CN106908597A - A kind of preparation method of immune affinity column - Google Patents
A kind of preparation method of immune affinity column Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of immune affinity column, wet method dress post obtains three kinds of immune affinity columns after being mainly coupled by three kinds of antibody and coupling matrices, and according to volume 1:1:1 mixing dress post, a kind of compound affinity column of preparation.Its column capacity is 2500 ng, and good purification can be recycled 3 times, and average recovery rate is between 67%~107%.The complex immunity affinity column at least has good specific adsorption to 12 kinds of sulfa drugs, including：Sulfaquinoxaline, sulphadiazine, sulfapryidine, 5-methoxysulfadiazine, sulfadimidine, Sulfamethoxazole, NU-445, madribon, sulphathiazole, sulfamethyldiazine, sulfachlorpyridazine sodium, daimeton.Prepare that this Conjugate ratio is high, specific adsorption strong, column capacity and rate of recovery immune affinity column high, and propose simultaneously detect sensitive, quick, the easy analysis method of above-mentioned 12 kinds of sulfa drug residues.
Description
Technical field
The present invention relates to a kind of preparation of the immune affinity column for detecting purifying sulpha drugs residual, belong to immunology and food
Quarantine Techniques field is surveyed in product examine.
Background technology
Sulfa drug residue in animal derived food is a class veterinary drug of extensive concern, after these residuals enter human body
The allergic reaction of human body can be caused, and there may be certain carcinogenicity.Therefore, the research to sulfa drug residue turns into inspection
The focus of food inspection worker.Than larger, the purification method of detection mainly has solid phase at present for the interference of detection process mesostroma
Extraction, liquid-liquid extraction etc., operate relatively complicated, the animal derived sample pre-treatments complexity of purification；Selectivity ratios are relatively low, and purification effect
Really undesirable, impurity interference is very big；The rate of recovery and repeatability are poor.
The content of the invention
Be to solve the technical problem that prior art is present, the invention provides a species specificity is high, good purification, operation
Easy immune affinity column（Immunoaffinity column, IAC）, and research and development are suitable for many animals such as meat, eggs and milk
Derived food matrix, can reduce the matrix effect existed when LC-MS instrument is detected, can simultaneously detect various sulfa drugs
Sensitive, quick, the easy analysis method of residual.
Technical solution of the present invention is as follows：A kind of preparation method of immune affinity column, described immune affinity column is net to detect
Change the immune affinity column of sulfa drug residue, described immune affinity column is according to volume 1 by three kinds of immune affinity columns:1:1
Mixing dress post, obtained compound affinity column, three kinds of described immune affinity columns are by three kinds of monoclonal antibody bar aspertoxin lists
Clonal antibody 5H4, NR3C2 monoclonal antibody 2B5 and PPBP monoclonal antibody 5C7 respectively with beaded agarose matrix cyanogen bromide
Activation Sepharose 4B couplings, three kinds of immune affinity columns obtained in wet method dress post, specific preparation process is as follows：
The first step：Two kinds of preparations of the artificial antigen TS-BSA and SM2-BSA of sulfa drugs, by the thio acyl of two carbon two of tetramethyl
Amine TS and SM2 sulfadimidine antigen is coupled and obtains with bovine serum albumin(BSA) BSA with ovalbumin；
Second step：Antigen obtained by the first step is immunogene to obtaining bone-marrow-derived lymphocyte, bone-marrow-derived lymphocyte and marrow after mouse immune
Oncocyte cell fusion obtains hybridoma, using inducing ascites method prepares above-mentioned 3 kinds of monoclonal antibodies in vivo,
3rd step：Three kinds of immune affinity columns, by kind monoclonal antibody and the beaded agarose matrix cyanogen bromide-activated of second step
Sepharose 4B are coupled, wet method dress post, the three kinds of immune affinity columns for obtaining.
