CN109824604A - Sulfonamide hapten and artificial antigen and the preparation method and application thereof - Google Patents
Sulfonamide hapten and artificial antigen and the preparation method and application thereof Download PDFInfo
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Abstract
The present invention relates to sulfonamide haptens and artificial antigen and the preparation method and application thereof.Shown in the structure of the sulfonamide hapten such as formula (1):
Description
Technical field
The present invention relates to field of immunodetection, specifically, being related to sulfonamide hapten and artificial antigen and its system
Preparation Method and application.
Background technique
Sulfa drugs (sulfonamides, SAs) has has a broad antifungal spectrum, cheap, chemical property stable and uses
The advantages that facilitating still is widely used in veterinary clinic, herding and aquaculture at present, to prevent and treat bacterial infection disease
Disease.But it is unreasonable using even abusing, easily cause SAs to remain in animal tissue, then by approach such as food chains in human body
Interior accumulation, endangers human health.Currently, China, European Union, the U.S., Japan and other countries or area define in animal food
The maximum residue limit (MRLs) of sulfa drugs.Other than controlling the unreasonable use of SAs from source, to animal-derived food
The residual of middle drug is detected and is monitored, and the important measures to ensure food safety.It is easy, fast it is therefore desirable to establish
Fast, sensitive, wide spectrum method for detecting residue.
Currently, the detection method of sulfa drug residue is mainly liquid chromatogram (HPLC) or chromatograph-mass spectrometer coupling (LC-
The physico-chemical analysis method such as MS).And these methods are required to expensive instrument, complicated sample pre-treatments and professional's operation,
It is cumbersome it is time-consuming, testing cost is higher, and is unable to execute-in-place, be unsuitable for the rapid screening of high-volume sample.
Immuno analytical method based on the specificity of antigen and antibody, invertibity association reaction, it is fast with its speed, at
The advantages that this is low, high sensitivity, is currently widely used for the quick screening of SAs in food-borne sample.But single sulfonamide
There is significant limitation in detection method, SAs multi-residue determination method is current research tendency, and conduct in practical applications
The antibody of the broad spectrum activity SAs of core reagent restricts the development of more residual immunoassay methods again.
The known key for preparing Broad specificity antibody is haptens.So far, have TS, SS, NS, SSS, HS, SA,
More than 10 sulfanilamide (SN) haptens such as BS, SA3, SA10, PS, SMX, SG and H1 is synthesized, and is prepared for more grams of corresponding broad spectrum activity SAs
Grand and monoclonal antibody, but there is the problems such as few identification SAs type, poor sensitivity, unequal cross reacting rate in these antibody, no
It can perform well in actual sample detection.
Summary of the invention
The object of the present invention is to provide sulfonamide haptens and artificial antigen and the preparation method and application thereof.
It in order to achieve the object of the present invention, is that sulfanilamide (SN) diformazan is phonetic in a first aspect, the present invention provides sulfonamide hapten
Pyridine valeric acid, shown in structure such as formula (1):
Wherein, n=0, or >=1 integer.
Preferably, n=1, the haptens are formula (I) compound represented:
Second aspect, the present invention provides the preparation method of the haptens, as n=1, the preparation side of formula (I) compound
Method includes the following steps:
1) 4- bromobutyrate reacts to obtain formula (2) compound with triphenylphosphine；
2) 2,4- pentanedione reacts to obtain formula (5) compound with guanidine hydrochloride；
3) formula (5) compound reacts to obtain formula (6) compound with N- bromo-succinimide；
4) formula (6) compound reacts to obtain formula (7) compound with benzyl chloride；
5) formula (7) compound reacts to obtain formula (8) compound with n-BuLi；
6) formula (8) compound reacts to obtain formula (9) compound with formula (2) compound；
7) formula (9) compound generation hydrogenation obtains formula (10) compound；
8) formula (10) compound reacts to obtain formula (12) compound with formula (11) compound；
9) formula (12) compound generation hydrolysis obtains formula (I) compound.
