CN109374809A - A kind of method of affine in immunity column purification-liquid chromatography-tandem mass spectrometry domoic acid toxoid - Google Patents
A kind of method of affine in immunity column purification-liquid chromatography-tandem mass spectrometry domoic acid toxoid Download PDFInfo
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Abstract
The present invention relates to a kind of domoic acid toxoid detection methods, more particularly, to a kind of affine in immunity column purification-liquid chromatography-tandem mass spectrometry domoic acid toxoid method, it utilizes high-performance, the DA toxin immuno-affinity column identified by force, and the analysis for combining high sensitivity and the liquid chromatography-tandem mass spectrometry of accuracy to detect DA toxin in different marine organisms measures, it can effectively solve the undesirable problem of sample purification in pre-treatment, and reduce sample substrate interference, shorten analysis time, the improvement method rate of recovery, affine in immunity technology is filled up in the blank of domoic acid toxoid detection field.
Description
Technical field
The present invention relates to a kind of domoic acid toxoid detection methods, more particularly, to a kind of affine in immunity column purification-liquid
Phase chromatography-tandem mass spectrometry measurement domoic acid toxoid method.
Background technique
Amnesic shellfish poisoning (ASP) is the toxin that toxic diatoms secretion generates, main active cartilage algae
Sour (domoic acid, DA) is a kind of nerve amino acids toxin.The toxin is Canadian Edward earliest in 1987
Prince island east coast due to eating Mytilus galloprovincialis caused by be poisoned to death and be found in event.Poisoner be mainly shown as abdominal pain,
Diarrhea, vomiting, and with symptoms such as obstinate headache, the loss of memory, Bewu βtseinstrubung, stupors.The study found that DA is mainly by multiple row
Pseudo nitzschia generates.Domoic acid content can cause eater to be poisoned when reaching 40mg/kg in shellfish tissue, and there is cause in when 150mg/kg
Dead dangerous, the mankind are 20mg/kg by feeding tolerable maximum limitation, and it is 20 that Canada has formulated threshold limit values standard first
This kind of toxin is also classified as shellfish conventional detection project in succession by μ g/g shellfish meat, Europe, Japan.China has still belonged to the research of DA
Step section still lacks effective detection means and monitoring network.Although also very rare in the report of China's detection DA at present,
Coastal area of china has been detected by a variety of Pseudo nitzschia algae strains for producing poison.In view of the toxin toxicity is big, reaction is fast, without antidote,
Carry out the detection research of DA toxin in marine organisms to ensuring that Safety of Aquatic Products and human health are particularly important.
Immune affinity column (IAC) is a kind of Novel chromatographic column based on antigen-antibody reaction, using specific antibody to mesh
The affine identification and Reversible binding characteristic for marking toxin are made, selection specificity and good adsorption cleaning with height
Energy.It is domestic at present that there has been no the relevant reports that means detection DA toxin is purified using immune affinity column as pre-treatment.
Summary of the invention
Goal of the invention of the invention is to provide for a kind of high sensitivity, and precision and the rate of recovery are able to satisfy detection and analysis
Requirement affine in immunity column purification-liquid chromatography-tandem mass spectrometry domoic acid toxoid method, this method utilize
High-performance, the DA toxin immuno-affinity column identified by force, and combine high sensitivity and the detection of the liquid chromatography-tandem mass spectrometry of accuracy
The analysis measurement of DA toxin, can effectively solve the undesirable problem of sample purification in pre-treatment, and reduce in different marine organisms
Sample substrate interference, shortens analysis time, and the improvement method rate of recovery fills up affine in immunity technology in the sky in Mycotoxin identification field
It is white.
