CN104391061B - The method of tetraodotoxin in immune affinity column purification-liquid chromatography tandom mass spectrometry determination sea life - Google Patents
The method of tetraodotoxin in immune affinity column purification-liquid chromatography tandom mass spectrometry determination sea life Download PDFInfo
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Abstract
The invention discloses the method for tetraodotoxin in a kind of immune affinity column purification-liquid chromatography tandom mass spectrometry determination sea life, its high sensitivity based on Liquid Chromatography-Tandem Mass Spectrometry and accuracy, utilize unique selective recognition of immune affinity column, can quick, easy, the efficient tetraodotoxin measured in sea life.<!-- 2 -->
Description
Technical field
The present invention relates to a kind of detection method of tetraodotoxin, particularly a kind of method of tetraodotoxin in immune affinity column purification-liquid chromatography tandom mass spectrometry determination sea life.
Background technology
Tetraodotoxin (Tetrodotoxin, TTX) is a kind of natural marine organism neurotoxin, widely distributed, is mainly present in globe fish, also has existence in other animal bodies such as rounded tail king crab, blue ring octopus, snail.Tetraodotoxin toxicity is extremely strong, edible 0.5mg just can causing death, the ion that its positively charged guanidine radicals can stretch into sodium channel selects filtrator, combine with the free carboxy in vias inner walls, hinder sodion to enter, thus suppress the conduction of nerve impulse, make people's sensoparalysis, quadriplegia, expiratory dyspnea, finally dead because of respiration inhibition.China has every year and to cause because the zone of eclipse has the sea life of TTX poisoning event to occur.In view of the serious harm of tetraodotoxin, be necessary to set up a kind of easy, method that effectively can detect tetraodotoxin.
At present, the detection method of tetraodotoxin has bioassay method, high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS) and euzymelinked immunosorbent assay (ELISA) (ELISA) etc.Wherein bioassay method generally needs special animal, poor for applicability, and complex operation, waste time and energy poor repeatability; Though euzymelinked immunosorbent assay (ELISA) high specificity, enzyme linked immunoassay length consuming time, operation easier is larger; Liquid chromatography tandem mass spectrometry is highly sensitive, but existing literature method detection limit is higher, complex pretreatment, and adopt solid phase extraction techniques purification sample undesirable, be subject to matrix interference, the recovery is low.Immune affinity column (IAC) is a kind of Novel chromatographic column based on antigen-antibody reaction, utilize biomacromolecule to have to be made to the characteristic of a class biomacromolecule specific recognition and Reversible binding, there is unique selection selectivity and good adsorption cleaning performance.At present, not yet have and purify using immune affinity column as pre-treatment the relevant report that means detect tetraodotoxin.
Summary of the invention
The object of the present invention is to provide the method for tetraodotoxin in a kind of immune affinity column purification-liquid chromatography tandom mass spectrometry determination sea life, its high sensitivity based on Liquid Chromatography-Tandem Mass Spectrometry and accuracy, utilize unique selective recognition of immune affinity column, can quick, easy, the efficient tetraodotoxin measured in sea life.
The technical solution adopted for the present invention to solve the technical problems is:
A method for tetraodotoxin in immune affinity column purification-liquid chromatography tandom mass spectrometry determination sea life, comprises the following steps:
(1) sample preparation: accurately take fully homogeneous samples 2.00g in 50mL tool plug centrifuge tube, add the methanol solution that 10mL contains 1% acetic acid, vortex oscillation 2min, 60 DEG C of water bath sonicator extract 15min, after being cooled to room temperature, and the centrifugal 5min of 6000r/min, pipette 5mL supernatant in another 50mL centrifuge tube, add 20mlPBS solution to dilute, regulate PH to 7 ~ 8 with 1mol/L sodium hydroxide solution, obtain sample liquid;
(2) immune affinity column purification: sample liquid on immune affinity column, after end of the sample, use water wash affinity column, extract Liquid Residue in post, and discard above whole efflux, finally with the methanol solution wash-out containing 1% acetic acid, the eluent of collection dries up in 60 DEG C of nitrogen, dissolves to obtain scavenging solution with 1mL mobile phase constant volume;
(3) LC-MS analysis: scavenging solution measures the peak area obtaining tetraodotoxin after membrane filtration for liquid chromatography-tandem mass spectrometer, peak area is substituted into typical curve analytical calculation, thus obtain the content of tetraodotoxin in sample.
