CN106543049A - The preparation method of sulfonamides hapten and its conjugated antigen and application - Google Patents
The preparation method of sulfonamides hapten and its conjugated antigen and application Download PDFInfo
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- C07C311/37—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
- C07C311/38—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton
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Abstract
The invention discloses the preparation method of sulfonamides hapten and its conjugated antigen and application.The structural formula of the sulfonamides hapten and its conjugated antigen is respectively as shown in formula I and formula II.The antiserum of high-titer, high-affinity and high specificity can be produced after the sulfonamides conjugated antigen immune animal, sulfonamides compound can quickly, sensitively, and conveniently be detected using ELISA adsorption analysis method.Sulfonamides standard substance are for the sulfonamides antiserum for adopting the conjugated antigen of the present invention to prepare and the half amount of suppression (IC of sulfonamides envelope antigen association reaction50) for 3.56ng/mL 50.78ng/mL, sulfonamides antiserum is 0.12ng/mL 1.22ng/mL to the lowest detectable limit (LOD) of sulfonamides standard substance.The hapten and conjugated antigen of the present invention is suitable for the detection to sulfonamides compound.
Description
Technical field
The present invention relates to the preparation method of sulfonamides hapten and its conjugated antigen and application in biological chemical field.
Background technology
Sulfa drugss refer to the general name of the class medicine with P-aminobenzene-sulfonamide structure, be a class be used for prevention and
The chemotherapeutic agent for the treatment of bacterial infection disease, its antimicrobial spectrum are wider, to most of gram positive bacterias and gram
Negative bacterium has inhibitory action.
Sulfa drugss are as stable in properties, antimicrobial spectrum is wide, small toxicity, it is oral easily absorb and cheap, extensively should
In for husbandry sector, there is in terms of animal diseases control significant curative effect.But unreasonable due to sulfa drugss makes
Even abused with, misuse, result in the generation of fastbacteria, especially the species of naphthalene plucked instrument Pseudomonas and gram positive bacteria increasingly increases
It is many.After pathogen produces drug resistance to a kind of sulfa drugss, it is also easy to produce cross resistance to other sulfa drugss,
The epidemic infection of multiple fastbacteria is thus resulted in.Additionally, the unreasonable of sulfa drugss is produced using also in livestock productss
Serious residual phenomena is given birth to, the sulfa drugss remained in food can endanger the healthy of people, in consideration of it, China and U.S.
State specifies that the off-drug period of sulfa drugss is 15 days, and MRL is 100ng/g, and EU countries also strictly limits sulfonamides
The residue limits of medicine.
The technical method of detection sulfa drug residue is mainly instrument confirmatory analysis and rapid screening analysis at present.Instrument
The features such as analysis and detection technology has high sensitivity, high specificity, however it is necessary that the behaviour of longer sample process time and specialty
Make personnel, be not suitable for the rapid screening of great amount of samples.Based on the immuno analytical method of Ag-Ab specific reaction, mainly
Including enzyme-linked absorption immunoassay and Sidestream chromatography immuno analytical method, because its speed is fast, low cost and other advantages, it is widely used
The rapid screening analysis of sulfa drugss in food samples.But the immune analysis method of detection sulfa drugss is special mostly
Different in naturely for a certain sulfa drugss, with increase of the sulfa drugss using species in animal husbandry, set up it is a kind of it is quick,
Method that is sensitive, accurate, easily detecting multiple types residual quantity of sulfonamide is current problem demanding prompt solution.
The content of the invention
The technical problem to be solved is how quick, accurately detection amine compound residual quantity.
To solve above-mentioned technical problem, present invention firstly provides a kind of sulfonamides hapten.
Sulfonamides hapten provided by the present invention, its structure is as shown in formula I：
To solve above-mentioned technical problem, present invention also offers the preparation method of compound shown in the Formulas I.
Shown in the Formulas I provided by the present invention, the preparation method of compound, comprises the steps：
1) compound A is carried out into reaction with N- acetylsulphanilyl chloride and obtains intermediate product III；The compound A is 2-
Methoxyl group-PABA methyl ester or 4- (4- aminophenyls) butanoic acid；
2) intermediate product III is hydrolyzed in the basic conditions, obtains compound shown in the Formulas I.
Above-mentioned preparation method is also included step 2) solution after hydrolysis carries out pH value regulation, concretely using dense
Spend the hydrochloric acid for 2mol/L pH value to be adjusted to 4.
