CN105315166A - Preparation methods of salbutamol hapten and salbutamol antigen, and application thereof in quantum dot immunofluorescence kit - Google Patents
Preparation methods of salbutamol hapten and salbutamol antigen, and application thereof in quantum dot immunofluorescence kit Download PDFInfo
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Abstract
The invention discloses a salbutamol hapten, a corresponding artificial antigen and a corresponding antibody marked by quantum dots, and discloses the preparation methods and applications of the salbutamol hapten, the corresponding artificial antigen and the corresponding antibody marked by quantum dots. The salbutamol hapten can be connected to a carrier protein to obtain the salbutamol antigen, which is applied for preparing a salbutamol specific antibody. The invention also protects a quantum dot immunofluorescence kit for detecting salbutamol. The kit is simple in operation, has good specificity and high sensitivity, is excellent in various performances and is very suitable for being popularized.
Description
Technical field
The present invention relates to a kind of haptens, antigen and its preparation method and application, be specifically related to the preparation method of a kind of salbutamol haptens and antigen and the application in quantum dot immune fluorescent test kit thereof, for detecting the salbutamolum residue amount in animal tissues and urine, belong to field of immunological detection.
Background technology
Salbutamol (SAL) is selectivity beta 2 receptor agonist, clinical medicine is mainly used in treat asthmatic bronchitis, bronchial asthma, bronchospasm caused by pulmonary emphysema.Meanwhile, salbutamol also has and promotes animals skeletal muscle growth, reduces lipopexia, the effect such as to improve food conversion ratio, and is often used as fodder additives by illegal businessman.People has eaten the animal food containing salbutamolum residue, there will be the symptom such as muscular tremor, palpitaition, allergy, headache, dizzy, Nausea and vomiting, expiratory dyspnea.Present European and American countries all forbids its application on husbandry sector, and U.S.'s regulation can only it for scientific research, and it is the medicine prohibitted the use that the Ministry of Agriculture of China 235 announces also clear stipulaties salbutamol and salt thereof, ester, must not detect in animal food.
The method of current detection salbutamol mainly contains high performance liquid chromatography (HPLC), gas chromatography-mass spectrography (GC-MS), liquid chromatography-mass spectrography (LC-MS) etc., but it is loaded down with trivial details that these methods also exist sample pre-treatments all to some extent, to shortcomings such as the requirement of plant and instrument and operating process are higher, be unsuitable for producing, grass-roots unit and Site Detection.
Quantum dot immune fluorescent detection method has high specificity, stablizes quick, simple to operate, level of automation advantages of higher, and its detectability is than ELISA and the several order of magnitude of Physico-chemical tests method height.
Summary of the invention
The object of this invention is to provide the preparation method of a kind of salbutamol haptens and antigen and the application in quantum dot immune fluorescent test kit thereof.
Salbutamol haptens provided by the invention is the compound shown in formula I;
formula I.
The invention also discloses the preparation method of compound shown in formula I, comprise the steps:
A: intermediate sal-1 synthesizes: add the bromo-1-of 2-[4-hydroxyl-3-(methylol) phenyl] second-1-ketone 1000mg in 250ml there-necked flask, methylene dichloride 200ml, tosic acid 3.8mg, ice bath is cooled to 0-5 degree, drip 2,2 methoxy propane 2208ul, room temperature reaction spends the night, the complete aftertreatment of TLC detection reaction, saturated sodium bicarbonate aqueous solution washing organic phase, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains off-white color solid 1211mg (sal-1); B: intermediate sal-2 synthesizes: add intermediate sal-1 in 100ml there-necked flask, 1140mg, anhydrous methanol 40ml, ice bath is cooled to 0 degree, add sodium borohydride 227mg, 0-5 degree reacts 2 hours, the complete aftertreatment of TLC detection reaction, reaction solution pours in frozen water, dichloromethane extraction, saturated sodium-chloride water solution washing organic phase, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains off-white color solid 1018mg(sal-2); C: intermediate sal-3 synthesizes: add intermediate sal-2 in 100ml there-necked flask, 1000mg, anhydrous methanol 40ml, ice bath is cooled to 0 degree, adds Anhydrous potassium carbonate 1396mg, room temperature reaction 2 hours, the complete aftertreatment of TLC detection reaction, filter, 5ml methanol wash filter cake, filtrate reduced in volume obtains oily object 700mg(sal-3); D: intermediate sal-4 synthesizes: add intermediate sal-3 in 100ml there-necked flask, 700mg, anhydrous methanol 40ml, add triethylamine 686mg, 6-amino-6-methyl isophthalic acid-enanthic acid 540mg, back flow reaction 12 hours, the complete aftertreatment of TLC detection reaction, remove methyl alcohol under reduced pressure, methylene dichloride dissolution residual substance, washing, saturated sodium-chloride water solution washing organic phase, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains oily matter 1008mg(sal-4); E: haptens sal-5 synthesizes: add intermediate sal-4 in 100ml there-necked flask, 1000mg, anhydrous methanol 40ml, ice bath is cooled to 0-5 degree, drips 1M sulfuric acid 10ml room temperature reaction 2 hours, the complete aftertreatment of TLC detection reaction, remove methyl alcohol under reduced pressure, be cooled to 10 degree and separate out white solid, filter, a small amount of water washing filter cake, obtains white solid 600mg(sal-5 after drying).