Wherein, step 1) -9) described in compound structure it is as follows:
Preferably, the preparation method of formula (I) compound includes the following steps:
1 ') 4- bromobutyrate is mixed with triphenylphosphine, is reacted in reflux in toluene；Petroleum is used in precipitating filtering, recycling
It is dry after ether washing, obtain formula (2) compound；
2 ') 2,4- pentanedione and guanidine hydrochloride is soluble in water, potassium carbonate is added, reaction mixture stirs at 36-44 DEG C
21.6-26.4h (is stirred for 24 hours) at preferably 40 DEG C；Precipitating is filtered, and is dried in vacuo after being washed with water, is obtained formula (5) compound；
3 ') formula (5) compound, N- bromo-succinimide are added in acetonitrile, 5.4-6.6h is heated under the conditions of 72-88 DEG C
(heating 6h under the conditions of preferably 80 DEG C)；Precipitating is filtered, and is dried in vacuo after being washed with acetonitrile, is obtained formula (6) compound；
4 ') formula (6) compound is dissolved in DMF, the mineral oil of containing hydrogenated sodium is then added portionwise at 0 DEG C, low
0.9-1.1h (preferably 1h) is stirred under the conditions of temperature, benzyl chloride is then added；21.6-26.4h is stirred at room temperature (preferably in mixture
For 24 hours), water is added, is extracted with ethyl acetate；Combined ethyl acetate phase is dried and concentrated after successively being cleaned with water, saturated brine,
Residue obtains formula (7) compound through silica gel column purification；
5 ') formula (7) compound is dissolved in tetrahydrofuran, -70.2~-85.8 DEG C (preferably -78 DEG C) is cooled to, in nitrogen
Under gas shielded, n-BuLi is added, stirs 1h, DMF is added, restores to room temperature, stirs 0.9-1.1h (preferably 1h)；Saturation is added
Ammonium chloride solution is extracted with methylene chloride, is collected methylene chloride phase, is dried and concentrated, residue is obtained through silica gel column purification
Formula (8) compound；
6 ') formula (2) compound is dissolved in tetrahydrofuran, is cooled to 0 DEG C, the mineral oil of containing hydrogenated sodium is added, room temperature is stirred
Mix 0.9-1.1h (preferably 1h)；Formula (8) compound for being dissolved in tetrahydrofuran is added, stirs 10.8- under the conditions of 45-55 DEG C
13.2h (preferably 50 DEG C stirring 12h)；Saturated ammonium chloride solution is added, is extracted with ethyl acetate, is repeated 3 times, merges organic phase,
It is dried and concentrated；Residue obtains formula (9) compound through silica gel column purification；
7 ') formula (9) compound, palladium-carbon catalyst are dissolved in ethyl alcohol, under hydrogen protection, 45-55 DEG C of heating 10.8-
13.2h (preferably 50 DEG C heating 12h), filtering remove catalyst, remove organic phase under vacuum；Residue is obtained through silica gel column purification
Formula (10) compound；
8 ') formula (10) compound, formula (11) compound and pyridine are dissolved in acetonitrile, stir 21.6- at 54-66 DEG C
26.4h (preferably 60 DEG C stirrings are for 24 hours)；Organic phase is removed under vacuum, is added water in residue, is extracted with methylene chloride, merges organic phase,
It is dried and concentrated.Residue obtains formula (12) compound through silica gel column purification；
9 ') formula (12) compound, NaOH are added in water and ethyl alcohol, are flowed back 21.6-26.4h (preferably for 24 hours), until formula
(12) compound reacts completely；Ethyl alcohol is removed under vacuum, adds water, adjusting pH value with acid is 2.7-3.3 (preferable ph 3)；It will precipitating
Filtering, it is dry to get sulfonamide hapten shown in formula (I).
In the specific embodiment of the present invention, (synthetic route is shown in Fig. 1) is made by the following method in the haptens:
1, by the 4- bromobutyrate (compound 1) of 100g, the triphenylphosphine of 150g is mixed, and is reacted in 1L reflux in toluene
24h.Precipitating filtering, recycling, and with petroleum ether, repetitive operation 3 times.Vacuum drying, obtains white solid product (compound
2)。
2, by 2, the 4- pentanedione (compound 3) of 200g, the guanidine hydrochloride (compound 4) of 200g is dissolved in the water of 2L, gradually
550g potassium carbonate is added.Reaction mixture stirs for 24 hours at 40 DEG C.Precipitating is filtered, and is washed with water, is repeated 3 times.It is done under vacuum
It is dry, obtain pale solid (compound 5).
3, by 150g compound 5, the N- bromo-succinimide of 200g is added in 1L acetonitrile, heats 6h under the conditions of 80 DEG C.
Precipitating is filtered, and is washed with cold acetonitrile, dry under vacuum condition, obtains white solid (compound 6).
4,100g compound 6 is dissolved in the DMF of 1L.The NaH that 50g is dissolved in 60% mineral oil divides at 0 DEG C
It criticizes and is added, under cryogenic, continue to stir 1h, be gradually added into 150g benzyl chloride.Mixture is stirred at room temperature for 24 hours.After be added
4L water makes to be extracted with ethyl acetate, be repeated 3 times.Combined ethyl acetate phase, washes with water, and is repeated 3 times；Saturated brine cleaning, weight
It is 3 times multiple；Use Na2SO4It is dried and concentrated.Residue obtains colorless oil (compound 7) through silica gel column purification.