In the preparation method of compound shown in above-mentioned Formulas I, step 1) reaction is in triethylamine and 4- lutidines
(DMAP) 2-3h is carried out at 60 DEG C under conditions of existing, concretely in 60 DEG C of reaction 2h of water-bath；Compound A, N- acetyl
The mol ratio of amino phenyl sulfonyl acyl chlorides, triethylamine and DMAP (DMAP) can be 2:2:2:1.
In the preparation method of compound shown in above-mentioned Formulas I, step 2) described being hydrolyzed in concentration be 24.03mg/mL-
95 DEG C of reaction 2-3h in the NaOH solution of 40.05mg/mL.
To solve above-mentioned technical problem, present invention also offers a kind of sulfonamides conjugated antigen.
Sulfonamides conjugated antigen provided by the present invention, its structure is as shown in formula II：
In formula II, K represents carrier protein；The carrier protein is concretely：Bovine serum albumin, human serum albumin,
Hemocyanin, ovalbumin, mouse serum albumin, Elityran or albumin rabbit serum etc..
To solve above-mentioned technical problem, present invention also offers the preparation method of compound shown in the formula II.
The preparation method of compound shown in the formula II provided by the present invention, including by compound shown in described Formulas I
Compound shown in the formula II is obtained with carrier protein couplet.
The preparation method of compound shown in above-mentioned formula II also includes to the step of shown in the formula II, compound carries out purification,
Can be the step of compound shown in described formula II is dialysed.The dialysis is carried out in distilled water；The dialysis is in room
Warm (20-30 DEG C) carries out 72h.
Wherein, compound shown in the Formulas I and carrier protein are in 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides
The coupling reaction that hydrochlorate (EDCHCl) and N-hydroxy-succinamide (NSH) are carried out under conditions of existing；Shown in the Formulas I
Compound, the carrier protein, 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDCHCl) and N- hydroxyls
The mol ratio of butanimide (NSH) concretely 40:1:80:120.
In the preparation method of compound shown in above-mentioned formula II, the concentration of the carrier protein can be 10.05-16.67mg/
ML, the carrier protein concretely bovine serum albumin, the concentration of the bovine serum albumin concretely 10.05mg/mL.
The preparation method of compound shown in the Formulas I or compound shown in the Formulas I is in compound shown in formula II is prepared
Application fall within the scope of protection of the invention.
Compound shown in the Formulas I or compound shown in the formula II, or, the preparation method of compound shown in the Formulas I
Or application of the preparation method of compound shown in the formula II in anti-amine compound antibody is prepared falls within protection of the present invention
Scope.
Using compound shown in the Formulas I or compound shown in the formula II, or, the preparation of compound shown in the Formulas I
The antibody of anti-amine compound prepared by the preparation method of method or compound shown in the formula II falls within the protection of the present invention
Scope, the antibody of the anti-amine compound can be monoclonal antibody, alternatively polyclonal antibody.
Compound shown in the Formulas I or compound shown in the formula II, or, the preparation method of compound shown in the Formulas I
Or the preparation method of compound shown in the formula II, or, the antibody of the anti-amine compound is in detection amine compound
Using falling within the scope of protection of the invention.
Above, the amine compound can be methoxy pyridazine, sulfametoxydiazine, sulfamethizole, sulfanilamide between sulfanilamide
First thiazole, sulfamonomethoxine, sulfaisodimidine, cistosulfa, sulfamethoxazole, sulfanilamide dimethoxy are phonetic
Pyridine, sulfamethyldiazine, sulfaquinoxaline, sulfaphenazole, sulfasalazine, sulfapyridine, sulfamethazole, sulfanilamide
Thiazole, sulfadiazine, sulfamethazine, phthalylsulfathiazole, sulfanitran, sulphaguanidine, sulfafurazole, sulphanilamide,
Sulfabenzamide, sulfadoxine, sulfacetamide and sulfoamido uracil.
It is demonstrated experimentally that so that height can be produced after the sulfonamides conjugated antigen immune animal of method provided by the present invention preparation
The antiserum of potency, high-affinity and high specificity, using the antiserum using ELISA adsorption analysis method can quickly,
Sulfonamides compound is detected sensitively, and conveniently.Sulfonamides standard substance are prepared for the sulfonamides conjugated antigen for adopting the present invention
Half amount of suppression (the IC of sulfonamides antiserum and sulfonamides envelope antigen association reaction50) for 3.56ng/mL-50.78ng/mL,
Adopt sulfonamides antiserum prepared by the sulfonamides conjugated antigen of the present invention to the lowest detectable limit (LOD) of sulfonamides standard substance for
0.12ng/mL-1.22ng/mL.The sulfonamides antiserum prepared using the sulfonamides conjugated antigen of the present invention can be specifically
Sulfonamides standard substance are had higher affinity by identification sulfonamides standard substance.The hapten and conjugated antigen of the present invention is suitable for
In the detection to sulfonamides compound.
