CN101987836A - Chlorpromazine hapten, artificial antigen, antibody as well as preparation method and application thereof - Google Patents
Chlorpromazine hapten, artificial antigen, antibody as well as preparation method and application thereof Download PDFInfo
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- CN101987836A CN101987836A CN 201010268994 CN201010268994A CN101987836A CN 101987836 A CN101987836 A CN 101987836A CN 201010268994 CN201010268994 CN 201010268994 CN 201010268994 A CN201010268994 A CN 201010268994A CN 101987836 A CN101987836 A CN 101987836A
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- ZPEIMTDSQAKGNT-UHFFFAOYSA-N chlorpromazine Chemical compound C1=C(Cl)C=C2N(CCCN(C)C)C3=CC=CC=C3SC2=C1 ZPEIMTDSQAKGNT-UHFFFAOYSA-N 0.000 title claims abstract description 101
- 229960001076 chlorpromazine Drugs 0.000 title claims abstract description 101
- 239000000427 antigen Substances 0.000 title claims abstract description 35
- 102000036639 antigens Human genes 0.000 title claims abstract description 35
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Abstract
The invention provides a chlorpromazine hapten, an artificial antigen and an antibody as well as a preparation method and application thereof. The chlorpromazine artificial hapten has a molecular structure (I); the chlorpromazine artificial antigen has molecular structures (II) or (III); the antiserum obtained by the invention has the titer of 1:3*105, the lowest detection limit of 3ng/mL and the half-inhibition concentration of 29ng/mL; and the generated antibody has high specificity, sensitivity and accuracy. The antigen and the antibody have wide application prospect in the detection of residual chlorpromazine in the food.
Description
Technical field
The invention belongs to the food safety technical field.Be specifically related to the preparation method and the application of chlorpromazine haptens, artificial antigen and antibody.
Background technology
(Chlorpromazine CPZ) claims chlorpromazine again to chlorpromazine, and molecular formula is: C
17H
19C
1N
2S, the medicinal hydrochloride form that is generally, chemical name is: N, N-dimethyl-2-chloro-10H-thiodiphenylamine-10-propylamin hydrochloride.Belong to one of phenothiazines medicine, be the blocker of central dopaminergic receptor, have calmness, antipsychotic, town tell, reduce body temperature and basal metabolism,
-adrenergic receptor and the blocking-up of m-cholinergic receptor, antihistaminic, influence effects such as internal secretion.Be used to control symptoms such as psychotic restless, nervous, the illusion of schizophrenia or other, vain hope clinically; The vomiting that the treatment a variety of causes causes; Also be used for hypothermic anesthesia and artificial hibernation; Share treatment cancer of late stage patient's severe pain with anodyne.
At present, because calmness, the hypnosis its special biological effects of chlorpromazine are often used as fodder additives.But chlorpromazine has anaphylaxiss such as oligoleukocythemia, postural hypotension, hepatic insufficiency, fash, contact dermatitis to human body.Tremble, cathisophobia, tardive dyskinesia such as hydrostomia, to the toxic side effect of human body.
European Union has just issued ban (EC) NO.17/97 that chlorpromazine forbids adding in feed as far back as 1997.China Ministry of Agriculture, the Ministry of Health, National Drug Administration unite issue " forbid use types of drugs catalogue " the 176th, No. 235 bulletins of the Ministry of Agriculture (2002) and also spell out in feed and animal drinking water: forbid chlorpromazine to be added in animal-feed and drinking-water, allow therapeutic dose, but in animal food, must not detect.The immune analysis technology is widely used at the drug residue detection range with its sensitivity, special, quick, easy advantage, compared with the physical and chemical inspection method a lot of advantages is arranged.
At present, the physical and chemical inspection method is mainly adopted in residual detection to chlorpromazine in the aquatic animal.Yet the existence of physical and chemical inspection method is complicated, loaded down with trivial details, the detection flux is little, need professional and defectives such as professional skill, detection cost costliness, can not realize the rapid detection analysis of batch samples.Therefore, be necessary to develop more simple and fast detection method easily.
The immune analysis technology is widely used at the drug residue detection range with its sensitivity, special, quick, easy advantage, artificial antigen synthetic be haptens material immune analysis method set up key, for chlorpromazine, because haptens design and synthetic reason, the specificity and the insight of the chlorpromazine antibody that prior art obtains are not enough, lack the correlation technique report of the immunological analysis method of chlorpromazine.