This invention exploits unique sample preparation technique and prepared the immune affinity column to the unique selective recognition of TTX, coordinate the LC-MS analysis method optimized, the tetraodotoxin in energy quick, easy, efficient mensuration sea life
As preferably, in step (2), proportion of mobile phase is: the 5mmol/L ammonium acetate solution containing 0.1% formic acid: acetonitrile=5%:95%.
As preferably, in step (3), chromatographic condition is: chromatographic column: ACQUITYUPLCBEHAmide post; Sample chamber temperature 10 DEG C; Column temperature 40 DEG C; Sampling volume 10 μ L; Flow velocity 0.3mL/min; Mobile phase A is the 5mmol/L ammonium acetate solution containing 0.1% formic acid, and B is acetonitrile, gradient elution: 0 ~ 1.5min, 5% ~ 80%A; 1.5 ~ 3.0min, 80%A; 3.0 ~ 3.5min, 80% ~ 5%A; 3.5 ~ 5min, 5%A.
As preferably, in step (3), Mass Spectrometry Conditions is: electric spray ion source, and positive ion scans; Detection mode: multiple-reaction monitoring; Capillary voltage: 3.0kV; Ion source temperature: 110 DEG C; Desolventizing temperature degree: 350 DEG C; Taper hole airshed: 50L/h; Desolventizing airshed: 600L/h.
As preferably, the method for making of step (3) standard curve is: compound concentration is the standard working solution of 0.3 μ g/L, 1.0 μ g/L, 2.0 μ g/L, 5.0 μ g/L, 10.0 μ g/L and 20.0 μ g/L, with TTX concentration for horizontal ordinate, peak area is ordinate, production standard curve.
As preferably, the preparation method of described immune affinity column is:
A, antigen synthesize
Immunogenic synthesis:
In little flask, add the BSA of 10mg successively, 2mL0.05MpH7.4 sodium acetate buffer, the TTX of 1mg, the formaldehyde of 37% (mass concentration) of 60 μ L, mixing, 37 DEG C are stirred 48h.Then use the PBS damping fluid of 0.1MpH7.3, in 4 DEG C of dialysis 3 days, change liquid every day 2 times, immunogene TTX-BSA; This antigen is used as immunity.
Detect former synthesis:
Substitute BSA with OVA, detect former TTX-OVA, for the bag quilt of ELISA experiment according to immunogenic synthetic method synthesis.
B, monoclonal antibody preparation and purifying
Get 5, the female Balb/c mouse in 6 ~ 8 week age, dorsal sc multi-point injection TTX-BSA, 20 μ g/ only, start booster immunization after immune 4 weeks, every 2 weeks booster immunizations 1 time; During initial immunity, with TTX-BSA and the emulsification of equal-volume Freund's complete adjuvant, TTX-BSA and the emulsification of equal-volume incomplete Freund's adjuvant during booster immunization, immunizing dose, immunization ways are constant;
2nd booster immunization is after 10 days, get mouse tail vein blood examination to survey, bag is by TTX-OVA, tiring of serum is detected with indirect competitive ELISA method, select to tire the highest BALB/c mouse for the preparation of hybridoma, merge first 3 days mouse peritoneal booster immunizations once, get and tire BALB/c mouse splenocyte the highest and myeloma cell Sp2/0 merges, screening obtains hybridoma;
BALB/c mouse lumbar injection 0.5mL whiteruss sensitization, injection 1 × 10 in 8 days backward mouse peritoneals
7individual hybridoma, starts after 10 days to extract ascites with syringe from mouse peritoneal, extracts ascites 1 time afterwards every 1 day, centrifugal, collect supernatant, adopt saturated ammonium sulfate method purifying supernatant, then be further purified acquisition TTX monoclonal antibody with ProteinG affinity chromatography;
Prepared by C, affinity column
The Sepharose4B dry powder 100mL1MHCl taking 0.5gCNBr activation is swelling, and washs; Use 100mL coupling buffer (0.1molL again
-1naHCO
3, 0.5molL
-1naCl, pH8.3) wash to obtain gel;
Get 1.5mL swelling after gel, add the TTX monoclonal antibody of 1.5mL10mg/mL, shaken at room temperature coupling reaction 2h, suction filtration, and by 30mL coupling buffer detergent gel, suction filtration, add 10mL Block buffer (0.1molL-1Tris-HCl, pH8.0), shaken at room temperature reaction 2h, suction filtration, use 100mLPBS (0.01MpH7.3) buffer solution gel again, and transfer in 5mL column jecket, add the 0.01MpH7.3PBS solution containing 0.05% thimerosal and 0.05%BSA, in 2 ~ 8 DEG C of storages.