Description of the drawings
Fig. 1 is flow chart prepared by sulfonamides hapten and sulfonamides conjugated antigen.
Fig. 2 is the haptenic mass spectrum of sulfonamides.
Fig. 3 is the haptenic hydrogen nuclear magnetic resonance spectrogram of sulfonamides.
Fig. 4 is schemed for the MALDI-TOF-MS of sulfonamides conjugated antigen.
Specific embodiment
The present invention is further described in detail with reference to specific embodiment, the embodiment for being given is only for explaining
The bright present invention, rather than in order to limit the scope of the present invention.
Experimental technique in following embodiments, if no special instructions, is conventional method.
In following embodiments, material used, reagent etc., if no special instructions, commercially obtain.
In following embodiments：2- methoxyl groups-PABA methyl ester (Sigma-Aldrich, 647616), 2- methoxies
Base-PABA methyl ester (Sigma-Aldrich, 335339), N- acetylsulphanilyl chloride (Aladdin reagent,
A114795), triethylamine (Aladdin reagent, T103285), DMAP (DMAP) (Aladdin reagent, D109207),
1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDCHCl) (Sigma-Aldrich, E6383), N- hydroxyls
Butanimide (NHS) (Aladdin reagent, H109330), bovine serum albumin (BSA) (Aladdin reagent, A116563),
HRP- goat anti-rabbit iggs (Jackson ImmunoReasearch Laboratories, 313-005-003), 3,3', 5,5'- tetra-
Methyl biphenyl amine (TMB) (Sigma-Aldrich, 860336).
Sulfanilamide standard substance in following embodiments：Sulfamethazine (SM2) (Sigma-Aldrich, S6256), sulphur
Amine first diazole (SMZ) (Sigma-Aldrich, S5632), sulfadimethoxine (SDM) (Sigma-Aldrich, S7385),
Sulfaquinoxaline (SQX) (Sigma-Aldrich, N13251), cistosulfa (SCP) (Sigma-Aldrich, S9882).
In following embodiments：Methoxy pyridazine (Sigma-Aldrich, S7257), sulfametoxydiazine (Sigma- between sulfanilamide
Aldrich, S7385), sulfamethylthiazole (Sigma-Aldrich, 46901), sulfamonomethoxine (Sigma-Aldrich,
32996), sulfaisodimidine (Sigma-Aldrich, 46908), sulfamethyldiazine (Sigma-Aldrich,
S6256), sulfadimethoxine (Sigma-Aldrich, S7385), sulfaphenazole (Sigma-Aldrich,
SS075802), sulfasalazine (Sigma-Aldrich, S0883), sulfamethoxazole (Sigma-Aldrich,
S7507), sulfapyridine (Sigma-Aldrich, S6252), sulfamethazole (Sigma-Aldrich, S5632), sulfanilamide thiophene
Azoles (Sigma-Aldrich, 46902), sulfadiazine (Sigma-Aldrich, S8626), phthalylsulfathiazole (Sigma-
Aldrich, 46901), sulfanitran (Sigma-Aldrich, 46882), sulphaguanidine (Aladdin reagent, S107128), sulfanilamide
Isoxazole (Sigma-Aldrich, S6377), sulphanilamide (Sigma-Aldrich, S9251), sulfabenzamide (Sigma-
Aldrich, 46762), sulfadoxine (Sigma-Aldrich, 31736), sulfacetamide (Aladdin reagent, S114284)) and
Sulfoamido uracil (CHEMSTEP, 17017-91-3).
New zealand white rabbit in following embodiments be Beijing Vital River Experimental Animals Technology Co., Ltd.'s product, product
Catalog number (Cat.No.) is S09045.
