CN104610079A - Salbutamol hapten derivative, and artificial salbutamol antigen and preparation method and application thereof - Google Patents
Salbutamol hapten derivative, and artificial salbutamol antigen and preparation method and application thereof Download PDFInfo
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Abstract
The present invention relates to biochemical industry technical field of biological, and in particular to a kind of salbutamol hapten derivant, salbutamol artificial antigen and its synthetic method and the application in fluorescence immune chromatography method detection salbutamol. It is diagram the present invention provides salbutamol hapten derivant structural formula. The derivant structure introduces a carboxyl-reactive structure of functional groups in three methyl one end of salbutamol, exposes the phenyl ring accessory structure of the specificity of salbutamol to greatest extent, while having can be with the functional group of carrier protein couplet. Salbutamol artificial antigen structural formula
, it is that salbutamol hapten derivant and arriving for carrier protein couplet are obtained into conjugate, wherein the carrier protein protein is bovine serum albumin (BSA) or ovalbumin (OVA); The method that the present invention establishes fluorescence immune chromatography method detection salbutamol, it is easy to operate, detection time is short, at low cost, specific high, reproducible.
Description
Technical field
The present invention relates to biochemical industry technical field of biological, particularly, relate to a kind of salbutamol hapten derivant, salbutamol artificial antigen and synthetic method thereof and detect the application in salbutamol in fluorescence immune chromatography method.
Background technology
Along with the development and progress of society, people's living standard constantly improves, and more and more payes attention to health diet.Residue of veterinary drug problem in food-safety problem, particularly food more and more causes the great attention of people.Improve the safety of livestock product, except strictly controlling and effectively managing, the monitoring residual to the toxic chemical substance such as additive and veterinary drug is vital, and therefore, science accurately detection of veterinary drugs in food method is the whether safe important leverage of animal-derived food.
Salbutamol (salbutamol, Sal), has another name called tetrahydro-isoquinolin, is a kind of receptor,β agonist, is widely used in treatment bronchial asthma clinically.Meanwhile, salbutamol has and promotes protein synthesis, increases animal carcasses lean ratio, repartitioning function, the effect of improving food conversion ratio, and often by the illegal production being used for livestock product as fodder additives, but it residually exists potential hazard to human health.The all successively legislation of the countries and regions such as European Union, the U.S., China is forbidden in Production of Livestock and Poultry, use the beta-stimulants of short growth of animal as fodder additives.Due to relatively lagging behind of salbutamol detection means research, and with clenbuterol, there is same growth-promoting effect, just progressively replace clenbuterol, become the new tool that lawless person seeks economic interests.Therefore, quick, simple, convenient, the effective method detecting SAL is set up, to guaranteeing that food safety is significant.
At present, in national standard and industry standard, HPLC and GC-MS method is often adopted to the detection of salbutamolum residue in feed and animal derived food.Although this method high specificity, highly sensitive, accurate, Sample pretreatment complex operation step, cost is high, adds that apparatus expensive, cost are large, operating skill requires high reason and is not suitable for the selective mechanisms of batch samples.Immunoassay (as euzymelinked immunosorbent assay (ELISA), colloidal gold method and fluorescence immune chromatography are sent out), due to the deficiency of the advantage of uniqueness and advantage the compensate for physico-chemical analysis such as analyzing samples easy and simple to handle is large in the qualitative, quantitative of antigen-antibody, play a part more and more important in the residue detection of beta-stimulants.Enzyme-linked immunosorbent assay can detect multiple sample simultaneously, has the advantages such as sensitive, high specificity, but is not suitable for Site Detection.Colloidal gold method and fluorescence immune chromatography method is simple to operate, quick, without the need to large-scale detecting instrument equipment, being more suitable for and on-the-spot detecting in time, is the trend of rapid detection development.
The basic reason affecting immunoassay Detection job is specificity and the affinity of antibody, these character depend on again immune haptenic molecular structure, so immune hapten molecule Design and synthesis produces specific antibody and sets up the step that small-molecule drug remains Fast Detection Technique most critical.