Salbutamol antigen provided by the invention is conjugate compound shown in formula I and carrier protein couplet obtained.
Common carrier albumen all can adopt, as bovine serum albumin (BSA), and ovalbumin (OVA), human serum albumin (HSA), mouse serum albumin (MSA), thyroprotein (TG) or hemocyanin (KLH) etc.
Shown in described formula I, the structure of the salbutamol antigen that compound and BSA coupling obtain is shown in formula II:
The invention also discloses the preparation method of described salbutamol antigen, comprise the steps:
1. take 7.3mg haptens and be dissolved in 2mlDMF, add EDC4.3mg, NHS5.2mg, stirring at room temperature reaction 2h;
2. get 50mgBSA and be dissolved in 5ml0.1M sodium carbonate buffer;
3. haptens dropwise step 1 obtained joins in the protein solution that step 2 obtains, and stirring at room temperature reaction 24h, PBS4 degree is dialysed 72 hours, and period changes dialyzate 6 times; Dialyzate is aseptically crossed the filter membrane in 0.22 μm of aperture, be sub-packed in ampere bottle ,-20 degree are preserved.
Described salbutamol antigen can prepare salbutamol specific antibody as immunogen, also can prepare microwell plate as coating antigen.
The specific antibody that application salbutamol antigen prepares specifically can be monoclonal antibody or polyclonal antibody.
Described salbutamol antigen, described specific antibody all can be applicable to detect salbutamol.
The present invention also protects a kind of quantum dot immune fluorescent test kit for detecting salbutamol, comprises quantum dot-labeled described antibody.
The preparation method of " quantum dot-labeled described antibody " is specific as follows:
1. get 2.5mg quantum dot, with the MES buffer solution of pH4.7,0.1M, then use the MES damping fluid of 1mlpH4.7,0.1M resuspended, add 0.96mgEDC and 1.15mgNHS, 37 DEG C are reacted 30 minutes, the centrifugal 5min of 20000rpm, collecting precipitation, is the quantum dot after activation;
2. the quantum dot after the activation that 1. step obtain is got, wash with the borate buffer solution of pH8.5,50mM, then by borate buffer solution mixing (cumulative volume is 0.8ml) of quantum dot, described antibody (protein content is 0.15mg) and pH8.5,50mM after 2.5mg activation, 25 DEG C are reacted 3.5 hours;
3. get completing steps liquid phase 2., add BSA and make its mass percentage be 5%, 37 DEG C of reactions 30 minutes, the centrifugal 5min of 20000rpm, collecting precipitation, with the PBS buffer solution of pH7.4,0.02M, resuspended with the PBS damping fluid of 1mlpH7.4,0.02M;
4. get the liquid phase that 3. step obtains, be diluted to 150000 times of volumes with the PBS damping fluid of pH7.4,0.02M.
Described quantum dot fluorescence immunoassay kit, comprising: be coated with the microwell plate of salbutamol antigen, antibody working fluid, salbutamol standardized solution, diluent, concentrated cleaning solution etc.
Whether the present invention also protects the above test kit detecting in sample to be tested containing the application in salbutamol.