The haptenic preparation of embodiment 1, sulfonamides and identification
First, the haptenic preparation of sulfonamides
1mmol2- methoxyl groups-PABA methyl ester (or 1mmol 4- (4- aminophenyls) butanoic acid) is dissolved in into 4mL
In tetrahydrofuran, the tetrahydrofuran solution of 2- methoxyl groups-PABA methyl ester is obtained.To 2- methoxyl group -4- aminobenzoics
The N- acetyl ammonia of 150 μ L triethylamines, the 4- lutidines of 0.5mmol and 1mmol is added in the tetrahydrofuran solution of sour methyl ester
Base benzene sulfonyl chloride, stirs and reacts 2h under the conditions of 60 DEG C of water-bath.The progress journey of reaction is determined using thin layer chromatography
Degree, as Rf value RfFor 0.5 or so when reaction it is complete, add suitable quantity of water that reaction is quenched when reacting complete, obtain the molten of reaction is quenched
Liquid.To in the solution for be quenched reaction, add dichloromethane to be extracted, carry out extracting operation 3 times altogether, obtain organic faciess；To organic
Na is added in phase2SO4It is dried, obtains dried organic faciess；Dried organic faciess are carried out at 35 DEG C it is concentrated in vacuo,
Obtain the intermediate product III shown in formula III：
Intermediate product III shown in formula III is added in the NaOH solution that 50mL concentration is 24.03mg/mL, in 95 DEG C of bars
Be hydrolyzed under part reaction 3h, obtains the solution after hydrolysis terminate.Adopt concentration for 2mol/L hydrochloric acid by hydrolysis
The pH value of the solution after end is adjusted to 4, is subsequently adding ethyl acetate and is extracted, and repeats extraction 3 times, collects organic mixing
Liquid.Na is added in organic faciess2SO4It is dried, obtains dried organic faciess；Dried organic faciess are carried out at 50 DEG C
Sample concentrated in vacuo, after being concentrated.Sample after concentration is dissolved in methanol solution, by silica gel chromatographic column (Protein
G affinity purification posts, media particle are 50-160 μm) isolated and purified, leacheate used in silica gel chromatography column separating purification
For Gly-HCl buffer (Ju Ti Pei Fang is dissolved in 500mL distilled water for 3.75gGly and 4.383gNaCl, with HCl adjust pH to
2.8) sample after, being isolated and purified.Sample after isolating and purifying is dissolved in into the mixed liquor (v of ethyl acetate and methanol：v
=1:1), in, magnetic agitation 1h finally carries out sucking filtration and is rotated under the conditions of 60 DEG C, obtains the sulfanilamide of light yellow crystal shape
Class hapten 80mg, yield is 24.8%.
2nd, the haptenic identification of sulfonamides
(molecular formula is C to the sulfonamides hapten of step one14H14N2O5S mass spectrum (Synapt, Waters) qualification result)：
MS m/z[M+H]+Theoretical value：322；Measured value：320.9 (being the molecular weight for removing a proton), the molecule with target product
Amount matches, mass spectrum such as Fig. 3.
The haptenic nuclear-magnetism qualification result of sulfonamides of step one：(Bruker, 300MHZ Advance nuclear magnetic resonance analyser,
Characteristic peaks are1HNMR (300MHz, DMSO-d6), δ (ppm) 11.43 (br, s, 1H, COOH), 7.52 (d, 2H, J=
8.61Hz, CHar), 6.20 (d, 2H, J=8.5Hz, CHar), 6.15 (d, 2H, J=8.5Hz, CHar), 5.88 (s, 2H,
NH2),3.73(m,2H,O-CH2)), hydrogen spectrum such as Fig. 3.
Above-mentioned qualification result shows that the haptenic structural formula of sulfonamides is Formulas I：
Embodiment 2, the preparation of sulfonamides conjugated antigen and identification
First, the preparation of sulfonamides conjugated antigen
The sulfonamides hapten that 0.04mmol embodiments 1 are prepared is dissolved in 1.8mL dimethylformamides (DMF)
In, obtain the haptenic dimethyl formamide solution of sulfonamides.Add in the haptenic dimethyl formamide solution of sulfonamides
1- (3- the dimethylamino-propyls) -3- ethyls carbon two of the N- hydroxy thiosuccinimides (NSH) and 0.08mmol of 0.12mmol
Inferior amine salt hydrochlorate (EDCHCl), is placed on magnetic stirring apparatuss under the conditions of room temperature (20-30 DEG C) and stirs 3h, and reaction obtains solution
A；The bovine serum albumin of 60.3mg is dissolved in into the Na that 6mL concentration is 0.05M2CO3In buffer solution (pH9.6), solution is obtained
B.Under magnetic stirring, solution A is added dropwise in solution B, (20-30 DEG C) stirring 4h of room temperature obtains reacted solution.