As preferably, coupling buffer consists of: 0.1mol/LNaHCO
3, 0.5mol/LNaCl, pH8.3.
As preferably, Block buffer is the Tris-HCl of 0.1mol/L, pH8.0.
The invention has the beneficial effects as follows: the high sensitivity and the accuracy that the present invention is based on Liquid Chromatography-Tandem Mass Spectrometry, utilize unique selective recognition of immune affinity column, can quick, easy, the efficient tetraodotoxin measured in sea life.
Accompanying drawing explanation
Fig. 1 is TTX first mass spectrometric full scan figure.
Fig. 2 extracts reagent acidity to the impact of TTX extraction effect.
Fig. 3 is the impact of sample liquid PH on the recovery of TTX in sample.
Fig. 4 is the MRM collection of illustrative plates of 10 μ g/kg mark-on negative samples after immune affinity column purification.
Fig. 5 is the MRM figure of the TTX standard solution of 0.3 μ g/mL.
Embodiment
Below by specific embodiment, and by reference to the accompanying drawings, technical scheme of the present invention is described in further detail.
In the present invention, if not refer in particular to, the raw material adopted and equipment etc. all can be buied from market or this area is conventional.Method in following embodiment, if no special instructions, is the conventional method of this area.
Instrument and reagent
ACQUITY Ultra Performance Liquid Chromatography-tandem mass spectrometer QuattroPremierXE (Waters, US); MS2 eddy mixer (German IKA company); Nitrogen dries up instrument (Organomatio company of the U.S.); Centrifuge5810 supercentrifuge (German Eppendorf company); 12 passage solid-phase extraction devices (supelco company of the U.S.).
TTX standard items (purity >=98.0%, German Dr.Ehrenstorfer company); Acetonitrile, methyl alcohol (chromatographically pure, German Merck company); Ammonium acetate, formic acid (Sigma Co., USA); Acetic acid, disodium hydrogen phosphate dodecahydrate, two hypophosphite monohydrate sodium dihydrogens, sodium chloride, NaOH (Shanghai traditional Chinese medicines group); Experimental water is ultrapure water.
The preparation of solution
(1) TTX standard solution: accurately take 5,00mgTTX standard items, be settled to 50mL with dissolving containing 0.1% formic acid acetonitrile-aqueous solution (1:1 (V/V)), 4 DEG C keep in Dark Place, and the pot-life is 6 months; 100ng/mL is diluted to containing 0.1% formic acid acetonitrile-aqueous solution (1:1 (V/V)) step by step during use.
(2) phosphate buffer (PBS): take disodium hydrogen phosphate dodecahydrate 6.45g respectively, two hypophosphite monohydrate sodium dihydrogen 1.09g, sodium chloride 4.25g, be settled to 500mL by water-soluble solution.
Embodiment:
A method for tetraodotoxin in immune affinity column purification-liquid chromatography tandom mass spectrometry determination sea life, comprises the following steps:
(1) sample preparation: accurately take fully homogeneous samples 2.00g in 50mL tool plug centrifuge tube, add the methanol solution that 10mL contains 1% acetic acid, vortex oscillation 2min, 60 DEG C of water bath sonicator extract 15min, after being cooled to room temperature, and the centrifugal 5min of 6000r/min, pipette 5mL supernatant in another 50mL centrifuge tube, add 20mlPBS solution to dilute, regulate PH to 7 ~ 8 with 1mol/L sodium hydroxide solution, obtain sample liquid.
(2) immune affinity column purification: sample liquid on immune affinity column, after end of the sample, use water wash affinity column, extract Liquid Residue in post, and discard above whole efflux, finally with the methanol solution wash-out containing 1% acetic acid, the eluent of collection dries up in 60 DEG C of nitrogen, dissolves to obtain scavenging solution with 1mL mobile phase (the 5mmol/L ammonium acetate solution containing 0.1% formic acid: acetonitrile=5%:95%) constant volume.
