation
(0.2mL) it is filled in solid-phase extraction column, adds natural sedimentation after the phosphate buffer of 0.01M, pH=6, with 0.01M, pH
After the phosphate buffer washing of=6, it is stored in the pH=6 containing 0.02% Sodium azide, the phosphate buffer of 0.01M,
Obtain Dihydrocapsaicin polyclonal antibody affinity column, stand-by in 4 DEG C of holdings.
Embodiment 4: Dihydrocapsaicin polyclonal antibody affinity column adsorbent and the preparation of immune affinity column
The present embodiment immune affinity column adsorbent include solid phase carrier (Ago-Gel) and with this solid phase carrier coupling
Dihydrocapsaicin polyclonal antibody, concrete preparation method is as follows:
Weigh 0.2g Ago-Gel, put in conical flask, repeatedly rinse 20min with 1mM HCl solution, agarose is coagulated
Peptization is in 5mL coupling buffer (0.1M Na2CO3, 0.5M NaCl, pH=8.3), add 0.6mg Dihydrocapsaicin Anti-TNF-α
Body, stirs 2h with 150rpm at ambient temperature, obtains Ago-Gel solution, and Ago-Gel solution is transferred to Sha Shi leakage
In bucket so that do not flow out with the antibody-solutions of agarose gel coupling, it is then that the 5 times of couplings of Ago-Gel solution are delayed with volume
Dissolved liquid rinses Ago-Gel, adds blocking buffer (0.1M, pH=8.0 that volume is Ago-Gel solution 2 times
Tris-HCl buffer solution) room temperature reaction 2h, then with high pH (the Tris-HCl buffer solution of 0.1M, pH=8.0) and low pH
After (the Tris-HCl buffer solution of 0.1M, pH=4.0) cushioning liquid alternately rinses gel three times, obtain Dihydrocapsaicin polyclone
Antibody mediated immunity affinity adsorbent.
The preparation of Dihydrocapsaicin polyclonal antibody affinity column: by the immune affinity sorbent of above-mentioned preparation
(0.2mL) it is filled in solid-phase extraction column, adds natural sedimentation after the phosphate buffer of 0.01M, pH=6, with 0.01M, pH
After the phosphate buffer washing of=6, it is stored in the pH=6 containing 0.02% Sodium azide, the phosphate buffer of 0.01M, obtain
Dihydrocapsaicin polyclonal antibody affinity column, stand-by in 4 DEG C of holdings.
Embodiment 5: the mensuration of Dihydrocapsaicin polyclonal antibody affinity column capacity:
Dihydrocapsaicin polyclone immune affinity column 10mL pure water rinsing embodiment 3 or embodiment 4 prepared, will
(concentration is 50ng/mL to the Dihydrocapsaicin standard solution that 10mL10% methanol/water is dissolved, and the content of Dihydrocapsaicin amounts to
For 500ng) cross post, remove unconjugated Dihydrocapsaicin with 10mL pure water rinsing pillar, finally elute with 5mL methanol solution,
Collection is in charge of by 1mL/ pipe, with Dihydrocapsaicin content in LC-MS/MS detection eluent.Result shows, Dihydrocapsaicin polyclone
The column capacity of antibody mediated immunity affinity column is 220-250ng.Cross reaction measurement result simultaneously shows, dihydro capsicum of the present invention
Element immune affine polyclonal antibody affinity column can specific binding Dihydrocapsaicin, and with fenvalerate, decis etc. change
Learn the equal no cross reaction of biotoxin pollutant such as residual contaminants and aflatoxin.