To achieve the goals above, the invention adopts the following technical scheme:
A kind of affine in immunity column purification-liquid chromatography-tandem mass spectrometry domoic acid toxoid side of the invention
Method, comprising the following steps:
(1) it sample pre-treatments: accurately weighs abundant homogeneous samples 2.00g and has in plug centrifuge tube in 50mL, 8mL body is added
The methanol aqueous solution that product concentration is 75%, vortex oscillation 2min, ultrasonic extraction 10min, 7000r/min is centrifuged 5min, by supernatant
Liquid moves in another 50mL centrifuge tube, obtains an extracting solution；The methanol that 8mL volumetric concentration is 75% is added into residual residue again
Aqueous solution repeats to extract once, obtains secondary raffinate；It is settled to 20mL after merging an extracting solution and secondary raffinate, must be mentioned
Take liquid；1mL extracting solution is pipetted into another 50mL centrifuge tube, 5mL PBS buffer solution is added and is diluted, obtains sample to be clean
Liquid；
(2) affine in immunity column purification: taking a DA monoclonal antibody immunity affinity column, removes after its recovery to room temperature affine
Column plug is released after saving liquid in column, upper sample liquid；After end of the sample, parent is eluted with 50% methanol aqueous solution of 6mL volumetric concentration
And column, Liquid Residue in column is extracted, and discard all of the above efflux, the methanol for finally containing 2% ammonium hydroxide of mass concentration with 3mL is molten
Liquid elution, the eluent of collection are dried with nitrogen in 50 DEG C, are dissolved with 1mL initial liquid phase constant volume, after 0.22 μm of membrane filtration
It is analyzed for liquid chromatography-mass spectrography.
Preferably, the PBS buffer solution is made by the following method in step (1): weighing two hypophosphite monohydrates two respectively
Hydrogen sodium 2.18g, disodium hydrogen phosphate dodecahydrate 12.90g, sodium chloride 8.50g are dissolved with water and are settled to 1000mL.
Preferably, the DA monoclonal antibody immunity affinity column is made by the following method in step (2):
(a) synthetic immunogen
Sequentially add the bovine serum albumin(BSA) of 10mg in small flask, the sodium acetate buffer of 2mL 0.05M, pH7.4,
The domoic acid of 1mg, the formalin that 60 μ L mass concentrations are 37% mix, 37 DEG C of stirring 48h, then use 0.1M, pH7.3
PBS buffer solution, in 4 DEG C dialyse 3 days, change daily liquid 2 times, obtain immunogene DA-BSA.
(b) synthesis detection is former
Sequentially add the chicken ovalbumin of 10mg in small flask, the sodium acetate buffer of 2mL 0.05M, pH7.4,
The domoic acid of 1mg, the formalin that 60 μ L mass concentrations are 37% mix, 37 DEG C of stirring 48h, then use 0.1M, pH7.3
PBS buffer solution, in 4 DEG C dialyse 3 days, change daily liquid 2 times, original DA-OVA must be detected.
(c) monoclonal antibody preparation and purifying
The female Balb/c mouse of 5 6~8 week old is taken, dorsal sc multi-point injection DA-BSA, 20 μ g/ is only, 4 weeks immune
After start booster immunization, every 2 weeks booster immunization 1 time；It is complete with immunogene DA-BSA and isometric Freund when initial immunity
Adjuvant emulsion is emulsified when booster immunization, immunizing dose, immunization ways with immunogene DA-BSA and isometric incomplete Freund's adjuvant
It is constant；
After 2nd time booster immunization 10 days, mouse tail vein blood examination is taken to survey, coating detection original DA-OVA uses indirect competition
The potency of ELISA method detection serum selects the highest BALB/c mouse of potency to be used to prepare hybridoma, merges preceding 3 days small
Mouse abdominal cavity booster immunization is primary, and the highest BALB/c mouse splenocyte of potency is taken to merge with myeloma cell Sp2/0, and screening obtains
Hybridoma；
0.5mL atoleine sensitization, injection 1 × 10 in 8 days backward mouse peritoneals is injected intraperitoneally in BALB/c mouse7A hybridization
Oncocyte starts to extract ascites from mouse peritoneal with syringe after 10 days, later every extraction in 1 day ascites 1 time, is centrifuged, collects
Supernatant purifies supernatant using saturated ammonium sulfate method, then is further purified with ProteinG affinity chromatography and obtains DA Dan Ke
Grand antibody.