The preparation method of immune affinity column is:
A, antigen synthesize
Immunogenic synthesis:
In little flask, add the BSA of 10mg successively, 2mL0.05MpH7.4 sodium acetate buffer, the TTX of 1mg, the formaldehyde of 37% (mass concentration) of 60 μ L, mixing, 37 DEG C are stirred 48h.Then use the PBS damping fluid of 0.1MpH7.3, in 4 DEG C of dialysis 3 days, change liquid every day 2 times, immunogene TTX-BSA; This antigen is used as immunity.
Detect former synthesis:
Substitute BSA with OVA, detect former TTX-OVA, for the bag quilt of ELISA experiment according to immunogenic synthetic method synthesis.
B, monoclonal antibody preparation and purifying
Get 5, the female Balb/c mouse in 6 ~ 8 week age, dorsal sc multi-point injection TTX-BSA, 20 μ g/ only, start booster immunization after immune 4 weeks, every 2 weeks booster immunizations 1 time; During initial immunity, with TTX-BSA and the emulsification of equal-volume Freund's complete adjuvant, TTX-BSA and the emulsification of equal-volume incomplete Freund's adjuvant during booster immunization, immunizing dose, immunization ways are constant.
2nd booster immunization is after 10 days, get mouse tail vein blood examination to survey, bag is by TTX-OVA, tiring of serum is detected with indirect competitive ELISA method, select to tire the highest BALB/c mouse for the preparation of hybridoma, merge first 3 days mouse peritoneal booster immunizations once, get and tire BALB/c mouse splenocyte the highest and myeloma cell Sp2/0 merges, screening obtains hybridoma (existing conventional method).
BALB/c mouse lumbar injection 0.5mL whiteruss sensitization, injection 1 × 10 in 8 days backward mouse peritoneals
7individual hybridoma, starts after 10 days to extract ascites with syringe from mouse peritoneal, extracts ascites 1 time afterwards every 1 day, centrifugal, collect supernatant, adopt saturated ammonium sulfate method purifying supernatant, then be further purified acquisition TTX monoclonal antibody with ProteinG affinity chromatography.
Prepared by C, affinity column
Sepharose4B dry powder (commercially available) 100mL1MHCl taking 0.5gCNBr activation is swelling, and washs; Use 100mL coupling buffer (0.1molL again
-1naHCO
3, 0.5molL
-1naCl, pH8.3) wash to obtain gel (swelling).
Get 1.5mL swelling after gel, add the TTX monoclonal antibody of 1.5mL10mg/mL, shaken at room temperature coupling reaction 2h, suction filtration, and by 30mL coupling buffer detergent gel, suction filtration, add 10mL Block buffer (0.1molL-1Tris-HCl, pH8.0), shaken at room temperature reaction 2h, suction filtration, use 100mLPBS (0.01MpH7.3) buffer solution gel again, and transfer in 5mL column jecket, add the 0.01MpH7.3PBS solution containing 0.05% thimerosal and 0.05%BSA, in 2 ~ 8 DEG C of storages.
(3) LC-MS analysis:
Chromatographic condition is: chromatographic column: ACQUITYUPLCBEHAmide post; Sample chamber temperature 10 DEG C; Column temperature 40 DEG C; Sampling volume 10 μ L; Flow velocity 0.3mL/min; Mobile phase A is the 5mmol/L ammonium acetate solution containing 0.1% formic acid, and B is acetonitrile, gradient elution: 0 ~ 1.5min, 5% ~ 80%A; 1.5 ~ 3.0min, 80%A; 3.0 ~ 3.5min, 80% ~ 5%A; 3.5 ~ 5min, 5%A.
Mass Spectrometry Conditions is: electric spray ion source, and positive ion scans; Detection mode: multiple-reaction monitoring; Capillary voltage: 3.0kV; Ion source temperature: 110 DEG C; Desolventizing temperature degree: 350 DEG C; Taper hole airshed: 50L/h; Desolventizing airshed: 600L/h.
The mass spectrum multiple-reaction monitoring experiment conditions such as taper hole voltage, collision energy, analysis thing parent ion and daughter ion are as shown in table 1.
The mass spectrum multiple-reaction monitoring experiment condition of table 1 tetraodotoxin
" * " is quota ion (Quantitativeion).
Scavenging solution measures the peak area obtaining tetraodotoxin after membrane filtration for liquid chromatography-tandem mass spectrometer, peak area is substituted into typical curve analytical calculation, thus obtains the content of tetraodotoxin in sample.