The present invention relies on immunology, immunochemistry ultimate principle and retention analysis technique means, design, synthesized micromolecule target analytes haptens, and and carrier protein couplet, prepare effective artificial antigen, immune animal preparation is for the specific antibody of small molecule analysis thing.The specificity immunology of antigen-antibody is utilized to react, micro-small molecules target analytes in quantitative detection sample.Preparation method of the present invention is simple and feasible, cost is lower, and yield of hapten is higher.Instant invention overcomes complicated to salbutamol sample pretreatment in existing detection technique, consuming time and need a large amount of organic solvent extraction, and accurate expensive detecting instrument will be used and be unsuitable for shortcomings such as promoting the use of in testing process.Salbutamol antigen of the present invention, can create the specific antibody for salbutamol by immune animal, for the salbutamolum residue in rapid detection food, have simple to operate, quick, processing sample amount is large, highly sensitive, the plurality of advantages such as high specificity.
Accompanying drawing explanation
Fig. 1 is the scintigram of quantum dot.
Fig. 2 is salbutamol quantum dot immune fluorescent kit standard graphic representation.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.Stirring in embodiment 1 all adopts magnetic stirring apparatus to carry out, test materials used, if no special instructions, is and purchases available from routine biochemistry reagent shop.
The structural formula of salbutamol is as follows:
。
The preparation of embodiment 1, immunogen and coating antigen
One, haptenic preparation
1, intermediate sal-1 synthesizes: add the bromo-1-of 2-[4-hydroxyl-3-(methylol) phenyl] second-1-ketone 1000mg in 250ml there-necked flask, methylene dichloride 200ml, tosic acid 3.8mg, ice bath is cooled to 0-5 degree, drip 2,2 methoxy propane 2208ul, room temperature reaction spends the night, the complete aftertreatment of TLC detection reaction, saturated sodium bicarbonate aqueous solution washing organic phase, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains off-white color solid 1211mg (sal-1).
2, intermediate sal-2 synthesizes: add intermediate sal-1 in 100ml there-necked flask, 1140mg, anhydrous methanol 40ml, ice bath is cooled to 0 degree, add sodium borohydride 227mg, 0-5 degree reacts 2 hours, the complete aftertreatment of TLC detection reaction, reaction solution pours in frozen water, dichloromethane extraction, saturated sodium-chloride water solution washing organic phase, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains off-white color solid 1018mg(sal-2).
3, intermediate sal-3 synthesizes: add intermediate sal-2 in 100ml there-necked flask, 1000mg, anhydrous methanol 40ml, ice bath is cooled to 0 degree, adds Anhydrous potassium carbonate 1396mg, and chambers temp reacts 2 hours, the complete aftertreatment of TLC detection reaction, filter, 5ml methanol wash filter cake, filtrate reduced in volume obtains oily object 700mg(sal-3).
4, intermediate sal-4 synthesizes: add intermediate sal-3 in 100ml there-necked flask, 700mg, anhydrous methanol 40ml, add triethylamine 686mg, 6-amino-6-methyl isophthalic acid-enanthic acid 540mg, back flow reaction 12 hours, the complete aftertreatment of TLC detection reaction, remove methyl alcohol under reduced pressure, methylene dichloride dissolution residual substance, washing, saturated sodium-chloride water solution washing organic phase, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains oily matter 1008mg(sal-4).
5, haptens sal-5 synthesizes: add intermediate sal-4 in 100ml there-necked flask, 1000mg, anhydrous methanol 40ml, ice bath is cooled to 0-5 degree, drip 1M sulfuric acid 10ml room temperature reaction 2 hours, the complete aftertreatment of TLC detection reaction, removes methyl alcohol under reduced pressure, is cooled to 10 degree and separates out white solid, filter, a small amount of water washing filter cake, obtains white solid 600mg(sal-5 after drying), the dry-matter obtained is haptens.
Haptenic structural formula is shown in formula I.
formula I.