The described first step：Two kinds of artificial antigen TS-BSA and SM2-BSA of sulfa drugs, preparation, it is specific to prepare
Step is as follows：
（1）The synthesis of SM2 haptens：SM2 is dissolved in the treated pyridine of anhydrous sodium sulfate, succinic anhydride be dissolved in anhydrous pyridine with
SM2 liquid mixes dissolving, and the h of stirring reaction 24, reaction solution immigration shakes up in being equipped with 1 separatory funnel of water, stands, entered with CH2Cl2
Row extraction jog, notes extraction 3 times of deflating, and collects lower floor's CH2Cl2 phases, merge 3 extraction CH2Cl2 phases, use 0.1mol/L
After HCl elutes 3 times, washed again with H2O and once collect lower floor's CH2Cl2 phases, a certain amount of anhydrous Na 2SO4 is added in extract
Dehydration, adds toluene low temperature to be evaporated in evaporating dish, filter vacuum is dried into recrystallization washs pastel with ethyl acetate and obtain
White crystal SM2-SA,
（2）The preparation of SM2-BSA coupling protein matter：Take step（1）Obtained 10 mg SM2-SA are dissolved in the anhydrous of 0.5 mL
In DMF, add 6 mg NHS and 10 mg DCC are stirred overnight at room temperature, after being centrifuged off precipitation with 4000r/min centrifugations 5min
Supernatant is added dropwise in 20 mg are dissolved in the BSA and solution of the carbonate buffer solution of 2 mL while stirring at leisure, stirring is anti-
Answer 4 h, reaction that supernatant is taken after terminating and load bag filter, dialysed 2 days with the phosphate buffer PBS of pH 7.4, often
It changes 3 dialyzates, obtains M2-BSA coupling protein matter.
（3）The synthesis of haptens TS
TS synthesis steps：Weigh the g of 2- amino -4- thiazolyl acetic acids 7.8 to be added in 100 mL absolute methanols, add 20mL front threes
Base chlorosilane, 0 DEG C of 2 d of backflow.Rotary evaporation removes organic solvent, is extracted with 250mL ethyl acetate, with 50 mL saturations
NaHCO3The aqueous solution is washed four times（To pH>7）, the 30 mL saturation NaCl aqueous solution wash 2 times, MgSO4Dry, filtering, rotary evaporation is used
30 mL ethyl acetate and 5 mL recrystallizing methanols, obtain（2-amino-4-thiazolyl）- methyl acetate.It is molten with 5 mL pyridines
Solution, add 3.6 g N-acetylsulfanilamide acyl chlorides, 24 h are stirred at room temperature, react, add 2 mL ethanol, with 4M HCl regulation pH to
Precipitation is produced after 3,10-20 minutes, is vacuum dried after filtering, obtain product, it is molten that product is dissolved into 25 mL 2M NaOH
In liquid, room temperature backflow 5h adjusts PH 4 with the hydrochloric acid of 4M, is extracted 4 times with 25mL ethyl acetate, is dried with magnesium sulfate, and rotation is steamed
It is dry to obtain TS；
（4）The synthesis of TS-BSA
4.1 weigh TS 62 mg, NHS 34 mg, DCC 44 mg, are dissolved into 1.5 mL DMF, and 24 h are stirred at room temperature；Respectively
Weigh BSA 20mg to be dissolved into the PBS of 3 mL pH 7.0, respectively by obtained 0.5 mL DMF solutions in above-mentioned steps, slowly
Be added drop-wise in protein solution, 24 h be stirred at room temperature, with the phosphate buffer PBS solutions of pH 7.0 dialyse 3 h,
By UV scanning, calculate TS-BSA coupling ratios and be respectively 10:1；SM2-BSA coupling ratios are respectively 15:1.
Described second step, prepares above-mentioned 3 kinds of monoclonal antibodies, respectively three kinds monoclonal antibody bar aspergillus poison
Plain monoclonal antibody 5H4, NR3C2 monoclonal antibody 2B5 and PPBP monoclonal antibody 5C7, its mini-bus aspertoxin monoclonal resists
Body 5H4 is the cell line obtained by SM2-BSA immunogen immune mouse, NR3C2 monoclonal antibody 2B5 and PPBP monoclonal antibodies
5C7 is the cell line obtained by ST-BSA immunogen immune mouse.