5,50g compound 7 is dissolved in the tetrahydrofuran of 500mL, is cooled to -78 DEG C.Under nitrogen protection, dropwise plus
The n-BuLi for entering the 1M of 200mL continues to stir 1h.50mL DMF is added, gradually restores to room temperature, and stir 1h.It is added full
And ammonium chloride solution, it is extracted using methylene chloride, collects methylene chloride phase, Na2SO4It is dried and concentrated.Residue is through silicagel column
Purifying, obtains white solid (compound 8).
6,50g compound 2 is dissolved in 500mL tetrahydrofuran, is cooled to 0 DEG C, 60% mineral of the sodium hydride containing 5g are added
Oil.1h is stirred at room temperature.The compound 8 that 20g is dissolved in 50mL tetrahydrofuran is added, stirs 12h under the conditions of 50 DEG C.Saturation is added
NH4Cl solution makes to be extracted with ethyl acetate, be repeated 3 times, and merges organic phase, uses Na2SO4It is dried and concentrated.Residue is through silica gel
Column purification obtains white solid (compound 9).
7, by 10g compound 9,5g palladium-carbon catalyst is dissolved in 50mL ethyl alcohol, under hydrogen protection, 50 DEG C of heating 12h.It crosses
Filter removes catalyst.Under vacuum, organic phase is removed.Residue obtains white powder (compound 10) through silica gel column purification.
8, by 2.5g compound 10,2.5g compound 11,1g pyridine is dissolved in the acetonitrile of 100mL, stirs for 24 hours at 60 DEG C.
Organic phase is removed under vacuum, is added water in residue, is extracted, be repeated 3 times with methylene chloride.Merge organic phase, uses Na2SO4Drying is simultaneously
Concentration.Residue obtains white powder (compound 12) through silica gel column purification.
9, by 1g compound 12, the NaOH of 1g is added in water and ethyl alcohol, and reflux for 24 hours, and is monitored using thin-layer chromatography,
Until compound 12 is reacted completely.Ethyl alcohol is removed under vacuum, adds water, is 3 with 1M HCl adjustment pH value.Precipitating is filtered, is done
It is dry, obtain white powder, as final product sulfadimidine valeric acid.Take the sulfonamide hapten of synthesis through hydrogen nuclear magnetic resonance
Spectrum (1H-NMR it) measures, structure is as shown in Figure 2.
The third aspect, the present invention provide sulfa drugs artificial antigen, are by the sulfonamide hapten and carrier
It is obtained after albumen coupling.The sulfa drugs artificial antigen can be used as immunogene and can also be used as coating antigen.
Wherein, the carrier protein is selected from bovine serum albumin(BSA) (BSA), ovalbumin (OVA), keyhole limpet hemocyanin
(KLH), thyroprotein, human serum albumins (HSA)；It is preferred that bovine serum albumin(BSA), keyhole limpet hemocyanin.
Fourth aspect, the present invention provide the preparation method of the artificial antigen, are made by carbodiimide or mixed anhydride method
Artificial antigen is made in the sulfonamide hapten and carrier protein couplet.Specifically, using active ester method by carrier protein
It is coupled in the carboxyl carbon of the haptens.It specifically, will be bad on the carboxyl and carrier protein of haptens using active ester method
Propylhomoserin coupling, generates amido bond.
Preferably, the coupling Mole Ratio of compound shown in formula (1) and carrier protein is 7-13:1.Preferably, shown in formula (I)
The coupling Mole Ratio of compound and carrier protein is 12.3:1.
5th aspect, the present invention provide the specific antibody prepared by the sulfa drugs artificial antigen, including more grams
Grand antibody and monoclonal antibody.
6th aspect, the present invention provide the following of the sulfonamide hapten or the sulfa drugs artificial antigen
Any application:
1. preparing the application in anti-sulfa drugs specific antibody；
2. detecting the application in anti-sulfa drugs specific antibody.
7th aspect, the present invention provides anti-sulfa drugs polyclonal antibody, by the artificial antigen immunization experiment animal
It obtains.
Preferably, the artificial antigen is coupled to obtain by the sulfonamide hapten with keyhole limpet hemocyanin.