CN102617516A(2012-8-1) disclose the antibody of a kind of salbutamol artificial antigen and preparation thereof, but the haptenic molecular structure of immunity used in the method and artificial antigen preparation still have much room for improvement.
Summary of the invention
An object of the present invention is to provide a kind of salbutamol hapten derivant.
Two of object of the present invention is to provide a kind of preparation method of described salbutamol hapten derivant.
Three of object of the present invention is to provide a kind of salbutamol artificial antigen.
Four of object of the present invention is to provide a kind of preparation method of described salbutamol artificial antigen.
Five of object of the present invention is to provide the application of a kind of described salbutamol artificial antigen in fluorescence immune chromatography method detection salbutamol.
The invention provides a kind of salbutamol haptens derivative compound, its chemical structural formula is:
。
Described salbutamol haptens derivative compound is the derivative according to the synthesis of salbutamol structure direction, consider that salbutamol and clenbuterol are structural similar, so select to introduce carboxyl-reactive structure of functional groups with one end design of clenbuterol structural similitude, farthest retain and expose salbutamol and the discrepant and part that immunogenicity is large of clenbuterol, having the active group had with carrier protein couplet simultaneously.
The present invention also protects the preparation method of described salbutamol haptens derivative compound; it is characterized in that; with the bromo-1-of compound 2-[4-hydroxyl-3-(methylol) phenyl],-ethyl-1-ketone (CAS:62932-94-9) is for raw material, and prepared by two-step reaction, it is as follows that it prepares path:
As preferably, described reaction conditions is specially:
S1. the bromo-1-of Weigh Compound 2-[4-hydroxyl-3-(methylol) phenyl]-ethyl-1-ketone 0.015-0.0028mol, add saturated sodium methylate as solvent, add amino-4 methvl-pentanoic acid of 4-of 0.01-0.03mol under ice-water bath condition after, normal-temperature reaction 1-2h, crystallization, filtration, washing drying obtain the first white solid 4-{ [2-(4-hydroxyl-3-(methylol) phenyl)-2-oxoethyl] is amino }-4-methylvaleric acid;
S2. the white solid 0.001-0.003mol obtained in S1 is dissolved in 20ml ethanol, adds the KBH of 0.001-0.003mol 95-98wt% successively
4and 0.001-0.003molZnCl
2, after normal-temperature reaction 12-18h, filter; Filtrate concentrates, and washed with isopropyl alcohol, obtains the second white solid specificity salbutamol hapten derivant.
More preferably, described reaction conditions is specially:
S1. the bromo-1-of Weigh Compound 2-[4-hydroxyl-3-(methylol) phenyl]-ethyl-1-ketone 0.02mol, add saturated sodium methylate as solvent, add amino-4 methvl-pentanoic acid of 4-of 0.02mol under ice-water bath condition after, normal-temperature reaction 1-2h, crystallization, filtration, washing drying obtain white solid 4-{ [2-(4-hydroxyl-3-(methylol) phenyl)-2-oxoethyl] is amino }-4-methylvaleric acid;
S2. the white solid 0.002mol obtained in S1 is dissolved in 20ml ethanol, adds the KBH of 0.002mol 95% successively
4and 0.002molZnCl
2, after normal-temperature reaction 15h, filter; Filtrate concentrates, and washed with isopropyl alcohol, obtains white solid specificity salbutamol hapten derivant.
A kind of salbutamol artificial antigen, described salbutamol artificial antigen is prepared by described salbutamol haptens derivative compound, and its chemical structural formula is
Described protein and carrier proteins are bovine serum albumin or ovalbumin.
