CN105272915B - Hapten compound and its more anti-, kits and application - Google Patents
Hapten compound and its more anti-, kits and application Download PDFInfo
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- CN105272915B CN105272915B CN201410334134.7A CN201410334134A CN105272915B CN 105272915 B CN105272915 B CN 105272915B CN 201410334134 A CN201410334134 A CN 201410334134A CN 105272915 B CN105272915 B CN 105272915B
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Abstract
The present invention relates to technical field of bioengineering, and more particularly to a kind of glutaryl aconitine of hapten compound 3, it has following structural formula (I)：
Description
Technical field
The present invention relates to technical field of bioengineering, more particularly to a kind of hapten compound and its polyclonal antibody,
Kit and application.
Background technology
Aconitine compounds containing is diterpene type alkaloid, and the different demarcation according to substituent on its structure parent nucleus is three classes：
1. diester-type, including Hypaconitine, aconitine, mesaconine etc., there is certain anti-inflammatory and analgesic effect, but it is also Chinese medicine monkshood
The major source of toxicity, its intoxicating dosage is minimum, as 1-2mg aconitines can causing death.2. monoester type, including benzoyl crow
Head alkali, benzoyl mesaconitine, benzoyl Hypaconitine etc., toxicity is relatively small, there is analgesia, and arrhythmia and anti-epileptic are made
With 3. without ester type, including aconine, new aconine etc., can anti-arrhythmia, and toxicity is lower.As can be seen here, it is different
Aconitine-type alkaloids has a more similar biological activity, but its toxicity has notable difference and closely related with structure.
On the one hand, the effective dose of aconitine-type alkaloids is relatively low, and safe-dosaging limits relative narrower, clinical individual are poor
The different effective and security implication to medication is obvious；On the other hand, the processing attenuation of monkshood, it is really most of raw with water-soluble removal
Alkaloids and diester-type alkaloids are converted into the process of monoester alkaloid to hydrolyze, the difference of crude drug source and concocting process
Control is directly connected to the quality evaluation of monkshood.Therefore, the detection to toxicity diester-type aconitine seems extremely necessary with monitoring
And key.
At present, to the detection of monkshood mesaconitine Alkaloid, more general method is HPLC methods and LC-MS methods.
Version Chinese Pharmacopoeia in 2010 uses HPLC methods, the analysis wherein mesaconine of diester-type, Hypaconitine, the content of aconitine and rule
Determine total content≤0.02% of three.However, the polarity of aconitine-type alkaloids is small, detection is controlled more multiple with chromatographic condition
It is miscellaneous, and analysis time overlength (65min), it is extremely inconvenient；At the same time, because the effective component and toxic ingredient content of detection are equal
Relatively low, total content is also only 2/10000ths, sample treatment, condition control or human users slightly difference or fluctuation during analysis,
The obvious deviation of measurement result can be brought.Statutory Specified Scientific Technology Research Institute of constitutional law portion then establishes to be detected with LC-MS methods
The content method and standard of Detection of aconitine in biological samples, mesaconine and Hypaconitine, however, quantitative LC-MS methods are set to detection
It is standby to require that high investment is big, it is difficult to popularity application, and also the pretreatment to detecting sample is also extremely cumbersome.Therefore, to monkshood
The effective monitoring of middle activity and toxicity aconitine-type alkaloids needs to research and develop easier, sensitiveer, more reliable detection side
Method.
Immunoassay is set up based on the high degree of specificity between antibody and antigen or haptens and sensitiveness
A kind of bioanalysis, compared to traditional chromatographic process, it has and can not compared for the accurate quick detection of traces composition
The technical advantage of plan：The pretreatment of sample is simple, the degree of accuracy is high, high sensitivity, and detection limit is up to ng even pg levels, detection
Time only needs 3-5min, is adapted to the live immediately monitoring of sample and the quick analysis of batch samples.
Carried out using antibody to pharmacokinetics inside the qualitative and quantitative analysis of traces traditional Chinese medicine ingredients, traditional Chinese medicine ingredients
Research is progressively applied, and specific method has affinity chromatography, immunochromatography, Western blot etc..Although antibody-immune
The research and development of analysis method, it is higher to put into larger technical requirements in advance, but once researches and develops the engineering that can successfully form characteristic property antibody
Bacterial strain, it is proprietary technology, not only seeks patent protection, but also can thus develop such as enzyme-linked immunologic detecting kit city
Applied product, thus have a good application prospect and market development value.
