CN106543049B - Sulfamido haptens and its preparation method and the application for combining antigen - Google Patents
Sulfamido haptens and its preparation method and the application for combining antigen 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
- C07C311/44—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 having the nitrogen atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
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Abstract
The invention discloses the preparation methods and application of sulfamido haptens and its combination antigen.The structural formula of the sulfamido haptens and its combination antigen is respectively as shown in formula I and formula II.The antiserum that high-titer, high-affinity and high specificity can be generated after the sulfamido combination antigen-immunized animal, using ELISA adsorption analysis method can quickly, sensitively, and conveniently detect sulfonamides compound.Half amount of suppression (the IC for the sulfamido antiserum and sulfamido envelope antigen association reaction that sulfamido standard items prepare combination antigen using the present invention50) it is 3.56ng/mL 50.78ng/mL, sulfamido antiserum is 0.12ng/mL 1.22ng/mL to the minimum detection limit (LOD) of sulfamido standard items.The haptens and combination antigen of the present invention is suitable for the detection to sulfonamides compound.
Description
Technical field
The present invention relates to the preparation methods and application of sulfamido haptens in biological chemical field and its combination antigen.
Background technology
Sulfa drugs refers to the general name of a kind of drug with P-aminobenzene-sulfonamide structure, be it is a kind of for preventing and
The chemotherapeutic agent of bacterial infection disease is treated, antimicrobial spectrum is wider, to most of gram-positive bacterias and gram
Negative bacterium has inhibiting effect.
Sulfa drugs due to property is stable, antimicrobial spectrum is wide, small toxicity, it is oral easily absorb and cheap, answered extensively
For in husbandry sector, to there is notable curative effect in terms of animal diseases control.But since the unreasonable of sulfa drugs makes
With, misuse even abuse, result in the generation of drug-fast bacteria, the especially type of naphthalene plucked instrument Pseudomonas and gram-positive bacteria increasingly increases
It is more.After pathogen generates drug resistance to a kind of sulfa drugs, it is also easy to generate cross resistance to other sulfa drugs,
Therefore the epidemic infection of multiple drug-fast bacteria is caused.In addition, unreasonable use of sulfa drugs is also produced in livestock products
Serious residual phenomena is given birth to, remaining sulfa drugs can endanger the health of people in food, in consideration of it, China and U.S.
State provides that the off-drug period of sulfa drugs is 15 days, and maximum residue limit 100ng/g, EU countries also strictly limits sulfamido
The residue limits of drug.
The technical method of detection sulfa drug residue is mainly that instrument confirmatory analysis and quick screening are analyzed at present.Instrument
Analysis and detection technology has the characteristics that high sensitivity, high specificity, but needs the behaviour of longer sample process time and profession
Make personnel, is not suitable for the quick 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, it is fast, at low cost because of its speed the advantages that, be widely used
The quick screening analysis of sulfa drugs in food samples.But the immunoassay method for detecting sulfa drugs is special mostly
Be directed to a certain sulfa drugs anisotropicly, as sulfa drugs uses the increase of type in animal husbandry, establish it is a kind of quickly,
Sensitive, accurate, easily detection multiple types residual quantity of sulfonamide method is current urgent problem to be solved.
Invention content
The technical problem to be solved by the present invention is to the residual quantities of how quick, accurate detection amine compound.
In order to solve the above technical problems, present invention firstly provides a kind of sulfamido haptens.
Sulfamido haptens provided by the present invention, structure is as shown in formula I：
In order to solve the above technical problems, the present invention also provides the preparation methods of compound shown in the Formulas I.
The preparation method of compound, includes the following steps shown in the Formulas I provided by the present invention：
1) it carries out compound A and N- acetamidobenzenesulfonyl chlorides that intermediate product III is obtained by the reaction；The compound A is 2-
Methoxyl group -4-aminobenzoic acid methyl esters or 4- (4- aminophenyls) butyric acid；
2) intermediate product III is hydrolyzed under alkaline condition, obtains compound shown in the Formulas I.
Above-mentioned preparation method further includes that the solution after step 2) hydrolysis is carried out pH value adjusting, is concretely used dense
Degree is that pH value is adjusted to 4 by the hydrochloric acid of 2mol/L.