Summary of the invention
An object of the present invention is to fill up the deficiency of the existing detection technique of chlorpromazine, a kind of chlorpromazine artificial semiantigen, artificial antigen and specific antibody are provided.
Another object of the present invention provides the preparation method of described chlorpromazine artificial semiantigen, artificial antigen and specific antibody.
A further object of the invention provides the application of described chlorpromazine artificial semiantigen, artificial antigen and specific antibody.
Purpose of the present invention is achieved by the following technical programs:
A kind of chlorpromazine artificial semiantigen, artificial antigen and specific antibody are provided, and described chlorpromazine artificial semiantigen has molecular structure shown in the formula (I):
Described chlorpromazine artificial antigen, have formula (II) or (III) shown in molecular structure:
The invention provides the haptenic preparation method of described chlorpromazine, be that chlorpromazine is dissolved in diacetyl oxide, splash into the acetyl nitrate of prepared fresh under the ice bath, carry out nitrated-oxidizing reaction, and logical reaction system bar soda acid crystallization method is obtained nitration oxidation product 2-oxygen-10-(3-(dimethylamino-propyl))-7-nitro-5-oxygen-10H-phenothiazine, adopt the reduction of palladium carbon catalytic hydrogenation then, the column chromatography of going forward side by side separation obtains described chlorpromazine haptens (I), i.e. 2-chloro-10-(3-(dimethylamino-propyl))-7-amino-5-oxygen-10H-phenothiazine.
Aforesaid method is realized by following steps:
(1) be chlorpromazine, acetyl nitrate to be dissolved in diacetyl oxide respectively in 1: 2 by molar ratio;
(2) acetyl nitrate solution is slowly splashed in the chlorpromazine solution, the ice bath reaction was regulated pH value to 8 with product with the saturated sodium hydroxide solution of ice after 1 hour, and crystallization obtains 2-chloro-10-(3-(dimethylamino-propyl))-7-nitro-5-oxygen-10H-phenothiazine;
(3) getting step (2) prepares 2-chloro-10-(3-(dimethylamino-propyl))-7-nitro-5-oxygen-10H-phenothiazine and is dissolved in the dehydrated alcohol, add and to be equivalent to the palladium-carbon catalyst that institute adds 2-chloro-10-(3-(dimethylamino-propyl))-7-nitro-5-oxygen-10H-phenothiazine 10% (calculation by mass percentage), feed hydrogen then continuously and reduce, further obtain target haptens (I) by the column chromatography purifying.
The antigenic preparation of chlorpromazine of the present invention has following two kinds of technical schemes:
Method one: is that mixing in 100: 1 is dissolved in 10mLPBS (phosphate buffered saline buffer) solution with described chlorpromazine haptens and carrier protein by feed ratio, dropwise add the volume by volume concentration be equal to the haptens molar weight and be 25% glutaraldehyde solution, the stirring at room reaction obtains described antigen.
Method two: described chlorpromazine haptens is dissolved in the hydrochloric acid (1mL 1mol/L), the sodium nitrite solution that dropwise adds 1.1mL 1mol/L, dropwise join after the diazotization reaction in carrier proteins-PBS solution, regulator solution pH value to 7.5,4 ℃ of back dialysis of spending the night obtain the purpose product ,-20 ℃ of preservations after the packing.
Described carrier protein can be bovine serum albumin white matter (BSA), human serum protein (HSA), hemocyanin (KLH) or ovalbumin (OVA), preferred bovine serum albumin (BSA).
The present invention provides described chlorpromazine monoclonal antibody specific and polyclonal antibody simultaneously, with described chlorpromazine antigen and antibody in the application aspect the chlorpromazine immunodetection.
The invention has the beneficial effects as follows:
The present invention to be having kept the space structure of chlorpromazine to greatest extent, redesign and synthetic haptens and antigen, compared with prior art, antigen of the present invention more high-quality, antibody efficiently behind animal immune.
A large amount of secular experiment showed, the antiserum titres that adopt antigen-immunized animal of the present invention to obtain can reach 1: 3 * 10
5Lowest detection is limited to 3ng/mL, 503nhibiting concentration is 29ng/mL, state antibody and have specificity height, highly sensitive, remarkable superiority that accuracy is high, therefore, antigen and antibody that invention provides can be used for setting up chlorpromazine competitive enzyme-linked immune adsorption analysis art, thereby the chlorpromazine that is used for rapid detection food is residual, has broad application prospects.