The content of the invention
The purpose of the present invention aims to provide a kind of polyclonal antibody for being capable of specific recognition diester-type aconitine and its examination
Agent box and application.
Specifically, the first aspect of the present invention there is provided a kind of hapten compound 3- glutaryl aconitines, its
It is characterised by, it has following structural formula (I)：
The second aspect of the present invention there is provided a kind of polyclonal antibody of diester-type aconitine, can be by haptens and ox blood
The conjugate of pure albumen is immunized new zealand rabbit and obtained, and the haptens is the 3- glutaryls crow with following structural formula (I)
Head alkali：
In a preference, the 3- glutaryls aconitine can be made via following methods：
(1) aconitine 50mg, glutaric anhydride 8.8mg and pyridine 2mL reaction 20h are weighed, with 3ml frozen water terminating reactions
Afterwards, extracted 3 times with the ethyl acetate of 3 times of amounts, evaporated under reduced pressure reactant；
(2) with the reactant obtained by Sephadex LH-20 column chromatographies purification steps (1), using ethanol/methylene to wash
De- liquid is eluted, and collects efflux, and evaporated under reduced pressure produces.
The third aspect of the present invention there is provided application of the above-mentioned polyclonal antibody in diester-type aconitine is detected.
Present invention also offers a kind of kit, the kit includes the polyclonal antibody of above-mentioned diester-type aconitine.
The diester-type aconitine polyclonal antibody of the present invention, has the following advantages that：
1. the diester-type aconitine of the present invention is more anti-can to dilute 80000 times of uses, so as to reduce cost；
2. the more anti-detection of the diester-type aconitine of the present invention is limited to 250pg, sensitivity is higher；
3. the how anti-high specificity of diester-type aconitine of the present invention, the cross reaction with monoester type aconitine are respectively less than 8%,
And with demethyl coclaurine no cross reaction.
The details of various aspects of the present invention will be able to detailed description in subsequent chapters and sections.By hereafter and claim
Description, the features of the present invention, purpose and advantage will become apparent from.
Brief description of the drawings
Fig. 1 is the ESI-MS collection of illustrative plates of haptens (AC-GS).
Fig. 2 is haptens (AC-GS)1H-NMR collection of illustrative plates.
Fig. 3 is the HMBC two-dimensional spectrums of haptens (AC-GS).
Fig. 4 is the HSQC two-dimensional spectrums of haptens (AC-GS).
Fig. 5 is haptens (AC-GS)13C-NMR collection of illustrative plates.
The MALDI-TOFMS collection of illustrative plates (molecular weight 66431.1Da) of Fig. 6 bovine serum albumin(BSA)s.
The MALDI-TOFMS collection of illustrative plates (molecular weight 79724.3Da) of Fig. 7 aconitine immunizing antigens.
The standard curve of Fig. 8 diester-type aconitine detection kits.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to conventional strip
Part or (wherein, aconitine reference substance is purchased from Chengdu Man Site biotech firms according to the condition proposed by manufacturer；N, N '-two
Ring ethyl carbonization imines (DCC), n-hydroxysuccinimide (NHS), Freund's complete adjuvant and incomplete Freund's adjuvant, TMB, ox
Seralbumin (BSA) and oralbumin OVA etc. are purchased from Sigma Co., USA；Other chemical reagent meet analysis
Standard；New zealand white rabbit 2, male, every body weight 2.5kg).Unless otherwise indicated, otherwise all percentage, ratio,
Ratio or number are by weight.
Unless otherwise defined, anticipated known to all specialties used in text and scientific words and one skilled in the art
Justice is identical.In addition, any method similar or impartial to described content and material all can be applied in the inventive method.Wen Zhong
Described preferable implementation only presents a demonstration with material to be used.
The features described above that the present invention mentions, or the feature that embodiment is mentioned can be in any combination.Patent specification is taken off
All features shown can be used in combination with any combinations thing form, each feature disclosed in specification, can provide phase with any
The alternative characteristics substitution of same, impartial or similar purpose.Therefore except there is special instruction, disclosed feature is only impartial or similar
The general example of feature.