The Sepharose 4B of the new 1.0g cyanogen bromide-activateds of this experiment are prepared for 3.5 mL matrix, slow with coupling respectively
10 mg, tri- kinds of sulfanilamide (SN) antibody couplings after fliud flushing dialysis, Conjugate ratio up to more than 99%.After coupling, wet method dress post is obtained
Three kinds of IAC respectively can only be to 3 kinds, 8 kinds and 7 kinds sulfa drugs（Totally 12 kinds）There is good specific adsorption；This experiment is by three
The Ago-Gel of coupling is planted according to volume 1:1:1 mixing dress post, prepares compound affinity column.Elution requirement to being combined pillar enters
Row optimization, and performance parameter to pillar tests.The elution requirement of pillar is：Eluted with the pure methyl alcohol of 2.0 mL, column performance
Test result is：The Conjugate ratio of antibody is more than 99%, and column capacity is 2500 ng/g matrix, and the compound IAC posts are to 12 kinds of sulfamidos
Medicine has good specific adsorption.
This 12 kinds of sulfa drugs are respectively：Sulfaquinoxaline, sulphadiazine, sulfapryidine, 5-methoxysulfadiazine, sulphur
Amine diformazan pyrimidine, Sulfamethoxazole, NU-445, madribon, sulphathiazole, sulfamethyldiazine, sulfanilamide (SN)
Chlorine pyridazine sodium, daimeton.
This method establishes 12 kinds of affine in immunities of residual quantity of sulfonamide detect pork, milk simultaneously in and purifies-surpass
High performance liquid chromatography and phase chromatographic tandem mass spectrography.
It is an advantage of the invention that：Prepare that Conjugate ratio is high, specific adsorption strong, column capacity and high immune of the rate of recovery
Affinity column, its high selectivity greatly simplifies Sample pretreatment process, is particularly suited for the animal derived foods such as rabbit meat, egg
The detection of middle Sulfonamides residual, analysis quality is improved；There is very strong reservation and concentrating capacity to component to be measured, especially
The separation and concentration of the extremely low component of concentration suitable for complex sample；Qualitative information is provided while purification to component；There is multicomponent
Disposal ability；Water is mutually operated, and can be reused, it is possible to reduce analysis cost；Analysis side is made to determinand selective enrichment
The test limit of method depends primarily on sample size, and this is that desired cleansing means are difficult to；As the purification of physics and chemistry Instrumental Analysis
Means, with reference to chromatography efficient detection residual quantity of sulfonamide, treat when can avoid using immunoassay direct detection sample
The problems such as survey thing information content is very little, influence factor is more, dosing accuracy is not enough, it is shown that immunological technique and conventional physiochemical techniques
Complementarity in terms of analysis mechanisms.
Brief description of the drawings
The immune affinity column of Fig. 1 detection purifying sulpha drugs residuals uses and regenerates schematic diagram.
Specific embodiment
In order to be able to be more clearly understood that technical scheme, further illustrated with reference to specific embodiment.
A kind of preparation method of immune affinity column, described immune affinity column is to detect exempting from for purifying sulpha drugs residual
Epidemic disease affinity column, described immune affinity column is according to volume 1 by three kinds of immune affinity columns:1:1 mixing dress post, obtained compound parent
And post, three kinds of described immune affinity columns are by three kinds of monoclonal antibody bar aspertoxin monoclonal antibody 5H4, NR3C2 Dan Ke
Grand antibody 2B5 and PPBP monoclonal antibodies 5C7 are coupled with beaded agarose matrix cyanogen bromide-activated Sepharose 4B respectively, wet
Three kinds of immune affinity columns obtained in method dress post, specific preparation process is as follows：
The first step：Two kinds of preparations of the artificial antigen TS-BSA and SM2-BSA of sulfa drugs, by the thio acyl of two carbon two of tetramethyl
Amine TS and SM2 sulfadimidine antigen is coupled and obtains with bovine serum albumin(BSA) BSA with ovalbumin；
Second step：Antigen obtained by the first step is immunogene to obtaining bone-marrow-derived lymphocyte, bone-marrow-derived lymphocyte and marrow after mouse immune
Oncocyte cell fusion obtains hybridoma, using inducing ascites method prepares above-mentioned 3 kinds of monoclonal antibodies in vivo,
3rd step：Three kinds of immune affinity columns, by kind monoclonal antibody and the beaded agarose matrix cyanogen bromide-activated of second step
Sepharose 4B are coupled, wet method dress post, the three kinds of immune affinity columns for obtaining.