Reacted solution is dialysed, the molecular cut off of dialyzer used is 10000-14000, room temperature (20-30
DEG C) with distilled water dialysis 72h, water is changed once per 12h, the solution collected in bag filter obtains sulfonamides conjugated antigen, is sub-packed in
In ampere bottle, -20 DEG C of preservations.
2nd, the identification of sulfonamides conjugated antigen
Mass spectrum (Synapt, the Waters) qualification result of the sulfonamides conjugated antigen of step one：MALDI-TOF-MS m/z
[M+H]+Measured value：69186.79, mass spectral results such as Fig. 4, the molecular weight of bovine serum albumin is 65867.88, it was demonstrated that combined anti-
The success of former synthesis, the haptenic molecular weight of the sulfonamides that embodiment 1 is prepared are 322, and its Conjugate ratio computing formula is：It is even
Connection rate=(molecular weight of sulfonamides conjugated antigen-bovine serum albumin molecular weight) haptenic molecular weight of/sulfonamides.Calculate
Conjugate ratio to sulfonamides conjugated antigen is the bovine serum albumin of 10.3, i.e., 1 and 10.3 sulfonamides hapten conjugations.
Above-mentioned qualification result shows the structural formula of sulfonamides conjugated antigen as shown in formula II, and K is bovine serum albumin：
The measure of embodiment 3, the preparation of sulfa drugss antibody serum and potency, susceptiveness and specificity
The formula of related solution is as follows：
Concentration is 0.1M, and pH value is the preparation of 7.2 PBS：Solution A：35.8g Na2HPO4·12H2O, 1L go
Ionized water, obtains the Na that concentration is 0.1M2HPO4Solution；Solution B：15.6g NaH2P04·2H2O, 1L deionized water, obtains dense
Spend the NaH for 0.1M2P04Solution；8.5g NaCl mix homogeneously will be added after 72mL solution As and the mixing of 28mL solution Bs.
Concentration is 0.01M, and pH value is the preparation of 7.4 PBS：8g NaCl、0.2g KCl、3.53g
Na2HPO4·12H2O、0.24g KH2PO4, 1L deionized waters.
Concentration is 0.02M, and pH value is the preparation of 7.2 PBS：It is 0.1M to take concentration, and pH value is that 7.2 PBS delays
Liquid 200mL is rushed, deionized water 800mL is added, is mixed.
Coating buffer：0.05mol/L, pH value are the aqueous solution of 9.6 sodium carbonate：Na2CO31.59g, NaHCO3
2.93g, plus deionized water is settled to 1 liter.
Cleaning mixture：Prepared per 1 liter of cleaning mixture as follows：Will be 0.5mL polysorbas20s, 5g Hydrazoic acid,sodium salt and 990mL dense
Spend for 0.01M, pH value is 7.4 phosphate buffer mixing, obtains the cleaning mixture.
Confining liquid：Prepared per 1 liter of confining liquid as follows：50mgBSA, 1g Hydrazoic acid,sodium salt, 30g caseins are mixed
Close, be 0.02M with concentration, pH value is that 7.2 phosphate buffer dissolves and be settled to 1000mL, obtains confining liquid.
First, the sero-fast preparation of sulfonamides
New zealand white rabbit 6, is randomly divided into experimental group and matched group (3 per group)；Experimental group is prepared with embodiment 2
Sulfonamides conjugated antigen solution (using coating buffer dilution) carries out immunity, and matched group is with bovine serum albumin (BSA) solution
(BSA is dissolved in coating buffer) carries out immunity, every rabbit immunity 1mg/mL every time, wherein, the sulfanilamide of the preparation of embodiment 2
BSA densitometer of the concentration of class conjugated antigen to be coupled with which, every two weeks immunity are once, immune 6 times altogether.The 6th immunity it
7 days afterwards, every group of rabbit ear edge vein exploitating blood prepared serum, obtains sulfonamides antiserum (from experimental group) and BSA antiserums
(from matched group).
2nd, the sero-fast titration of sulfonamides
The sulfonamides antiserum and BSA antiserums for respectively being prepared by step one carries out indirect competitive ELISA analysis (per Kong Jun
If three repeating holes), comprise the following steps that：
1st, it is coated with：
The preparation of 1.1 sulfonamides envelope antigens
The structural formula of sulfonamides envelope antigen for embodiment 2 formula II, wherein K be hemocyanin (KLH), its preparation process
It is identical with sulfonamides conjugated antigen preparation process in embodiment 2, the BSA of embodiment 2 is replaced with into hemocyanin (KLH).
