CN105652005B - Quantitatively detect time-resolved fluoroimmunoassay chromatograph test strip, the preparation method and applications of Capsaicinoids - Google Patents
Quantitatively detect time-resolved fluoroimmunoassay chromatograph test strip, the preparation method and applications of Capsaicinoids Download PDFInfo
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Abstract
The present invention relates to the time-resolved fluoroimmunoassay chromatograph test strip of quantitative detection Capsaicinoids, preparation method and applications.Anti- capsaicine, Dihydrocapsaicin, the general monoclonal antibody reagent of synthetic capsaicin and the reaction bulb that it includes fluorescent test paper strip, europium marks, wherein：The fluorescent test paper strip includes cardboard, the adhesive faces of cardboard paste adsorptive pads, detecting pad and sample pad successively from top to bottom, adjacent each pad is in the overlapping connection in junction, described detecting pad is using nitrocellulose filter as base wad, horizontal nature controlling line and detection line are set from top to bottom on nitrocellulose filter, described nature controlling line is coated with rabbit-anti mouse polyclonal antibody, and Capsaicinoids envelope antigen is coated with described detection line；The anti-capsaicine, Dihydrocapsaicin, the general monoclonal antibody of synthetic capsaicin are secreted by the hybridoma cell strain that deposit number is CCTCC NO.C201534 to be produced.It can be used for quantitatively detect Capsaicinoids content, and it is simple to operate, quick, the degree of accuracy is high.
Description
Technical field
The present invention relates to the quantitative detection time-resolved fluoroimmunoassay chromatograph test strip of Capsaicinoids, preparation method and
It is applied.
Background technology
Capsaicinoid compounds are the dominant chemicals for making capsicum have pungent stimulation, and its composition mainly includes capsicum
The compounds such as element, Dihydrocapsaicin, high Dihydrocapsaicin, nordihydrocapsaicin, high capsaicine.Wherein capsaicine and dihydro capsicum
Element accounts for the 90% of Capsaicinoids total amount, and provides about 90% peppery thermal sensation stimulation.Synthetic capsaicin have with it is natural
Capsaicine similar structural portion and function, and it is more dominant than natural capsicum element in price and peppery degree, it is also widely used for
The fields such as medicine, insecticide, antifouling paint, military weapon.In addition to assigning food acid and being used as flavouring, capsaicine
Class material also has anti-inflammatory analgesic, Anti-bacterium, antitumor action, can promote fat combustion, reduce blood fat, gastric acid secretion inhibiting,
There are the medical values such as protective effect to mucosal lesion.Pesticide field, Capsaicinoids are considered as one kind preferably without public affairs
Evil agricultural chemicals, has been reported and is applied on the crops such as water fruits and vegetables, cereal, eliminates drug resistance and residual is examined.In light industry
Field can be used for high-grade special antifouling paint, prevent attachment of the marine aquatic biological to hull；For being driven in cable material
Mouse bites.In field of medicaments, the pharmacokinetic study of Capsaicinoids is be unable to do without to animal tissue and blood serum sample
The detection of middle Capsaicinoids.Additionally it is reported that, Capsaicinoids can be used as anti-depressant main component, quilt
Illegally it is applied in various sports races, particularly in horse racing activity.Therefore, Capsaicinoids analysis determining method in sample
Research it is significant to the Normalization rule of Capsaicinoids and its safety evaluation.
Existing Capsaicinoids analyzing detecting method is mainly precision instrument analytic approach, mainly including high-efficient liquid phase color
Spectrometry, chromatograph-mass spectrometer coupling, these method high sensitivities, accuracy is good, but expensive equipment be present, it is desirable to Capsaicinoids
Sample Purification on Single degree is high, and sample pretreatment process is cumbersome, and time-consuming, and the deficiencies of high is required to experimental situation, it is difficult to realizes quick
Detection, also, Capsaicinoids total amount is measured, it is necessary to calculate the content sum of various Capsaicinoids.It is fast in recent years
The immunoassay method that speed grows up has high specificity, high sensitivity, analysis due to the shortcomings that overcoming the above method
Capacity is big, convenient and swift, cost is cheap, suitable for live batch detection the advantages that, be widely used in each neck such as medical science, agronomy
Domain.
The content of the invention
Problem to be solved by this invention is to provide a kind of time-resolved fluoroimmunoassay for quantitatively detecting Capsaicinoids
Chromatograph test strip, preparation method and applications.
In order to solve the above technical problems, the technical solution adopted by the present invention is：
Quantitatively detect the time-resolved fluoroimmunoassay chromatograph test strip of Capsaicinoids, it is characterised in that：It includes glimmering
Anti- capsaicine, Dihydrocapsaicin, the general monoclonal antibody reagent of synthetic capsaicin and the reaction bulb of light test strips, europium mark, its
In：The fluorescent test paper strip includes cardboard, and the adhesive faces of cardboard paste adsorptive pads, detecting pad and sample pad successively from top to bottom,
Adjacent each pad overlaps connection in junction, and described detecting pad is using nitrocellulose filter as base wad, from upper on nitrocellulose filter
And it is lower horizontal nature controlling line and detection line be set, described nature controlling line is coated with rabbit-anti mouse polyclonal antibody, in described detection line
It is coated with Capsaicinoids envelope antigen；The anti-capsaicine, Dihydrocapsaicin, the general monoclonal antibody of synthetic capsaicin are by protecting
The hybridoma cell strain secretion that numbering is CCTCC NO.C201534 is hidden to produce.Described hybridoma cell strain YQQD8 in
On April 23rd, 2015 is preserved in China typical culture collection center (CCTCC), and preservation address is China, Wuhan, and Wuhan is big
Learn, deposit number is CCTCC NO.C201534.
By such scheme, the anti-Capsaicinoids monoclonal antibody of the europium mark is to be prepared in accordance with the following methods
's：After europium labelled reagent is activated with the concussion of anti-Capsaicinoids monoclonal antibody overnight, target product europium mark is produced
Anti- Capsaicinoids monoclonal antibody.
By such scheme, the preparation method of the anti-Capsaicinoids monoclonal antibody of the europium mark：Europium is marked and tried
Agent ultrasonic disperse adds EDC vibration activation, centrifugation, removes supernatant, it is mixed to add borate buffer vibration in borate buffer
It is even, ultrasonic disperse, then add the general monoclonal antibody of anti-capsaicine, Dihydrocapsaicin, synthetic capsaicin, the wherein examination of europium mark
Agent and the mass ratio of anti-capsaicine, Dihydrocapsaicin, the general monoclonal antibody of synthetic capsaicin are 1:(0.04-0.3), mix
Afterwards, overnight, centrifugation removes uncombined part for shaking table concussion, then adds the bound site of OVA closing europium labelled reagent excess surfaces
Point, obtain the anti-Capsaicinoids monoclonal antibody of target product europium mark.