The described first step：Two kinds of artificial antigen TS-BSA and SM2-BSA of sulfa drugs, preparation, it is specific to prepare
Step is as follows：
（1）The synthesis of SM2 haptens：SM2 is dissolved in the treated pyridine of anhydrous sodium sulfate, succinic anhydride be dissolved in anhydrous pyridine with
SM2 liquid mixes dissolving, and the h of stirring reaction 24, reaction solution immigration shakes up in being equipped with 1 separatory funnel of water, stands, entered with CH2Cl2
Row extraction jog, notes extraction 3 times of deflating, and collects lower floor's CH2Cl2 phases, merge 3 extraction CH2Cl2 phases, use 0.1mol/L
After HCl elutes 3 times, washed again with H2O and once collect lower floor's CH2Cl2 phases, a certain amount of anhydrous Na 2SO4 is added in extract
Dehydration, adds toluene low temperature to be evaporated in evaporating dish, filter vacuum is dried into recrystallization washs pastel with ethyl acetate and obtain
White crystal SM2-SA,
（2）The preparation of SM2-BSA coupling protein matter：Take step（1）Obtained 10 mg SM2-SA are dissolved in the anhydrous of 0.5 mL
In DMF, add 6 mg NHS and 10 mg DCC are stirred overnight at room temperature, after being centrifuged off precipitation with 4000r/min centrifugations 5min
Supernatant is added dropwise in 20 mg are dissolved in the BSA and solution of the carbonate buffer solution of 2 mL while stirring at leisure, stirring is anti-
Answer 4 h, reaction that supernatant is taken after terminating and load bag filter, dialysed 2 days with the phosphate buffer PBS of pH 7.4, often
It changes 3 dialyzates, obtains M2-BSA coupling protein matter.
（3）The synthesis of haptens TS
TS synthesis steps：Weigh the g of 2- amino -4- thiazolyl acetic acids 7.8 to be added in 100 mL absolute methanols, add 20mL front threes
Base chlorosilane, 0 DEG C of 2 d of backflow.Rotary evaporation removes organic solvent, is extracted with 250mL ethyl acetate, with 50 mL saturations
NaHCO3The aqueous solution is washed four times（To pH>7）, the 30 mL saturation NaCl aqueous solution wash 2 times, MgSO4Dry, filtering, rotary evaporation is used
30 mL ethyl acetate and 5 mL recrystallizing methanols, obtain（2-amino-4-thiazolyl）- methyl acetate.It is molten with 5 mL pyridines
Solution, add 3.6 g N-acetylsulfanilamide acyl chlorides, 24 h are stirred at room temperature, react, add 2 mL ethanol, with 4M HCl regulation pH to
Precipitation is produced after 3,10-20 minutes, is vacuum dried after filtering, obtain product, it is molten that product is dissolved into 25 mL 2M NaOH
In liquid, room temperature backflow 5h adjusts PH 4 with the hydrochloric acid of 4M, is extracted 4 times with 25mL ethyl acetate, is dried with magnesium sulfate, and rotation is steamed
It is dry to obtain TS；
（4）The synthesis of TS-BSA
4.1 weigh TS 62 mg, NHS 34 mg, DCC 44 mg, are dissolved into 1.5 mL DMF, and 24 h are stirred at room temperature；Respectively
Weigh BSA 20mg to be dissolved into the PBS of 3 mL pH 7.0, respectively by obtained 0.5 mL DMF solutions in above-mentioned steps, slowly
Be added drop-wise in protein solution, 24 h be stirred at room temperature, with the phosphate buffer PBS solutions of pH 7.0 dialyse 3 h,
By UV scanning, calculate TS-BSA coupling ratios and be respectively 10:1；SM2-BSA coupling ratios are respectively 15:1.
Described second step, prepares above-mentioned 3 kinds of monoclonal antibodies, respectively three kinds monoclonal antibody bar aspergillus poison
Plain monoclonal antibody 5H4, NR3C2 monoclonal antibody 2B5 and PPBP monoclonal antibody 5C7, its mini-bus aspertoxin monoclonal resists
Body 5H4 is the cell line obtained by SM2-BSA immunogen immune mouse, NR3C2 monoclonal antibody 2B5 and PPBP monoclonal antibodies
5C7 is the cell line obtained by ST-BSA immunogen immune mouse.
One, sulfa drugs antigen synthesizes and Antibody preparation
With bovine serum albumin(BSA)（BSA, 99.9%）It is carrier protein, sulfonamides is successfully synthesized with the coupling of SM2 and TS antigens
Thing artificial antigen TS-BSA and SM2-BSA.The coupling ratio of TS-BSA is respectively 10:1；The coupling ratio of SM2-BSA is respectively 15:1.