The preparation and identification of 1 haptens of embodiment -3- glutaryls aconitine (AC-GS)
1. aconitine 50mg, glutaric anhydride 8.8mg, pyridine 2mL reaction 20h are weighed, after 3ml frozen water terminating reactions, with 3
The ethyl acetate of amount extracts 3 times again, evaporated under reduced pressure.
2. reactant Sephadex LH-20 column chromatographies are purified, using ethanol/methylene as eluent, outflow is collected
Liquid, evaporated under reduced pressure obtain sterling.
3. process ESI-MS,13C-NMR、1H-NMR, HMBC, HSQC two-dimensional spectrum identify type I compound chemical constitution.ESI-
MS determining instruments：MSD-Trap-XCT, Q-Tof micro (ESI-MS) types mass spectrograph (Agilent companies),1H-NMR、13C-
NMR and HMBC, HSQC measure use AVANCE III-400 NMRs (Bruker companies)
3.1. find out from ESI-MS, type I compound molecular ion peak m/z signals be 760.4 [M]+, illustrate reaction product phase
It is 759 to molecular mass, increase by 114, this and aconitine and a molecule glutaric anhydride list with aconitine relative molecular mass compared with
The increased relative molecular mass of product after esterification is just consistent, and it is anti-to illustrate that glutaric anhydride generates esterification with aconitine
Answer (Fig. 1).
3.2.1There is δ in H-NMR spectrums (Fig. 2)H=4.98 H, and δ in HMBC spectrums (Fig. 3)HC on 4.94 H and C=O
It is the H on the C to react to have correlation, δ in HSQC two-dimensional spectrums (Fig. 4)Hδ corresponding to=4.98C=71.84, and δH=
4.94 H and C4And C19Correlation, infer that glutaric acid is connected to C3On position, i.e., structure shown in formula I is 3- glutaryl aconitines.
1H-NMR compose (Fig. 2) and13The specific analysis result of C-NMR spectrums (Fig. 5) is as shown in Table 1 and Table 2：
Table 11The analysis result of H-NMR spectrums
Table 213C-NMR composes analysis result
The preparation of the immunizing antigen of embodiment 2 (AC-GS-BSA) and envelope antigen (AC-GS-OVA)
1. accurately weigh 3- glutaryl aconitines 10mg, n-hydroxysuccinimide 1.5mg, N, the ring ethyl carbon of N '-two
Change imines 30mg to be added in 1mL DMF, put lucifuge on magnetic stirring apparatus and 4h is stirred at room temperature.20mg BSA are dissolved in
2mL0.01mol·L-1In PBS (pH7.4), then it is added dropwise under agitation in above-mentioned reaction solution, in 4 DEG C of stirring reaction mistakes
Night.
2. reactant distilled water dialysis 72h is freeze-dried to obtain white powder, -20 DEG C of preservations after completion of the reaction.
3. using MALDI-TOF-MS (MALDI-TOFMS) identification immunogenic conjugates
(AC-GS-BSA) coupling situation：
MALDI-TOFMs can directly display the hapten number (Hapten Number) of immune operation, and method is succinctly fast
Speed.
Fig. 6 is BSA MALDI-TOFMS collection of illustrative plates, and BSA molecular weight is 66431.1Da as known in the figure.
Fig. 7 is the MALDI-TOFMS collection of illustrative plates of AC-BSA compounds, and it is attached that AC-BSA main peak comes across m/z80348.5Da
Closely.
Oneself knows that the molecular weight of aconitine is 759.4, and the Conjugate ratio that AC-BSA can be calculated is 18.
Calculation formula=(AC-BSA-BSA)/AC=(79724.3/80348.5-66431.1)/759.4.
4. conjugate (the AC- of envelope antigen i.e. 3- glutaryls aconitine and chicken serum albumin is prepared with same procedure
GS-OVA)。
Embodiment 3 is immunized new zealand rabbit acquisition diester-type aconitine with immunizing antigen and resisted more
First, the configuration of immunizing antigen：AC-GS-BSA solution adds the milkiness that Freund's complete adjuvant forms white water-in-oil type
Liquid.During booster immunization, the AC-GS-BSA of same concentrations is added to isometric incomplete Freund's adjuvant.