Two, immunogenic preparation
1, take 7.3mg haptens and be dissolved in 2mlDMF, add EDC4.3mg, NHS5.2mg, stirring at room temperature reaction 2h;
2, get 50mgBSA and be dissolved in 5ml0.1M sodium carbonate buffer;
3, haptens dropwise step 1 obtained joins in the protein solution that step 2 obtains, and stirring at room temperature reaction 24h, PBS4 degree is dialysed 72 hours, and period changes dialyzate 6 times; Dialyzate is aseptically crossed the filter membrane in 0.22 μm of aperture, be sub-packed in ampere bottle ,-20 degree are preserved.
Immunogenic structural formula is shown in formula II.
formula II.
Three, the preparation of coating antigen
Replace BSA with OVA, other same step 2, obtains coating antigen solution.
The preparation of embodiment 2, polyclonal antibody
Immunogen solution prepared by Example 1, adopts the PBS damping fluid dilution of pH7.4,0.01M, obtains immunogen diluent, for the preparation of polyclonal antibody.Adopt new zealand white rabbit as immune animal.
Immunologic process following (immunizing dose is with albumen gauge):
First immunisation: immunogen diluent and isopyknic Freund's complete adjuvant are mixed and made into emulsifying agent, neck dorsal sc multi-point injection, immunizing dose is 1mg/kgb.w.;
Booster immunization: after first immunisation 4 weeks, 8 weeks afterwards and after 12 weeks, respectively carries out a booster immunization, by immunogen diluent and isopyknic Freund's incomplete adjuvant mixing and emulsifying, and neck dorsal sc multi-point injection, single immunization dosage is 1mg/kgb.w.;
Final immunization: first immunisation carried out final immunization after 16 weeks, direct neck dorsal sc multi-point injection immunogen diluent, immunizing dose is 1mg/kgb.w..
Final immunization is after 1 week, and blood sampling is separation of serum also, is polyclonal antibody corresponding to immunogen (being called for short polyclonal antibody first).
The assembling of embodiment 3, quantum dot immune fluorescent detection kit
Quantum dot is purchased from Shenzhen Taylor, this Science and Technology Ltd., and catalog number is TLS
?lumiQD
tM20.The sign of this quantum dot is as follows: particle diameter is 20nm, the CV of particle diameter is 15%, and quantum yield is 60%, and surface-bound carboxylic content is 5 × 10
-3mmol/mg, water-soluble, CdSe/ZnS nucleocapsid structure, excitation wavelength is 345nm, and emission wavelength is 620nm; Red fluorescence quantum dot.The scintigram of quantum dot as shown in Figure 1.Carboxyl on quantum dot and the amino on protein form peptide bond and couple together.
Quantum dot immune fluorescent detection kit comprises following assembly:
1, bag is by the microwell plate of coating antigen
Coating antigen solution prepared by Example 1, is buffered liquid dilution (bag is buffered the carbonate buffer solution of liquid and pH9.6,0.05M) with bag, obtains the coating antigen diluent that protein concentration is 5ng/mL.The phosphate buffered saline buffer of 10g bovine serum albumin, 0.1mLproclin300 and 1000mLpH7.4,0.01M is mixed, obtains confining liquid.
(1) coating antigen diluent is added microwell plate (100 μ L/ hole), hatch 16 hours for 37 DEG C.
(2), after completing steps (1), the liquid inclined in hole, washing, pats dry.
(3), after completing steps (2), every hole adds 200 μ L confining liquids, 37 DEG C of incubation 2h.
(4), after completing steps (3), the liquid inclined in hole, preserves with the vacuum-sealing of aluminium film after dry.
2, quantum dot-labeled polyclonal antibody working fluid
1. get 2.5mg quantum dot, with the MES buffer solution of pH4.7,0.1M, then use the MES damping fluid of 1mlpH4.7,0.1M resuspended, add 0.96mgEDC and 1.15mgNHS, 37 DEG C are reacted 30 minutes, the centrifugal 5min of 20000rpm, collecting precipitation, is the quantum dot after activation.
2. the quantum dot after the activation that 1. step obtain is got, wash with the borate buffer solution of pH8.5,50mM, then by borate buffer solution mixing (cumulative volume is 0.8ml) of quantum dot after 2.5mg activation, polyclonal antibody first (protein content is 0.15mg) prepared by embodiment 2 and pH8.5,50mM, 25 DEG C of reactions 3.5 hours (polyclonal antibody and quantum dot form stable covalent peptide bonds combination).