The immune bone-marrow-derived lymphocyte for obtaining is carried out to mouse and obtains hybridoma with myeloma cell's cell fusion, using body
Inside induce ascites method and prepare three kinds of monoclonal clonal antibodies 5H4,2B5 and 5C7.5H4 antibody is to 3 kinds of sulfonamides（Sulfanilamide (SN)
Diformazan pyrimidine, madribon, sulfamethyldiazine）Suppression preferably, 2B5 antibody is to 8 kinds of sulfonamides（Sulfanilamide (SN) quinoline
Evil quinolines, sulphadiazine, sulfapryidine, 5-methoxysulfadiazine, Sulfamethoxazole, NU-445, madribon,
Daimeton）Suppression preferably, 5C7 antibody to 7 kinds of sulfonamides (sulfaquinoxaline, sulphadiazine, sulfapryidine,
Sulfamethoxazole, madribon, sulphathiazole, sulfachlorpyridazine sodium) suppression preferably, IC50 is respectively less than 20 ng/
mL。
The preparation of two, sulfa drugs immune affinity columns and its performance study
1. prepared by matrix：Weigh the Sepharose 4B of 1.0 g CNBr activation（1g lyophilized matrix powder can form 3.5mL end bodies
Long-pending swollen matrix）, it is dissolved in 1mM HCl（25 DEG C, 15min）.Matrix will be swelling immediately, is subsequently placed in sintered glass filtering
Device（Porosity：G3）Middle use 1mM HCl wash 15 min.
2. ligand coupling：
（1）With 0.1 M NaHCO38.3 coupling buffers of pH, 4 DEG C of h of antibody 24 of dialysis coupling, then use coupling buffer
Three kinds of sulfanilamide (SN) ACs of control, about 2 mg/mL, in case coupling is used.Use base after 10 mL coupling buffers washing swelling
, after washing, be transferred to matrix in antibody-solutions rapidly by matter.1 g matrix Sepharose 4B and 15 ~ 35 mg antibody couplings.
（2）Room temperature condition（20~25℃）It is lower by the way of end-over-end（The soft stirring means for relaxing）Fully
Mix the above-mentioned h of mixture 2, or the mixing overnight at 4 DEG C.
（3）2000 r/min are centrifuged 1 min, and Sepharose is centrifuged to ttom of pipe, and supernatant is transferred into new centrifuge tube
In, ice bath is preserved, and determines supernatant AC value, and calculate Conjugate ratio.
（4）The Sepharose 4B at centrifuge tube bottom are taken, is washed using the coupling buffer of at least 5 times of matrix volumes, removed
Unnecessary part.
3. close：
（1）Close the active group of all residuals, transfer medium to 0.1 M Tris-HCl buffer solutions, in pH 8.0.Room temperature bar
16 h under the conditions of 2 h or 4 DEG C are stood under part.
（2）To remove the unnecessary part not being coupled after coupling, matrix is entered with the buffer solution of two kinds of pH low, high successively
Row washing, at least washs 3 circulations, at least 5 times matrix volumes of usage amount of every kind of buffer solution.
Each wash cycle step：First with 0.1 M Acetic acid-sodium acetates, the buffer solution that pH 4.0 includes 0.5 M NaCl is washed
Wash, the buffer solution for then including 0.5 M NaCl with 0.1 M Tris-HCl, pH 8.0 again is washed.
4. post is filled：Using wet method dress post, after pillar is installed, with 5 times of 0.01% NaN of the aseptic filtration of bed volume3-
PBS crosses post, and uses 0.01% NaN3- PBS is preserved.
5. three kinds of affinity columns of Antibody preparation are specific：With three kinds of sulfa drugs IAC posts to 12 kinds of sulfa drugs spies
Opposite sex absorption.The mL of sulfa drugs standard liquid 10.0 of 100 ng is taken respectively, it is pure with 10 mL respectively by immune affinity column
After water wash, eluted with 5.0 mL methyl alcohol, collect eluent, detected with HPLC.Result shows that three kinds of affinity columns are to 12 kinds of sulfanilamide (SN)
The situation of Drug absorbability is different, and this experiment purpose is to prepare the affinity column that can be adsorbed well to 12 kinds of sulfa drugs, because
This by three kinds of immune affinity columns, it is necessary to mix dress post.