Utilize described salbutamol haptens derivative compound to prepare a method for salbutamol artificial antigen as claimed in claim 3, it utilizes carbodlimide method will state salbutamol haptens derivative compound and carrier protein couplet obtains, and comprises the following steps:
P1, described salbutamol hapten derivant is dissolved in DMSO obtains derivative compound solution;
P2, in P1, first add EDC1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, then react 15-60 minute after adding NHS N-hydroxy-succinamide Homogeneous phase mixing;
P3, the reaction solution obtained in P2 is carried out linked reaction 1-5 hour in the 25-30 DEG C of solution joined containing carrier proteins, wherein said carrier proteins is bovine serum albumin or ovalbumin;
P4, P3 gained reaction solution to be dialysed, collect dialyzate and obtain salbutamol artificial antigen.
As preferably, carrier proteins described in described step P3 is bovine serum albumin or ovalbumin; When described carrier proteins is bovine serum albumin, the mol ratio of derivative compound and bovine serum albumin is 45 ~ 60:1, and the artificial antigen of synthesis is salbutamol immunogen; When carrier proteins is ovalbumin, the mol ratio of derivative compound and ovalbumin is 35 ~ 50:1, and the artificial antigen of synthesis is coating antigen.
As preferably, described in described step P2, EDC and NHS is dissolved in DMF or DMSO, and EDC, NHS and derivative compound mole dosage ratio are 1 ~ 2:1 ~ 1.5:1 ~ 2.
As preferably, the damping fluid of carrier protein solution described in described step P3 is the NaHCO of 0.1mol/L
3damping fluid; Dialyzate described in P4 to be 0.01mol/L, PH be 7.2 ~ 7.9 phosphoric acid buffer.Described NaHCO
3the compound method of damping fluid is: take 8.4g NaHCO
3, be dissolved in and be settled in 1000ml ultrapure water, regulate PH to 7.4; Phosphoric acid buffer: take NaH
2pO
42H
2o:0.3g, NaCL: 9g, Na
2hPO
412H
2o: 0.3g is settled to 1000ml, regulates PH to be 7.1 ~ 7.8.
The present invention also protects the application of salbutamol artificial antigen described above in the preparation of salbutamol specific antibody; Described antibody is monoclonal antibody or polyclonal antibody.
The present invention also protects the application of described salbutamol artificial antigen in salbutamol fluorescence immune chromatography detection method.
As preferably, described detection method comprises the steps:
P11. described salbutamol immunogen immune animal Dispersal risk is utilized;
P12. cytogamy and screening;
P13. ascites preparation and antibody purification;
P14. by the antibody obtained in step P 13 with anti-rabbit IgG bis-is anti-carries out fluorochrome label respectively, and detection analytic liquid is mixed with;
P15. described salbutamol coating antigen and the rabbit igg antibody rear and front end be sprayed at respectively on nitrocellulose filter bar are prepared detection line and nature controlling line, wherein near thieving paper end be front end, near sample pad be rear end;
P16. the nitrocellulose filter containing detection line and nature controlling line described in glass fiber sample pad, step P15, thieving paper and PVC black floor are assembled into immuno-chromatographic test paper strip;
P17. the cutting of test strip described in step P16 proper width is loaded in plastics cartridges, and detect analytic liquid described in step P14 and support the use and detect on fluorescence immune chromatography detector.
Advantage of the present invention and feature;
The invention provides a kind of salbutamol derivative compound of controlled syntheses, according to salbutamol and the constitutional features of cloning special sieve, one end (alkyl end) orientation identical in its structure derives active function groups structure (carboxyl), farthest retains the specificity structure exposing the two and the strong structure (i.e. the structure division of phenyl ring end) of immunity; Based on method for preparing artificial antigen of the present invention, salbutamol derivative compound described above is utilized to prepare specific salbutamol artificial antigen; Described specificity artificial antigen is prepared specific antibody, establishes the method detecting salbutamol with artificial antigen as above and the antibody fluorescence immune chromatography method that is raw material.
Antigen of the present invention and antibody starting material all belong to self-control, and raw materials cost reduces greatly, and antigen and antibodies specific are by force, and combined with fluorescent immunochromatography technique sets up high specific and highly sensitive salbutamol method for quick.