Description of drawings
The haptenic synthetic route chart of Fig. 1 chlorpromazine
Fig. 2 chlorpromazine haptens mass spectrum
Fig. 3 chlorpromazine haptens mass spectrum
Fig. 4 chlorpromazine ELISA competition test graphic representation
Embodiment
Further describe the present invention below in conjunction with the drawings and specific embodiments.The employed test method of following embodiment is ordinary method if no special instructions; Employed material, agent etc. if no special instructions, are the reagent and the material that can obtain from commercial channels.
Execute example 1: the synthetic and evaluation of haptens
, chlorpromazine is haptenic synthetic
The haptenic synthetic route of chlorpromazine as shown in Figure 1.
(1) take by weighing 319mg (1mmol) chlorpromazine and be dissolved in the 5mL diacetyl oxide, stirring and dissolving is abundant in the ice bath;
(2) acetyl nitrate (preparation method is with reference to the routine) 2mmol that gets prepared fresh is dissolved in the 1mL diacetyl oxide and slowly splashes in the described solution of step (1), ice bath reaction 1 hour, after reaction finishes product is regulated pH value to 8 with the saturated sodium hydroxide solution of ice, crystallization obtains 2-chloro-10-(3-(dimethylamino-propyl))-7-nitro-5-oxygen-10H-phenothiazine, and productive rate is 61%;
(3) getting above-mentioned 2-chloro-10-(3-(dimethylamino-propyl))-7-nitro-5-oxygen-10H-phenothiazine is dissolved in the 20mL ethanol, add and to be equivalent to the palladium-carbon catalyst that institute adds 2-chloro-10-(3-(dimethylamino-propyl))-7-nitro-5-oxygen-10H-phenothiazine 10% (according to mass percent calculating), and feed hydrogen reducing continuously and obtain target haptens (I) through the chromatographic column separation and purification.
Two, the haptenic evaluation of chlorpromazine
Get the above-mentioned purpose product and identify that through the ESI mass spectrum as seen its molecular ion peak is 350 (M+1), sees shown in accompanying drawing 2 and the accompanying drawing 3.The results of elemental analyses of hapten compound (%): C, 58.36; H, 5.76; N, 12.01; S, 9.16, element ratio is consistent with haptens (I) structure, illustrates that the chlorpromazine haptens synthesizes successfully, and the molecular structural formula of purpose product is suc as formula shown in (I):
Embodiment 2: the synthetic and evaluation of artificial antigen
One, the synthetic and evaluation of chlorpromazine antigen (III)
Chlorpromazine antigen (III) glutaraldehyde method is synthetic:
(1) take by weighing 35mg chlorpromazine haptens and 68mg BSA and be dissolved in together in 10mL phosphate buffered saline buffer (PBS) solution, stirring at room is even;
(2)) 40 μ L glutaraldehyde are dropwise splashed in the described mixing solutions of step (1), dropwise back stirring at room reaction 3 hours;
(3) will be loaded on dialysis tubing through step (2) reacted solution, 4 ℃ with PBS solution dialysis 72 hours, during change dialyzate 6 times, promptly obtain the purpose product, after the packing in-20 ℃ of preservations.
Also can adopt human serum protein, hemocyanin or ovalbumin in the aforesaid method, the preparation method is the same with reference to present embodiment method chlorpromazine envelope antigen, just used carrier albumen difference.
The evaluation of chlorpromazine antigen (III):
Get chlorpromazine haptens, carrier proteins BSA and chlorpromazine antigen and carry out ultraviolet (200nm-400nm) scanning evaluation respectively, the highest light absorption value that compares the three, identify whether haptens and carrier proteins coupling takes place, find that the antigenic light absorption value of chlorpromazine is obviously different with chlorpromazine haptens and BSA, illustrate that haptens makes chlorpromazine antigen with BSA success coupling.As calculated, the chlorpromazine haptens is 24: 1 with the mol ratio that combines of BSA.