(d) prepared by affinity column
The HCl solution for weighing the hydrogen bromide activated sepharose 4B dry powder 100mL 1M of 0.5g CNBr activation is molten
It is swollen, and wash；Gel is washed to obtain with 100mL coupling buffer again；
The DA monoclonal antibody of 1.5mL 10mg/mL, shaken at room temperature coupling reaction is added in gel after taking 1.5mL to be swollen
2h, filter, and use 30mL coupling buffer detergent gel, filter, be added 10mL Block buffer, shaken at room temperature react 2h, pumping
Filter, then use the PBS buffer solution detergent gel of 100mL 0.01M, pH7.3, and is transferred in 5mL column tube, is added that contain quality dense
Degree be respectively 0.05% thimerosal, 0.05% bovine serum albumin(BSA) 0.01M, pH7.3 PBS solution, in 2~8 DEG C store.
Preferably, in step (d), the formula of the coupling buffer are as follows: 0.1molL-1NaHCO3, 0.5mol
L-1NaCl, pH8.3.
Preferably, the formula of the Block buffer are as follows: 0.1molL-1Tris-HCl, pH8.0.
Preferably, the liquid phase chromatogram condition are as follows: chromatographic column: ACQUITY UPLC BEH C18Column (2.1mm ×
50mm, 1.7 μm)；2 μ L of sampling volume；10 DEG C of sample room temperature；40 DEG C of column temperature；Flow velocity 0.2mL/min；Mobile phase A is acetonitrile,
B is the 2mmol/L ammonium acetate solution containing 0.1% formic acid, gradient elution: 0~1.0min, 90%A；1.0~3.0min, 90%~
10%A；3.0~4.0min, 10%A；4.0~4.1min, 10%~90%A；4.1~5.5min, 90%A.
Preferably, the Mass Spectrometry Conditions are as follows: electric spray ion source, cation scan (Electrospray
Ionization, ESI-)；Detection mode: multiple-reaction monitoring pattern (Multiple Reaction Monitor, MRM)；Capillary
Tube voltage: 3.0kV；Ion source temperature: 150 DEG C；Desolvation temperature: 600 DEG C；Taper hole throughput: 150L/h；Desolventizing gas
Flow: 1000L/h；Orifice potential: 12V；Collision energy: 16eV；Analyte qualitative ion pair: m/z 312.2 > 248.2；Analysis
Object quota ion pair: m/z 312.2 > 266.2.
Therefore, the invention has the following beneficial effects: a kind of high sensitivity is provided, precision and the rate of recovery are able to satisfy
The affine in immunity column purification of the requirement of detection and analysis-liquid chromatography-tandem mass spectrometry domoic acid toxoid method, this
Method combines the liquid chromatography-tandem of high sensitivity and accuracy using high-performance, the DA toxin immuno-affinity column identified by force
The analysis measurement of DA toxin, can effectively solve that sample purification in pre-treatment is undesirable to ask in Mass Spectrometer Method difference marine organisms
Topic, and sample substrate interference is reduced, shorten analysis time, the improvement method rate of recovery fills up affine in immunity technology in domoic acid
The blank of toxoid detection field.
Detailed description of the invention
Fig. 1 is domoic acid (DA) standard solution second order ms full scan figure.
Fig. 2 is 0.025 μ g mL-1Domoic acid (DA) standard solution MRM figure.
Specific embodiment
The present invention will be further described with reference to the accompanying drawings and detailed description.
In the present invention, if not refering in particular to, used raw material and equipment etc. are commercially available or commonly used in the art.
Method in following embodiments is unless otherwise instructed the conventional method of this field.
(1) instrument and reagent
ACQUITY ultra performance liquid chromatography-tandem mass spectrometer Xevo TQ-S (Waters, US)；MS2 whirlpool is mixed
Clutch (German IKA company)；It is dried with nitrogen instrument (Organomatio company of the U.S.)；Centrifuge5810 supercentrifuge
(German Eppendorf company)；12 channel solid-phase extraction devices (supelco company of the U.S.).
Domoic acid (DA) standard items (purity >=98.0%, Canadian National Research Council)；Acetonitrile, methanol (chromatography
It is pure, German Merck company)；Ammonium acetate, formic acid (Sigma Co., USA)；Disodium hydrogen phosphate dodecahydrate, two hypophosphite monohydrates two
Hydrogen sodium, sodium chloride, ammonium hydroxide (Shanghai Chinese medicines group)；Experimental water is ultrapure water.
(2) PBS buffer solution is prepared
Sodium dihydrogen phosphate dihydrate 2.18g, disodium hydrogen phosphate dodecahydrate 12.90g, sodium chloride 8.50g are weighed respectively,
It is dissolved with water and is settled to 1000mL.