By such scheme, the molecular structural formula of described Capsaicinoids envelope antigen is as shown in formula II：
Pr carrier proteins.
By such scheme, the carrier protein is bovine serum albumin(BSA) BSA, oralbumin OVA or keyhole limpet hemocyanin
KLH, preferably oralbumin OVA.
By such scheme, adsorptive pads length 35~45mm, wide 3~5mm in the fluorescent test paper strip；Detecting pad length 23~
28mm, wide 3~5mm；Sample pad grows 10~15mm, wide 3~5mm, and the adjacent overlapping length respectively padded is 1~3mm；The fluorescence examination
The spacing on edge is 5~10mm, nature controlling line and detection line spacing in detection line and nitrocellulose filter in paper slip on detecting pad
For 5~10mm.
By such scheme, the Capsaicinoids coating in the fluorescent test paper strip on detecting pad needed for detection line per cm
Antigen coat amount is 0.125~0.6 μ g；The package amount of rabbit-anti mouse polyclonal antibody needed for nature controlling line per cm is 0.1~0.6
μg；The anti-capsaicine of europium mark, Dihydrocapsaicin, the content of the general monoclonal antibody of synthetic capsaicin are in the reaction bulb
0.31~0.53 μ g.
By such scheme, the time-resolved fluoroimmunoassay chromatograph test strip of described quantitative detection Capsaicinoids also wraps
Include sample diluting liquid and sample diluting liquid suction pipe, described sample diluting liquid is that quality volume fraction is 0.01~0.30% to tell
Temperature -20,0.5~1.5% sucrose and 0.1~1% ovalbumin (OVA) aqueous solution.
By such scheme, the preparation process of described fluorescent test paper strip is as follows：
(1) blotting paper is cut out into obtain adsorptive pads；
(2) preparation of detecting pad：
Capsaicinoids envelope antigen is configured to the coating buffer that concentration is 0.2~1.0mg/mL, in away from cellulose nitrate
Along 5~10mm position on plain film, its transverse direction is coated on nitrocellulose filter with line spray mode, obtains detection line, every li
The package amount of Capsaicinoids envelope antigen is 0.125~0.6 μ g needed for rice detection line, and 30 are then dried under the conditions of 37 DEG C
~60 minutes；
Rabbit-anti mouse polyclonal antibody is made into the coating buffer that concentration is 0.15~1.0mg/mL, in away from 5~10mm of detection line
Position, with line spray mode its transverse direction is coated on nitrocellulose filter, obtain nature controlling line, the rabbit-anti needed for nature controlling line per cm
The package amount of mouse 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 pad：
Glass fibre membrane is put into confining liquid and soaked, is taken out, is dried 6~12 hours under the conditions of 37 DEG C, obtains sample pad,
Then room temperature preservation in drier is put；
(4) assembling of fluorescent test paper strip：
Adsorptive pads, detecting pad, sample pad are pasted successively from top to bottom in the adhesive faces of cardboard, and adjacent each pad is handed in junction
Folded connection, overlapping length is 1~3mm, produces fluorescent test paper strip.
Prepared by such scheme, in the preparation of the fluorescent test paper strip Capsaicinoids envelope antigens (4- [(4- hydroxyls-
3- methoxyl groups) benzamido group] -4- oxo carboxylic acids-OVA) the coating buffer solution used in coating buffer is：Contain egg white in per 10mL
Albumen OVA 0.1g, sodium azide 0.002g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g,
Potassium dihydrogen phosphate 0.002g；
Preparing the coating buffer solution used in rabbit-anti mouse polyclonal antibody coating buffer is：It is pure containing ox blood in per 10mL
Albumen 0.1g, sodium azide 0.002g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, phosphoric acid
Potassium dihydrogen 0.002g；
The confining liquid used in the preparation of the fluorescent test paper strip is：Contain disodium hydrogen phosphate in per 100mL
2.9g, two hypophosphite monohydrate sodium dihydrogen 0.3g, tween (Tween) -201.0g, polyvinylpyrrolidone (PVPK-30) 1.0g,
EDTA0.25g, bovine serum albumin(BSA) BSA0.5g, Sodium azide 0.02g.
Application of the above-mentioned time-resolved fluoroimmunoassay chromatograph test strip in Capsaicinoids quantitatively detect：By europium mark
The anti-Capsaicinoids monoclonal antibody of note is added in example reaction bottle, after then adding testing sample solution, is mixed, will be glimmering
In light test strips insertion example reaction bottle so that a part of sample pad is immersed in liquid, after 37 DEG C are reacted 10 minutes, with the time point
Distinguish that fluorometric investigation instrument is detected, it is glimmering to obtain detection line time-resolved fluorescence intensity and nature controlling line time resolution on fluorescent test paper strip
The ratio of luminous intensity；It is glimmering based on the fluorescent test paper strip detection line time-resolved fluorescence intensity being obtained ahead of time and nature controlling line time resolution
The ratio of luminous intensity and the relation curve of Capsaicinoids concentration, obtain containing for Capsaicinoids in testing sample solution
Amount, the content of Capsaicinoids in testing sample is most produced through converting afterwards.
By such scheme, described fluorescent test paper strip detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence
The ratio (T/C) of intensity is obtained with the relation curve of Capsaicinoids concentration using following methods：
(1) prepare and obtain a series of Capsaicinoids standard solution of concentration；
(2) the Capsaicinoids standard solution of appropriate above-mentioned each concentration is added separately in example reaction bottle, added
Enter in example reaction bottle, mix, fluorescent test paper strip is inserted in example reaction bottle, 37 DEG C are reacted 10 minutes, glimmering with time resolution
Light immunity analysis instrument detects to obtain the time-resolved fluorescence intensity level of detection line and nature controlling line on each fluorescent test paper strip, is derived from
The ratio of each fluorescent test paper strip detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity；
(3) fluorescent test paper strip detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity are obtained through fitting
Ratio and Capsaicinoids concentration relation curve.
It is fixed that the time-resolved fluoroimmunoassay chromatograph test strip provided by the invention for quantitatively detecting Capsaicinoids can be used for
Amount detection Capsaicinoids content, and it is simple to operate, quick, the degree of accuracy is high.
Brief description of the drawings
Fig. 1 is glimmering in the time-resolved fluoroimmunoassay chromatograph test strip provided by the invention for quantitatively detecting Capsaicinoids
The structural representation of light test strips.In figure：1 sample pad, 2 detecting pads, 3 detection lines, 4 nature controlling lines, 5 adsorptive pads.
Fig. 2 is the general artificial complete antigen ultraviolet spectrogram of Capsaicinoids that the present invention synthesizes.
The general artificial complete antigen of Capsaicinoids-envelope antigen ultraviolet spectrogram that Fig. 3 present invention synthesizes.