Antigenic preparation of embodiment 5 chlorpromazine and evaluation
Two, the synthetic and evaluation of chlorpromazine antigen (II)
The diazotization method of chlorpromazine antigen (II) is synthetic:
(1) the chlorpromazine haptens that takes by weighing 35mg is dissolved in the hydrochloric acid of 1mL 1mol/L, is 2 with the hydrochloric acid conditioning solution pH value of 1mol/L;
(2) get 1.1mL 1mol/L sodium nitrite solution dropwise join in half an hour in the haptens solution, 0～5 ℃ of diazotization reaction 1 hour;
(3) take by weighing 68mgBSA and be dissolved in 10mLPBS (the conventional phosphate buffered saline buffer that uses in the laboratory) solution, step (2) reacted solution is dropwise joined in the BSA-PBS solution, with the sodium hydroxide solution regulator solution pH value to 7.5 of 1mol/L, 4 ℃ are spent the night;
(4) will be loaded on dialysis tubing through step (3) reacted solution, 4 ℃ with PBS solution dialysis 72 hours, during change dialyzate 6 times, promptly obtain the purpose product ,-20 ℃ of preservations after the packing.
The evaluation of chlorpromazine antigen (II):
Get chlorpromazine haptens, carrier proteins BSA and chlorpromazine antigen and carry out ultraviolet (200nm～400nm) scanning evaluation respectively, and obviously different by the antigenic light absorption value of the highest light absorption value discovery chlorpromazine that compares the three with chlorpromazine haptens and BSA, illustrate that haptens makes chlorpromazine antigen with BSA success coupling.As calculated, the chlorpromazine haptens is 15: 1 with the mol ratio that combines of BSA.
Embodiment 3: mono-clonal, polyclonal antibody or the preparation of chlorpromazine genetic engineering antibody
(1) MONOCLONAL ANTIBODIES SPECIFIC FOR
Animal immune: haptens and carrier protein couplet thing with the carboxyl spacerarm that contains 2 carbon are that immunogen is carried out the interval immunity to the Balb/c mouse, and immunoassay detects and obtain containing in the blood mouse spleen of chlorpromazine specific antibody indirectly.
Cytogamy and clone: get the Balb/c mouse boosting cell and the myeloma cell SP20 that produce specific antibody and merge, adopt indirect competition temporal resolution immune analysis method to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay or microclone method that cloning is carried out in positive hole, obtain and set up the hybridoma cell strain that produces monoclonal antibody.
Cell cryopreservation and recovery: get the hybridoma that is in logarithmic phase and make cell suspension, be sub-packed in frozen pipe, in the medium-term and long-term preservation of liquid nitrogen with frozen storing liquid.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal frozen storing liquid, move in the culturing bottle and cultivate.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying: adopt in the body and induce method, Balb/c mouse (8 age in week) abdominal cavity is injected the sterilization paraffin oil, 7～14 days pneumoretroperitoneum injection hybridomas were gathered ascites after 7～10 days.Carry out the ascites purifying through sad-saturated sulfuric acid amine method or affinity chromatography, purity is identified through the SDS-PAGE electrophoresis, bottle packing ,-20 ℃ of preservations.
(2) preparation of chlorpromazine rabbit polyclonal antibody
Adopting new zealand white rabbit as immune animal, is that immunogen is carried out immunity to new zealand white rabbit with chlorpromazine and carrier protein couplet thing, and repeatedly serum antibody titer is measured in the immunity back, and heart is taken a blood sample, and obtains the polyclonal antibody of purifying through sulfuric acid amine fractionation precipitation.
(3) preparation of chlorpromazine genetic engineering antibody
Genetic engineering antibody mainly refers to small molecular antibody, comprising: Fab (being made of complete light chain and Fd), Fv is (by V
HAnd V
LConstitute), ScFv (single-chain antibody, V
HAnd V
LBetween be formed by connecting by a connection peptides), single domain antibody is (only by V
HForm) etc. through the improved antibody of genetic engineering technique.