(3) 25 μ g/mL domoic acid (DA) standard solution
Suitable domoic acid standard solution is accurately pipetted, is settled to 50mL with methanol dilution, -20 DEG C are kept in dark place,
Pot-life is 3 months.
(4) method parameter optimizes
1. chromatography and Mass Spectrometry Conditions selection
ACQUITY UPLC BEH C18Column is strong to domoic acid (DA) retention, and preferable chromatographic peak profile, fully meets
The needs of domoic acid (DA) liquid-phase chromatographic analysis.Present invention research compares multiple groups different volumes score formic acid and various concentration
Influence of the ammonium acetate solution to chromatography.The result shows that being stream with the 5mmol/L ammonium acetate solution of 0.1% formic acid and acetonitrile
It is dynamic that mutually there is good chromatographic isolation effect, and peak shape is sharply symmetrical.
Present invention selection is under cation scan pattern with peristaltic pump with domoic acid (DA) standard solution of 3.0 μ g/mL
Flow Injection Analysis is carried out, mass spectrometry parameters are optimized.The molecular ion of DA is obtained by first mass spectrometric scanning analysis, is adjusted
Examination selects best orifice potential and capillary voltage, wherein [M+H]+(m/z 312.2) abundance highest, so that ion may be selected
M/z 312.2 is detected.Second mass analysis is carried out to analyte ions, is obtained fragment ion information (as shown in Figure 1), and
The mass spectrometry parameters such as collision energy are optimized.
2. extracting the selection of reagent
DA is a kind of non-protein amino acid, is dissolved in water, is slightly soluble in methanol.In view of methanol extracts reagent as common,
Tissue can be readily penetrated through, precipitating proteins, experimental selection methanol aqueous solution (volumetric concentration 50%~90%) is as extraction examination
Agent relatively respectively extracts reagent to the extraction efficiency of negative mark-on sample.As a result as shown in table 1, when extracting one time, compare
90% methanol aqueous solution, the sample DA rate of recovery extracted using 50% and 75% methanol aqueous solution have been respectively increased 11.2%
With 17.2%；After carrying out second extraction to sample, the DA rate of recovery can further improve 9.5%~16.3%, it is contemplated that 50% first
Alcohol solution extract loading takes a long time, and 75% methanol aqueous solution of final choice extracts reagent as DA.
The influence of 1 methanol content of table and extraction time to the DA rate of recovery
3. immune affinity column condition optimizing
The affine combination of immune affinity column acts on, excessive organic reagent directly related with the organic reagent content of sample solution
Affinity interaction can be made to weaken or destroy.Methanol-PBS mixed solution the discovery for comparing different proportion (1:9,1:3 and 1:1), works as first
Alcohol content is between 10%~25%, and the DA rate of recovery is close to 100%；And after methanol content rises to 50%, the DA rate of recovery is aobvious
Write decline；Finally select loading after the methanol ratio in extracting solution is diluted to 25%.
Shellfish structural constituent complexity is different, often quantifies because matrix interference influences mass spectrum, methanol aqueous solution (body is selected in experiment
Product 10%~70%) it is leacheate, compare its clean-up effect to DA in shellfish extracting solution.As a result, it has been found that when methanol ratio reaches
When to 60%~70%, DA rate of recovery decline 18%~80%；Compared to 10% and 30% methanol aqueous solution, 50% methanol aqueous solution
Clean-up effect is best, and the DA rate of recovery in scallop, oyster, mussel and mud blood clam sample is respectively 91.8 ± 7.4%, 90.5 ±
2.3%, 88.2 ± 5.2%, 94.0 ± 2.1%.Final choice is using 6 mL, 50% methanol aqueous solution as leacheate.
In view of DA in acid condition be easy decompose, experiment compare the methanol solution containing 1% formic acid of mass concentration,
Methanol solution containing 1% ammonium hydroxide of mass concentration, the methanol solution containing 2% ammonium hydroxide of mass concentration are to the elution effect of DA.Knot
Fruit finds (table 2), and under the methanol solution elution containing 2% ammonium hydroxide of mass concentration, the DA rate of recovery is up to 97.1%, and optimum
Elution volume is 3mL.