Accompanying drawing explanation
Fig. 1 is salbutamol derivative compound structural formula;
Fig. 2 is salbutamol artificial antigen structural formula;
Fig. 3 is salbutamol and clenbuterol structural formula comparison schematic diagram;
Fig. 4 is the UV scanning figure of salbutamol artificial antigen, BSA and salbutamol derivative;
Fig. 5 is the structure of the salbutamol fluorescence immune chromatography test card of dress plastic clip; Fig. 6 is the salbutamol fluorescence immune chromatography test card structure with plastics cartridge;
Fig. 7 is that fluorescence immune chromatography method detects salbutamol standard substance suppression graphic representation;
Fig. 8 is that salbutamol fluorescence immune chromatography detects negative findings collection of illustrative plates;
Fig. 9 is that salbutamol fluorescence immune chromatography detects positive findings collection of illustrative plates.
Embodiment
Here further describes the present invention by reference to the accompanying drawings.Except specified otherwise, the present invention in an embodiment used kit raw material is conventional reagent.
Embodiment 1
The controlled syntheses of salbutamol derivative compound:
S1. faint yellow solid 4.9g(0.02mol in the bromo-1-of Weigh Compound 2-[4-hydroxyl-3-(methylol) phenyl]-ethyl-1-ketone (CAS:62932-94-9)) in 50ml there-necked flask, add the saturated sodium methylate of 20ml as solvent, 2.63g(0.02mol is added under ice-water bath condition) after amino-4 methvl-pentanoic acid (import brand) of 4-, normal-temperature reaction 1-2h, crystallization, filtration, washing drying obtain white solid, that is: 4-{ [2-(4-hydroxyl-3-(methylol) phenyl)-2-oxoethyl] is amino }-4-methylvaleric acids.
S2. the white solid 0.59g(0.002mol will obtained in S1) be dissolved in 20ml ethanol, add 0.11g(0.002mol successively) KBH of 95%
4, 0.27g(0.002mol) and ZnCl
2, after normal-temperature reaction 15h, filter.Filtrate concentrates, and washed with isopropyl alcohol, obtains white solid, that is: be salbutamol derivative compound.
Embodiment 2
The preparation of salbutamol artificial antigen (immunogen and coating antigen):
Take 40mg salbutamol derivative compound, be dissolved in 2ml DMF, form the final concentration of 20mg/ml, get wherein 90ul and 6ul EDC(1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) (100mg/ml) mixing, add again 10ul NHS (N-hydroxy-succinamide) (50mg/ml), after Homogeneous phase mixing, react 15 ~ 60min under agitation.
To join in 1ml 6mg/mlBSA solution (or joining in 1ml 4.5mg/mlOVA solution) after centrifugal for above-mentioned reaction mixture (1600rmp) under 25 ~ 30 DEG C of agitation conditions after linked reaction 1 ~ 5h, dialyse 4 times with phosphoric acid buffer, 12h changes a not good liquor.Collect dialyzate, the concentration measuring immunogen and coating antigen with the Quick Start Bradford Protein Assay Kit of U.S. Bole (BIO-RAD) company is respectively 5.4mg/ml and 3.8mg/ml.Structural formula as shown in Figure 2 for the salbutamol artificial antigen (immunogen and coating antigen) obtained.In Fig. 2, protein is BSA or OVA.
Described BSA solution (or OVA solution) compound method: take 60mg BSA(or 45mg OVA) be dissolved in the NaHCO of the 0.1mol/L of 10ml
3damping fluid;
Described NaHCO
3damping fluid preparation method: take 8.4g NaHCO
3, be dissolved in and be settled in 1000ml ultrapure water, regulate PH to 7.1 ~ 7.9;
Described phosphoric acid buffer liquid and preparation method thereof: take two hypophosphite monohydrate sodium dihydrogens: 0.3g, NaCL: 9g, disodium hydrogen phosphate dodecahydrate: 0.3g is settled to 1000ml, regulates PH to be 7.3 ~ 7.8.