Embodiment
The acquisition of the anti-Capsaicinoids monoclonal antibody of embodiment 1
Anti- Capsaicinoids monoclonal antibody is by hybridoma cell strain that deposit number is CCTCC NO.C201534
Monoclonal antibody caused by YQQD8 secretions, specific preparation method：
The BALB/c mouse that hybridoma cell strain YQQD8 injections are treated with freund 's incomplete adjuvant in advance, collecting should
The ascites of mouse, using caprylic acid-ammonium antibody purification, concrete operations are：With double-layer filter paper filter mouse ascites, 4 DEG C,
12000r/min centrifuges 30min, draws supernatant, gained ascites supernatant is mixed with the acetate buffer of 3 times of volumes, under stirring
Caprylic acid is slowly added to, the caprylic acid volume needed for every milliliter of ascites is 33 μ L, mixed at room temperature 30min, 4 DEG C of standing more than 2h；4
DEG C, 12000r/min centrifugation 30min, precipitation is abandoned, after obtained supernatant is filtered with double-layer filter paper, adds 1/10 filtrate volume
Molar concentration be 0.1mol/L and phosphate buffer that pH value is 7.4, it is mixed to adjust this with 2mol/L sodium hydroxide solution
The pH value of liquid is closed to 7.4,4 DEG C of precoolings, ammonium sulfate is slowly added under condition of ice bath to the final concentration of 0.277g/mL of ammonium sulfate, 4 DEG C
More than 2h is stood, then 4 DEG C, 12000r/min centrifugation 30min, abandons supernatant, by the former ascites volume 1/10 of gained precipitation
0.01mol/L phosphate buffers are resuspended, and load bag filter, and 0.01mol/L phosphate buffers are dialysed, will fully be dialysed
Protein solution put -70 DEG C of refrigerator freezings, freezed afterwards with freeze drier, collect freeze-dried powder, produce purified anti-capsicum
Element, Dihydrocapsaicin, synthetic capsaicin total amount monoclonal antibody, antibody freeze-dried powder are put standby in -20 DEG C of refrigerators；
Described acetate buffer is 0.29g sodium acetates, and 0.141mL acetic acid adds water to be settled to obtained by 100mL；Described
0.1mol/L phosphate buffer is 0.8g sodium chloride, 0.29g disodium hydrogen phosphates, 0.02g potassium chloride, biphosphate
Potassium 0.02g, water is added to be settled to obtained by 100mL.
With the anti-capsaicine, Dihydrocapsaicin, conjunction of the identification hybridoma cell strain YQQD8 secretions of commercially available hypotype identification kit
Hypotype into the general monoclonal antibody of capsaicine is IgG1。
The potency for the mouse hydroperitoneum antibody of RGDV for measuring YQQD8 with conventional non-competing Enzyme Linked Immunoadsorbent Assay (ELISA) method is reachable
2.56×106, i.e. mouse hydroperitoneum antibody of RGDV dilution 2.56 × 106Times when solution measurement result for the positive.With conventional indirect competition
ELISA method identifies that its 50% inhibition concentration IC50 to Capsaicinoids is 5.8ng/mL, to capsaicine, Dihydrocapsaicin,
50% inhibition concentration IC of synthetic capsaicin508.5ng/mL, 5.0ng/mL, 13.5ng/mL are followed successively by, it is peppery to capsaicine, dihydro
Green pepper element, the cross reacting rate scope of synthetic capsaicin are 62.9%-170%.
Hybridoma cell strain YQQD8 acquisition：
The preparation of the general artificial immunity antigen of a Capsaicinoids
The preparation of the general artificial semiantigen of Capsaicinoids：
Weigh Vanillylamine hydrochloride 0.28g and be dissolved in 6ml tetrahydrofurans, agitation and dropping 0.15g triethylamines, be stirred at room temperature
30min.The accurate succinic anhydride 0.15g (0.0015mol) that weighs is added in above-mentioned reaction solution, is stirred overnight at room temperature.To reaction solution
2min is stirred at room temperature in middle addition 3mL ethyl acetate, and gained precipitation is that haptens is 4- [(4- hydroxy-3-methoxies) after filtering
Benzamido group] -4- oxo carboxylic acids (4- [(4-hydroxy-3-methoxybenzyl) amino] -4-oxobutanoic acid),
Molecular formula is C12H15NO5。
It is through nuclear-magnetism qualification result：1H NMR(400MHz,DMSO)δ12.17(s,1H),8.87(s,1H),8.31(d,J
=6.0Hz, 1H), 4.21 (d, J=5.8Hz, 2H), 3.80 (s, 3H), 2.52 (t, J=6.9Hz, 2H), 2.42 (t, J=
6.7Hz,2H).It is consistent with the theoretical value of its result, show that the hapten compound successfully synthesizes.
The preparation of the general artificial complete antigen-immunizing antigen of Capsaicinoids
Weigh the general artificial semiantigen 20.3mg (about 0.08mmol) of above-mentioned Capsaicinoids and 11.6mg (about
0.1mmol) NHS adds 400ulDMF and is dissolved in reaction bulb, 30min is stirred at room temperature, weighs 20.6mg (about in reaction bulb
0.1mmol) DCC is dissolved in 100ulDMF, and DCC/DMF solution is added dropwise into above-mentioned reaction bulb dropwise, and 4h is stirred at room temperature, and 4 DEG C quiet
Put overnight.8000rpm/5min, take the active ester liquid of supernatant.
By the active ester liquid of supernatant, it is added drop-wise in 6ml 7mg/ml BSA solution and reacts dropwise, reaction buffer 0.2M
PH8.0 phosphate buffer.Room temperature carries out 4h under magnetic stirring for reaction.Reaction solution is put in bag filter, uses 0.01M
4 DEG C of stirring dialysis, a dialyzate are changed per 4h, dialyse 72h altogether in pH7.4 PBS.Obtain capsaicine artificial antigen-exempt from
Epidemic disease antigen.The continuous scanning spectra of ultraviolet-visible spectrum is shown in Fig. 1, and qualification result shows that artificial antigen is coupled successfully.
The preparation of the general artificial complete antigen-envelope antigen of Capsaicinoids
Deserve to be called and state the general artificial semiantigen 4.55mg of Capsaicinoids and be dissolved in 200 μ L dry DMFs, then in order plus
Enter 4.27 μ L tri-n-butylamines, 2.34 μ L isobutyl chlorocarbonates, at room temperature stirring reaction 1h, the Capsaicinoids half for obtaining activation are anti-
Original solution.
The Capsaicinoids haptens solution of activation is added drop-wise in 10ml 4.5mg/ml OVA solution dropwise and reacted,
Reaction buffer is 0.2M pH8.0 phosphate buffer.Room temperature carries out 4h under magnetic stirring for reaction.Reaction solution is put
Analyse in bag, with 4 DEG C of stirring dialysis in 0.01M pH7.4 PBS, a dialyzate is changed per 4h, dialyse 72h altogether.Obtain peppery
Green pepper element artificial antigen-envelope antigen.The continuous scanning spectra of ultraviolet-visible spectrum is shown in Fig. 3, and qualification result shows that artificial antigen is coupled
Success.