In the preparation method of compound shown in above-mentioned Formulas I, the step 1) reaction is in triethylamine and 4- lutidines
(DMAP) 2-3h is carried out under the conditions of existing at 60 DEG C, concretely in 60 DEG C of reaction 2h of water-bath；Compound A, the N- acetyl
The molar ratio of amino phenyl sulfonyl acyl chlorides, triethylamine and 4-dimethylaminopyridine (DMAP) can be 2:2:2:1.
In the preparation method of compound shown in above-mentioned Formulas I, step 2) is described to be hydrolyzed in a concentration of 24.03mg/mL-
95 DEG C of reaction 2-3h in the NaOH solution of 40.05mg/mL.
In order to solve the above technical problems, the present invention also provides a kind of sulfamido combination antigens.
Sulfamido combination antigen provided by the present invention, structure is as shown in formula II：
In formula II, K indicates carrier protein；The carrier protein is concretely：Bovine serum albumin(BSA), human serum albumins,
Hemocyanin, ovalbumin, mouse serum albumin, thyroglobulin or albumin rabbit serum etc..
In order to solve the above technical problems, the present invention also provides the preparation methods of compound shown in the formula II.
The preparation method of compound shown in the formula II provided by the present invention includes by compound shown in the 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 further includes the steps that being purified to compound shown in the formula II,
Can be the step of compound shown in the formula II is dialysed.The dialysis carries out in distilled water；The dialysis is in room
(20-30 DEG C) progress 72h of temperature.
Wherein, compound shown in the Formulas I and carrier protein are in 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides
The coupling reaction carried out under the conditions of hydrochloride (EDCHCl) and n-hydroxysuccinimide (NSH) are existing；Shown in the Formulas I
Compound, the carrier protein, 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDCHCl) and N- hydroxyls
The molar ratio of succinimide (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(BSA), the concentration of the bovine serum albumin(BSA) concretely 10.05mg/mL.
In preparation method compound shown in formula II of compound shown in compound or the Formulas I shown in the Formulas I
Application also belong to the scope of protection of the invention.
Compound shown in compound or the formula II shown in the Formulas I, 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 preparing anti-amine compound antibody also belongs to protection of the present invention
Range.
Using compound shown in compound shown in the Formulas I or 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 compound shown in method or the formula II also belongs to the protection of the present invention
The antibody of range, the anti-amine compound can be monoclonal antibody or polyclonal antibody.
Compound shown in compound or the formula II shown in the Formulas I, 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 detecting amine compound
Using also belonging to the scope of protection of the invention.
Above, the amine compound can between sulfanilamide (SN) methoxy pyridazine, sulfametoxydiazine, sulfamethizole, sulfanilamide (SN)
First thiazole, daimeton, sulfaisodimidine, cistosulfa, sulfamethoxazole, sulfanilamide (SN) dimethoxy are phonetic
Pyridine, sulfamethyldiazine, sulfaquinoxaline, sulfaphenazolum, salicylazosulfapyridine, sulfapryidine, sulfamethazole, sulfanilamide (SN)
Thiazole, sulphadiazine, sulfamethazine, phthalylsulfathiazol, sulfanitran, sulphaguanidine, sulfonamidoxazole, sulphanilamide,
Sulfabenzamide, sulfadoxine, sulfacetamide and sulfoamido urea pyrimidine.
It is demonstrated experimentally that height can be generated after the sulfamido combination antigen-immunized animal prepared in method provided by the present invention
The antiserum of potency, high-affinity and high specificity, using the antiserum using ELISA adsorption analysis method can quickly,
Sensitively, and conveniently detect sulfonamides compound.Sulfamido standard items prepare sulfamido combination antigen using the present invention
Half amount of suppression (the IC of sulfamido antiserum and sulfamido envelope antigen association reaction50) it is 3.56ng/mL-50.78ng/mL,
Sulfamido antiserum prepared by sulfamido combination antigen using the present invention is to the minimum detection limit (LOD) of sulfamido standard items
0.12ng/mL-1.22ng/mL.Sulfamido antiserum prepared by sulfamido combination antigen using the present invention can be specifically
It identifies sulfamido standard items, there is higher affinity to sulfamido standard items.The haptens and combination antigen of the present invention is applicable in
In the detection to sulfonamides compound.