1, chromatogram and Mass Spectrometry Conditions are selected
TTX due to water wettability and polarity stronger, general reverse chromatograms post not easily retains, and ACQUITYUPLCBEHAmide chromatographic column is as the hydrophilic chromatographic post being exclusively used in the analysis of strong polar compound, strong to TTX retention, and good chromatographic peak profile, meet the needs of TTX liquid-phase chromatographic analysis completely.Have positively charged guanidine radicals in TTX molecular structure, ammonium acetate solution can promote that guanidine ionizes, and provides proton source for cationic formation, improves TTX Ionization Efficiency; Formic acid reaches suitable separating effect by regulating mobile phase PH, improves chromatographic peak profile.More than the present invention's research compares, group different volumes mark formic acid and variable concentrations ammonium acetate solution are on the impact of stratographic analysis.Result shows, with the 5mmol/L ammonium acetate solution of 0.1% formic acid and the acetonitrile chromatographic resolution effect good for mobile phase has, and peak shape is sharply symmetrical.
Owing to having positively charged guanidine radicals in TTX molecular structure, the present invention selects to carry out Flow Injection Analysis with peristaltic pump with the TTX standard solution of 3.0 μ g/mL under positive ion scan pattern, is optimized mass spectrometry parameters.By first mass spectrometric scanning analysis, obtain the molion of TTX, best taper hole voltage and capillary voltage, wherein [M+H] are selected in debugging
+(m/z320) abundance is the highest, thus can detect by Selective ion mode m/z320, as shown in Figure 1.Second mass analysis is carried out to analyte ions, obtains fragmention information, and the mass spectrometry parameters such as collision energy are optimized, in table 1.
2, the selection of reagent is extracted
TTX is a kind of amino perhydro quinazoline type compound, is only dissolved in acidulous water or alcoholic solution.Consider that methyl alcohol is as conventional extraction reagent, energy is penetrate tissue rapidly, precipitating proteins, and tool dehydration property, and the present invention selects methyl alcohol as extraction reagent.For investigating the appropriate acidity needed for TTX extraction, obtained the extraction reagent of different acidity by the acetic acid adding different volumes mark, extract the extraction efficiency of reagent to the negative mark-on samples of 5 μ g/kg under more each acidity, result as shown in Figure 2.When acetic acid volume fraction is less than 1% in extraction reagent, extracts the peak area obtained and increase with the volume fraction increase of acetic acid; After volume fraction is greater than 1%, peak area remains unchanged substantially, and excessive acetic acid is unfavorable for regulating sample liquid PH, therefore selects to extract reagent containing 1% acetic acid methanol as TTX.
3, immune affinity column condition optimizing
The affine combination of immune affinity column is directly related with the PH of sample solution, and unfavorable PH can make affinity interaction weaken or destroy.The present invention adopts hydrochloric acid and NaOH to regulate sample liquid PH, and by the recovery of TTX under more different PH, investigate the affinity interaction ability of immune affinity column under different PH, result as shown in Figure 3.When PH is 7 ~ 8 time, affinity interaction ability is best, and the TTX recovery is the highest; When PH be less than 6.5 or be greater than 9 time, affinity obviously weakens, and determines that PH is the optimum PH of TTX immune affinity column 7 ~ 8 time thus.When using 8mL water wash affinity column, impurity can remove substantially, and TTX is not eluted; When 4mL contains 1% acetic acid methanol wash-out affinity column, TTX almost wash-out completely, the recovery also tends towards stability.Therefore the present invention adopts 8mL water as leacheate, and 4mL contains 1% acetic acid methanol as eluent.Marine Biological Samples is after the purification of immune affinity column, and target peak energy is effectively separated with assorted peak, and response intensity is high, and the recovery is good, as shown in Figure 4.
4, the range of linearity and quantitative limit
Be 0.3,1.0,2.0,5.0,10.0 and 20.0 μ g/L standard working solution with TTX standard solution compound concentration, with TTX concentration for horizontal ordinate, peak area is ordinate, production standard curve.Its equation of linear regression: y=72.2835x+13.5344 (r
2=0.9972), TTX is good in 0.3 ~ 20.0 μ g/L internal linear.Being 0.1 μ g/kg with the detection limit (LOD) of 3 times of signal to noise ratio (S/N ratio) determination this method, is 0.3 μ g/kg with the quantitative limit (LOQ) of 10 times of signal to noise ratio (S/N ratio) determination this method, as shown in Figure 5.More existing pertinent literature (Liu Xi, magnificent pretty young woman, Dai Yue etc. adopt correlation analysis method to measure detecting of TTX and are limited to 7.6 ~ 30 μ g/kg), this method detection limit is lower, and sensitivity is higher, the mensuration of TTX in marine organism preferably.