B hybridoma cell strains YQQD8 preparation
1. animal immune
7 week old BALB/c mouse 5 is bought, above-mentioned Capsaicinoids artificial antigen-immunizing antigen is immunized.Exempt from for the first time
Epidemic disease is (limited purchased from Beijing Bo Aolong biotechnologys by immunizing antigen and isometric fast adjuvant-free QuickAntibody-Mouse5W
Company) it is rapid mix after, in mouse back leg Calf muscle injecting immune mouse.Strengthen respectively by the same manner within 21st day, 42 days
It is immune.The dosage of 3 immune immunizing antigens used is identical, is every μ g of mouse 12.5.It is immune latter 8 days every time, tail vein blood, point
From serum, mice serum potency is monitored using indirect elisa method.3rd time immune latter 8 days, tail vein blood, separates serum, adopts
With indirect elisa method monitor mice serum potency, and with indirect competitive ELISA method determine mice serum sensitivity, select potency,
Mouse corresponding to the of a relatively high serum of sensitivity carries out last time booster immunization, during booster immunization, using mouse peritoneal
Inject peak mode, the dosage of immunizing antigen be before 2 times, it is not necessary to mixed with adjuvant.
2. cell fusion
After last time booster immunization 3 days, use 50% (percetage by weight) polyethylene glycol i.e. PEG (molecular weight for
1500) make fusion agent, carry out cell fusion, specific steps according to a conventional method：Immune mouse is killed under aseptic condition, separation spleen is thin
Born of the same parents, mixed with mouse source myeloma cell SP2/0 with about 5-10 ︰ 1 number ratio, it is thin to wash mixing with RPMI-1640 basic culture solutions
Born of the same parents, merged with 50%PEG, merge 1 minute, then fill it up with RPMI-1640 basic culture solutions, centrifuged, remove supernatant, used
Mouse boosting cell and mouse source myeloma cell the SP2/0 fused cell formed are resuspended 72mLRPMI-1640 complete culture solutions, will
Cell is resuspended to be added drop-wise in 96 porocyte culture plates, 2 drops/hole, puts 37 DEG C of CO2gas incubator cultures, described RPMI-
1640 complete culture solutions contain 20% (percentage by volume) hyclone, 10% (percentage by volume)Culture medium
It is HAT with 1% (percetage by weight) hypoxanthine-aminopterin-thymidine；Semi-solid RPMI-1640 is cultivated completely
Base refers to, the methylcellulose of 1% (mass percent) is added in RPMI-1640 complete culture solutions.Above-mentioned SP2/0 is purchased from
Ingression Ke bio tech ltd；Culture medium is purchased from Beijing Bo Aolong Immune Technology Corp.；RPMI-
1640 basic culture solutions are purchased from Hyclone companies；1% hypoxanthine-aminopterin-thymidine is that HAT is purchased from
Sigma-Aldrich companies.
3. screening and the clone of cell line
Treat that the cell colony on 96 orifice plates is grown to and account for the size of bottom hole 1/2, nutrient solution turns yellow, you can carry out antibody test.Adopt
The culture hole for having Growth of Hybridoma Cell is screened with ELISA method, screening is carried out in two steps, and the first step is using indirect
ELISA method filters out positive hole of the anti-capsaicine without anti-carrier protein BSA；Second step is using indirect competitive ELISA method to the
The positive hole that one step filters out is detected, former by the use of capsaicine as competition, with the antigenic competition that is fixed on elisa plate
With reference to the antibody in supernatant, wherein the antibody combined with capsaicine will be washed off afterwards the step of, combined with envelope antigen
Antibody be then fixed on elisa plate, add HRP mark sheep anti-mouse antibody and nitrite ion after can then develop the color.Competition is former peppery
Green pepper element concentration is higher, and colour developing is more shallow, and the absorbance at 450nm is lower.Light absorption value is higher and sensitive when selection is not plus competition is former
The higher hole of degree (the higher finger of light absorption value herein, competition be originally 0 hole measured by light absorption value, the higher inhibiting rate that refers to of sensitivity is
Competition original content IC when 50%50Value is smaller).The culture of subcloned cells is carried out using semisolid culturemedium, is treated after clone thin
During born of the same parents' colony length to the visible size of naked eyes, each cell colony is chosen into fluid nutrient medium respectively, sieved using two same steps
Method is selected to be detected, after such repeated cloning 1 time, final screening obtains hybridoma cell strain YQQD8.Cell line YQQD8 is
China typical culture collection center (CCTCC) is preserved on April 23rd, 2015, preservation address is China, Wuhan, Wuhan
University, deposit number are CCTCC NO.C201534, and Classification And Nomenclature is hybridoma cell strain YQQD8.
The general monoclonal antibody hybridoma cell strain system YQQD8 antibody of the anti-capsaicines of c, Dihydrocapsaicin, synthetic capsaicin
Variable region sequences determine
(1) total serum IgE is extracted：Using Tiangeng company total RNA extraction reagent box and to specifications extraction can produce hybridization
Oncocyte strain YQQD8 total serum IgE；
(2) cDNA is synthesized：For the total serum IgE obtained using step 1 as template, oligo (dT) 15 is primer, according to
The reverse transcriptase specifications of SuperScriptTM-2 II carry out reverse transcription, synthesize the chains of cDNA first；Primer oligo (dT) 15 by
Invitrogen is bought；
(3) PCR methods clone variable region gene：Drawn according to the conserved positions design of mouse antibody gene sequence in GENEBANK
Thing, antibody light and heavy chain variable region gene is expanded by masterplate of cDNA.PCR programs are：94 DEG C of 30s, 55 DEG C of 45s, 72 DEG C of 1min,
30 circulations of amplification, last 72 DEG C of extensions 10min.Agarose gel electrophoresis separation of the PCR primer Jing Guo 1% (percetage by weight)
Afterwards, DNA fragmentation is reclaimed with kits, be connected in carrier pMD18-T, converted bacillus coli DH 5 alpha competent cell, choose
Positive colony is taken, Shanghai Sani bio tech ltd is delivered to and is sequenced.The sequence of wherein primer is respectively：Weight chain variable
Area's primer is 5 ,-GAV GTG AWG STG GTG GAG TC-3, (20mer) and 5 ,-GAG GAG ACG GTG ACT GAG
GT-3, (20mer) wherein S, R and W are degeneracy base, M=A/C, R=A/G, S=C/G, W=A/T, and light chain variable district primer is
5 ,-RTT KTG ATG ACC CAR AC-3, (17mer) and 5 ,-ACG TTT KAT TTC CAG CTT GG-3, (20mer).