The preparation method is: the RNA that extracts chlorpromazine monoclonal cell or the mouse boosting cell after the chlorpromazine immunogen immune, reverse transcription is cDNA, designerantibodies weight chain amplimer, utilize round pcr to amplify the weight chain gene of antibody, insert suitable expression plasmid (TG1 bacterial strain, commercial), at expression in escherichia coli, utilize immune affine method to carry out purifying, purity is identified by the SDS-PAGE electrophoresis.Concrete steps are as follows:
V wherein
H(Back), V
H(For) two primers antibody heavy chain variable region gene V that is used to increase
HV
L(Back), V
L(For) two primers antibody chain variable region gene V that is used to increase
LTwo primers of Linker1, Linker2 are used for overlapping extension PCR method splicing V as the joint primer
H, V
LGene fragment becomes the scFv gene fragment, and this joint primer includes coding connection peptides (Gly
4Ser)
3Gene fragment, 5 ' end and V
H3 ' the complementation of fragment end, 3 ' end and V
LFragment 5 ' end is complementary.RS (Back), two primers of RS (For) are used for secondary PCR amplification total length scFv gene fragment, introduce Bgl I and NotI restriction enzyme site simultaneously.Two primers of R1, R2 are primer on the carrier, are used for carrier and insert segmental PCR evaluation.
V
H(Back) 5?
-CAG?GTS?MAR?CTG?CAG?SAG?TCW?GG-3?
V
H(For) 5?
-TGA?GGA?GAC?GGT?GAC?CGT?GGT?GCC-3?
V
L(Back) 5?
-GAC?ATC?GAG?CTC?ACT?CAG?TCT?CCA-3?
V
L(For) 5?
-CCG?TTT?TAT?TTC?CAG?CCT?GGT?CCC-3?
GGC?GGT?TCA?GGC?GGA?GGT?GGC?TCT?GG-3?
ACC?GCC?AGA?GCC?ACC?TCC?GCC-3?
RS(Back) 5?
-GTC?CTC?GCA?ACT?GCG?
GCC?CAG?CCG?GCC?ATG
(containing Bgl I site) GCC CAG GTC AAA CTG CAG GAG TCA GG-3
RS(For) 5?
-GAG?TCA?TTC?T
GC?GGC?CGC?CCG?TTT?TAT?TTC
(containing Not I site) CAG CCT GGT CCC-3
V
LAmplification condition: reaction soln vortex mixing, of short duration centrifugal after, carry out pcr amplification reaction, reaction process and condition are: 94 ℃ * 5min sex change, add 1 μ L high-fidelity Pfu enzyme (2.5U), and carry out following circulation: 94 ℃ * 30s, 54 ℃ * 1min, 72 ℃ * 1min, totally 25 circulations, last 72 ℃ are extended 10min.After reaction finished, the VL reaction product was got 5 μ L and is carried out the detection of 1% agarose gel electrophoresis.
V
HAmplification condition: reaction soln vortex mixing, of short duration centrifugal after, carry out pcr amplification reaction, reaction process and condition are: 94 ℃ * 5min sex change, add 1 μ L high-fidelity Pfu enzyme (2.5U), and carry out following circulation: 94 ℃ * 30s, 60 ℃ * 1min, 72 ℃ * 1min, totally 30 circulations, last 72 ℃ are extended 10min.After reaction finishes, V
HReaction product is got 5 μ L and is carried out the detection of 1% agarose gel electrophoresis.
(1) splicing of scFv gene fragment and pcr amplification:
Overlapping extension
At first carry out the pre-splicing of linker by following reaction system:
Linker1 primer (10 μ mol/L) 0.5 μ L
Linker2 primer (10 μ mol/L) 0.5 μ L
10×PCR?Buffer 5μL
dNTP(2.5mmol/L) 4μL
Deionized water 34.5 μ L
Cumulative volume 44.5 μ L
Reaction soln vortex mixing, of short duration centrifugal.The pre-sex change of 94 ℃ * 5min on the PCR instrument adds 0.5 μ L high-fidelity Pfu enzyme, carries out following circulation: 94 ℃ * 45s, and 50 ℃ * 1min, 72 ℃ * 1min, totally 3 circulations.
Add V in the system in the above
H, V
LCarry out the splicing of scFv:
V
HPurified product (50ng) 3 μ L
V
LPurified product (50ng) 2.5 μ L
Linker splices solution 44.5 μ L in advance
Cumulative volume 50 μ L
Successively with V
H, V
LAfter the adding, with rifle mixing gently.0.5 μ L high-fidelity Pfu enzyme is added in the pre-sex change of 94 ℃ * 5min on the PCR instrument, carries out following circulation: 94 ℃ * 45s, and 50 ℃ * 1min, 72 ℃ * 1min, totally 30 circulations, 72 ℃ are extended 10min.