The rate of recovery of DA under the different elution requirements of table 2
4. the range of linearity and quantitative limit
It is 0.005,0.025,0.125,0.5,1.0,2.0 and 5.0 μ g/mL standards with DA standard solution compound concentration
Working solution, using DA concentration as abscissa, peak area is ordinate, makes standard curve, equation of linear regression: y=7611X
+1952.57(R2=0.999), DA is linear good in 0.005~5.0 μ g/mL.The detection of this method is determined with 3 times of signal-to-noise ratio
Limit (LOD) be 0.02 μ g/g, with 10 times of signal-to-noise ratio determine this method quantitative limit (LOQ) be 0.05 μ g/g, as shown in Figure 2.
5. the rate of recovery and precision
Accurately weigh 2.00g feminine gender shellfish samples, the DA standard solution of pitch-based sphere 0.25,2.5 and 25 μ g/g, each
Pitch-based sphere is measured in parallel 5 times, is calculated the rate of recovery and precision, be the results are shown in Table 3.DA is returned 0.25-25 μ g/g pitch-based sphere
Yield is 92.71-103.62%, and in a few days and in the daytime RSD is respectively 5.92-7.16% and 4.81-5.91%.
The average recovery rate and relative standard deviation (n=5) of 3 DA of table
| Pitch-based sphere | In a few days RSD | In the daytime RSD | The rate of recovery ± SD |
| (ng/g) | (%, n=5) | (%, n=3) | (%, n=5) |
| 10 | 7.16 | 5.91 | 92.71±6.39 |
| 50 | 5.92 | 3.29 | 94.01±4.72 |
| 500 | 6.57 | 4.81 | 103.62±5.57 |
Embodiment
(1) it sample pre-treatments: accurately weighs abundant homogeneous samples 2.00g and has in plug centrifuge tube in 50mL, 8mL body is added
The methanol aqueous solution that product concentration is 75%, vortex oscillation 2min, ultrasonic extraction 10min, 7000r/min is centrifuged 5min, by supernatant
Liquid moves in another 50mL centrifuge tube, obtains an extracting solution；The methanol that 8mL volumetric concentration is 75% is added into residual residue again
Aqueous solution repeats to extract once, obtains secondary raffinate；It is settled to 20mL after merging an extracting solution and secondary raffinate, must be mentioned
Take liquid；1mL extracting solution is pipetted into another 50mL centrifuge tube, 5mL PBS buffer solution is added and is diluted, obtains sample to be clean
Liquid；
(2) affine in immunity column purification: taking a DA monoclonal antibody immunity affinity column, removes after its recovery to room temperature affine
Column plug is released after saving liquid in column, upper sample liquid；After end of the sample, parent is eluted with 50% methanol aqueous solution of 6mL volumetric concentration
And column, Liquid Residue in column is extracted, and discard all of the above efflux, the methanol for finally containing 2% ammonium hydroxide of mass concentration with 3mL is molten
Liquid elution, the eluent of collection are dried with nitrogen in 50 DEG C, are dissolved with 1mL initial liquid phase constant volume, after 0.22 μm of membrane filtration
It is analyzed for liquid chromatography-mass spectrography；Liquid phase chromatogram condition are as follows: chromatographic column: ACQUITY UPLC BEH C18Column (2.1mm ×
50mm, 1.7 μm)；2 μ L of sampling volume；10 DEG C of sample room temperature；40 DEG C of column temperature；Flow velocity 0.2mL/min；Mobile phase A is acetonitrile,
B is the 2mmol/L ammonium acetate solution containing 0.1% formic acid, gradient elution: 0~1.0min, 90%A；1.0~3.0min, 90%
~10%A；3.0~4.0min, 10%A；4.0~4.1min, 10%~90% A；4.1~5.5min, 90%A；
Mass Spectrometry Conditions are as follows: electric spray ion source, cation scan (Electrospray ionization, ESI-)；Detection
Mode: multiple-reaction monitoring pattern (Multiple Reaction Monitor, MRM)；Capillary voltage: 3.0kV；Ion source temperature
Degree: 150 DEG C；Desolvation temperature: 600 DEG C；Taper hole throughput: 150L/h；Desolventizing gas flow: 1000L/h；Orifice potential:
12V；Collision energy: 16eV；Analyte qualitative ion pair: m/z 312.2 > 248.2；Analyte quota ion pair: m/z
312.2>266.2；
Wherein, DA monoclonal antibody immunity affinity column is made by the following method:
(a) synthetic immunogen
Sequentially add the bovine serum albumin(BSA) of 10mg in small flask, the sodium acetate buffer of 2mL 0.05M, pH7.4,
The domoic acid of 1mg, the formalin that 60 μ L mass concentrations are 37% mix, 37 DEG C of stirring 48h, then use 0.1M, pH7.3
PBS buffer solution, in 4 DEG C dialyse 3 days, change daily liquid 2 times, obtain immunogene DA-BSA；
(b) synthesis detection is former
Sequentially add the chicken ovalbumin of 10mg in small flask, the sodium acetate buffer of 2mL 0.05M, pH7.4,
The domoic acid of 1mg, the formalin that 60 μ L mass concentrations are 37% mix, 37 DEG C of stirring 48h, then use 0.1M, pH7.3
PBS buffer solution, in 4 DEG C dialyse 3 days, change daily liquid 2 times, original DA-OVA must be detected；
(c) monoclonal antibody preparation and purifying
The female Balb/c mouse of 5 6~8 week old is taken, dorsal sc multi-point injection DA-BSA, 20 μ g/ is only, 4 weeks immune
After start booster immunization, every 2 weeks booster immunization 1 time；It is complete with immunogene DA-BSA and isometric Freund when initial immunity
Adjuvant emulsion is emulsified when booster immunization, immunizing dose, immunization ways with immunogene DA-BSA and isometric incomplete Freund's adjuvant
It is constant；
After 2nd time booster immunization 10 days, mouse tail vein blood examination is taken to survey, coating detection original DA-OVA uses indirect competition
The potency of ELISA method detection serum selects the highest BALB/c mouse of potency to be used to prepare hybridoma, merges preceding 3 days small
Mouse abdominal cavity booster immunization is primary, and the highest BALB/c mouse splenocyte of potency is taken to merge with myeloma cell Sp2/0, and screening obtains
Hybridoma；
0.5mL atoleine sensitization, injection 1 × 10 in 8 days backward mouse peritoneals is injected intraperitoneally in BALB/c mouse7A hybridization
Oncocyte starts to extract ascites from mouse peritoneal with syringe after 10 days, later every extraction in 1 day ascites 1 time, is centrifuged, collects
Supernatant purifies supernatant using saturated ammonium sulfate method, then is further purified with ProteinG affinity chromatography and obtains DA Dan Ke
Grand antibody；
(d) prepared by affinity column
The HCl solution for weighing the hydrogen bromide activated sepharose 4B dry powder 100mL 1M of 0.5g CNBr activation is molten
It is swollen, and wash；Gel, the formula of coupling buffer are as follows: 0.1molL are washed to obtain with 100mL coupling buffer again-1NaHCO3,
0.5mol·L-1NaCl, pH8.3；
The DA monoclonal antibody of 1.5mL 10mg/mL, shaken at room temperature coupling reaction is added in gel after taking 1.5mL to be swollen
2h is filtered, and with 30mL coupling buffer detergent gel, the formula of coupling buffer are as follows: 0.1 molL-1NaHCO3,
0.5mol·L-1NaCl, pH8.3 are filtered, and 10mL Block buffer, the formula of Block buffer are as follows: 0.1molL is added- 1Tris-HCl, pH8.0, shaken at room temperature react 2h, filter, then are washed and coagulated with the PBS buffer solution of 100mL 0.01M, pH7.3
Glue, and be transferred in 5mL column tube, being added containing mass concentration is respectively 0.05% thimerosal, 0.05% bovine serum albumin(BSA)
The PBS solution of 0.01M, pH7.3 are stored in 2~8 DEG C.
Are amounted to by 59 portions of shellfishes and is divided for 5 coastal cities mussel, blood clam, mud blood clam, scallop, oyster kinds using this method
Analysis detection.Obtain that the results are shown in Table 3.DA recall rate 10%, including 5 batch scallops and 1 batch oyster, wherein scallop detects
Rate highest, and DA level of pollution is differed in 0.9-11.8 μ g/g；Oyster recall rate 7%, DA content are 0.07 μ g/g.