Obtained gene order result：The long 313bp of weight chain variable district coding gene sequence, sequence such as SEQ ID NO:1 institute
Show, the weight chain variable district according to coded by the gene order obtained derives the gene order is made up of 104 amino acid, sequence
Row such as SEQ ID NO:Shown in 3.The long 299bp of light chain variable district coding gene sequence, sequence such as SEQ ID NO:Shown in 2, according to
The gene order obtained derives that the light chain variable district coded by the gene order is made up of 99 amino acid, sequence such as SEQ
ID NO:Shown in 4.
Embodiment 2：Quantitatively detect time-resolved fluoroimmunoassay chromatograph test strip and its preparation of Capsaicinoids
The time-resolved fluoroimmunoassay chromatograph test strip of Capsaicinoids is quantitatively detected, it includes fluorescent test paper strip, contained
There are anti-Capsaicinoids monoclonal antibody, reaction bulb, sample diluting liquid and the sample diluting liquid suction pipe that europium marks, described is glimmering
Light test strips include cardboard, and the one side of cardboard pastes adsorptive pads, detecting pad and sample pad successively from top to bottom, and adjacent each pad is even
Connect and locate overlapping connection, overlapping length 1mm, wherein：Adsorptive pads long 38mm, wide 3.8mm；Detecting pad long 25mm, wide 3.8mm；Sample
Product pad long 15mm, wide 3.8mm.The detecting pad is set from top to bottom using nitrocellulose filter as base wad on nitrocellulose filter
Horizontal nature controlling line and detection line, the nature controlling line are coated with rabbit-anti mouse polyclonal antibody, and its package amount is 0.25 μ g/cm Quality Controls
Line, in the detection line coating Capsaicinoids envelope antigen be the general artificial complete antigen of above-mentioned Capsaicinoids-
Envelope antigen, its package amount are 0.4 μ g/cm detection lines, and the spacing on detection line and edge on nitrocellulose filter is 9mm, nature controlling line
Spacing with detection line is 8mm.
The acquisition of the fluorescent test paper strip：
(1) preparation of adsorptive pads
Blotting paper is cut out into growth 38mm, wide 3.8mm specification, produces adsorptive pads；
(2) preparation of detecting pad
The coating of detection line：
The coating buffer for being 0.5mg/mL into concentration with coating buffer by Capsaicinoids envelope antigen；In away from nitre
Along 9mm position on acid cellulose film, its transverse direction is coated on nitrocellulose filter with line spray mode, obtains detection line, often
The package amount of centimetre envelope antigen needed for detection line is 0.4 μ g, is then dried 60 minutes under the conditions of 37 DEG C；
Described coating buffer solution is：Contain ovalbumin OVA 0.1g, sodium azide 0.002g, sodium chloride in per 10mL
0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g；
The coating of nature controlling line：
Rabbit-anti mouse polyclonal antibody is made into the coating buffer that concentration is 0.25mg/mL with coating buffer solution；In away from detection line
8mm position, its transverse direction is coated on nitrocellulose filter with line spray mode, obtains nature controlling line, needed for nature controlling line per cm
The package amount of rabbit-anti mouse polyclonal antibody is 0.25 μ g, is then dried 2 hours under the conditions of 37 DEG C；
Described coating buffer solution is：
Contain bovine serum albumin(BSA) 0.1g, sodium azide 0.002g, sodium chloride 0.08g, ten hydrogen phosphate dihydrates in per 10mL
Disodium 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g；
Described nitrocellulose filter long 25mm, wide 3.8mm.
(3) preparation of sample pad：
Glass fibre membrane is cut out into growth 15mm, wide 3.8mm specification, is put into confining liquid and soaks, take out, in 37 DEG C of bars
Dried 6 hours under part, obtain sample pad, then put room temperature preservation in drier；
Described confining liquid is 2.9g disodium hydrogen phosphates, the hypophosphite monohydrate sodium dihydrogens of 0.3g bis-, 1.0g Tween-
20,1.0g polyvinylpyrrolidones (PVPK-30), 0.25g EDTA, 0.5g bovine serum albumin(BSA) BSA, 0.02g Sodium azides, add
Water is settled to obtained by 100mL；
(4) assembling of fluorescent test paper strip：
Adsorptive pads, detecting pad and sample pad are pasted successively from top to bottom in the one side of cardboard, and adjacent each pad is handed in junction
Folded connection, overlapping length 2mm, produces fluorescent test paper strip.
The acquisition of the anti-Capsaicinoids monoclonal antibody of the europium mark：
Take the 0.2mol/L pH 8.18 μ L of borate buffer 800, add 200 μ L europiums labelled reagents (particle diameter 100nm, Gu
Shape thing content 1%), vibration mixes.It is cleaned by ultrasonic instrument, ultrasonic 10min with 800W.40 μ L 15mg/mL EDC solutions are added, are shaken
Swing and mix 15min.14000r/min, 10 DEG C, 10min is centrifuged, removes supernatant, added 1mL borate buffers and redissolve, vibration is mixed
It is even, ultrasonic 10min.80 μ g Capsaicinoids monoclonal antibodies are added, are mixed, 250r/min, incubator overnight at 25 DEG C.Again
Supernatant is removed in centrifugation, adds borate buffers of the 1mL containing 0.5%OVA and redissolves, and vibration mixes, ultrasonic 10min, shaking table 2h at 25 DEG C,
Obtain the anti-Capsaicinoids monoclonal antibody of target product europium mark.Above-mentioned europium labelled reagent can be purchased from Shanghai uricytin thing section
Skill Co., Ltd, but not limited to this.
Application of the above-mentioned time-resolved fluoroimmunoassay chromatograph test strip in Capsaicinoids quantitatively detect：
The ratio (T/C) of I fluorescent test paper strip detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity
With the foundation of the relation curve of Capsaicinoids concentration：
(1) to being detected as Capsaicinoids through high performance liquid chromatography (HPLC) as negative balb/c mice serum samples
Product examine survey liquid carry out Capsaicinoids mark-on, with 62.5ng/mL, 31.25ng/mL, 15.625ng/mL, 7.8125ng/
ML, 3.90625ng/mL, 1.95313ng/mL, 0.97656ng/mL and 0ng/mL Capsaicinoids standard solution, its
Middle capsaicine, Dihydrocapsaicin, the ratio of synthetic capsaicin are 1:1:1；
(2) after the anti-Capsaicinoids monoclonal antibody sample sustained-release liquid for marking above-mentioned europium dilutes 100 times, 18 are taken
μ L (about 0.37 μ g label probes) are put in 300 μ L reaction bulbs, described sample diluting liquid be quality volume fraction be 0.01~
0.30% Tween-20,0.5~1.5% sucrose and 0.1~1% ovalbumin (OVA) aqueous solution.