5, the recovery and precision
Accurately take the negative shrimp samples of 2.00g, Pitch-based sphere is the TTX standard solution of 0.30,1.50,5.00 μ g/kg, each Pitch-based sphere replicate determination 6 times, calculates the recovery and precision, the results are shown in Table 2.TTX is at the average recovery rate 88.7% ~ 102.3% of 0.30 ~ 5.00 μ g/kg Pitch-based sphere, and relative standard deviation is 1.99% ~ 6.36%.
The average recovery rate of table 2TTX and relative standard deviation (n=6)
6, actual sample analysis
Adopt this method that Trachypenaeus Curvirostris, prawn, conger pile, dog whelk, monodentate spiral shell, basket whelk, squid, Sepiella maindroni, mussel, flower clam, Portunus trituberculatus Miers, Ovalipes punctatus, rounded tail king crab etc. are amounted to 13 kinds of samples and detect, in basket whelk, flower clam, rounded tail king crab sample, detect TTX, concentration is followed successively by 56.4 μ g/kg, 2.43 μ g/kg and 174 μ g/kg.Analysis result shows, this method adopts immune affinity column purification-Liquid Chromatography-Tandem Mass Spectrometry to detect tetraodotoxin in sea life, highly sensitive, and precision and the recovery all can meet the requirement detecting and analyze.
Above-described embodiment is one of the present invention preferably scheme, not does any pro forma restriction to the present invention, also has other variant and remodeling under the prerequisite not exceeding the technical scheme described in claim.
Claims (7)
1. the method for tetraodotoxin in immune affinity column purification-liquid chromatography tandom mass spectrometry determination sea life, is characterized in that, comprise the following steps:
(1)
sample preparation:accurately take fully homogeneous samples 2.00g in 50mL tool plug centrifuge tube, add the methanol solution that 10mL contains 1% acetic acid, vortex oscillation 2min, 60 DEG C of water bath sonicator extract 15min, after being cooled to room temperature, and the centrifugal 5min of 6000r/min, pipette 5mL supernatant in another 50mL centrifuge tube, add 20mlPBS solution to dilute, regulate PH to 7 ~ 8 with 1mol/L sodium hydroxide solution, obtain sample liquid;
(2)
immune affinity column purifies:sample liquid on immune affinity column, after end of the sample, uses water wash affinity column, extract Liquid Residue in post, and discard above whole efflux, finally with the methanol solution wash-out containing 1% acetic acid, the eluent collected dries up in 60 DEG C of nitrogen, dissolves to obtain scavenging solution with 1mL mobile phase constant volume;
(3)
lC-MS analysis: scavenging solution measures the peak area obtaining tetraodotoxin after membrane filtration for liquid chromatography-tandem mass spectrometer, peak area is substituted into typical curve analytical calculation, thus obtains the content of tetraodotoxin in sample; In step (3), chromatographic condition is: chromatographic column: ACQUITYUPLCBEHAmide post; Sample chamber temperature 10 DEG C; Column temperature 40 DEG C; Sampling volume 10 μ L; Flow velocity 0.3mL/min; Mobile phase A is the 5mmol/L ammonium acetate solution containing 0.1% formic acid, and B is acetonitrile, gradient elution: 0 ~ 1.5min, 5% ~ 80%A; 1.5 ~ 3.0min, 80%A; 3.0 ~ 3.5min, 80% ~ 5%A; 3.5 ~ 5min, 5%A.
2. method according to claim 1, is characterized in that: in step (2), proportion of mobile phase is: the 5mmol/L ammonium acetate solution containing 0.1% formic acid: acetonitrile=5%:95%.
3. method according to claim 1, is characterized in that: in step (3), Mass Spectrometry Conditions is: electric spray ion source, and positive ion scans; Detection mode: multiple-reaction monitoring; Capillary voltage: 3.0kV; Ion source temperature: 110 DEG C; Desolventizing temperature degree: 350 DEG C; Taper hole airshed: 50L/h; Desolventizing airshed: 600L/h.