The level of pollution of DA in 3 positive sample of table
| Kind | DA concentration (μ g/g) |
| Scallop | 11.8 |
| Scallop | 14.9 |
| Scallop | 0.7 |
| Scallop | 9.3 |
| Scallop | 0.9 |
| Oyster | 0.07 |
From the analysis of table 3, the result shows that, this method rate of recovery is high, and precision and sensitivity are able to satisfy domoic acid class poison
The detection of element needs.
Above-mentioned embodiment is only a preferred solution of the present invention, not the present invention is made in any form
Limitation, there are also other variations and modifications on the premise of not exceeding the technical scheme recorded in the claims.
Claims (7)
1. a kind of affine in immunity column purification-liquid chromatography-tandem mass spectrometry domoic acid toxoid method, feature exist
In, comprising the following steps:
(1) it sample pre-treatments: accurately weighs abundant homogeneous samples 2.00g and has in plug centrifuge tube in 50mL, it is dense that 8mL volume is added
The methanol aqueous solution that degree is 75%, vortex oscillation 2min, ultrasonic extraction 10min, 7000r/min centrifugation 5min move supernatant
To in another 50mL centrifuge tube, an extracting solution is obtained；It is water-soluble that the methanol that 8mL volumetric concentration is 75% is added into residual residue again
Liquid repeats to extract once, obtains secondary raffinate；It is settled to 20mL after merging an extracting solution and secondary raffinate, obtains extracting solution；
1mL extracting solution is pipetted into another 50mL centrifuge tube, 5mL PBS buffer solution is added and is diluted, obtains sample liquid to be clean；
(2) affine in immunity column purification: taking a DA monoclonal antibody immunity affinity column, and it is stifled to remove affinity column after its recovery to room temperature
Head is released after saving liquid in column, upper sample liquid；It is that the elution of 50% methanol aqueous solution is affine with 6mL volumetric concentration after end of the sample
Column extracts Liquid Residue in column, and discards all of the above efflux, and the methanol solution of 2% ammonium hydroxide of mass concentration is finally contained with 3mL
Elution, the eluent of collection are dried with nitrogen in 50 DEG C, are dissolved with 1mL initial liquid phase constant volume, are supplied after 0.22 μm of membrane filtration
Liquid chromatography-mass spectrography is analyzed.
2. a kind of affine in immunity column purification-liquid chromatography-tandem mass spectrometry domoic acid class according to claim 1
The method of toxin, which is characterized in that in step (1), the PBS buffer solution is made by the following method: weighing two hydrations respectively
Sodium dihydrogen phosphate 2.18g, disodium hydrogen phosphate dodecahydrate 12.90g, sodium chloride 8.50g are dissolved with water and are settled to 1000mL.
3. a kind of affine in immunity column purification-liquid chromatography-tandem mass spectrometry domoic acid class according to claim 1
The method of toxin, which is characterized in that in step (2), the DA monoclonal antibody immunity affinity column is made by the following method:
(a) synthetic immunogen
Sequentially add the bovine serum albumin(BSA) of 10mg in small flask, the sodium acetate buffer of 2mL 0.05M, pH7.4,1mg's
Domoic acid, the formalin that 60 μ L mass concentrations are 37% mix, 37 DEG C of stirring 48h, then use the PBS of 0.1M, pH7.3
Buffer dialyses 3 days in 4 DEG C, changes liquid 2 times daily, obtain immunogene DA-BSA；
(b) synthesis detection is former
Sequentially add the chicken ovalbumin of 10mg in small flask, the sodium acetate buffer of 2mL 0.05M, pH7.4,1mg's
Domoic acid, the formalin that 60 μ L mass concentrations are 37% mix, 37 DEG C of stirring 48h, then use the PBS of 0.1M, pH7.3
Buffer dialyses 3 days in 4 DEG C, changes liquid 2 times daily, must detect original DA-OVA；
(c) monoclonal antibody preparation and purifying
The female Balb/c mouse of 5 6~8 week old is taken, only, 4 Zhou Houkai are immunized in dorsal sc multi-point injection DA-BSA, 20 μ g/