3. completing steps liquid phase is 2. got, adding BSA and making its mass percentage be 5%(object is close remaining activity amino sites), 37 DEG C are reacted 30 minutes, the centrifugal 5min of 20000rpm, collecting precipitation, with the PBS buffer solution of pH7.4,0.02M, resuspended with the PBS damping fluid of 1mlpH7.4,0.02M, 4 DEG C of preservations are stand-by.
4. get the liquid phase that 3. step obtains, be diluted to 150000 times of volumes with the PBS damping fluid of pH7.4,0.02M, obtain primary antibodie working fluid first.
3, standard solution
Salbutamol is dissolved in the phosphate buffered saline buffer of pH7.4,0.02M, obtains the standard solution that concentration is 0.002ng/mL, 0.01ng/mL, 0.05ng/mL, 0.5ng/mL and 5ng/mL respectively.Using the negative control solution of the phosphate buffered saline buffer of pH7.4,0.02M as standard solution, be called 0 solution.
4, diluent
The PBS damping fluid of pH7.4,0.02M.
5,20 × concentrated cleaning solution
1000mL phosphate buffered saline buffer (pH7.4,0.5M) is mixed with 1mLproclin300, obtains 20 × concentrated cleaning solution (20 × concentrated cleaning solution is diluted with water to 20 times of volumes, is washings).
The using method of embodiment 4, quantum dot immune fluorescent detection kit
Standard solution or sample to be tested solution 50 μ L is added by the microwell plate of coating antigen to bag, primary antibodie working fluid adds 50 μ L, room temperature reaction 30min, abandon supernatant, (each washing process is all as follows: add 250 μ L washingss in every hole to wash 3 times, supernatant is abandoned after 30 seconds), pat dry with thieving paper, detect its florescent intensity value with fluorescence microplate reader.Fluorescence microplate reader is set to excitation wavelength 345nm, emission wavelength 620nm.The mean value (B) of the luminous intensity of the standard solution of each concentration is divided by the luminous intensity values (B of 0 solution
0), then be multiplied by 100%, i.e. combination rate.Calculation formula: combination rate (%)=B/B
0× 100%.With the salbutamol concentration (ng/mL) in standard solution for X-axis, B/B
0for Y-axis, drawing standard graphic representation (see figure 2).The concentration of salbutamol in sample to be tested solution can be obtained according to the regression equation of typical curve.In the present invention, the analysis of detected result can utilize professional software, can realize the real-time analysis of great amount of samples, and whole testing process only needs just can complete for 60 minutes.Combination rate (B/B
0) salbutamol concentration in standard solution corresponding when being 50% is IC
50value.According to canonical plotting, IC
50=0.028ng/mL.
The Cleaning Principle of test kit: when the conjugate of haptens pre-coated on microwell plate and carrier proteins, add sample solution, add quantum dot-labeled antibody subsequently, salbutamol residual in sample and microwell plate wrap the antibody that the coating antigen competition binding of quilt is quantum dot-labeled, sample fluorescence intensity becomes negative correlation with salbutamol content in sample, compares the residual quantity that can draw salbutamol in sample with typical curve.
The specificity of embodiment 5, quantum dot immune fluorescent detection kit
Detect the cross reacting rate of 11 kinds of medicines to be measured (with salbutamol structure or functionally similar 11 kinds of medicines) and salbutamol respectively.
1, medicine to be measured is dissolved in the phosphate buffered saline buffer of pH7.4,0.02M, obtains the solution of different concns.
2, to wrapping in the quilt microwell plate of coating antigen the solution 50 μ L adding step 1 and prepare, primary antibodie working fluid adds 50 μ L, room temperature reaction 30min, abandon supernatant, (each washing process is all as follows: add 250 μ L washingss in every hole to wash 3 times, supernatant is abandoned after 30 seconds), pat dry with thieving paper, detect its florescent intensity value with fluorescence microplate reader.Fluorescence microplate reader is set to excitation wavelength 345nm, emission wavelength 620nm.
Cross reacting rate (%)=(IC of salbutamol
50the IC of value/medicine to be measured
50value) × 100%.The results are shown in Table 1.The specificity of test kit is good.