Polyclonal antibody provided by the invention is wide spectrum antibody, can detect monocycle class sulfanilamide (SN) (sulphanilamide, sulfanilamide (SN) simultaneously
Vinegar acyl, sulphaguanidine), five-ring heterocycles bicyclic class sulfanilamide (SN) (sulfaphenazolum, phthalylsulfathiazol, sulfanilamide (SN) Sulfafurazole, sulfanilamide (SN) thiophene
Azoles, sulfamethoxazole, sulfamethazole, sulfamethizole) and hexa-member heterocycle bicyclic class sulfanilamide (SN) (sulfanitran, willow nitrogen
Sulfapryidine, sulfapryidine, cistosulfa, sulfamethoxypyridazine, sulfaethoxypyridazine, sulfaquinoxaline, sulfaclozine,
Sulfadoxine, sulfabenzamide, sulfalene, sulfaisodimidine, daimeton, 5-methoxysulfadiazine, sulfanilamide (SN)
Pyrimidine, sulfamethazine, madribon, sulfamethyldiazine, sulfabromomethazine) etc..
Eighth aspect, the present invention provide the sulfonamides quality testing prepared by the specific antibody or the polyclonal antibody
Test agent or kit.
9th aspect, the present invention provide following any application of the specific antibody or the polyclonal antibody:
(1) application in detection sulfa drugs；
(2) application in the immuno-chromatographic test paper strip for preparing sulfa drugs；
(3) application in the colloidal gold colloidal gold detection test paper strip for preparing sulfa drugs.
In the present invention, the sulfa drugs includes but is not limited to monocycle class sulfanilamide (SN) (sulphanilamide, sulfacetamide, sulfanilamide (SN)
Guanidine), five-ring heterocycles bicyclic class sulfanilamide (SN) (sulfaphenazolum, phthalylsulfathiazol, sulfanilamide (SN) Sulfafurazole, sulphathiazole, methylene sulfonamide
Isoxazole, sulfamethazole, sulfamethizole) and hexa-member heterocycle bicyclic class sulfanilamide (SN) (sulfanitran, salicylazosulfapyridine, sulphur
Amine pyridine, cistosulfa, sulfamethoxypyridazine, sulfaethoxypyridazine, sulfaquinoxaline, sulfaclozine, sulfadoxine, sulphur
Amine benzoyl, sulfalene, sulfaisodimidine, daimeton, 5-methoxysulfadiazine, sulphadiazine, sulfanilamide (SN) two
Methylpyrimidine, madribon, sulfamethyldiazine, sulfabromomethazine).
Tenth aspect, the present invention provide quick, sensitive, wide spectrum the sulphur established based on the polyclonal antibody and coating antigen
The enzyme-linked immune analytic method of amine drug.
Preferably, the coating antigen is coupled to obtain by the sulfonamide hapten with bovine serum albumin(BSA).
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
Present invention firstly discloses new sulfonamide haptens, artificial antigen and preparation method thereof, with the sulfanilamide (SN)
Animal is immunized in class drug artificial antigen, and potency height, the specific antibody of high sensitivity can be obtained.Sulfonamides provided by the invention
Object haptens and its antibody of preparation, the sulfonamides object detecting method to establish quick, easy, inexpensive, sensitive, special provide
New tool.
Sulfa drugs antibody, preparation process letter are prepared using the conjugate of haptens provided by the invention and carrier protein
Single, economical, the detection sensitivity of antibody is high, practical value is high.Before the present invention has good application in detection of veterinary drugs in food
Scape.
Detailed description of the invention
Fig. 1 is sulfonamide hapten synthetic route in the embodiment of the present invention 1.
Fig. 2 is the sulfonamide hapten nuclear magnetic resonance spectroscopy prepared in the embodiment of the present invention 1.
Fig. 3 is sulfa drugs coating antigen MALDI-TOF spectrogram in the embodiment of the present invention 3.
Fig. 4 is sulfa drugs ELISA standard curve in the embodiment of the present invention 5.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
The preparation and authentication of 1 sulfonamide hapten of embodiment
1, by the 4- bromobutyrate (compound 1) of 100g, the triphenylphosphine of 150g is mixed, and is reacted in 1L reflux in toluene
24h.Precipitating filtering, recycling, and with petroleum ether, repetitive operation 3 times.Vacuum drying, obtains white solid product (compound
2)。
2, by 2, the 4- pentanedione (compound 3) of 200g, the guanidine hydrochloride (compound 4) of 200g is dissolved in the water of 2L, gradually
550g potassium carbonate is added.Reaction mixture stirs for 24 hours at 40 DEG C.Precipitating is filtered, and is washed with water, is repeated 3 times.It is done under vacuum
It is dry, obtain pale solid (compound 5).
3, by 150g compound 5, the N- bromo-succinimide of 200g is added in 1L acetonitrile, heats 6h under the conditions of 80 DEG C.
Precipitating is filtered, and is washed with cold acetonitrile, dry under vacuum condition, obtains white solid (compound 6).