(2) pcr amplification of scFv gene
Adopt following reaction system:
5×PCR?Buffer 10μL
dNTP(2.5mmol/L) 4μL
RS (Back) primer (10 μ mol/L) 1 μ L
RS (For) primer (10 μ mol/L) 1 μ L
Sterile pure water 30 μ L
Cumulative volume 50 μ L
Mix, of short duration centrifugal after, on the PCR instrument, the pre-sex change of 94 ℃ * 2min adds 0.5 μ L high-fidelity Pfu enzyme, carries out following circulation: 94 ℃ * 45s, 60 ℃ * 1min, 72 ℃ * 1min, totally 30 circulations, last 72 ℃ are extended 10min.Get 5 μ L reaction product and carry out electrophoresis detection.
Embodiment 4: the chlorpromazine enzyme-linked immunoassay method is set up
Adopt aforementioned polyvalent antibody, determine the working concentration of antigen (carrier proteins is an ovalbumin, and the preparation method is with reference to embodiment 2) and chlorpromazine antibody by the square formation volumetry, antigenic working concentration is 1 μ g/mL, and the working concentration of chlorpromazine antibody is 1/30000.
Chlorpromazine standard solution with different concns is done experimental solutions, and its concentration is as follows: 0ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1 μ g/mL, adopt 8 groups of parallel laboratory tests (n=8).
Indirect competitive ELISA method: with the coated elisa plate of above-mentioned working concentration, experimental solutions and antibody-solutions are joined in the enzyme plate aperture simultaneously, blank well and negative control hole are set simultaneously, 37 ℃ of incubation 40min, pour out liquid in the hole, wash 6 times with washings, enzyme plate is upside down on the thieving paper pats: add the good ELIAS secondary antibody solution of dilution, 37 ℃ of incubation 20min, with washings washing 6 times, pat dry: add the substrate chromophoric solution, 37 ℃ of incubation 10min, add the stop buffer color development stopping, measuring light absorption value A at wavelength 450nm place with microplate reader, is ordinate zou with light absorption value A, with the log of chlorpromazine experimental solutions concentration
10Value is X-coordinate, draws the semilog canonical plotting, sees the competition test of chlorpromazine ELISA shown in the accompanying drawing 4 curve.The result shows that typical curve has complete anti-S shape, and upper mounting plate and lower platform are arranged, the replicate(determination) number of times of typical curve 8 times, and experimental repeatability is good, and relative standard deviation is in 15%.
Calculate the lowest detectable limit (LOD value) of chlorpromazine antibody in buffered soln by formula and be 3ng/mL, half amount of suppression (IC
50Value) be 28.83ng/mL.
Embodiment 5: chlorpromazine enzyme-linked immunoassay method actual sample detects
(1) detection of flesh of fish sample
Take by weighing the good flesh of fish sample of 1.0g homogeneous, put into the 15mL centrifuge tube, the extracting solution A (the 0.1mol/L HCl that the 80mL acetonitrile adds 20mL mixes as extracting solution) that adds 4mL, the centrifugal 10min of room temperature behind the mixing, get the 2ml supernatant and add 4mL1mol/L NaOH again, the cyclohexane that adds 10mL again, vortex 1 minute; Centrifugal 5 minutes of 3000rpm gets upper solution 5mL and joins the 50mL round-bottomed flask, removes solvent under reduced pressure, detects after redissolving with 1mL PBST.The results are shown in Table 1
Table 1 (Luo Fei) fish sample detection result (n=3)
(2) detection of water sample
Get fish pond water, with filter paper filtering fish pond water, get 50 μ L after centrifugal and use haptens of the present invention, artificial antigen and antibody and make elisa assay, analytical procedure is with reference to routine.Experimental result is shown in Table 2.
Table 2 fish pond water sample detected result (n=3)
By table 1 and table 2 result as seen, the rate of recovery that detects through test kit after the pre-treatment of fish pond water sample and tissue sample is between 79%～96%.
Claims (10)
1. chlorpromazine haptens, the molecular structure shown in (I) that it is characterized in that having formula:
2. the preparation method of the described chlorpromazine artificial semiantigen of claim 1, it is characterized in that it being that chlorpromazine is dissolved in diacetyl oxide, splash into the acetyl nitrate of prepared fresh under the ice bath, reaction obtains chlorpromazine haptens intermediate 2-chloro-10-(3-(dimethylamino-propyl))-7-nitro-5-oxygen-10H-phenothiazine, and then adopts the reduction of palladium carbon catalytic hydrogenation method to obtain described chlorpromazine haptens (I).