Embodiment 6: the mensuration of the Dihydrocapsaicin polyclonal antibody affinity column recovery of standard addition of embodiment 3:
Take the 20mL vegetable oil blank sample without Dihydrocapsaicin to be placed in 50mL centrifuge tube, be separately added into dihydro capsicum
Element standard concentration, to 2,10,20 μ g/kg, adds 20mL methyl alcohol vibration 10min, and under the conditions of 4 DEG C, 4500rpm is centrifuged 10min, takes
Supernatant liquor, with the 0.45 organic membrane filtration of μm, takes 1.0mL filtrate pure water and is settled to 20mL, obtain sample extracting solution stand-by.Will
Immune affinity column 10mL pure water that embodiment 3 prepares wash-out, adds 10mL sample extracting solution, with 10mL pure water drip washing,
Elute with 0.5mL methanol aqueous solution afterwards, collect machine testing on eluent LC-MS/MS.Above-mentioned experiment repeats 5 times, detection
Result is as shown in table 1: the Dihydrocapsaicin of vegetable oil sample subscripts the rate of recovery all between 83.1-96.3%, and RSD (marks relatively
Quasi-deviation) it is respectively less than 6%, its result shows: the method for the present invention meets vegetable oil sample Dihydrocapsaicin content detection requirement.
Table 1 vegetable oil sample Dihydrocapsaicin subscripts the rate of recovery and relative standard deviation
Embodiment 7: the mensuration of the Dihydrocapsaicin polyclonal antibody affinity column recovery of standard addition of embodiment 4:
Take the 20mL vegetable oil blank sample without Dihydrocapsaicin to be placed in 50mL centrifuge tube, be separately added into dihydro capsicum
Element standard concentration, to 2,10,20 μ g/kg, adds 20mL methyl alcohol vibration 10min, and under the conditions of 4 DEG C, 4500rpm is centrifuged 10min, takes
Supernatant liquor, with the 0.45 organic membrane filtration of μm, takes 1.0mL filtrate pure water and is settled to 20mL, obtain sample extracting solution stand-by.Will
Immune affinity column 10mL pure water that embodiment 4 prepares wash-out, adds 10mL sample extracting solution, with 10mL pure water drip washing,
Elute with 0.5mL methanol aqueous solution afterwards, collect machine testing on eluent LC-MS/MS.Above-mentioned experiment repeats 5 times, detection
Result is as shown in table 1: the Dihydrocapsaicin of vegetable oil sample subscripts the rate of recovery all between 86.5-97.1%, and RSD (marks relatively
Quasi-deviation) it is respectively less than 5%, its result shows: the method for the present invention meets vegetable oil sample Dihydrocapsaicin content detection requirement.
Table 2 vegetable oil sample Dihydrocapsaicin subscripts the rate of recovery and relative standard deviation
Claims (10)
1. a Dihydrocapsaicin polyclonal antibody adsorbent, it is characterised in that: described immunosorbent includes that solid phase carries
The Dihydrocapsaicin polyclonal antibody of coupling on body and this solid phase carrier, described Dihydrocapsaicin polyclonal antibody is employing formula I
Described Dihydrocapsaicin artificial semiantigen (E)-4-[2-methoxyl group-4-(pelargonamide methyl) phenoxy group]-4-oxygen-2-butenoic acid
The Dihydrocapsaicin artificial antigen immune animal obtained with bovine serum albumin(BSA) (BSA) coupling, and isolated and purified postlyophilization
The Dihydrocapsaicin polyclonal antibody obtained,
Dihydrocapsaicin polyclonal antibody adsorbent the most according to claim 1, it is characterised in that: described immunity
Immune animal is repeated for interval after Dihydrocapsaicin artificial antigen is used Freund's complete adjuvant or incomplete Freund's adjuvant emulsification,
Wherein；Initial immunity Freund's complete adjuvant, follow-up immunization incomplete Freund's adjuvant, to antiserum titre reach 4000 with
On, i.e. put to death Balb/c mouse, take whole antiserum；Described purifying is that octanoic acid ammonium sulfate method purifies.
Dihydrocapsaicin polyclonal antibody adsorbent the most according to claim 1, it is characterised in that: described dihydro
After capsicim artificial semiantigen is activated by N, N '-dicyclohexylcarbodiimide and N-hydroxy-succinamide (NHS), then with ox
Seralbumin carries out coupling and obtains.
Dihydrocapsaicin polyclonal antibody adsorbent the most according to claim 1, it is characterised in that: described solid phase carries
Body is Ago-Gel or polyacrylamide silica gel microball.