Salbutamol artificial antigen (conjugate), BSA and salbutamol derivative UV scanning figure are shown in shown in accompanying drawing 4, salbutamol derivative maximum absorption band is at 274nm, BSA absorption peak is at 278nm, the absorption peak of conjugate 276nm place (BSA and conjugate obtain protein concentration and are 1.5mg/ml) and peak value higher than BSA, illustrate coupling success.
Embodiment 3
The application of salbutamol artificial antigen:
P11. immune animal
The salbutamol immunogen (SAL-BSA) obtained in embodiment 2 is diluted to 0.2mg/ml, and get after 500ul immunogen mixes with isopyknic Freund's complete adjuvant, emulsification is complete, immune BALB/c mouse, and at mouse back, subcutaneous and foot carries out injecting immune.First time immunity complete Freund's adjuvant, cannot be used up full freund's adjuvant afterwards.After 4th immunity, one week eye socket gets blood, separation of serum, surveys tiring of anti-salbutamol antibody.Detect through ELISA, the antibody titer after mouse four immunity is 1:436,000.
P12. cytogamy and screening
Mouse after four immunity, through abdominal injection about 100 μ g immunogen booster immunization again, after 3 days, gets this mouse spleen for merging.Get SP2/0 cell to mix with splenocyte, add serum-free medium, centrifugal (1500rpm, 3min), gets sedimentation cell, dropwise adds 1ml 50% Macrogol 4000, leaves standstill 90 seconds.Then dropwise add the serum-free medium 10ml of 37 DEG C of pre-temperature, leave standstill 5min.Cell suspension centrifugal (1000rpm, 3min) after merging, with complete culture solution, is inoculated in and is added with in 96 orifice plates of feeder layer cells, every hole 2 × 10
4/ ml myeloma cell.Put 37 DEG C, 5%CO
2cultivate two days in incubator, add the complete culture solution of 2 × HAT, make final concentration in hole be 1 × HAT.When treating that hybridoma colonies grows to 1/10-1/5 area at the bottom of hole, namely with ELISA method screening fused cell antibody positive wells.
P13. ascites preparation and antibody purification
Get BALB/c mouse, in abdominal injection 0.5ml paraffin oil, after 7 days, abdominal injection 0.5ml 1 × 10
6positive hybridoma cell.Observe mouse growth situation, about 7 days visible belly protuberances, gather ascites in time.Utilize affinity chromatography technology (Protein G Resin affinity purification) purifying to obtain highly purified monoclonal antibody, protein content is 4mg.
P14. analytic liquid preparation is detected
A. the monoclonal antibody obtained in the above-mentioned P13 of fluorescent mark respectively and anti-rabbit IgG antibody.
B. with the phosphoric acid buffer containing BSA, the antibody dilution after fluorescent mark is mixed with detection analytic liquid.
P15. the blocking of salbutamol fluorescence immune chromatography test paper is standby
A. be buffered with bag salbutamol coating antigen (SAL-OVA) that liquid (phosphoric acid buffer) just obtains in embodiment 2 respectively and rabbit IgG antibody is diluted to proper concn (0.4-3.0 mg/ml).At the temperature of 25 ± 5 DEG C, the SAL-OVA after dilution and rabbit IgG antibody are evenly sprayed on nitrocellulose membrane and (form detection line and nature controlling line respectively), under the humidity condition of 12%-30%, dry 5 ~ 8 hours, kept dry is for subsequent use;
B. the bag that gluing steps a obtains successively respectively on black PVC substrate is formed test card (Fig. 5) by good nitrocellulose filter, glass fiber paper and thieving paper, cuts into proper width as requested.Wherein 1: black PVC substrate; 2: thieving paper: 3: nitrocellulose filter; 4: glass fibre; 5: Quality Control tape (C line); 6: detect tape (T line)
C. the test card obtained by step b loads in the lower cover of cartridge, covers upper cover, forms complete band cartridge test card (Fig. 6).Wherein, 11: lower cover; 12: upper cover; 13: application of sample window; 14: glass fibre; 15: detection window; 16: Quality Control tape (C line); 17: detect tape (T line); 18: nitrocellulose filter; 19: salbutamol Item Mark (SAL); 110: bar code recognition region.