Each 180 μ L of Capsaicinoids standard solution of above-mentioned each concentration are taken, are added separately to containing the anti-of europium mark
In the example reaction bottle of Capsaicinoids monoclonal antibody, mix, insert fluorescent test paper strip, 37 DEG C are reacted 10 minutes, with suction
Water paper blots sample pad residual liquid, detects (excitation wavelength with time-resolved fluorescence immunoassay instrument at once：365nm, determine ripple
It is long：The time-resolved fluorescence intensity level at (T) and nature controlling line (C) place at detection line on each fluorescent test paper strip 615nm) is obtained, thus
Obtain the ratio (T/C) of each fluorescent test paper strip detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity；
(3) using Capsaicinoids concentration as abscissa, the Capsaicinoids standard solution of each concentration is examined accordingly
The ratio of survey line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity is that T/C values are ordinate, and fitting obtains glimmering
The ratio (T/C) and capsaicine class thing of light ELISA test strip line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity
The relation curve of matter concentration.The valid analysing range of this method is 2.3~62.5ng/mL.
Ⅱ：The recovery testu of Capsaicinoids content in mouse blood serum sample：
High performance liquid chromatography of learning from else's experience (HPLC) is detected as Capsaicinoids as negative mouse blood serum sample, with 10% first
4 times of dilutions of alcohol/PBS (v/v), produce mouse blood serum sample detection liquid.Thereto respectively accurate addition 20ng/mL, 40ng/mL and
(wherein capsaicine, Dihydrocapsaicin, the ratio of synthetic capsaicin are 1 to 80ng/mL Capsaicinoids standard items:1:1), mix
It is even, obtain each test serum sample detection liquid；
Take the above-mentioned μ L of test serum sample detection liquid 180 to add in example reaction bottle respectively, mix, insert fluorescent test paper
Bar, after 37 DEG C are reacted 10 minutes, detect (excitation wavelength with time-resolved fluorescence immunoassay instrument immediately：365nm, determine wavelength：
615nm), the ratio of each fluorescent test paper strip detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity is obtained
(T/C) fluorescent test paper strip detection line time-resolved fluorescence intensity obtained above and nature controlling line time resolution, are then substituted into
The ratio (T/C) of fluorescence intensity and the relation curve of Capsaicinoids concentration, obtain the Capsaicinoids in sample solution
Concentration, Capsaicinoids content in sample then can be tried to achieve according to extension rate.Measure the capsaicine in mouse blood serum sample
Class content of material recovery of standard addition is followed successively by：94.1%, 90.3.5%, 108.5%.
By this method testing result compared with the testing result of high performance liquid chromatography standard method, obtain：This method is examined
Survey result and the testing result of high performance liquid chromatography standard method is highly consistent, coincidence rate is up to 98%.
Embodiment 3：Quantitatively detect time-resolved fluoroimmunoassay chromatograph test strip and its application of Capsaicinoids
The time-resolved fluoroimmunoassay chromatograph test strip of Capsaicinoids is quantitatively detected, it includes fluorescent test paper strip, contained
There are example reaction bottle, sample diluting liquid and the sample diluting liquid suction pipe of the anti-Capsaicinoids monoclonal antibody of europium mark, institute
The fluorescent test paper strip stated includes cardboard, and the one side of cardboard pastes adsorptive pads, detecting pad and sample pad successively from top to bottom, adjacent each
Pad is overlapped in junction and connected, overlapping length 1mm, wherein：Adsorptive pads long 41mm, wide 4mm；Detecting pad long 25mm, wide 4mm；
Sample pad long 13mm, wide 4mm.The detecting pad is set from top to bottom using nitrocellulose filter as base wad on nitrocellulose filter
Horizontal nature controlling line and detection line, the nature controlling line are coated with rabbit-anti mouse polyclonal antibody, and its package amount is 0.15 μ g/cm Quality Controls
Line, Capsaicinoids envelope antigen is coated with the detection line, its package amount is 0.3 μ g/cm detection lines, detection line and nitric acid
The spacing on edge is 10mm on cellulose membrane, and the spacing of nature controlling line and detection line is 5mm.
The acquisition of the fluorescent test paper strip：
(1) preparation of adsorptive pads
Blotting paper is cut out into growth 41mm, wide 4mm specification, produces adsorptive pads；
(2) preparation of detecting pad
The coating of detection line：
The coating buffer for being 0.5mg/mL into concentration with coating buffer by Capsaicinoids envelope antigen；In away from nitre
Along 10mm position on acid cellulose film, its transverse direction is coated on nitrocellulose filter with line spray mode, obtains detection line, often
The package amount of centimetre envelope antigen needed for detection line is 0.3 μ g, is then dried 30 minutes under the conditions of 37 DEG C；
Described coating buffer solution is：Contain ovalbumin OVA 0.1g, sodium azide 0.002g, sodium chloride in per 10mL
0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g；
The coating of nature controlling line：
Rabbit-anti mouse polyclonal antibody is made into the coating buffer that concentration is 0.25mg/mL with coating buffer solution；In away from detection line
5mm position, its transverse direction is coated on nitrocellulose filter with line spray mode, obtains nature controlling line, needed for nature controlling line per cm
The package amount of rabbit-anti mouse polyclonal antibody is 0.15 μ g, is then dried 2 hours under the conditions of 37 DEG C；
Described coating buffer solution is：
Contain bovine serum albumin(BSA) 0.1g, sodium azide 0.002g, sodium chloride 0.08g, ten hydrogen phosphate dihydrates in per 10mL
Disodium 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g；
Described nitrocellulose filter long 25mm, wide 4mm.
(3) preparation of sample pad：
Glass fibre membrane is cut out into growth 13mm, wide 4mm specification, is put into confining liquid and soaks, take out, in 37 DEG C of conditions
Lower drying 8 hours, obtains sample pad, then puts room temperature preservation in drier；
Described confining liquid is 2.9g disodium hydrogen phosphates, the hypophosphite monohydrate sodium dihydrogens of 0.3g bis-, 1.0g Tween-
20,1.0g polyvinylpyrrolidones (PVPK-30), 0.25g EDTA, 0.5g bovine serum albumin(BSA) BSA, 0.02g Sodium azides, add
Water is settled to obtained by 100mL；
(4) assembling of fluorescent test paper strip：
Adsorptive pads, detecting pad and sample pad are pasted successively from top to bottom in the one side of cardboard, and adjacent each pad is handed in junction
Folded connection, overlapping length 1mm, produces fluorescent test paper strip.