Description of the drawings
Fig. 1 is flow chart prepared by sulfamido haptens and sulfamido combination antigen.
Fig. 2 is the mass spectrogram of sulfamido haptens.
Fig. 3 is the hydrogen nuclear magnetic resonance spectrogram of sulfamido haptens.
Fig. 4 is that the MALDI-TOF-MS of sulfamido combination antigen schemes.
Specific implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
In following embodiments：2- methoxyl groups -4-aminobenzoic acid methyl esters (Sigma-Aldrich, 647616), 2- methoxies
Base -4-aminobenzoic acid methyl esters (Sigma-Aldrich, 335339), N- acetamidobenzenesulfonyl chlorides (Aladdin reagent,
A114795), triethylamine (Aladdin reagent, T103285), 4-dimethylaminopyridine (DMAP) (Aladdin reagent, D109207),
1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDCHCl) (Sigma-Aldrich, E6383), N- hydroxyls
Succinimide (NHS) (Aladdin reagent, H109330), bovine serum albumin(BSA) (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 (SN) standard items 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 (SN)
Aldrich, S7385), Sulfamethylthiazole (Sigma-Aldrich, 46901), daimeton (Sigma-Aldrich,
32996), sulfaisodimidine (Sigma-Aldrich, 46908), sulfamethyldiazine (Sigma-Aldrich,
S6256), sulfadimethoxine (Sigma-Aldrich, S7385), sulfaphenazolum (Sigma-Aldrich,
SS075802), salicylazosulfapyridine (Sigma-Aldrich, S0883), sulfamethoxazole (Sigma-Aldrich,
S7507), sulfapryidine (Sigma-Aldrich, S6252), sulfamethazole (Sigma-Aldrich, S5632), sulfanilamide (SN) thiophene
Azoles (Sigma-Aldrich, 46902), sulphadiazine (Sigma-Aldrich, S8626), phthalylsulfathiazol (Sigma-
Aldrich, 46901), sulfanitran (Sigma-Aldrich, 46882), sulphaguanidine (Aladdin reagent, S107128), sulfanilamide (SN)
Isoxazole (Sigma-Aldrich, S6377), sulphanilamide (Sigma-Aldrich, S9251), sulfabenzamide (Sigma-
Aldrich, 46762), sulfadoxine (Sigma-Aldrich, 31736), sulfacetamide (Aladdin reagent, S114284)) and
Sulfoamido urea pyrimidine (CHEMSTEP, 17017-91-3).
New zealand white rabbit in following embodiments is Beijing Vital River Experimental Animals Technology Co., Ltd.'s product, product
Catalog number (Cat.No.) is S09045.
The preparation and identification of embodiment 1, sulfamido haptens
One, the preparation of sulfamido haptens
1mmol2- methoxyl groups -4-aminobenzoic acid methyl esters (or 1mmol 4- (4- aminophenyls) butyric acid) is dissolved in 4mL
In tetrahydrofuran, the tetrahydrofuran solution of 2- methoxyl groups -4-aminobenzoic acid methyl esters 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 esters
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-layered chromatography
Degree, as Rf value RfThe reaction was complete when being 0.5 or so, adds suitable quantity of water that reaction is quenched when the reaction was complete, obtains that the molten of reaction is quenched
Liquid.Dichloromethane is added into the solution for be quenched reaction to be extracted, carries out extracting operation altogether 3 times, obtains organic phase；To organic
Na is added in phase2SO4It is dried, the organic phase after being dried；Organic phase after drying is concentrated in vacuo at 35 DEG C,
Obtain intermediate product III shown in formula III：
Intermediate product III shown in formula III is added in the NaOH solution of a concentration of 24.03mg/mL of 50mL, in 95 DEG C of items
Reaction 3h is hydrolyzed under part, obtains the solution after hydrolysis.Using the hydrochloric acid of a concentration of 2mol/L by hydrolysis
After the pH value of solution adjust to 4, ethyl acetate is then added and is extracted, extraction 3 times is repeated, collects organic mixes
Liquid.Na is added into organic phase2SO4It is dried, the organic phase after being dried；Organic phase after drying is carried out at 50 DEG C
It is concentrated in vacuo, the sample after being concentrated.Sample after concentration is dissolved in methanol solution, silica gel chromatographic column (Protein is passed through
G affinity purification columns, media particle are 50-160 μm) it is isolated and purified, leacheate used in silica gel chromatograph column separating purification
For Gly-HCl buffer solutions (specific formula is that 3.75gGly and 4.383gNaCl is dissolved in 500mL distilled water, with HCl adjust pH to
2.8) sample after, being isolated and purified.Sample after isolating and purifying is dissolved in the mixed liquor (v of ethyl acetate and methanol：v
=1:1) in, magnetic agitation 1h finally carries out filtering and being rotated under the conditions of 60 DEG C, obtains the sulfanilamide (SN) of light yellow crystal shape
Class haptens 80mg, yield 24.8%.