4,100g compound 6 is dissolved in the DMF of 1L.The NaH that 50g is dissolved in 60% mineral oil divides at 0 DEG C
It criticizes and is added, under cryogenic, continue to stir 1h, be gradually added into 150g benzyl chloride.Mixture is stirred at room temperature for 24 hours.After be added
4L water makes to be extracted with ethyl acetate, be repeated 3 times.Combined ethyl acetate phase, washes with water, and is repeated 3 times；Saturated brine cleaning, weight
It is 3 times multiple；Use Na2SO4It is dried and concentrated.Residue obtains colorless oil (compound 7) through silica gel column purification.
5,50g compound 7 is dissolved in the tetrahydrofuran of 500mL, is cooled to -78 DEG C.Under nitrogen protection, dropwise plus
The n-BuLi for entering the 1M of 200mL continues to stir 1h.50mL DMF is added, gradually restores to room temperature, and stir 1h.It is added full
And ammonium chloride solution, it is extracted using methylene chloride, collects methylene chloride phase, Na2SO4It is dried and concentrated.Residue is through silicagel column
Purifying, obtains white solid (compound 8).
6,50g compound 2 is dissolved in 500mL tetrahydrofuran, is cooled to 0 DEG C, 60% mineral of the sodium hydride containing 5g are added
Oil.1h is stirred at room temperature.The compound 8 that 20g is dissolved in 50mL tetrahydrofuran is added, stirs 12h under the conditions of 50 DEG C.Saturation is added
NH4Cl solution makes to be extracted with ethyl acetate, be repeated 3 times, and merges organic phase, uses Na2SO4It is dried and concentrated.Residue is through silica gel
Column purification obtains white solid (compound 9).
7, by 10g compound 9,5g palladium-carbon catalyst is dissolved in 50mL ethyl alcohol, under hydrogen protection, 50 DEG C of heating 12h.It crosses
Filter removes catalyst.Under vacuum, organic phase is removed.Residue obtains white powder (compound 10) through silica gel column purification.
8, by 2.5g compound 10,2.5g compound 11,1g pyridine is dissolved in the acetonitrile of 100mL, stirs for 24 hours at 60 DEG C.
Organic phase is removed under vacuum, is added water in residue, is extracted, be repeated 3 times with methylene chloride.Merge organic phase, uses Na2SO4Drying is simultaneously
Concentration.Residue obtains white powder (compound 12) through silica gel column purification.
9, by 1g compound 12, the NaOH of 1g is added in water and ethyl alcohol, and reflux for 24 hours, and is monitored using thin-layer chromatography,
Until compound 12 is reacted completely.Ethyl alcohol is removed under vacuum, adds water, is 3 with 1M HCl adjustment pH value.Precipitating is filtered, is done
It is dry, obtain white powder, as final product sulfadimidine valeric acid.Take the sulfonamide hapten of synthesis through hydrogen nuclear magnetic resonance
Spectrum (1H-NMR it) measures, structure is as shown in Figure 2.
The preparation of 2 sulfa drugs immunizing antigen sulfadimidine valeric acid of embodiment-hemocyanin
1,0.1mmol sulfadimidine valeric acid is taken, 40mg N, N '-dicyclohexylcarbodiimide (DCC),
25mg N-hydroxysuccinimide (NHS) is added in 1mL DMF, and stirred overnight at room temperature is esterified haptens carboxyl
(activation).
2, activation products are taken out, 4000rpm is centrifuged 10min.
3, the KLH for weighing 100mg is dissolved in the PBS of 20mL pre-cooling.
4, the supernatant after taking activation products to be centrifuged, is slowly added in KLH solution dropwise, is stirred overnight under the conditions of 4 DEG C.
5, above-mentioned reaction product is placed in bag filter, the dialysed overnight in 5L dialyzate.
6, product is taken out, is frozen after packing in -70 DEG C of refrigerator-freezers, as immunogene sulfadimidine valeric acid-KLH.Implement
The coating antigen sulfadimidine valeric acid-bovine serum albumin(BSA) preparation of 3 sulfonamides analyte detection of example
1,0.5mmol sulfadimidine valeric acid is taken, 200mg N, N '-dicyclohexylcarbodiimide (DCC),
100mg N-hydroxysuccinimide (NHS) is added in 1mL DMF, stirred overnight at room temperature, carries out ester to haptens carboxyl
Change (activation).
2, activation products are taken out, 4000rpm is centrifuged 10min.
3, the BSA for weighing 500mg is dissolved in the PBS of 20mL pre-cooling.
4, the supernatant after being centrifuged in activation products is taken, is slowly added in BSA solution, is stirred overnight under the conditions of 4 DEG C dropwise.