2nd, animal is immunized：Rabbit single cage before immune is raised 7-10 days, is immunized after observing health status.Immune
Vestibule edge venous blood sampling 1.5-2mL is as negative serum.During first immunisation, every rabbit is subcutaneously injected for 6 points in backbone both sides point,
1 booster immunization was carried out every 14 days.The 7th day after final immunization, rabbit arteria carotis takes blood, separates polyclonal antibody.
3rd, immunosurveillance：Using immune preceding rabbit anteserum as negative control, 7d takes blood from rabbit auricular vein after each booster immunization
1mL, titration is carried out by serum titer assay method.
Determined using indirect ELISA method：
(1) envelope antigen (AC-GS-OVA, preparing as described in Example 2) that concentration is 25 μ g/ml is coated with enzyme mark
Plate, 4 DEG C overnight；
(2) closed after using washing lotion board-washing with confining liquid, 150 μ L/ holes, 37 DEG C of incubation 1h；
(3) after washing lotion board-washing, 100 μ L are added per hole from 1:1000 start the antibody serum of doubling dilution, separately use blank blood
Make negative control clearly, 2h is incubated in 37 DEG C.
(4) washing lotion board-washing, the μ L of HRP marks mountain sheep anti mouse ELIAS secondary antibody 100 of dilution 10000,37 DEG C of incubations are added per hole
1h；
(5) washing lotion board-washing, 50 μ L terminate liquids are added per hole after nitrite ion 100 μ L, 37 DEG C of incubation 20min are added per hole
Each hole OD is determined on ELIASA afterwards450nmValue.Negative control OD450It is worth for N, positive control OD450It is worth for P.If P/N >=2.1 and
P-N ＞ 0.2 are the positive, if 1.5 ＜ P/N ＜ 2.1 are suspicious, P/N≤1.5 are feminine gender.
4th, serum processing method：Rabbit arteria carotis takes blood after immune end, is stored at room temperature 1h, and 4 DEG C stand overnight, and treats that it is solidifying
Gu slightly supernatant separates out, 3000rpm centrifugation 10min, upper strata antiserum is taken out.- 20 DEG C of refrigerators are protected after being mixed with equivalent glycerine
Deposit.
The more anti-purifying of the diester-type aconitine of embodiment 4
1. with 50% saturated ammonium sulphate：
(1) saturated ammonium sulfate 5ml is added dropwise, to wait early stage the precipitation of appearance to be further continued for after dissolving, until be not redissolved,
Continue to add to 50%, be put into refrigerator, 2h is stood at 4 DEG C；
(2) gained serum sediment is transferred in centrifuge tube, 10000r/min centrifuges 30min at 4 DEG C, and supernatant is abandoned
Go, taking precipitate is dissolved in 2ml PBS.
2. with 33% saturated ammonium sulphate：
The above-mentioned PBS solution containing precipitation is taken, 1.2ml saturated ammonium sulfate is added dropwise to 33%, 4 DEG C of standing 2h, equally
Under the conditions of centrifuge, taking precipitate, with 1mLPBS dissolve precipitate.
The more anti-CHARACTERISTICS IDENTIFICATION of the diester-type aconitine of embodiment 5
The more anti-identification of one, diester-types aconitine
(1) determining the protein quantity：25ul albumen mark product are taken to be added to sample in each hole of 96 orifice plates；Added per hole
200ul BCA working solutions, rock and mix 30s, 30min is incubated at 37 DEG C；Room temperature is cooled to, is detected using ELIASA in 562nm
Light absorption value；Using BSA concentration as abscissa, using light absorption value as ordinate, standard curve is drawn.In the range of linearity of standard curve
Interior, according to the correction light absorption value of each albumen sample, and sample dilution, the protein concentration for calculating sample is 23.12mg/
ml。
(2) specificity identification of diester-type aconitine antibody：It is serially diluted aconitine, mesaconitine, Hypaconitine, benzene first
Acyl aconitine, benzoyl mesaconitine, benzoyl Hypaconitine, intersected with indirect competitive ELISA method detection is more anti-with it
Reaction：Take out the ELISA Plate that 10 μ g/ml envelope antigen has been coated with, after washing lotion board-washing, add 37 DEG C of sealer and close 1 hour；
After washing lotion board-washing, add 50 μ L competition medicine per hole and 50 μ L be diluted to the antibody-solutions of optimum concentration and are at war with reaction,
37 DEG C are incubated 2 hours；After washing lotion board-washing, HRP mark sheep anti mouse ELIAS secondary antibodies, 100 μ L/ holes, 37 DEG C of incubations are added；Washing lotion board-washing
Afterwards, add substrate to develop the color, 100 μ L/ holes, 2M H are directly added into after 37 DEG C of colour developings2SO4,50 μ l/ holes；Terminated with ELIASA measure anti-
Light absorption value of the ELISA Plate at 450nm after answering.Cross reaction calculation formula is as follows：
Cross reaction (%)=[IC50(haptens)/IC50(competition medicine)] × 100%
As a result show (table 3)：Aconitine (AC) and diester-type aconitine compounds containing mesaconitine (MC), Hypaconitine
(HC) cross reaction is 59.71%, 78.81%, with monoester type aconitine compounds containing benzoyl aconite alkali (BAC), benzene first
The cross reaction of acyl mesaconitine (BMC), benzoyl Hypaconitine (BHC) is respectively 7.6%, 1.85%, 4.47%, and with going
First coclaurine no cross reaction.