Two, the identification of sulfamido haptens
Sulfamido haptens (the molecular formula C of step 114H14N2O5S mass spectrum (Synapt, Waters) qualification result)：
MS m/z[M+H]+Theoretical value：322；Measured value：320.9 (to remove the molecular weight of a proton), the molecule with target product
Amount matches, mass spectrum such as Fig. 3.
The nuclear-magnetism qualification result of the sulfamido haptens of step 1：(Bruker, 300MHZ Advance Nuclear Magnetic Resonance,
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 structural formula of sulfamido haptens is Formulas I：
The preparation and identification of embodiment 2, sulfamido combination antigen
One, the preparation of sulfamido combination antigen
The sulfamido haptens that 0.04mmol embodiments 1 are prepared is dissolved in 1.8mL dimethylformamides (DMF)
In, obtain the dimethyl formamide solution of sulfamido haptens.It is added into the dimethyl formamide solution of sulfamido haptens
The N- hydroxy thiosuccinimides (NSH) of 0.12mmol and 1- (3- the dimethylamino-propyls) -3- ethyls carbon two of 0.08mmol
Inferior amine salt hydrochlorate (EDCHCl) is placed on magnetic stirring apparatus under the conditions of room temperature (20-30 DEG C) and stirs 3h, solution is obtained by the reaction
A；The bovine serum albumin(BSA) of 60.3mg is dissolved in the Na of a concentration of 0.05M of 6mL2CO3In 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, the solution after being reacted.
Solution after reaction is dialysed, the molecular cut off of dialysis membrane used is 10000-14000, room temperature (20-30
DEG C) dialysed 72h with distilled water, change that water is primary per 12h, the solution collected in bag filter obtains sulfamido combination antigen, is sub-packed in
In ampere bottle, -20 DEG C of preservations.
Two, the identification of sulfamido combination antigen
Mass spectrum (Synapt, Waters) qualification result of the sulfamido combination antigen of step 1：MALDI-TOF-MS m/z
[M+H]+Measured value：The molecular weight of 69186.79, mass spectral results such as Fig. 4, bovine serum albumin(BSA) are 65867.88, it was demonstrated that in conjunction with anti-
The molecular weight of the success of original synthesis, the sulfamido haptens that embodiment 1 is prepared is 322, and Conjugate ratio calculation formula is：It is even
The molecular weight of connection rate=(molecular weight of sulfamido combination antigen-bovine serum albumin(BSA) molecular weight)/sulfamido haptens.It calculates
Conjugate ratio to sulfamido combination antigen is 10.3, i.e. 1 bovine serum albumin(BSA) and 10.3 sulfamido hapten conjugations.
Above-mentioned qualification result shows the structural formula of sulfamido combination antigen as shown in formula II, and K is bovine serum albumin(BSA)：
Embodiment 3, sulfa drugs antibody serum prepares and the measurement of potency, sensitivity and specificity
The formula of related solution is as follows：
A concentration of 0.1M, the preparation for the PBS buffer solution that pH value is 7.2：Solution A：35.8g Na2HPO4·12H2O, 1L are gone
Ionized water obtains the Na of a concentration of 0.1M2HPO4Solution；Solution B：15.6g NaH2P04·2H2O, 1L deionized water obtain dense
Degree is the NaH of 0.1M2P04Solution；8.5g NaCl are added after 72mL solution As and 28mL solution Bs are mixed to be uniformly mixed.