Beginning booster immunization, every 2 weeks booster immunization 1 time；When initial immunity, with immunogene DA-BSA and isometric Freund's complete adjuvant cream
Change, is emulsified with immunogene DA-BSA and isometric incomplete Freund's adjuvant when booster immunization, immunizing dose, immunization ways are constant；
After 2nd time booster immunization 10 days, mouse tail vein blood examination is taken to survey, coating detection original DA-OVA uses indirect competitive ELISA method
The potency of serum is detected, the highest BALB/c mouse of potency is selected to be used to prepare hybridoma, preceding 3 days mouse peritoneals is merged and adds
It is strong immune primary, take the highest BALB/c mouse splenocyte of potency to merge with myeloma cell Sp2/0, it is thin that screening obtains hybridoma
Born of the same parents；
0.5mL atoleine sensitization, injection 1 × 10 in 8 days backward mouse peritoneals is injected intraperitoneally in BALB/c mouse7A hybridoma is thin
Born of the same parents started to extract ascites from mouse peritoneal with syringe after 10 days, and later every extraction in 1 day ascites 1 time, supernatant is collected in centrifugation
Liquid purifies supernatant using saturated ammonium sulfate method, then acquisition DA monoclonal is further purified with ProteinG affinity chromatography and resists
Body；
(d) prepared by affinity column
The HCl solution swelling of the hydrogen bromide activated sepharose 4B dry powder 100mL 1M of 0.5g CNBr activation is weighed, and is washed
It washs；Gel is washed to obtain with 100mL coupling buffer again；
Gel after taking 1.5mL to be swollen, is added the DA monoclonal antibody of 1.5mL 10mg/mL, and shaken at room temperature coupling reaction 2h takes out
Filter, and use 30mL coupling buffer detergent gel, filter, be added 10mL Block buffer, shaken at room temperature react 2h, suction filtration, then
It with the PBS buffer solution detergent gel of 100mL 0.01M, pH7.3, and is transferred in 5mL column tube, is added and distinguishes containing mass concentration
For the PBS solution of 0.05% thimerosal, 0.01M, pH7.3 of 0.05% bovine serum albumin(BSA), stored in 2~8 DEG C.
4. a kind of affine in immunity column purification-liquid chromatography-tandem mass spectrometry domoic acid class according to claim 3
The method of toxin, which is characterized in that in step (d), the formula of the coupling buffer are as follows: 0.1molL-1NaHCO3,
0.5mol·L-1NaCl, pH8.3.
5. a kind of affine in immunity column purification-liquid chromatography-tandem mass spectrometry cartilage algae according to claim 3 or 4
The method of acids toxin, which is characterized in that the formula of the Block buffer are as follows: 0.1molL-1Tris-HCl, pH8.0.
6. a kind of affine in immunity column purification-liquid chromatography-tandem mass spectrometry domoic acid class according to claim 1
The method of toxin, which is characterized in that the liquid phase chromatogram condition are as follows: chromatographic column: ACQUITY UPLC BEH C18Column (2.1mm ×
50mm, 1.7 μm)；2 μ L of sampling volume；10 DEG C of sample room temperature；40 DEG C of column temperature；Flow velocity 0.2mL/min；Mobile phase A is acetonitrile, B
For the 2mmol/L ammonium acetate solution containing 0.1% formic acid, gradient elution: 0~1.0min, 90%A；1.0~3.0min, 90%~
10%A；3.0~4.0min, 10%A；4.0~4.1min, 10%~90%A；4.1~5.5min, 90%A.
7. a kind of affine in immunity column purification-liquid chromatography-tandem mass spectrometry domoic acid class according to claim 1
The method of toxin, which is characterized in that the Mass Spectrometry Conditions are as follows: electric spray ion source, cation scan (Electrospray
Ionization, ESI-)；Detection mode: multiple-reaction monitoring pattern (Multiple Reaction Monitor, MRM)；Capillary
Tube voltage: 3.0kV；Ion source temperature: 150 DEG C；Desolvation temperature: 600 DEG C；Taper hole throughput: 150L/h；Desolventizing air-flow
Amount: 1000L/h；Orifice potential: 12V；Collision energy: 16eV；Analyte qualitative ion pair: m/z 312.2 > 248.2；Analyte
Quota ion pair: m/z 312.2 > 266.2.
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