4. the method according to claim 1-3 any one, it is characterized in that: the method for making of step (3) standard curve is: compound concentration is the standard working solution of 0.3 μ g/L, 1.0 μ g/L, 2.0 μ g/L, 5.0 μ g/L, 10.0 μ g/L and 20.0 μ g/L, with TTX concentration for horizontal ordinate, peak area is ordinate, production standard curve.
5. method according to claim 1, is characterized in that: the preparation method of described immune affinity column is:
a, antigen synthesize
Immunogenic synthesis:
In little flask, add the BSA of 10mg successively, 2mL0.05MpH7.4 sodium acetate buffer, the TTX of 1mg, 60 μ L 37% formaldehyde, mixing, 37 DEG C are stirred 48h, then use the PBS damping fluid of 0.1MpH7.3, in 4 DEG C of dialysis 3 days, change liquid every day 2 times, immunogene TTX-BSA;
Detect former synthesis:
Substitute BSA with OVA, detect former TTX-OVA according to immunogenic synthetic method synthesis;
b, monoclonal antibody preparation and purifying
Get 5, the female Balb/c mouse in 6 ~ 8 week age, dorsal sc multi-point injection TTX-BSA, 20 μ g/ only, start booster immunization after immune 4 weeks, every 2 weeks booster immunizations 1 time; During initial immunity, with TTX-BSA and the emulsification of equal-volume Freund's complete adjuvant, TTX-BSA and the emulsification of equal-volume incomplete Freund's adjuvant during booster immunization, immunizing dose, immunization ways are constant;
2nd booster immunization is after 10 days, get mouse tail vein blood examination to survey, bag is by TTX-OVA, tiring of serum is detected with indirect competitive ELISA method, select to tire the highest BALB/c mouse for the preparation of hybridoma, merge first 3 days mouse peritoneal booster immunizations once, get and tire BALB/c mouse splenocyte the highest and myeloma cell Sp2/0 merges, screening obtains hybridoma;
BALB/c mouse lumbar injection 0.5mL whiteruss sensitization, injection 1 × 10 in 8 days backward mouse peritoneals
7individual hybridoma, starts after 10 days to extract ascites with syringe from mouse peritoneal, extracts ascites 1 time afterwards every 1 day, centrifugal, collect supernatant, adopt saturated ammonium sulfate method purifying supernatant, then be further purified acquisition TTX monoclonal antibody with ProteinG affinity chromatography;
prepared by C, affinity column
The Sepharose4B dry powder 100mL1MHCl taking 0.5gCNBr activation is swelling, and washs; Gel is washed to obtain again with 100mL coupling buffer;
Get 1.5mL swelling after gel, add the TTX monoclonal antibody of 1.5mL10mg/mL, shaken at room temperature coupling reaction 2h, suction filtration, and by 30mL coupling buffer detergent gel, suction filtration, adds 10mL Block buffer, shaken at room temperature reaction 2h, suction filtration, then use 100mLPBS buffer solution gel, and transfer in 5mL column jecket, add the 0.01MpH7.3PBS solution containing 0.05% thimerosal and 0.05%BSA, in 2 ~ 8 DEG C of storages.
6. method according to claim 5, is characterized in that: coupling buffer consists of: 0.1mol/LNaHCO
3, 0.5mol/LNaCl, pH8.3.
7. method according to claim 5, is characterized in that: Block buffer is the Tris-HCl of 0.1mol/L, pH8.0.
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| CN104820033A (en) * | 2015-05-04 | 2015-08-05 | 钦州学院 | Detection method of tetrodotoxin |
| CN106596790A (en) * | 2016-12-28 | 2017-04-26 | 山东出入境检验检疫局检验检疫技术中心 | Method for rapidly detecting content of tetrodotoxin in fish flesh through liquid chromatography-mass spectrometry |
| CN106645496A (en) * | 2016-12-30 | 2017-05-10 | 山东出入境检验检疫局检验检疫技术中心 | Method for quickly detecting tetrodotoxin content in fish liver employing liquid-mass chromatography |
| CN109374809B (en) * | 2018-11-22 | 2020-07-31 | 浙江省海洋水产研究所 | Method for determining chondroacystis toxin by immunoaffinity column purification-liquid chromatography-tandem mass spectrometry |
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