Table 3 and the cross reaction table of other compounds
The preparation and its application of the diester-type aconitine enzyme-linked immunologic detecting kit of embodiment 6
First, enzyme linked immunological kit is made up of following substances：
(1) ELISA Plate of pre-coated antigen：Envelope antigen is diluted to 5 μ g/ml with coating buffer solution, 100 μ are added per hole
L, 4 DEG C overnight, and next day inclines coating buffer, with wash liquid dilute 3 times, pats dry, 150 μ l are then added in every hole and are closed
Liquid, 37 DEG C of incubation 2h, liquid in hole of inclining, is sealed after drying with aluminium foil bag.
(2) diester-type aconitine reference standards and negative control standard items.
(3) diester-type aconitine is more anti-
(4) the rabbit secondary antibody of horseradish peroxidase-labeled.
(5) antibody diluent：0.01M PBS, pH7.6.
(6) 10 × concentration washing lotion：0.1M phosphate buffers containing 0.5% Tween-20, pH7.4.
(7) nitrite ion A liquid, nitrite ion B liquid.A liquid is mixed in equal volume with B liquid using preceding.
(8) terminate liquid：2M sulfuric acid solutions.
2nd, the application of kit
(1) detection method
1. sample pre-treatments
Take unprocessed Radix Aconiti Lateralis powder 2.0g, it is accurately weighed, be placed in plug conical flask, ammonification test solution 3ml, precision add isopropanol-
Ethyl acetate (1:1) mixed solution 50ml, weighed weight, it is ultrasonically treated 30 minutes, lets cool, then weighed weight, with isopropanol-second
Acetoacetic ester (1:1) mixed solution supplies reduced weight, shakes up, filtration.Precision measures subsequent filtrate 25ml, is recovered under reduced pressure at 40 DEG C
For solvent to doing, residue is accurate to add alcohol mixed solution 6ml dissolvings, filters and produces.10-50 times of solution is diluted to ethanol.
Above-mentioned solution is diluted to the testing sample solution containing 10% ethanol with PBS.
2. diester-type aconitine standard liquid is prepared
Aconitine standard items are prepared to 500000-50ng/mL various concentrations solution with ethanol, by above-mentioned solution PBS
It is diluted to the standard liquid containing 10% ethanol.
3. detected with kit
(1) detecting step
10min is placed under the micropore and all reagent normal temperature of taking-up requirement.First add the dibasic acid esters of 50 each concentration of μ L respectively
Type titer and testing sample are added in micropore, add the 50 μ L how anti-dilution (1 of diester-type aconitine：80000) it is, positive
Property control wells be the 50 how anti-dilutions of μ L add 50 μ L contain 10% ethanol PBS, blank well be 50 μ L PBS add 50 μ L to contain 10% second
The PBS of alcohol, 37 DEG C, it is incubated 120min.Remove liquid in hole, add 400 μ l washing lotions in each micropore, gently rock the several seconds,
Quick upset by liquid in micropore to the greatest extent, to a folded clean blotting paper clap it is several under, repeat operation common board-washing 3 times.Add
100 μ l ELIAS secondary antibody working solutions, 37 DEG C, it is incubated 60min.Go liquid simultaneously board-washing 3 times in hole.Will colour developing A liquid and colour developing B liquid etc.