Table 1 cross reacting rate result
The sensitivity of embodiment 6, test kit, precision, accuracy
One, the method (preparing dummy) of Sample pretreatment
Urine sample (as pig urine): get not containing the urine sample of salbutamol, the urine of clarification can be directly used in detection, if cloudy urine needs the centrifugal 5min of first 3000g, gets 50 μ L supernatant liquors for analyzing.
Muscle tissue or viscera tissue (as pork or pork liver): take 5g not containing muscle or the internal organ of salbutamol, add the homogenate of 10mL0.1mol/L aqueous hydrochloric acid, then 30 minutes are hatched in 80 DEG C of water-baths, the centrifugal 15min of 3000g after cooling, being 7-9 by 2mL supernatant liquor 1MNaOH aqueous solution adjust pH, getting 50 μ L for detecting.
Two, test kit preservation period experiment
Test kit preservation condition is 2-8 DEG C, preserves after 6 months, measures the IC of salbutamol
50value.Consider in transport and use procedure, have improper preservation condition and occur, test kit is placed 6 days under 37 DEG C of preservation conditions, carries out hot Acceleration study.Result shows that this test kit indices meets the requirements completely.
Table 2 cryopreservation experimental result
The hot Acceleration study of table 3
Three, sensitivity, precision, accuracy
1, sensitivity
Sensitivity index using lowest detectable limit as test kit of the present invention.Get 20 parts of dummies, detected signal value, calculate the mean value of dummy fluorescence intensity, and this mean value is brought into the testing concentration that typical curve obtains correspondence, calculate the standard deviation (SD) of each corresponding concentration value, add by mean value the lowest detectable limit that three times of standard deviations are this sample, the results are shown in Table 4.
The lowest detectable limit of table 4 salbutamol in pig urine and pork
| Tissue sample | The average measurement (ng/ml, ng/g, n=20) of dummy | SD（n=20） | LOD（ng/ml，ng/g） |
| Pig urinates | 0.007 | 0.003 | 0.016 |
| Pork | 0.009 | 0.004 | 0.021 |
2, precision
Extraction five test kits are often criticized from three batches of test kits (01 batch, 02 batch, 03 batch), measure 0.2ng/ml, 0.5ng/ml two concentration (namely adding salbutamol in pig urine dummy), each concentration arrange 5 parallel, repeat 5 times, according to typical curve, calculate the concentration value that each RLU is corresponding, and the variation coefficient between computing board inner panel, the results are shown in Table 5, batch variation < 15% in crowd, illustrate that the accuracy of test kit is good.
The precision of table 5 test kit
3, accuracy
Its accuracy of interpolation experiment reflection of test kit.In pig urine dummy, add salbutamol, make its concentration be 0.2ng/ml or 0.5ng/ml.Add salbutamol in pork dummy, make its concentration be 0.3ng/ml or 0.5ng/ml.Each concentration 5 is parallel.After sample process, measure the concentration of salbutamol, consider extension rate simultaneously, substitute into typical curve and calculate the rate of recovery, calculate the variation coefficient (test kits of three batches) simultaneously.The results are shown in Table 6 and table 7.
Salbutamol TIANZHU XINGNAO Capsul and the variation coefficient in table 6 pig urine
Salbutamol TIANZHU XINGNAO Capsul and the variation coefficient in table 7 pork
Claims (10)
1. a salbutamol haptens is compound shown in formula I.