P16. detect
After getting the detection analytic liquid Homogeneous phase mixing of 100uL sample and 100uL, get the application of sample window that 100uL joins test card, after reaction 5-10min, detect, according to T line signal value and C line signal ratio (T/C value) and the Cutoff value contrast judgement yin and yang attribute result of sample FCR fluorescence immunity analyzer (being produced by SeaBird bio tech ltd, Huzhou) is upper.
P17. fluorescence immune chromatography method detects the sensitivity of salbutamol
By adding salbutamol standard substance (purchased from German DR company) in blank diaper, be configured to 0ng/ml, 0.25ng/ml, 0.5ng/ml, 1ng/ml, 2ng/ml, 4ng/ml, 8ng/ml series concentration sample.Above-mentioned series concentration sample is detected according to the step in P16, each sample repeats 3 times, the experimental result detected is as shown in table 1, according to table 1 data with SAl concentration for X-coordinate, T/C value be ordinate zou drawing standard curve (four parametric regression methods recurrence) as shown in Figure 7.The equation that in Fig. 7, curve is corresponding is as shown in table 2, calculates IC
50=0.86ng/ml.
Table 1 fluorescence immune chromatography method detects different concns clenbuterol sample
The equation that table 2 suppresses Curves corresponding
By 0ng/ml sample duplicate detection 10 times, calculate the mean value (X) of its T/C value, standard deviation (SD) and precision (CV value) respectively.The account form of sensitivity is, the SAL concentration value of T/C value corresponding in the typical curve of Fig. 7 of X-2*SD.
Table 3 fluorescence immune chromatography method duplicate detection 0ng/ml clenbuterol sample results
The T/C value of the X-2*SD in table 3 data is substituted into (equation: y=(A-D)/[1+(x/C) in equation corresponding to Fig. 6 typical curve as y value
b]+D) and the x value arrived be 0.15ng/ml, namely sensitivity is 0.15ng/ml.
P18. salbutamol fluorescence immune chromatography Test paper card Cleaning Principle
Employing competition law detects, and salbutamol antigen (coating antigen) competition binding on the salbutamol antigen in sample and detection line (T line) detects the fluorescently-labeled anti-salbutamol antibody in analytic liquid.When antigen concentration in sample is very low, the fluorescence antibody that detection line combines increases, and then fluorescent signal on detection line is just strong, and therefore, the ratio (T/C value) of detection line (T line) fluorescent signal and the upper fluorescent signal of nature controlling line (C line) is with regard to greatly; Otherwise when in sample, salbutamol antigen concentration is higher, T/C value is just very little.So salbutamol content is higher in sample, T/C value is lower.According to the yin and yang attribute of the multilevel iudge detected result of T/C value and Cut off value.
As shown in FIG. 8 and 9, be respectively salbutamol fluorescence immune chromatography method detect feminine gender and positive findings collection of illustrative plates.As seen from the figure, compare with negative sample detected result collection of illustrative plates, in positive sample detected result collection of illustrative plates, T line peak value is far smaller than the peak value of negative sample.Negative and positive sample is distinguished obviously.
P19. fluorescence immune chromatography method detects the cross reaction of salbutamol
Several frequently seen beta 2 receptor agonist is configured to respectively the sample of 30ng/ml with negative pig urine, carries out fluorescence immune chromatography detection, all present negative findings compared with normal negative urine sample and Cutoff value.Detected result is as shown in table 4:
The fluorescence immune chromatography detected result of the common beta 2 receptor agonist of table 4
Note: the T/C value of Cut off value corresponding to 1ng/ml salbutamol adds 2 times of SD scopes.That is: 0.35 ~ 0.78.