Amount mixing is made into substrate nitrite ion.Each micropore adds 100 μ l substrate nitrite ion, room temperature lucifuge colour developing 15-20min.Add
The μ l of terminate liquid 100, absorbance is determined under 450nm with ELIASA.
(2) kit repeatability and stability test and result calculate
Precision is tested in plate：6 micropores on same ELISA Plate are taken, are tested with testing sample, experiment repeats 3
It is secondary.
Precision is tested between plate：Take with a batch of 3 pieces of ELISA Plates, tested with testing sample, experiment is repeated 3 times.
The computational methods of the coefficient of variation：The standard deviation of the coefficient of variation (CV)=measurement result and the percentage of its average value.
As a result calculate：Using light absorption value when adding diester-type aconitine standard items as B, light absorption value when being not added with standard items is
B0, B/B0 are ordinate, and lg (standard items, ng/mL) is abscissa；With ln ((B/B0)/(1-B/B0)) for ordinate, lg (marks
Quasi- product, ng/mL) it is abscissa, as shown in figure 8, linear equation is Y=-1.6768x+5.5858, R2=0.9982.Linear model
Enclose and be limited to 250pg for the μ g/ml of 25ng/ml~25, detection, sensitivity is higher, calculates diester-type aconitine in unprocessed Radix Aconiti Lateralis medicinal material
Content is 1.303mg/g (table 4 and table 5).
Reperformance test result in the plate of table 4
Reperformance test result between the plate of table 5
Many aspects involved in the present invention have been explained as above.However, it should be understood that without departing from spirit of the invention
Under the premise of, those skilled in the art can carry out equivalent change and modification to it, and the change and modification equally fall into the application
The coverage of appended claims.
Claims (5)
1. a kind of hapten compound 3- glutaryl aconitines, it is characterised in that it has following structural formula (I)：
2. a kind of polyclonal antibody of diester-type aconitine, new zealand rabbit is immunized by the conjugate of haptens and bovine serum albumin(BSA)
After obtain, it is characterised in that the haptens is the 3- glutaryl aconitines with following structural formula (I)：
3. polyclonal antibody as claimed in claim 2, it is characterised in that the 3- glutaryls aconitine can be via with lower section
Method is made：
(1) aconitine 50mg, glutaric anhydride 8.8mg and pyridine 2mL reaction 20h are weighed, after 3ml frozen water terminating reactions, with 3
The ethyl acetate of amount extracts 3 times again, evaporated under reduced pressure reactant；
(2) reactant obtained by Sephadex LH-20 column chromatographies purification steps (1) is used, using ethanol/methylene as eluent
Eluted, collect efflux, evaporated under reduced pressure produces.
4. application of the polyclonal antibody as claimed in claim 2 in diester-type aconitine is detected.
5. a kind of kit, it is characterised in that the kit includes more grams of diester-type aconitine as claimed in claim 2
Grand antibody.
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| CN113968818A (en) * | 2020-07-22 | 2022-01-25 | 中国农业大学 | 10-hydroxy mesaconine hapten, artificial antigen and antibody as well as preparation method and application thereof |
| CN113881638B (en) * | 2021-09-16 | 2023-10-13 | 江南大学 | De-himbine hapten, monoclonal antibody, hybridoma cell strain and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010063638A1 (en) * | 2008-12-01 | 2010-06-10 | Universite Victor Segalen Bordeaux 2 | Bis [o-(14-benzoylaconine-8-yl)] esters |
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2014
- 2014-07-14 CN CN201410334134.7A patent/CN105272915B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010063638A1 (en) * | 2008-12-01 | 2010-06-10 | Universite Victor Segalen Bordeaux 2 | Bis [o-(14-benzoylaconine-8-yl)] esters |
Non-Patent Citations (2)
| Title |
|---|
| An enzyme-linked immunosorbent assay for aconitine-type alkaloids using an anti-aconitine monoclonal antibody;Katsumi Kido et al;《analytica chimica acta》;20080408;第616卷;第109-114页 * |
| Screening and identification of multi compounds in Radix aconiti using combination of liquid chromatography/time-of-flight tandem mass spectrometry;Wang, Jianhua et al;《Asian Journal of Chemistry 》;20121231;第24卷(第1期);第303-308页 * |
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