2. the present invention goes back the preparation method of compound shown in protection I, comprises the steps:
Intermediate sal-1 synthesizes: add the bromo-1-of 2-[4-hydroxyl-3-(methylol) phenyl] second-1-ketone 1000mg in 250ml there-necked flask, methylene dichloride 200ml, tosic acid 3.8mg, ice bath is cooled to 0-5 degree, drip 2,2 methoxy propane 2208ul, room temperature reaction spends the night, the complete aftertreatment of TLC detection reaction, saturated sodium bicarbonate aqueous solution washing organic phase, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains off-white color solid 1211mg (sal-1); Intermediate sal-2 synthesizes: add intermediate sal-1 in 100ml there-necked flask, 1140mg, anhydrous methanol 40ml, ice bath is cooled to 0 degree, add sodium borohydride 227mg, 0-5 degree reacts 2 hours, the complete aftertreatment of TLC detection reaction, reaction solution pours in frozen water, dichloromethane extraction, saturated sodium-chloride water solution washing organic phase, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains off-white color solid 1018mg(sal-2); Intermediate sal-3 synthesizes: add intermediate sal-2 in 100ml there-necked flask, 1000mg, anhydrous methanol 40ml, ice bath is cooled to 0 degree, adds Anhydrous potassium carbonate 1396mg, and chambers temp reacts 2 hours, the complete aftertreatment of TLC detection reaction, filter, 5ml methanol wash filter cake, filtrate reduced in volume obtains oily object 700mg(sal-3); Intermediate sal-4 synthesizes: add intermediate sal-3 in 100ml there-necked flask, 700mg, anhydrous methanol 40ml, add triethylamine 686mg, 6-amino-6-methyl isophthalic acid-enanthic acid 540mg, back flow reaction 12 hours, the complete aftertreatment of TLC detection reaction, remove methyl alcohol under reduced pressure, methylene dichloride dissolution residual substance, washing, saturated sodium-chloride water solution washing organic phase, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure obtains oily matter 1008mg(sal-4); Haptens sal-5 synthesizes: add intermediate sal-4 in 100ml there-necked flask, 1000mg, anhydrous methanol 40ml, ice bath is cooled to 0-5 degree, drip 1M sulfuric acid 10ml room temperature reaction 2 hours, the complete aftertreatment of TLC detection reaction, removes methyl alcohol under reduced pressure, is cooled to 10 degree and separates out white solid, filter, a small amount of water washing filter cake, obtains white solid 600mg(sal-5 after drying), the dry-matter obtained is haptens.
3. a salbutamol antigen is conjugate compound shown in formula I and carrier protein couplet obtained.
4. salbutamol antigen according to claim 3, it is characterized in that, described carrier proteins is human serum albumin, bovine serum albumin, ovalbumin, mouse serum albumin or rabbit serum proteins.
5. the present invention also protects the preparation method of salbutamol antigen according to claim 3, comprises the steps:
Take 7.3mg haptens and be dissolved in 2mlDMF, add EDC4.3mg, NHS5.2mg, stirring at room temperature reaction 2h; Get 50mgBSA and be dissolved in 5ml0.1M sodium carbonate buffer; Join in protein solution by the haptens dropwise obtained, stirring at room temperature reaction 24h, PBS4 degree is dialysed 72 hours, and period changes dialyzate 6 times; Dialyzate is aseptically crossed the filter membrane in 0.22 μm of aperture, be sub-packed in ampere bottle ,-20 degree are preserved.
6. salbutamol antigen described in claim 3 or 4 is preparing the application in salbutamol specific antibody.
7. application rights requires the quantum dot-labeled specific antibody that described in 3 or 4, salbutamol antigen prepares.
8. specific antibody described in salbutamol antigen, claim 7 described in claim 3 or 4 is detecting the application in salbutamol.
9. application rights requires the quantum dot immune fluorescent test kit that described in 3 or 4, described in salbutamol antigen, claim 7, specific antibody prepares.
10. quantum dot immune fluorescent test kit described in claim 9, it is characterized in that, it comprises: be coated with the microwell plate of salbutamol antigen, salbutamol standardized solution, diluent, concentrated cleaning solution, antibody working fluid.
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| CN107860724A (en) * | 2017-11-09 | 2018-03-30 | 陈军 | A kind of salbutamol detection method based on optical biosensor |
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| CN103145831A (en) * | 2013-03-28 | 2013-06-12 | 江南大学 | Synthesis method of specific salbutamol artificial antigen |
| CN103235117A (en) * | 2013-04-15 | 2013-08-07 | 天津康利缘生物工程有限公司 | Beta 2-acceptor stimulant ELISA kit and usage method and application thereof |
| CN103755802A (en) * | 2014-01-16 | 2014-04-30 | 江南大学 | Synthesis method of structurally specific salbutamol complete antigen |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106526188A (en) * | 2016-11-09 | 2017-03-22 | 百奥森（江苏）食品安全科技有限公司 | Detection kit for salbutamol in foods |
| CN107860724A (en) * | 2017-11-09 | 2018-03-30 | 陈军 | A kind of salbutamol detection method based on optical biosensor |
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