1.2 coating
With the sulfonamides envelope antigen of coating buffer solution step 1.1, the sulfonamides envelope antigen of following concentration is obtained
Solution：100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL、3.125μg/mL、1.5625μg/mL、0μg/
ML, each concentration are coated with a line, 100 μ L/ holes, and 4 DEG C overnight.
2nd, washing and closing：Liquid in hole in coating of inclining hole, is washed with cleaning mixture 3 times, each 3min；Add per hole
150 μ L confining liquids, 37 DEG C of constant temperature close 1h, and then liquid in hole of inclining is washed with cleaning mixture 3 times, each 3min.
3rd, it is loaded：By 50 μ L using coating buffer according to following ratio 1:204800、1:2048000、1:20480000、
1:204800000、1:2048000000 and 1：20480000000 rabbit anti-serums for carrying out doubling dilution (sulfonamides antiserum or
BSA antiserums) it is added in ELISA Plate and reacts：, per 100 μ L of hole, then 37 DEG C of reaction 1h, liquid in hole of inclining are washed with cleaning mixture
Wash 3 times, each 3min；Then 100 μ L HRP- goat anti-rabbit iggs are added in every hole, 37 DEG C of reaction 1h, liquid in hole of inclining, so
Washed with cleaning mixture 3 times afterwards, each 3min.
4th, chromogenic assay：100 μ L of TMB solution, 37 DEG C of colour developing 20min are added per hole, then 50 μ L concentration of addition are per hole
The H of 2M2SO4With terminating reaction, the OD in each hole is finally determined with microplate reader450Nm values.
In elisa assay：Replace rabbit anteserum with high purity water as blank.
Experimental group：The sulfonamides antiserum of step one is the effect of the sulfonamides envelope antigen solution of 12.5 μ g/mL to concentration
Valency is 1:20480000.
Matched group：The BSA antiserums of step one, without chromogenic reaction in measure, illustrate that matched group does not produce specificity pin
Antiserum to sulfonamides envelope antigen.
As a result show, with the sulfonamides conjugated antigen immune rabbit of embodiment 2, it is possible to obtain the anti-blood of sulfonamides of high-titer
Clearly.
3rd, lowest detectable limit (LOD) and half amount of suppression (IC50) determine
With coating buffer solution sulfanilamide standard substance：Sulfamethazine (SM2), sulfamethizole (SMZ), sulfanilamide two
Sulfamonomethoxine (SDM), sulfaquinoxaline (SQX) and cistosulfa (SCP), are each configured to the solution of following Concentraton gradient：
10000ng/mL, 1000ng/mL, 100ng/mL, 10ng/mL, 1ng/mL and 0.1ng/mL, as experimental solutions.It is flat using 6 groups
Row test (n=6).
The blood sample that respectively step one is adopted prepares yellow amine antiserum, is tested as follows：
1st, it is coated with：The sulfonamides envelope antigen prepared with coating buffer solution step 2, obtains sulfonamides envelope antigen
Concentration is the sulfonamides envelope antigen solution of 12.5 μ g/mL, is coated with experimental port, 100 μ L/ with the sulfonamides envelope antigen solution
Hole, 4 DEG C overnight.
2nd, washing and closing：Incline liquid in hole, is washed with cleaning mixture 3 times, each 3min；150 μ L closings are added per hole
Liquid, 37 DEG C of constant temperature close 1h, and then liquid in hole of inclining is washed with cleaning mixture 3 times, and each 3min obtains ELISA Plate.
3rd, it is loaded：
3.1st, standard sample wells
The sulfonamides antiserum of step one is diluted into 20480000 times with coating buffer and obtains the dilution of sulfonamides antiserum
The sulfonamides standard solution of 50 μ L sulfonamides antiserum diluents and 50 any of the above-described kind of concentration of μ L is added to ELISA Plate by liquid
On, then 37 DEG C of reaction 1h, liquid in hole of inclining are washed with cleaning mixture 3 times, each 3min；Then 100 μ L are added in every hole
Then HRP- goat anti-rabbit iggs, 37 DEG C of reaction 1h, liquid in hole of inclining is washed with cleaning mixture 3 times, each 3min.
3.2nd, negative control hole
Differ only in sulfonamides standard solution is replaced with into isopyknic high purity water with 3.1, other steps are constant.