"-" indicates negative.
Claims (10)
1. a salbutamol hapten derivant, is characterized in that: described salbutamol hapten derivant is applicable to prepare salbutamol artificial antigen, and the chemical structural formula of described salbutamol hapten derivant is:
。
2. a preparation method for salbutamol hapten derivant as claimed in claim 1, is characterized in that: with the bromo-1-of compound 2-[4-hydroxyl-3-(methylol) phenyl]-ethyl-1-ketone for raw material, prepared by two-step reaction, and it is prepared path and is:
。
3. a preparation method for salbutamol hapten derivant as claimed in claim 1, is characterized in that: described reaction conditions is specially:
S1. the bromo-1-of Weigh Compound 2-[4-hydroxyl-3-(methylol) phenyl]-ethyl-1-ketone 0.015-0.0028mol, add saturated sodium methylate as solvent, add amino-4 methvl-pentanoic acid of 4-of 0.01-0.03mol under ice-water bath condition after, normal-temperature reaction 1-2h, crystallization, filtration, washing drying obtain the first white solid 4-{ [2-(4-hydroxyl-3-(methylol) phenyl)-2-oxoethyl] is amino }-4-methylvaleric acid;
S2. the white solid 0.001-0.003mol obtained in S1 is dissolved in 20ml ethanol, adds KBH4 and 0.001-0.003molZnCl2 of 0.001-0.003mol 95-98wt% successively, after normal-temperature reaction 12-18h, filter; Filtrate concentrates, and washed with isopropyl alcohol, obtains the second white solid specificity salbutamol hapten derivant.
4. the salbutamol artificial antigen prepared by salbutamol hapten derivant described in claim 1, is characterized in that: described salbutamol artificial antigen chemical structural formula is:
Described protein and carrier proteins are bovine serum albumin or ovalbumin.
5. a preparation method for salbutamol artificial antigen as claimed in claim 3, is characterized in that: described salbutamol artificial antigen utilizes carbodlimide method described salbutamol hapten derivant and carrier protein couplet to be obtained, and comprises the following steps:
P1, described salbutamol hapten derivant is dissolved in DMSO obtains derivative compound solution;
P2, in P1, first add EDC1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, then react 15-60 minute after adding NHS N-hydroxy-succinamide Homogeneous phase mixing;
P3, the reaction solution obtained in P2 is carried out linked reaction 1-5 hour in the 25-30 DEG C of solution joined containing carrier proteins, wherein said carrier proteins is bovine serum albumin or ovalbumin;
P4, P3 gained reaction solution to be dialysed, collect dialyzate and obtain salbutamol artificial antigen.
6. preparation method according to claim 4, is characterized in that: carrier proteins described in described step P3 is bovine serum albumin or ovalbumin; When described carrier proteins is bovine serum albumin, the mol ratio of derivative compound and bovine serum albumin is 45 ~ 60:1, and the artificial antigen of synthesis is salbutamol immunogen; When carrier proteins is ovalbumin, the mol ratio of derivative compound and ovalbumin is 35 ~ 50:1, and the artificial antigen of synthesis is coating antigen.
7. preparation method according to claim 5, it is characterized in that: described in described step P2, EDC and NHS is dissolved in DMF or DMSO, and the mole dosage ratio of EDC, NHS and described salbutamol hapten derivant is 1 ~ 2:1 ~ 1.5:1 ~ 2.
8. preparation method according to claim 5, is characterized in that: the damping fluid of carrier protein solution described in described step P3 is the NaHCO3 damping fluid of 0.1mol/L; Dialyzate described in P4 to be 0.01mol/L, PH be 7.2 ~ 7.9 phosphoric acid buffer.
9. the application of salbutamol artificial antigen in salbutamol fluorescence immune chromatography detection method described in a claim 4.