6. IAC specificity is combined：By three kinds of Ago-Gels of antibody coupling according to 1:1:1 volume mixture fills post, prepares
Compound affinity column.12 kinds of mL of sulfa drugs standard liquid 10.0 of 100 ng are taken respectively, by complex immunity affinity column, are used
After 10 mL pure water drip washing, eluted with 5.0 mL methyl alcohol, collect eluent, detected with HPLC.The complex immunity affinity column is to 12 kinds
Sulfa drugs has good specific adsorption.Therefore, the immune affinity column can apply to 12 kinds of inspections of sulfa drugs
Survey.
7. the determination of elution requirement：This experiment is molten using the reduction of organic solvent methyl alcohol using the elution process for changing polarity
The polarity of liquid, methyl alcohol eluting power is strong, and it is easy to concentration and the detection of HPLC.With the increase of methanol concentration, eluting rate by
It is cumulative big；When being eluted with 100% methyl alcohol, eluting rate reaches 100%.Detection and raising detection for the ease of HPLC is sensitive
Degree, so selecting pure methyl alcohol as eluent.With the increase of methyl alcohol volume, eluting rate gradually increases；When methanol usage exceedes
After 1.5 mL, eluting rate reaches maximum 100%.Therefore, in order to improve detection sensitivity, the volume of the selected eluent of experiment is 2
mL。
8. column capacity：This experiment calculates column capacity using saturation, and will exceed the determinand of theoretical capacity makes IAC satisfy
With measure after scrubbed and wash-out.Take the madribon containing 5000 ng（It is excessive）The mL of working solution 10, passes through
Immune affinity column, with 10 mL pure water drip washing after, with 2.0 mL methyl alcohol elute, collect eluent, detected with HPLC.In eluent
The content of madribon is 2500 ng, therefore the column capacity of the immune affinity column is 2500 ng.
9. the tolerance test of methyl alcohol：200 ng madribons are dissolved separately in 10 mL a certain proportion of
In methanol aqueous solution（Respectively 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% methanol-water and acetonitrile water）, respectively by IAC,
After with 10 mL pure water drip washing, 2.0 mL methyl alcohol are eluted and calculate the rate of recovery.IAC is resistant to methanol-water of the methanol content less than 20%
Solution.Therefore, we carry out sample purification experiment, and the content of methyl alcohol can not be more than 20% in the sample extracting solution of upper prop.
Using three kinds of antibody of sulfamido and cyanogen bromide for obtaining（CNBr）The agarose of activation（Sepharose 4B）Carry out
Coupling, wet method dress post, the three kinds of IAC for obtaining can only have specificity well to inhale to 3 kinds, 8 kinds and 7 kinds sulfa drugs respectively
It is attached；This experiment is by three kinds of Ago-Gels of coupling according to volume 1:1:1 mixing dress post, prepares compound affinity column.To combined column
The elution requirement of son is optimized, and performance parameter to pillar is tested.The elution requirement of pillar is：With 2.0 mL methyl alcohol
Elute, column performance test result is：The Conjugate ratio of antibody is more than 99%, and column capacity is 2500 ng/g matrix, the compound IAC posts pair
12 kinds of sulfa drugs have good specific adsorption.
The three, immune affinity columns methods of purification determine the various sulfa drugs in animal derived food simultaneously
1. establish and detect 12 kinds of affine in immunity purification-ultra high efficiency liquid phases of residual quantity of sulfonamide in pork, milk simultaneously
Chromatography., through ultrasonic extraction, sulfa drugs affine in immunity column purification, selectivity is high, good purification for sample, and with ultra high efficiency
Liquid chromatogram-PDA carries out detection confirmation.Result shows:The method is easy, quick, sensitive, accurate, good purification, recovery
Rate is high, it is adaptable to detected while 12 kinds of residual quantity of sulfonamide in pork and milk.