3.3rd, blank control wells
With 3.1 to differ only in blank well be that the sulfonamides antiserum diluent of addition is replaced with high purity water, its
Its step is constant.
4th, chromogenic assay：100 μ L of TMB solution, 37 DEG C of reaction 20min are added per hole, then 50 μ L concentration of addition are per hole
The H of 2M2SO4With terminating reaction, the OD in each hole is finally determined with microplate reader450Nm values.
With OD450nmIt is worth for vertical coordinate, with the 1og of sulfonamides standard solution concentration10It is worth for abscissa, drafting semilog
Canonical plotting.Standard curve has complete reverse-s shape shape, and has upper mounting plate and lower platform, the parallel assay of standard curve
Number of times 6 times, experimental repeatability are good, and relative standard deviation (coefficient of variation) is within 20%.
10% amount of suppression and half amount of suppression (IC are drawn according to standard curve50), compare detection sensitivity.
Suppression ratio is with being calculated as follows：
In formula：ODmaxFor the light absorption value (i.e. negative control) being not added with during standard substance, ODxFor standard concentration be x when suction
Light value, ODminFor the light absorption value of blank control wells.
As a result as shown in table 1, sulfamethazine, sulfamethizole, sulfadimethoxine, sulfaquinoxaline and sulphur
Amine chlorine pyridazine is to sulfonamides antiserum and the half amount of suppression (IC of sulfonamides envelope antigen association reaction50) it is respectively 50.78ng/
ML, 3.56ng/mL, 10.34ng/mL, 12.18ng/mL and 9.75ng/mL；Sulfonamides antiserum is to sulfamethazine, sulphur
Amine first diazole, sulfadimethoxine, sulfaquinoxaline and cistosulfa lowest detectable limit (LOD) respectively 1.22ng/mL,
0.12ng/mL, 0.97ng/mL, 3.98ng/mL and 0.15ng/mL.With negative control (not plus sulfonamides standard solution) phase
Than after adding sulfonamides standard solution, light absorption value is in obvious gradient decline trend, illustrates the sulfonamides antiserum energy for obtaining
It is enough specifically to recognize sulfonamides standard substance, there is higher affinity to sulfonamides standard substance.
Table 1, the lowest detectable limit (LOD) of sulfonamides standard substance and half amount of suppression (IC50) measurement result
| Sulfonamides compound | Lowest detectable limit (LOD)/(ng/mL) | Half amount of suppression (IC50)/(ng/mL) |
| Sulfamethazine | 1.22 | 50.78 |
| Sulfamethizole | 0.12 | 3.56 |
| Sulfadimethoxine | 0.97 | 10.34 |
| Sulfaquinoxaline | 3.98 | 12.18 |
| Cistosulfa | 0.15 | 9.75 |
4th, sulfonamides antiserum specific assay
According to the step three in embodiment 3, sulfonamides antiserum is determined to following sulfonamides compound to the anti-blood of sulfonamides
Half amount of suppression (IC clearly with sulfonamides envelope antigen association reaction50)：Methoxy pyridazine, sulphur between sulfamethazine, sulfanilamide
Amine Sulfamonomethoxine, sulfamethylthiazole, sulfamonomethoxine, sulfaisodimidine, sulfamethyldiazine, sulfanilamide dimethoxy
Pyrimidine, sulfaphenazole, sulfasalazine, sulfamethoxazole, sulfapyridine, sulfamethazole, sulfathiazole,
Sulfadiazine, phthalylsulfathiazole, sulfanitran, sulphaguanidine, sulfafurazole, sulphanilamide, sulfabenzamide, sulfadoxine, sulphur
Amine acetyl and sulfoamido uracil, Concentraton gradient are as follows：10000ng/mL、1000ng/mL、100ng/mL、10ng/mL、
1ng/mL and 0.1ng/mL.The IC of each competitor is calculated according to the following formula50。
Cross reacting rate=IC50(sulfamethazine)/IC50(competitor) × 100%
As a result show, sulfonamides antiserum is to participating in 22 kinds of medicines in the 27 kinds of sulfa drugss for detecting, including sulfanilamide
Between methoxy pyridazine, sulfametoxydiazine, sulfamethizole, sulfamethylthiazole, sulfamonomethoxine, sulfanilamide dimethyl it is different phonetic
Pyridine, cistosulfa, sulfamethyldiazine, sulfadimethoxine, sulfaphenazole, sulfasalazine, sulfaquinoxaline,
Sulfamethoxazole, sulfapyridine, sulfamethazole, sulfathiazole, sulfadiazine, sulfamethazine, phthalein sulfanilamide
Thiazole, sulfanitran, sulphaguanidine, sulfafurazole have preferable cross reaction, and (cross reacting rate is in 30.86%-5519%
Between), it is general (between 1%-10%) to the cross reacting rate of sulphanilamide and sulfabenzamide, but to sulfadoxine, sulfanilamide
The cross reacting rate of acetyl and sulfoamido uracil it is then relatively low (<1%).