The acquisition of the anti-Capsaicinoids monoclonal antibody of the europium mark：
The 0.2mol/L pH8.18 μ L of borate buffer 800 are taken, add 200 μ L europiums labelled reagent (particle diameter 100nm, solids
Thing content 1%), vibration mixes.It is cleaned by ultrasonic instrument, ultrasonic 10min with 800W.40 μ L 15mg/mL EDC solutions are added, are vibrated
Mix 15min.14000r/min, 10 DEG C, 10min being centrifuged, removes supernatant, added 1mL borate buffers and redissolve, vibration mixes,
Ultrasonic 10min.160 μ g Capsaicinoids monoclonal antibodies are added, are mixed, 250r/min, incubator overnight at 25 DEG C.Again from
The heart removes supernatant, adds borate buffers of the 1mL containing 0.5%OVA and redissolves, vibration mixes, ultrasonic 10min, shaking table 2h at 25 DEG C, obtains
The anti-Capsaicinoids monoclonal antibody of target product europium mark.Above-mentioned europium labelled reagent can be purchased from Shanghai uricytin thing science and technology
Co., Ltd, but not limited to this.
Described sample diluting liquid is the Tween-20 that quality volume fraction is 0.01~0.30%, 0.5~1.5% sucrose
With 0.1~1% ovalbumin (OVA) aqueous solution.
Application of the above-mentioned time-resolved fluoroimmunoassay chromatograph test strip in Capsaicinoids quantitatively detect：
The ratio (T/C) of I fluorescent test paper strip detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity
With the foundation of the relation curve of Capsaicinoids concentration：
(1) to being detected as Capsaicinoids through high performance liquid chromatography (HPLC) as negative rabbit anteserum sample detection liquid
Carry out Capsaicinoids mark-on, with 62.5ng/mL, 31.25ng/mL, 15.625ng/mL, 7.8125ng/mL,
3.90625ng/mL, 1.95313ng/mL, 0.97656ng/mL and 0ng/mL Capsaicinoids standard solution, wherein；
(2) after the anti-Capsaicinoids monoclonal antibody sample sustained-release liquid for marking above-mentioned europium dilutes 100 times, 20 are taken
μ L (about 0.43 μ g label probes) are put in 300 μ L reaction bulbs, take the Capsaicinoids standard solution of above-mentioned each concentration each
180 μ L, are added separately in example reaction bottle, mix, and insert fluorescent test paper strip, and 37 DEG C are reacted 10 minutes, are blotted with blotting paper
Sample pad residual liquid, detects (excitation wavelength with time-resolved fluorescence immunoassay instrument at once：365nm, determine wavelength：
The time-resolved fluorescence intensity level at (T) and nature controlling line (C) place at detection line on each fluorescent test paper strip 615nm) is obtained, is derived from
The ratio (T/C) of each fluorescent test paper strip detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity；
(3) using Capsaicinoids concentration as abscissa, the Capsaicinoids standard solution of each concentration is examined accordingly
The ratio of survey line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity is that T/C values are ordinate, and fitting obtains glimmering
The ratio (T/C) and capsaicine class thing of light ELISA test strip line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity
The relation curve of matter concentration.The valid analysing range of this method is 1.9~62.5ng/mL.
The recovery testu of Capsaicinoids content in II rabbit anteserum sample：
High performance liquid chromatography of learning from else's experience (HPLC) is detected as Capsaicinoids as negative rabbit anteserum sample, with 10% first
4 times of dilutions of alcohol/PBS (v/v), produce rabbit anteserum sample detection liquid.Thereto respectively accurate addition 20ng/mL, 40ng/mL and
(wherein capsaicine, Dihydrocapsaicin, the ratio of synthetic capsaicin are 1 to 80ng/mL Capsaicinoids standard items:1:1), mix
It is even, obtain each test serum sample detection liquid；
Take the above-mentioned μ L of test serum sample detection liquid 180 to add in example reaction bottle respectively, mix, insert fluorescent test paper
Bar, after 37 DEG C are reacted 10 minutes, detect (excitation wavelength with time-resolved fluorescence immunoassay instrument immediately：365nm, determine wavelength：
615nm), the ratio of each fluorescent test paper strip detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity is obtained
(T/C) fluorescent test paper strip detection line time-resolved fluorescence intensity obtained above and nature controlling line time resolution, are then substituted into
The ratio (T/C) of fluorescence intensity and the relation curve of Capsaicinoids concentration, obtain the Capsaicinoids in sample solution
Concentration, Capsaicinoids content in sample then can be tried to achieve according to extension rate.Measure the capsaicine class in blood serum sample
Content of material recovery of standard addition is followed successively by：91.0%, 89.5%, 111.5%.
By this method testing result compared with the testing result of high performance liquid chromatography standard method, obtain：This method is examined
Survey result and the testing result of high performance liquid chromatography standard method is highly consistent, coincidence rate is up to 96%；In addition, this method sample detection
Complicated pre-treatment is not needed, also the standard method of more efficient liquid chromatogram is few for required time, significantly improves detection speed.
Claims (10)
1. quantitatively detect the time-resolved fluoroimmunoassay chromatographic test paper bar assembly of Capsaicinoids, it is characterised in that：It includes
Anti- capsaicine, Dihydrocapsaicin, the general monoclonal antibody reagent of synthetic capsaicin and the example reaction of fluorescent test paper strip, europium mark
Bottle, wherein：The fluorescent test paper strip includes cardboard, and the adhesive faces of cardboard paste adsorptive pads, detecting pad and sample successively from top to bottom
Product pad, adjacent each pad overlap connection in junction, and described detecting pad is using nitrocellulose filter as base wad, on nitrocellulose filter
Horizontal nature controlling line and detection line are set from top to bottom, and described nature controlling line is coated with rabbit-anti mouse polyclonal antibody, described detection
Capsaicinoids envelope antigen is coated with line；The anti-capsaicine, Dihydrocapsaicin, the general monoclonal antibody of synthetic capsaicin
Secreted and produced by the hybridoma cell strain that deposit number is CCTCC NO.C201534.
2. the time-resolved fluoroimmunoassay chromatograph test strip group according to claim 1 for quantitatively detecting Capsaicinoids
Part, it is characterised in that：The anti-capsaicine of europium mark, Dihydrocapsaicin, the general monoclonal antibody of synthetic capsaicin be according to
What following methods were prepared：By europium labelled reagent activate after with anti-capsaicine, Dihydrocapsaicin, synthetic capsaicin general purpose single gram
Grand antibody mixing, concussion overnight, produce the anti-capsaicine, Dihydrocapsaicin, synthetic capsaicin general purpose single of target product europium mark
Clonal antibody.
3. the time-resolved fluoroimmunoassay chromatograph test strip group according to claim 2 for quantitatively detecting Capsaicinoids
Part, it is characterised in that：The preparation of the anti-capsaicine, Dihydrocapsaicin, the general monoclonal antibody of synthetic capsaicin of the europium mark
Method：By europium labelled reagent ultrasonic disperse in borate buffer, EDC vibration activation is added, centrifugation, supernatant is removed, adds
Borate buffer vibration mixes, and ultrasonic disperse, then adds the general monoclonal of anti-capsaicine, Dihydrocapsaicin, synthetic capsaicin
Antibody, mix, overnight, centrifugation removes uncombined part for shaking table concussion, then adds OVA closing europium labelled reagent excess surfaces
Binding site, obtain the anti-capsaicine, Dihydrocapsaicin, the general monoclonal antibody of synthetic capsaicin of target product europium mark.