A concentration of 0.01M, the preparation for the PBS buffer solution that pH value is 7.4：8g NaCl、0.2g KCl、3.53g
Na2HPO4·12H2O、0.24g KH2PO4, 1L deionized waters.
A concentration of 0.02M, the preparation for the PBS buffer solution that pH value is 7.2：A concentration of 0.1M is taken, the PBS that pH value is 7.2 is slow
Deionized water 800mL, mixing is added in fliud flushing 200mL.
It is coated with buffer solution：The aqueous solution of 0.05mol/L, the sodium carbonate that pH value is 9.6：Na2CO31.59g NaHCO3
2.93g adds deionized water to be settled to 1 liter.
Cleaning solution：Every 1 liter of cleaning solution is prepared as follows：0.5mL polysorbas20s, 5g sodium azide and 990mL is dense
Degree is 0.01M, and the phosphate buffer that pH value is 7.4 mixes, and obtains the cleaning solution.
Confining liquid：Every 1 liter of confining liquid is prepared as follows：50mgBSA, 1g sodium azide, 30g caseins are mixed
It closes, with a concentration of 0.02M, the phosphate buffer that pH value is 7.2 dissolves and is settled to 1000mL, obtains confining liquid.
One, the sero-fast preparation of sulfamido
New zealand white rabbit 6 is randomly divided into experimental group and control group (every group 3)；Experimental group is prepared with embodiment 2
Sulfamido combination antigenic solution is immunized (using the dilution of coating buffer solution), and control group is with bovine serum albumin(BSA) (BSA) solution
(BSA is dissolved in coating buffer solution) is immunized, and 1mg/mL is immunized in every rabbit every time, wherein sulfanilamide (SN) prepared by embodiment 2
BSA densimeter of the concentration of class combination antigen to be coupled with it is immunized once, is immunized 6 times altogether every two weeks.It is immunized at the 6th time
7 days afterwards, every group of rabbit ear edge vein exploitating blood prepared serum, obtained sulfamido antiserum (coming from experimental group) and BSA antiserums
(coming from control group).
Two, the sero-fast titration of sulfamido
The sulfamido antiserum and BSA antiserums respectively prepared by step 1 carries out indirect competitive ELISA analysis (per Kong Jun
If three repeating holes), it is as follows：
1, it is coated with：
The preparation of 1.1 sulfamido envelope antigens
The structural formula of sulfamido envelope antigen is the formula II of embodiment 2, and wherein K is hemocyanin (KLH), preparation process
It is identical as sulfamido combination antigen preparation process in embodiment 2, the BSA of embodiment 2 is replaced with into hemocyanin (KLH).
1.2 coating
With the sulfamido envelope antigen of coating buffer solution step 1.1, the sulfamido 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, and 100 holes μ L/, 4 DEG C overnight.
2, washing and closing：Liquid in hole in coating of inclining hole, is washed 3 times, each 3min with cleaning solution；It is added per hole
150 μ L confining liquids, 37 DEG C of constant temperature close 1h, then liquid in hole of inclining is washed 3 times, each 3min with cleaning solution.
3, it is loaded：By 50 μ L using coating buffer solution according to following ratio 1:204800、1:2048000、1:20480000、
1:204800000、1:2048000000 and 1：20480000000 carry out doubling dilutions rabbit anti-serum (sulfamido antiserum or
BSA antiserums) it is added on ELISA Plate and reacts：, per 100 μ L of hole, then 37 DEG C of reaction 1h, liquid in hole of inclining is washed with cleaning solution
It washs 3 times, each 3min；Then it is added 100 μ L HRP- goat anti-rabbit iggs into every hole, 37 DEG C of reaction 1h, liquid in hole of inclining, so
It is washed 3 times with cleaning solution afterwards, each 3min.
4, chromogenic assay：TMB solution 100 μ L, 37 DEG C of colour developing 20min are added per hole, it is a concentration of that 50 μ L are then added per hole
The H of 2M2SO4To terminate reaction, the OD in each hole is finally measured with microplate reader450Nm values.
In elisa assay：High purity water is used to replace rabbit anteserum as blank control.