5, above-mentioned reaction product is placed in bag filter, is dialysed three days, replace a dialyzate for every eight hours.
6, product is taken out, is frozen after packing in -20 DEG C of refrigerator-freezers, as coating antigen sulfadimidine valeric acid-BSA.
7, the sulfa drugs coating antigen sulfadimidine valeric acid-BSA of synthesis is taken to measure through MALDI-TOF, such as Fig. 3 institute
Show, the m/z of main peak is 71759.7, shows that haptens sulfadimidine valeric acid and carrier protein BSA are coupled successfully, idol
Connection molar ratio is 12.3:1.
The preparation of 4 sulfonamides analyte detection polyclonal antibody of embodiment
50 6-8 week old females are immunized as immunogene in the conjugate sulfadimidine valeric acid-KLH prepared using embodiment 2
BALB/c mouse (SPF rank), immune programme booster immunization for a fundamental immunity and for several times.
100 μ g immunogenes are mixed with isometric Freund's complete adjuvant when first immunisation, are emulsified.Protein emulsion is more
The nape of the neck that point is injected in mouse is subcutaneous, the 200 progress fundamental immunities of μ L every.
50 μ g immunogenes are mixed with isometric incomplete Freund's adjuvant, are emulsified.It carries out within 4 weeks after first immunisation
First time booster immunization, second of the booster immunization of progress in latter 3 weeks, latter 3 weeks progress third time booster immunizations, volume is 200 μ L
Every.
From be immunized it is secondary, it is immune every time after eye socket blood sampling in the 8th day, separate serum, it is anti-that indirect competitive ELISA detects serum
The potency of body identifies the type and half-inhibitory concentration (IC of sulfonamide50)。
Potency height, identification is selected to compose wide, IC50It is worth low mouse, extracts eyeball, collect blood, centrifugation obtains antiserum, i.e.,
For sulfa drugs polyclonal antibody.
The foundation of 5 sulfa drugs enzyme-linked immune analytic method of embodiment
1, preparation of reagents
It is coated with buffer: Na2CO31.59g NaHCO32.93g adds distilled water to 1L, mixes.
Block buffer: calf serum 50mL, sucrose 50g, casein 2.5g, Na2HPO4·12H2O 5.8g,
NaH2PO4·2H2300 300 μ L of O 0.593g, NaCl 8g, proclin, adds distilled water to be settled to 1L, mixes, sets 4 DEG C of refrigerators
It saves.
Antibody diluent: NaH2PO4·2H2O 0.6g, Na2HPO41.072g, NaCl 16g, KCl 0.4g,
200 μ L, triton X-100 of proclin300,500 μ L adds distilled water to 1L, mixes, set 4 DEG C of refrigerators and save.
Standard dilutions: Na2HPO4·12H2O 2.9g, NaH2PO4·2H2O 0.59g, NaCl 8.5g, KCl
0.2g adds distilled water water to be settled to 1L, mixes.
Enzyme labelled antibody dilution: NaH2PO4·2H2O 0.6g, Na2HPO41.072g, NaCl 16g, KCl 0.4g,
Proclin 300 200 μ L, distilled water 950mL are added 50mL and have inactivated calf serum, mix, set 4 DEG C of refrigerators and protect after dissolution
It deposits.
Washing buffer (20 ×): Na2HPO4·12H2O 23.2g, KH2PO42g, NaCl 64g, KCl 0.036g,
300 300 μ L of Tween-20 20mL, proclin, adds water 980mL to be settled to 1L.Dilute 20 times of uses.
Substrate buffer solution: Na2HPO4·12H2O 3.68g, citric acid 0.933g, adds distilled water to 100mL.
TMB storing liquid: it takes 10mg TMB to be dissolved in 4mL ethylene glycol, is kept in dark place.
Substrate solution: TMB storing liquid 0.4mL, substrate buffer solution 10mL, 30%H are taken2O210 μ L, are kept in dark place.
Stop buffer: taking 98% concentrated sulfuric acid 22.2mL, adds distilled water 177.8mL, mixes.