4. the time-resolved fluoroimmunoassay chromatograph test strip group according to claim 1 for quantitatively detecting Capsaicinoids
Part, it is characterised in that：The molecular structural formula of described Capsaicinoids envelope antigen is as shown in formula II：
Pr carrier proteins, the carrier protein are bovine serum albumin(BSA) BSA, oralbumin OVA or keyhole limpet hemocyanin KLH.
5. the time-resolved fluoroimmunoassay chromatograph test strip group according to claim 1 for quantitatively detecting Capsaicinoids
Part, it is characterised in that：The time-resolved fluoroimmunoassay chromatographic test paper bar assembly for quantitatively detecting Capsaicinoids also includes
Sample diluting liquid and sample diluting liquid suction pipe, described sample diluting liquid are that quality volume fraction is 0.01~0.30% to tell
Temperature -20, the aqueous solution that quality volume fraction is 0.5~1.5% sucrose and quality volume fraction is 0.1~1% ovalbumin；
Adsorptive pads length 35~45mm, wide 3~5mm in the fluorescent test paper strip；Detecting pad grows 23~28mm, wide 3~5mm；Sample
Product pad grows 10~15mm, wide 3~5mm, and the adjacent overlapping length respectively padded is 1~3mm；In the fluorescent test paper strip on detecting pad
The spacing on edge is 5~10mm on detection line and nitrocellulose filter, and the spacing of nature controlling line and detection line is 5~10mm.
6. the time-resolved fluoroimmunoassay chromatograph test strip group according to claim 1 for quantitatively detecting Capsaicinoids
Part, it is characterised in that：Capsaicinoids envelope antigen in the fluorescent test paper strip on detecting pad needed for detection line per cm
Package amount is 0.125~0.6 μ g；The package amount of rabbit-anti mouse polyclonal antibody needed for nature controlling line per cm is 0.1~0.6 μ g；
The anti-capsaicine of europium mark, Dihydrocapsaicin, the content of the general monoclonal antibody of synthetic capsaicin are in the example reaction bottle
0.31~0.53 μ g.
7. the time-resolved fluoroimmunoassay chromatograph test strip group according to claim 1 for quantitatively detecting Capsaicinoids
Part, it is characterised in that：The preparation process of described fluorescent test paper strip is as follows：
(1) blotting paper is cut out into obtain adsorptive pads；
(2) preparation of detecting pad：
Capsaicinoids envelope antigen is configured to the coating buffer that concentration is 0.2~1.0mg/mL, in away from nitrocellulose filter
On along 5~10mm position, its transverse direction is coated on nitrocellulose filter with line spray mode, obtains detection line, inspection per cm
The package amount of Capsaicinoids envelope antigen needed for survey line is 0.125~0.6 μ g, and 30~60 are then dried under the conditions of 37 DEG C
Minute；
Rabbit-anti mouse polyclonal antibody is made into the coating buffer that concentration is 0.15~1.0mg/mL, in the position away from 5~10mm of detection line
Put, its transverse direction is coated on nitrocellulose filter with line spray mode, obtains nature controlling line, the rabbit-anti mouse needed for nature controlling line per cm is more
The package amount of clonal 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 pad：
Glass fibre membrane is put into confining liquid and soaked, is taken out, is dried 6~12 hours under the conditions of 37 DEG C, obtains sample pad, then
Put room temperature preservation in drier；
(4) assembling of fluorescent test paper strip：
Adsorptive pads, detecting pad, sample pad are pasted successively from top to bottom in the adhesive faces of cardboard, and adjacent each pad is in the overlapping company in junction
Connect, overlapping length is 1~3mm, produces fluorescent test paper strip.
8. the time-resolved fluoroimmunoassay chromatograph test strip group according to claim 1 for quantitatively detecting Capsaicinoids
Part, it is characterised in that：Used in the coating buffer that Capsaicinoids envelope antigen is prepared in the preparation of the fluorescent test paper strip
Coating buffer solution be：Contain ovalbumin OVA 0.1g, sodium azide 0.002g, sodium chloride 0.08g, Shi Ershui in per 10mL
Disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g；
Preparing the coating buffer solution used in rabbit-anti mouse polyclonal antibody coating buffer is：Contain bovine serum albumin(BSA) in per 10mL
0.1g, sodium azide 0.002g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, biphosphate
Potassium 0.002g；
The confining liquid used in the preparation of the fluorescent test paper strip is：Per in 100mL containing disodium hydrogen phosphate 2.9g, two
Hypophosphite monohydrate sodium dihydrogen 0.3g, tween (Tween) -20 1.0g, polyvinylpyrrolidone (PVP) K-30 1.0g,
EDTA0.25g, bovine serum albumin(BSA) BSA 0.5g, Sodium azide 0.02g.
9. the time-resolved fluoroimmunoassay chromatographic test paper bar assembly of the quantitative detection Capsaicinoids described in claim 1 is peppery
Green pepper element class material quantitatively detect in application：By the anti-capsaicine, Dihydrocapsaicin, the general monoclonal of synthetic capsaicin of europium mark
Antibody is added in example reaction bottle, after then adding testing sample solution, is mixed, and fluorescent test paper strip is inserted into example reaction bottle
In so that a part of sample pad is immersed in liquid, after 37 DEG C are reacted 10 minutes, is detected with time-resolved fluorescence tester,
Obtain the ratio of detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity on fluorescent test paper strip；Based on advance
The fluorescent test paper strip detection line time-resolved fluorescence intensity of acquisition and the ratio and capsaicine of nature controlling line time-resolved fluorescence intensity
The relation curve of class material concentration, obtain testing sample solution in Capsaicinoids content, most afterwards through convert produce it is to be measured
The content of Capsaicinoids in sample.
10. application according to claim 9, it is characterised in that：Described fluorescent test paper strip detection line time-resolved fluorescence
Intensity is to use following methods with the ratio of nature controlling line time-resolved fluorescence intensity and the relation curve of Capsaicinoids concentration
Obtain：
(1) prepare and obtain a series of Capsaicinoids standard solution of concentration；
(2) the Capsaicinoids standard solution of appropriate above-mentioned each concentration is added separately in example reaction bottle, adds sample
In product reaction bulb, mix, fluorescent test paper strip is inserted in example reaction bottle, 37 DEG C are reacted 10 minutes, are exempted from time-resolved fluorescence
Epidemic disease analyzer detects to obtain the time-resolved fluorescence intensity level of detection line and nature controlling line on each fluorescent test paper strip, is derived from each glimmering
The ratio of light ELISA test strip line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity；
(3) ratio of fluorescent test paper strip detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity is obtained through fitting
The relation curve of value and Capsaicinoids concentration.
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