Experimental group：Effect of the sulfamido antiserum of step 1 to the sulfamido envelope antigen solution of a concentration of 12.5 μ g/mL
Valence is 1:20480000.
Control group：The BSA antiserums of step 1 illustrate that control group does not generate specific needle without chromogenic reaction in measurement
To the antiserum of sulfamido envelope antigen.
The result shows that with the sulfamido combination antigen immune rabbit of embodiment 2, the anti-blood of sulfamido of high-titer can be obtained
Clearly.
Three, minimum detection limit (LOD) and half amount of suppression (IC50) measure
With coating buffer solution sulfanilamide (SN) standard items：Sulfamethazine (SM2), sulfamethizole (SMZ), sulfanilamide (SN) two
Sulfamonomethoxine (SDM), sulfaquinoxaline (SQX) and cistosulfa (SCP), are each configured to the solution of following concentration 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 experiment (n=6).
The blood sample that step 1 is adopted respectively prepares yellow amine antiserum, is tested as follows：
1, it is coated with：The sulfamido envelope antigen prepared with coating buffer solution step 2, obtains sulfamido envelope antigen
The sulfamido envelope antigen solution of a concentration of 12.5 μ g/mL is coated with experimental port, 100 μ L/ with the sulfamido envelope antigen solution
Hole, 4 DEG C overnight.
2, washing and closing：Incline liquid in hole, is washed 3 times with cleaning solution, each 3min；150 μ L closings are added per hole
Liquid, 37 DEG C of constant temperature close 1h, then liquid in hole of inclining is washed 3 times, each 3min obtains ELISA Plate with cleaning solution.
3, it is loaded：
3.1, standard sample wells
The sulfamido antiserum of step 1 is diluted 20480000 times with coating buffer solution and obtains the dilution of sulfamido antiserum
The sulfamido standard solution of any of the above-described kind of concentration of 50 μ L sulfamido antiserum dilutions and 50 μ L is added to ELISA Plate by liquid
On, then 37 DEG C of reaction 1h, liquid in hole of inclining is washed 3 times, each 3min with cleaning solution；Then 100 μ L are added into every hole
Then HRP- goat anti-rabbit iggs, 37 DEG C of reaction 1h, liquid in hole of inclining are washed 3 times, each 3min with cleaning solution.
3.2, negative control hole
It differs only in sulfamido standard solution is replaced with to isometric high purity water with 3.1, other steps are constant.
3.3, blank control wells
With 3.1 to differ only in blank well be that the sulfamido antiserum dilution of addition replaced with high purity water,
Its step is constant.
4, chromogenic assay：TMB solution 100 μ L, 37 DEG C of reaction 20min are added per hole, it is a concentration of that 50 μ L are then added per hole
The H of 2M2SO4To terminate reaction, the OD in each hole is finally measured with microplate reader450Nm values.
With OD450nmValue is ordinate, with the 1og of sulfamido standard solution concentration10Value is abscissa, draws semilog
Canonical plotting.Standard curve has complete reverse-s shape shape, and has upper mounting plate and lower platform, the parallel determination of standard curve
Number 6 times, experimental repeatability is good, and relative standard deviation (coefficient of variation) is within 20%.
10% amount of suppression and half amount of suppression (IC are obtained according to standard curve50), compare detection sensitivity.
Inhibiting rate is with being calculated as follows：
In formula：ODmaxTo be not added with the light absorption value (i.e. negative control) when standard items, ODxSuction when for standard concentration being x
Light value, ODminFor the light absorption value of blank control wells.
The results are shown in Table 1, sulfamethazine, sulfamethizole, sulfadimethoxine, sulfaquinoxaline and sulphur
Half amount of suppression (IC of the amine chlorine pyridazine to sulfamido antiserum and sulfamido envelope antigen association reaction50) it is respectively 50.78ng/
ML, 3.56ng/mL, 10.34ng/mL, 12.18ng/mL and 9.75ng/mL；Sulfamido antiserum is to sulfamethazine, sulphur
Amine first diazole, sulfadimethoxine, sulfaquinoxaline and cistosulfa minimum detection limit (LOD) be respectively 1.22ng/mL,
0.12ng/mL, 0.97ng/mL, 3.98ng/mL and 0.15ng/mL.With negative control (not plus sulfamido standard solution) phase
Than after sulfamido standard solution is added, light absorption value is in apparent gradient decline trend, illustrates the sulfamido antiserum energy obtained
It is enough specifically to identify sulfamido standard items, there is higher affinity to sulfamido standard items.