10. the application of salbutamol artificial antigen in salbutamol fluorescence immune chromatography detection method according to claim 9, it is characterized in that, described detection method comprises the steps:
P11. described salbutamol immunogen immune animal Dispersal risk is utilized;
P12. cytogamy and screening;
P13. ascites preparation and antibody purification;
P14. by the antibody obtained in step P 13 with anti-rabbit IgG bis-is anti-carries out fluorochrome label respectively, and detection analytic liquid is mixed with;
P15. described salbutamol coating antigen and the rabbit igg antibody rear and front end be sprayed at respectively on nitrocellulose filter bar are prepared detection line and nature controlling line, wherein near thieving paper end be front end, near sample pad be rear end;
P16. the nitrocellulose filter containing detection line and nature controlling line described in glass fiber sample pad, step P15, thieving paper and PVC black floor are assembled into immuno-chromatographic test paper strip;
P17. the cutting of test strip described in step P16 proper width is loaded in plastics cartridges, and detect analytic liquid described in step P14 and support the use and detect on fluorescence immune chromatography detector.
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Cited By (4)
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|---|---|---|---|---|
| CN107860724A (en) * | 2017-11-09 | 2018-03-30 | 陈军 | A kind of salbutamol detection method based on optical biosensor |
| CN107868066A (en) * | 2017-07-07 | 2018-04-03 | 中国农业科学院北京畜牧兽医研究所 | A kind of preparation method of chaff propylhomoser and its salt |
| CN109678707A (en) * | 2019-01-22 | 2019-04-26 | 东莞暨南大学研究院 | A kind of deuterium-labeled D3The synthetic method of salbutamol |
| CN110045105A (en) * | 2018-11-09 | 2019-07-23 | 广州市妇女儿童医疗中心 | Coxsack A16 virus IgA antibody quantum dot immune fluorescent chromatograph test strip and kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102617516A (en) * | 2012-02-23 | 2012-08-01 | 中国农业大学 | Albuterol artificial antigens and antibodies prepared by same |
| CN103145831A (en) * | 2013-03-28 | 2013-06-12 | 江南大学 | Synthesis method of specific salbutamol artificial antigen |
| CN103755802A (en) * | 2014-01-16 | 2014-04-30 | 江南大学 | Synthesis method of structurally specific salbutamol complete antigen |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102617516A (en) * | 2012-02-23 | 2012-08-01 | 中国农业大学 | Albuterol artificial antigens and antibodies prepared by same |
| CN103145831A (en) * | 2013-03-28 | 2013-06-12 | 江南大学 | Synthesis method of specific salbutamol artificial antigen |
| CN103755802A (en) * | 2014-01-16 | 2014-04-30 | 江南大学 | Synthesis method of structurally specific salbutamol complete antigen |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107868066A (en) * | 2017-07-07 | 2018-04-03 | 中国农业科学院北京畜牧兽医研究所 | A kind of preparation method of chaff propylhomoser and its salt |
| CN107868066B (en) * | 2017-07-07 | 2019-11-12 | 中国农业科学院北京畜牧兽医研究所 | A kind of preparation method of chaff propylhomoser and its salt |
| CN107860724A (en) * | 2017-11-09 | 2018-03-30 | 陈军 | A kind of salbutamol detection method based on optical biosensor |
| CN110045105A (en) * | 2018-11-09 | 2019-07-23 | 广州市妇女儿童医疗中心 | Coxsack A16 virus IgA antibody quantum dot immune fluorescent chromatograph test strip and kit |
| CN110045105B (en) * | 2018-11-09 | 2022-04-26 | 广州市妇女儿童医疗中心 | Coxsackie A16 virus IgA antibody quantum dot immunofluorescence chromatography test strip and kit |
| CN109678707A (en) * | 2019-01-22 | 2019-04-26 | 东莞暨南大学研究院 | A kind of deuterium-labeled D3The synthetic method of salbutamol |
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| CN104610079B (en) | 2016-05-25 |
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