3. preparation method according to claim 2 is characterized in that may further comprise the steps:
(1) chlorpromazine, acetyl nitrate are dissolved in diacetyl oxide respectively;
(2) acetyl nitrate solution is slowly splashed in the chlorpromazine solution, the ice bath reaction, after reaction finishes product is regulated pH value to 8 with the saturated sodium hydroxide solution of ice, crystallization gets chlorpromazine haptens intermediate 2-chloro-10-(3-(dimethylamino-propyl))-7-nitro-5-oxygen-10H-phenothiazine;
(3) get 2-chloro-10-(3-(dimethylamino-propyl))-7-nitro-5-oxygen-10H-phenothiazine that step (2) prepares and be dissolved in the dehydrated alcohol, add palladium-carbon catalyst, feed hydrogen reducing continuously, separate obtaining the target haptens by column chromatography.
4. chlorpromazine artificial antigen, it is characterized in that having formula (II) or (III) shown in molecular structure:
5. the preparation method of the described chlorpromazine artificial antigen of claim 4 is characterized in that it being to adopt glutaraldehyde method or diazotization method that described chlorpromazine haptens of claim 1 and protein coupling are prepared.
6. preparation method according to claim 5 is characterized in that may further comprise the steps:
(1) takes by weighing described chlorpromazine haptens and carrier proteins and be dissolved in together in the PBS solution, stir;
(2) glutaraldehyde is dropwise splashed in the described mixing solutions of step (1), dropwise back stirring at room reaction;
(3) will obtain the purpose product through step (2) reacted solution dialysis, after the packing in-20 ℃ of preservations;
Perhaps may further comprise the steps:
(1) takes by weighing described chlorpromazine haptens and be dissolved in the hydrochloric acid, regulator solution pH value;
(2) get sodium nitrite solution and dropwise join in step (1) the chlorpromazine haptens solution diazotization reaction;
(3) carrier proteins is dissolved in the PBS solution, the solution after step (2) diazotization reaction is dropwise joined in the carrier proteins solution, regulator solution pH value is spent the night;
(4) will obtain the purpose product ,-20 ℃ of preservations after the packing through the dialysis of step (3) reacted solution.
7. according to claim 5 or 6 described preparation methods, it is characterized in that described carrier proteins is bovine serum albumin white matter, human serum protein, hemocyanin or ovalbumin, preferred bovine serum albumin.
8. the described artificial antigen of claim 4 is at preparation chlorpromazine antibody or the application aspect in the chlorine detection promazine.
9. chlorpromazine monoclonal antibody, polyclonal antibody or genetic engineering antibody that adopts the described artificial antigen of claim 4 to prepare.
10. the application of the described antibody of claim 9 in the chlorine detection promazine.
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| CN103018451A (en) * | 2011-09-20 | 2013-04-03 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for chloramphenicol detection, and applications thereof |
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| CN101412697A (en) * | 2008-11-21 | 2009-04-22 | 华南农业大学 | Preparation of 1-amino hydantoin derivative hapten, antigen and antibody |
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| CN103018451A (en) * | 2011-09-20 | 2013-04-03 | 北京勤邦生物技术有限公司 | Enzyme-linked immunoassay kit for chloramphenicol detection, and applications thereof |
| CN103018451B (en) * | 2011-09-20 | 2016-04-20 | 北京勤邦生物技术有限公司 | The enzyme linked immunological kit of chlorine detection mycin and application thereof |
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| CN104530239A (en) * | 2014-12-26 | 2015-04-22 | 华中农业大学 | Monoclonal antibody and enzyme linked immunosorbent assay and kit for detecting promethazine drugs |
| CN104530239B (en) * | 2014-12-26 | 2017-12-05 | 华中农业大学 | For detecting the monoclonal antibody and enzyme-linked immunoassay method and kit of phenothiazines |
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| CN107607721A (en) * | 2017-09-16 | 2018-01-19 | 北京勤邦生物技术有限公司 | A kind of magnetic immunochemiluminescence detection kit of chlorpromazine and its application |
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| CN113372295A (en) * | 2021-07-08 | 2021-09-10 | 河北大学 | Phenothiazine hapten, complete antigen, preparation method and application thereof |
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