2. establish and detect 12 kinds of affine in immunities of sulfa drugs in pork, beef, 4 kinds of matrix of milk and egg simultaneously
Post-Ultra Performance Liquid Chromatography tandem mass spectrometry (IAC-UHPLC-MS/MS).With Waters ACQUITY UPLC BEH C18
Post is separated with methyl alcohol and 0.1% aqueous formic acid as flowing, and mass spectrum is ionized with positive ion mode, and multiple-reaction monitoring (MRM) is fixed
It is fixed to measure.Sample IAC-SULF affine in immunity column purifications, good purification, average recovery rate between 67%~107%, relatively
Standard deviation is less than 10.0%.12 kinds of quantitative limit scopes of sulfonamide are 2.3~10 μ g kg-1, method has wider linear
Scope, coefficient R 2>0.99, it is adaptable to detected while various sulfa drugs in animal derived food.
The above is only better embodiment of the invention, therefore all constructions according to described in present patent application scope,
The equivalent change or modification that feature and principle are done, is included in the range of present patent application.
Claims (3)
1. a kind of preparation method of immune affinity column, it is characterised in that described immune affinity column is detection purification sulfonamides
The immune affinity column of thing residual, described immune affinity column is according to volume 1 by three kinds of immune affinity columns:1:1 mixing dress post, system
Compound affinity column, three kinds of described immune affinity columns be by three kinds of monoclonal antibody bar aspertoxin monoclonal antibody 5H4,
NR3C2 monoclonal antibody 2B5 and PPBP monoclonal antibodies 5C7 respectively with beaded agarose matrix cyanogen bromide-activated Sepharose
4B is coupled, and three kinds of immune affinity columns obtained in wet method dress post, specific preparation process is as follows：The first step：Two kinds of sulfa drugs
The preparation of artificial antigen TS-BSA and SM2-BSA, by thio two carbon diamides TS and SM2 the sulfadimidine antigen of tetramethyl with
Bovine serum albumin(BSA) BSA and ovalbumin are coupled and obtain；Second step：Antigen obtained by the first step for immunogene to mouse immune after
Bone-marrow-derived lymphocyte is obtained, bone-marrow-derived lymphocyte obtains hybridoma with myeloma cell's cell fusion, using inducing ascites legal system in vivo
It is standby to obtain above-mentioned 3 kinds of monoclonal antibodies, the 3rd step：Three kinds of immune affinity columns, by kind monoclonal antibody and the pearl fine jade of second step
Lipolysaccharide matrix cyanogen bromide-activated Sepharose 4B are coupled, wet method dress post, the three kinds of immune affinity columns for obtaining.
2. the preparation method of immune affinity column according to claim 1, it is characterised in that the described first step：Two kinds of sulphurs
The artificial antigen TS-BSA and SM2-BSA of amine drug, preparation, specific preparation process is as follows：（1）The synthesis of SM2 haptens：
SM2 is dissolved in the treated pyridine of anhydrous sodium sulfate, and succinic anhydride is dissolved in anhydrous pyridine and mixes dissolving, stirring reaction 24 with SM2 liquid
H, reaction solution immigration is shaken up in being equipped with 1 separatory funnel of water, stands, and extraction jog is carried out with CH2Cl2, notes extraction 3 of deflating
It is secondary, lower floor's CH2Cl2 phases are collected, merge 3 extraction CH2Cl2 phases, after eluting 3 times with 0.1mol/L HCl, one is washed again with H2O
Secondary collection lower floor CH2Cl2 phases, add a certain amount of anhydrous Na2SO4 dehydration in extract, add toluene low in evaporating dish
Temperature is evaporated, and filter vacuum is dried into recrystallization pastel is washed with ethyl acetate and obtain white crystal SM2-SA,（2）SM2-BSA
The preparation of coupling protein matter：Take step（1）Obtained 10 mg SM2-SA are dissolved in the dry DMF of 0.5 mL, add 6
Mg NHS and 10 mg DCC are stirred overnight at room temperature, by supernatant after being centrifuged off precipitating with 4000r/min centrifugations 5min, slowly
Ground is added dropwise in 20 mg are dissolved in the BSA and solution of the carbonate buffer solution of 2 mL while stirring, and the h of stirring reaction 4, reaction terminates