2, square matrix titrates
Optimal primary concentration of envelope and antibody dilution are screened using square matrix titration.Using coating buffer by coating antigen
Doubling dilution is at 1:5 000,1:10 000,1:20 000,1:40 000,1:80 000 and 1:160 000, from the 1st to the 12nd
Leie time is longitudinal to be added in 96 hole elisa Plates, and 1 repetition is arranged in coating, each dilution.It will be polyclonal using antibody diluent
Antibody doubling dilution is at 1:2 000,000 He of 1:4 000,1:8 000,1:16 000,1:32 000,1:64 000,1:128
1:256 000 is successively laterally added 96 hole elisa Plates from the 1st to eighth row, does indirect ELISA.Meanwhile it is each coating antigen, anti-
Bulk concentration combination, is added the sulfamethazine of 1ng/mL (1ppb), be at war with reaction.Square matrix titration results are shown in Table 1, just
For step selection OD value close to 2.0, adjacent two holes OD value has large change, and competes preferable peridium concentration and the dilution of corresponding antibody
Degree combination.The concentration of coating antigen 1:80000 and the dilution combination of antibody 1:64000 are selected from table 1.
1 square matrix titration of table
3, standard curve is established
By monocycle class sulfanilamide (SN) (sulphanilamide, sulfacetamide, sulphaguanidine), the bicyclic class sulfanilamide (SN) of five-ring heterocycles (sulfaphenazolum,
Phthalylsulfathiazol, sulfanilamide (SN) Sulfafurazole, sulphathiazole, sulfamethoxazole, sulfamethazole, sulfamethizole) and
Hexa-member heterocycle bicyclic class sulfanilamide (SN) (sulfanitran, salicylazosulfapyridine, sulfapryidine, cistosulfa, sulfamethoxypyridazine, sulphur
Amine ethoxy pyridazine, sulfaquinoxaline, sulfaclozine, sulfadoxine, sulfabenzamide, sulfalene, sulfaisodimidine, sulphur
Sulfamonomethoxine, 5-methoxysulfadiazine, sulphadiazine, sulfamethazine, madribon, methylene sulfonamide between amine
Pyrimidine, sulfabromomethazine) etc. standard items prepare 8 concentration gradients, 3 parallel holes of each concentration do indirect competition
ELISA draws standard curve, calculates IC50Value, wherein the standard curve of sulfamethyldiazine is as shown in Figure 4.
4, cross reacting rate measures
By above-mentioned IC50The IC of value and sulfaquinoxaline50Value comparison, obtains cross reacting rate, the results are shown in Table 2.Originally it grinds
There is a degree of cross reacting rate to the 32 kinds of sulfa drugs measured in the indirect competitive ELISA method for studying carefully foundation.
2 ELISA method IC of table50Value and antibody cross reaction rate
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. sulfonamide hapten, which is characterized in that shown in its structure such as formula (1):
Wherein, n=0, or >=1 integer；
Preferably, n=1, the haptens are formula (I) compound represented:
2. the preparation method of haptens described in claim 1, which is characterized in that as n=1, the preparation method of formula (I) compound
Include the following steps:
1) 4- bromobutyrate reacts to obtain formula (2) compound with triphenylphosphine；
2) 2,4- pentanedione reacts to obtain formula (5) compound with guanidine hydrochloride；
3) formula (5) compound reacts to obtain formula (6) compound with N- bromo-succinimide；
4) formula (6) compound reacts to obtain formula (7) compound with benzyl chloride；
5) formula (7) compound reacts to obtain formula (8) compound with n-BuLi；
6) formula (8) compound reacts to obtain formula (9) compound with formula (2) compound；
7) formula (9) compound generation hydrogenation obtains formula (10) compound；
8) formula (10) compound reacts to obtain formula (12) compound with formula (11) compound；
9) formula (12) compound generation hydrolysis obtains formula (I) compound；
Wherein, step 1) -9) described in compound structure it is as follows:
Preferably, the preparation method of formula (I) compound includes the following steps:
1 ') 4- bromobutyrate is mixed with triphenylphosphine, is reacted in reflux in toluene；Precipitating filtering, recycling are washed with petroleum ether
It is dry after washing, obtain formula (2) compound；
2 ') 2,4- pentanedione and guanidine hydrochloride is soluble in water, potassium carbonate is added, reaction mixture stirs 21.6- at 36-44 DEG C
26.4h；Precipitating is filtered, and is dried in vacuo after being washed with water, is obtained formula (5) compound；
3 ') formula (5) compound, N- bromo-succinimide are added in acetonitrile, 5.4-6.6h is heated under the conditions of 72-88 DEG C；It is heavy
Shallow lake is filtered, and is dried in vacuo after being washed with acetonitrile, is obtained formula (6) compound；
4 ') formula (6) compound is dissolved in DMF, the mineral oil of containing hydrogenated sodium is then added portionwise at -1~1 DEG C, low