Table 1, the minimum detection limit (LOD) of sulfamido standard items and half amount of suppression (IC50) measurement result
| Sulfonamides compound | Minimum detection 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 |
Four, sulfamido antiserum specific assay
According to the step three in embodiment 3, sulfamido antiserum is measured to following sulfonamides compound to the anti-blood of sulfamido
Clearly with the half amount of suppression (IC of sulfamido envelope antigen association reaction50)：Methoxy pyridazine, sulphur between sulfamethazine, sulfanilamide (SN)
Amine Sulfamonomethoxine, Sulfamethylthiazole, daimeton, sulfaisodimidine, sulfamethyldiazine, sulfanilamide (SN) dimethoxy
Pyrimidine, sulfaphenazolum, salicylazosulfapyridine, sulfamethoxazole, sulfapryidine, sulfamethazole, sulphathiazole,
Sulphadiazine, phthalylsulfathiazol, sulfanitran, sulphaguanidine, sulfonamidoxazole, sulphanilamide, sulfabenzamide, sulfadoxine, sulphur
Amine vinegar acyl and sulfoamido urea pyrimidine, concentration 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%
The results show that 22 kinds of drugs in 27 kinds of sulfa drugs of the sulfamido antiserum to participating in detection, including sulfanilamide (SN)
Between methoxy pyridazine, sulfametoxydiazine, sulfamethizole, Sulfamethylthiazole, daimeton, sulfanilamide (SN) dimethyl it is different phonetic
Pyridine, cistosulfa, sulfamethyldiazine, sulfadimethoxine, sulfaphenazolum, salicylazosulfapyridine, sulfaquinoxaline,
Sulfamethoxazole, sulfapryidine, sulfamethazole, sulphathiazole, sulphadiazine, sulfamethazine, phthalein sulfanilamide (SN)
Thiazole, sulfanitran, sulphaguanidine, sulfonamidoxazole 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 (SN)
The cross reacting rate of vinegar acyl and sulfoamido urea pyrimidine it is then relatively low (<1%).
Table 2, sulfamido antiserum are to the half amount of suppression (IC of sulfonamides compound50) measurement result
Claims (10)
1. compound shown in formula I：
2. the preparation method of compound, includes the following steps shown in Formulas I described in claim 1：
1) compound A and N- acetamidobenzenesulfonyl chlorides be obtained by the reaction intermediate product III shown in formula III；The chemical combination
Object A is 2- methoxyl groups -4-aminobenzoic acid methyl esters；
2) intermediate product III is hydrolyzed under alkaline condition, obtains compound shown in Formulas I described in claim 1.
3. according to the method described in claim 2, it is characterized in that：Step 1) the reaction is in triethylamine and 4- lutidines
2-3h is carried out under the conditions of existing at 60 DEG C.
4. compound shown in formula II：
In formula II, K indicates carrier protein.
5. the preparation method of compound shown in the formula II described in claim 4, including：It will change shown in Formulas I described in claim 1
It closes object and obtains compound shown in the formula II with carrier protein couplet.
6. according to the method described in claim 5, it is characterized in that：The method further include to compound shown in the formula II into
The step of row purifying.
7. compound shown in Formulas I described in claim 1, or, preparation method according to claim 2 or 3 is wanted in preparation right
Ask the application in compound shown in 4 formula II.
8. compound shown in the formula II described in compound or claim 4 shown in Formulas I described in claim 1, or, claim
Application of the preparation method in the antibody for preparing anti-amine compound described in 2 or 3 or 5 or 6.
9. using compound shown in the formula II described in compound or claim 4 shown in Formulas I described in claim 1, or, right
It is required that 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 formula II described in compound or claim 4 shown in Formulas I described in claim 1, or, right is wanted
The preparation method described in 2 or 3 or 5 or 6 is sought, or, application of the antibody described in claim 9 in detecting amine compound.
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