Supernatant being taken later and loading bag filter, dialysed 2 days with the phosphate buffer PBS of pH 7.4,3 dialysis are changed daily
Liquid, obtains M2-BSA coupling protein matter；（3）The synthesis of haptens TS, TS synthesis steps：Weigh the g of 2- amino -4- thiazolyl acetic acids 7.8
It is added in 100 mL absolute methanols, adds 20mL trim,ethylchlorosilanes, 0 DEG C of 2 d of backflow, rotary evaporation to remove organic solvent,
Extracted with 250mL ethyl acetate, with 50 mL saturations NaHCO3The aqueous solution is washed four times（To pH>7）, the 30 mL saturation NaCl aqueous solution
Wash 2 times, MgSO4Dry, filtering, rotary evaporation, with 30 mL ethyl acetate and 5 mL recrystallizing methanols, is obtained（2-amino-4
- thiazolyl）- methyl acetate, with 5 mL pyridinium dissolutions, adds 3.6 g N-acetylsulfanilamide acyl chlorides, and 24 h are stirred at room temperature, and reacts
It is complete, 2 mL ethanol are added, precipitation is produced after being adjusted pH to 3,10-20 minutes with 4M HCl, it is vacuum dried after filtering, produced
Thing, product is dissolved into 25 mL 2M sodium hydroxide solutions, room temperature backflow 5h, and PH 4 is adjusted with the hydrochloric acid of 4M, uses 25mL second
Acetoacetic ester is extracted 4 times, is dried with magnesium sulfate, and rotation is evaporated and obtains TS；（4）The synthesis of TS-BSA, 4.1 weigh TS 62 mg, NHS
The mg of 34 mg, DCC 44, is dissolved into 1.5 mL DMF, and 24 h are stirred at room temperature；BSA 20mg are weighed respectively is dissolved into 3 mL pH
In 7.0 PBS, respectively by obtained 0.5 mL DMF solutions in above-mentioned steps, it is slowly dropped in protein solution, is stirred at room temperature
24 h, with the phosphate buffer PBS solutions of pH 7.0 3 h of dialysis, by UV scanning, calculate TS-BSA coupling ratios and are respectively
10:1；SM2-BSA coupling ratios are respectively 15:1.
3. the preparation method of immune affinity column according to claim 1, described second step, prepare above-mentioned 3 kinds of lists
Clonal antibody, respectively three kinds monoclonal antibody bar aspertoxin monoclonal antibody 5H4, NR3C2 monoclonal antibody 2B5 and
PPBP monoclonal antibody 5C7, its mini-bus aspertoxin monoclonal antibody 5H4 is obtained by SM2-BSA immunogen immune mouse
Cell line, NR3C2 monoclonal antibody 2B5 and PPBP monoclonal antibodies 5C7 be by ST-BSA immunogen immune mouse obtain it is thin
Born of the same parents' strain.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109425734A (en) * | 2017-08-26 | 2019-03-05 | 北京维德维康生物技术有限公司 | A kind of complex immunity affinity column of bisphenol-A and the like and its preparation and application |
| CN110007023A (en) * | 2019-04-15 | 2019-07-12 | 陕西科技大学 | The high resolution mass spectrum screening method of sulfa drugs and its analysis method with protein macromolecule interaction in fish body |
| CN113252754A (en) * | 2021-05-19 | 2021-08-13 | 郑州大学 | Electrochemical immunosensor for detecting sulfadimethoxine and preparation method thereof |
| CN113281517A (en) * | 2021-05-10 | 2021-08-20 | 洛阳师范学院 | Method for detecting 2, 4-diaminopyrimidine medicine residues by combining immunomagnetic bead pretreatment with HPLC-UV |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109425734A (en) * | 2017-08-26 | 2019-03-05 | 北京维德维康生物技术有限公司 | A kind of complex immunity affinity column of bisphenol-A and the like and its preparation and application |
| CN110007023A (en) * | 2019-04-15 | 2019-07-12 | 陕西科技大学 | The high resolution mass spectrum screening method of sulfa drugs and its analysis method with protein macromolecule interaction in fish body |
| CN113281517A (en) * | 2021-05-10 | 2021-08-20 | 洛阳师范学院 | Method for detecting 2, 4-diaminopyrimidine medicine residues by combining immunomagnetic bead pretreatment with HPLC-UV |
| CN113281517B (en) * | 2021-05-10 | 2024-01-30 | 洛阳师范学院 | Method for detecting 2, 4-diaminopyrimidine medicine residues by combining immune magnetic bead pretreatment with HPLC-UV |
| CN113252754A (en) * | 2021-05-19 | 2021-08-13 | 郑州大学 | Electrochemical immunosensor for detecting sulfadimethoxine and preparation method thereof |
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