0.9-1.1h is stirred under the conditions of temperature, and benzyl chloride is then added；21.6-26.4h is stirred at room temperature in mixture, and water is added, uses acetic acid
Ethyl ester extraction；Combined ethyl acetate phase is dried and concentrated after successively being cleaned with water, saturated brine, and residue is obtained through silica gel column purification
To formula (7) compound；
5 ') formula (7) compound is dissolved in tetrahydrofuran, is cooled to -70.2~-85.8 DEG C, under nitrogen protection, be added just
Butyl lithium stirs 0.9-1.1h, and DMF is added, and restores to room temperature, stirs 0.9-1.1h；Saturated ammonium chloride solution is added, uses dichloro
Methane is extracted, and is collected methylene chloride phase, is dried and concentrated, residue obtains formula (8) compound through silica gel column purification；
6 ') formula (2) compound is dissolved in tetrahydrofuran, is cooled to -1~1 DEG C, the mineral oil of containing hydrogenated sodium is added, room temperature is stirred
Mix 0.9-1.1h；Formula (8) compound for being dissolved in tetrahydrofuran is added, stirs 10.8-13.2h under the conditions of 45-55 DEG C；It is added
Saturated ammonium chloride solution is extracted with ethyl acetate, and is repeated 3 times, and merges organic phase, is dried and concentrated；Residue through silica gel column purification,
Obtain formula (9) compound；
7 ') formula (9) compound, palladium-carbon catalyst are dissolved in ethyl alcohol, under hydrogen protection, 45-55 DEG C of heating 10.8-13.2h,
Filtering removes catalyst, removes organic phase under vacuum；Residue obtains formula (10) compound through silica gel column purification；
8 ') formula (10) compound, formula (11) compound and pyridine are dissolved in acetonitrile, stir 21.6-26.4h at 54-66 DEG C；Very
Sky is lower to remove organic phase, adds water in residue, is extracted with methylene chloride, merges organic phase, be dried and concentrated；Residue is pure through silicagel column
Change, obtains formula (12) compound；
9 ') formula (12) compound, NaOH are added in water and ethyl alcohol, flow back 21.6-26.4h, until formula (12) compound is complete
Reaction；Ethyl alcohol is removed under vacuum, adds water, adjusting pH value with acid is 2.7-3.3；Precipitating is filtered, it is dry to get formula (I) compound.
3. sulfa drugs artificial antigen, which is characterized in that sulfonamide hapten and carrier protein as described in claim 1
It is obtained after coupling；
Wherein, the carrier protein is selected from bovine serum albumin(BSA), ovalbumin, keyhole limpet hemocyanin, thyroprotein, human serum
Albumin；It is preferred that bovine serum albumin(BSA), keyhole limpet hemocyanin.
4. the preparation method of artificial antigen described in claim 3, which is characterized in that make institute by carbodiimide or mixed anhydride method
It states sulfonamide hapten and artificial antigen is made in carrier protein couplet.
5. the specific antibody of the preparation of the artificial antigen as described in claim 3, including polyclonal antibody and monoclonal antibody.
6. following any application of artificial antigen described in haptens or claim 3 described in claim 1:
1. preparing the application in anti-sulfa drugs specific antibody；
2. detecting the application in anti-sulfa drugs specific antibody.
7. anti-sulfa drugs polyclonal antibody, which is characterized in that the artificial antigen immunization experiment animal as described in claim 3 is obtained
?；
Preferably, the artificial antigen is coupled to obtain by the sulfonamide hapten with keyhole limpet hemocyanin.
8. the sulfonamides analyte detection of the preparation of polyclonal antibody described in specific antibody or claim 7 as described in claim 5
Reagent or kit.
9. following any application of polyclonal antibody described in specific antibody or claim 7 described in claim 5:
(1) application in detection sulfa drugs；
(2) application in the immuno-chromatographic test paper strip for preparing sulfa drugs；
(3) application in the colloidal gold colloidal gold detection test paper strip for preparing sulfa drugs.
10. application according to claim 9, which is characterized in that the sulfa drugs includes sulphanilamide, sulfanilamide (SN) vinegar
Acyl, sulphaguanidine, sulfaphenazolum, phthalylsulfathiazol, sulfanilamide (SN) Sulfafurazole, sulphathiazole, sulfamethoxazole, sulfanilamide (SN) two
First oxazole, sulfamethizole, sulfanitran, salicylazosulfapyridine, sulfapryidine, cistosulfa, sulfamethoxypyridazine, sulfanilamide (SN)
Ethoxy pyridazine, sulfaquinoxaline, sulfaclozine, sulfadoxine, sulfabenzamide, sulfalene, sulfaisodimidine, sulfanilamide (SN)
Between Sulfamonomethoxine, 5-methoxysulfadiazine, sulphadiazine, sulfamethazine, madribon, methylene sulfonamide it is phonetic
Pyridine, sulfabromomethazine.
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