Half amount of suppression (the IC of table 2, sulfonamides antiserum to sulfonamides compound50) measurement result
Claims (10)
1. compound shown in formula I：
2. the preparation method of compound shown in the Formulas I described in claim 1, comprises the steps：
1) compound A is carried out reacting the intermediate product III obtained shown in formula III with N- acetylsulphanilyl chloride；The chemical combination
Thing A is 2- methoxyl groups-PABA methyl ester or 4- (4- aminophenyls) butanoic acid；
2) intermediate product III is hydrolyzed in the basic conditions, obtains compound shown in the Formulas I described in claim 1.
3. method according to claim 2, it is characterised in that：Step 1) it is described reaction in triethylamine and 4- lutidines
2-3h is carried out at 60 DEG C under conditions of presence.
4. compound shown in formula II：
In formula II, K represents carrier protein.
5. the preparation method of compound shown in the formula II described in claim 4, including：To change shown in Formulas I described in claim 1
Compound obtains compound shown in the formula II with carrier protein couplet.
6. method according to claim 5, it is characterised in that：Methods described also includes entering compound shown in the formula II
The step of row purification.
7. compound shown in the Formulas I described in claim 1, or, the preparation method described in Claims 2 or 3 prepare right will
Ask the application in compound shown in 4 formula II.
8. compound shown in the Formulas I described in claim 1 or compound shown in the formula II described in claim 4, or, claim
Application of the preparation method described in 2 or 3 or 5 or 6 in the antibody for preparing anti-amine compound.
9. compound shown in the Formulas I described in claim 1 is utilized or compound shown in the formula II described in claim 4, or, right
Require the antibody of anti-amine compound prepared by the preparation method described in 2 or 3 or 5 or 6.
10. compound shown in the Formulas I described in claim 1 or compound shown in the formula II described in claim 4, or, right will
The preparation method described in 2 or 3 or 5 or 6 is sought, or, application of the antibody described in claim 9 in detection amine compound.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061155A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of test strips and its preparation method and application detecting metalaxyl |
| CN109824604A (en) * | 2019-02-25 | 2019-05-31 | 中国农业大学 | Sulfonamide hapten and artificial antigen and the preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1904613A (en) * | 2006-08-08 | 2007-01-31 | 天津科技大学 | Artificial antigen used for immune analysis of sulfanilamide multi kind residue and antibody and its preparation |
-
2016
- 2016-10-25 CN CN201610941345.6A patent/CN106543049B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1904613A (en) * | 2006-08-08 | 2007-01-31 | 天津科技大学 | Artificial antigen used for immune analysis of sulfanilamide multi kind residue and antibody and its preparation |
Non-Patent Citations (3)
| Title |
|---|
| ESTELA CLIMENT ET AL.: "Controlled Delivery Systems Using Antibody-Capped Mesoporous Nanocontainers", 《JOURNAL OF THE AMERICAN SOCIETY》 * |
| HONGYUAN ZHANG ET AL.: "Hapten Synthesis and Development of Polyclonal Antibody-Based Multi-Sulfonamide Immunoassays", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
| 段振娟等: "磺胺母核结构半抗原的合成与表征", 《天津科技大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061155A (en) * | 2018-09-21 | 2018-12-21 | 中国烟草总公司郑州烟草研究院 | A kind of test strips and its preparation method and application detecting metalaxyl |
| CN109061155B (en) * | 2018-09-21 | 2021-05-11 | 中国烟草总公司郑州烟草研究院 | Test strip for detecting metalaxyl and preparation method and application thereof |
| CN109824604A (en) * | 2019-02-25 | 2019-05-31 | 中国农业大学 | Sulfonamide hapten and artificial antigen and the preparation method and application thereof |
| CN109824604B (en) * | 2019-02-25 | 2020-06-23 | 中国农业大学 | Sulfonamide hapten and artificial antigen as well as preparation method and application thereof |
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