5. the preparation method of the Dihydrocapsaicin polyclonal antibody adsorbent described in claim 1, it is characterised in that:
When described solid phase carrier selects polyacrylamide silica gel microball, preparation method is: by polyacrylamide silica gel microball with pure
The phosphate buffer of water and pH=6 alternately rinses, with the phosphate buffer of pH=6 join polyacrylamide silica gel microball hangs
Supernatant liquid；Dissolve Dihydrocapsaicin polyclonal antibody with the phosphate buffer of pH=6, be then added dropwise to polyacrylamide silicon
In glue microballoon aaerosol solution, add carbodiimide (EDC), after 4 DEG C of condition stirring reaction 20-22h, obtain with polyacrylamide
Amine silica gel microball is the Dihydrocapsaicin polyclonal antibody adsorbent of solid phase carrier；
When described solid phase carrier selects Ago-Gel, preparation method is: through hydrochloric acid, Ago-Gel is washed removal of impurities repeatedly
It is dissolved in coupling buffer after matter activation, adds above-mentioned Dihydrocapsaicin polyclonal antibody, stir at ambient temperature, obtain fine jade
Sepharose solution, after then not filtering with the antibody-solutions of agarose gel coupling in Ago-Gel solution, uses coupling
Wash buffer Ago-Gel, adds the Tris-HCl buffer solution of 0.1M, pH=8.0, room temperature reaction 1.5-2.5h, then
Alternately rinse agarose with the Tris-HCl buffer solution of 0.1M, pH=8.0 and the Tris-HCl buffer solution of 0.1M, pH=4.0 to coagulate
Glue, removes Dihydrocapsaicin polyclonal antibody and other impurity of non-coupling, obtains the Dihydrocapsaicin polyclonal antibody purified
Immunosorbent.
The preparation method of Dihydrocapsaicin polyclonal antibody adsorbent the most according to claim 5, it is characterised in that:
When described solid phase carrier selects polyacrylamide silica gel microball: polyacrylamide silica gel microball, Dihydrocapsaicin polyclone
Antibody, the mass ratio of carbodiimide are 0.4-1.2g:0.8-4.0mg:28-84mg；The body of the phosphate buffer of described pH=6
Long-pending and polyacrylamide silica gel microball quality ratio is 1-5mL/g；
When described solid phase carrier selects Ago-Gel: described Ago-Gel and the mass ratio of Dihydrocapsaicin polyclonal antibody
For 0.2-0.6g:0.4-2mg；The volume of the coupling buffer of described dissolving Ago-Gel and the ratio of the quality of Ago-Gel
Example is 20-40mL/g.
7. it is mounted with the Dihydrocapsaicin Anti-TNF-α of Dihydrocapsaicin polyclonal antibody adsorbent described in claim 1
Body immune affinity column.
8. the preparation method of the Dihydrocapsaicin polyclonal antibody affinity column described in claim 7, it is characterised in that: concrete
Step is as follows: first the Dihydrocapsaicin polyclonal antibody adsorbent described in claim 1 is filled in solid-phase extraction column
In, add natural sedimentation after the phosphate buffer of 0.01M, pH=6, then wash with the phosphate buffer of 0.01M, pH=6
Wash, be stored in the pH 6 containing 0.02wt% Sodium azide, the phosphate buffer of 0.01M.
9. the application of the Dihydrocapsaicin polyclonal antibody affinity column described in claim 7, it is characterised in that utilize right
Require that the sample extracting solution before upper machine is enriched with and purifies by the Dihydrocapsaicin polyclonal antibody affinity column described in 7.
Application the most according to claim 9, it is characterised in that: concrete operations are: first by two described in claim 7
Hydrogen capsicim polyclonal antibody affinity column pure water rinsing, adds pending sample extracting solution, then uses pure water drip washing,
After dried liquid stream, by methanol-eluted fractions, collecting eluent, described eluent is the sample extracting solution purified and after enrichment.
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| CN106084059B (en) * | 2016-05-26 | 2019-10-25 | 中国农业科学院油料作物研究所 | The general specific antibody of anti-Capsaicinoids, test strips and kitchen waste grease immunochromatography method for quick identification |
| CN112707839B (en) * | 2019-10-24 | 2022-03-15 | 中国农业大学 | Capsaicin hapten and artificial antigen as well as preparation method and application thereof |
| CN110981744B (en) * | 2019-11-08 | 2020-09-22 | 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 |
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