CN104109112B - Cyproheptadine semiantigen, artificial antigen, antibody, preparation methods of cyproheptadine semiantigen and artificial antigen, and application of artificial antigen and antibody - Google Patents
Cyproheptadine semiantigen, artificial antigen, antibody, preparation methods of cyproheptadine semiantigen and artificial antigen, and application of artificial antigen and antibody Download PDFInfo
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- CN104109112B CN104109112B CN201310138339.3A CN201310138339A CN104109112B CN 104109112 B CN104109112 B CN 104109112B CN 201310138339 A CN201310138339 A CN 201310138339A CN 104109112 B CN104109112 B CN 104109112B
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Classifications
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- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/68—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
- C07D211/70—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- Toxicology (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a cyproheptadine semiantigen, an artificial antigen, an antibody, preparation methods of cyproheptadine semiantigen and the artificial antigen, and application of the artificial antigen and the antibody. The cyproheptadine semiantigen has a molecular structure shown as a formula (I), and the cyproheptadine artificial antigen has a molecular structure shown as a formula (II). The titer of a monoclonal antibody obtained from the antigen reaches 1:300000, and the 50% inhibiting concentration is 0.20 mu g/L. The generated antibody is high in specificity, high in sensitivity and high in accuracy. The antigen and the antibody have wide application prospect on detection of cyproheptadine residue in food.
Description
Technical field
The invention belongs to technical field of food safety is and in particular to the preparation of Cyproheptadine hapten, artificial antigen and antibody
Method and application.
Background technology
Cyproheptadine（Cyproheptadine, molecular formula：C21H21N）, also known as cyproheptadine, it is a kind of antihistaminic, tool
There is anti-5-HT, block H1Receptor and cholinolytic effect, be mainly used in treat urticaria, eczema, contact dermatitis, skin pruritus,
The anaphylactic diseases such as allergic rhinitises are it can also be used to improve the appetite of patient.
Cyproheptadine is a kind of people's prescription drugss, but finds to add in current some at home feed products, and analysis reason can
Can be to reach the purpose of weightening by promoting animal appetite.The edible animal derived product containing Cyproheptadine residual, to human body
Health has certain hazardness, and the effect to child becomes apparent from, and high concentration can directly cause stupor, rapid breathing, whole body
The poisoning symptom such as powerless, severe patient then directly results in death of child.Therefore, the requirement according to european union directive 2001/82, Cyproheptadine
Forbid using in animal as veterinary drug；The Ministry of Agriculture of China also issues No. 1519 bulletins on December 27th, 2010, forbids raising
Using Cyproheptadine in material and drinking water for animals.
Research to Cyproheptadine is concentrated mainly on field of medicaments at present, the examination criteria method of Cyproheptadine only in feedstuff,
And in animals urine and animal tissue residual corresponding method of detection research report less, and mostly instrument detection, exist complexity,
Loaded down with trivial details, detection flux little, need the defect such as professional and professional skill, testing cost costliness it is impossible to realize batch samples
Quick detection analysis.Therefore, it is necessary to develop simpler fast and easily detection method.
Immunological assay techniques are extensive in drug residue detection field with its sensitive, special, quick, easy advantage
Application, the synthesis of artificial antigen is the key that hapten material immune analysis method is set up, for Cyproheptadine, because hapten sets
The reason meter and synthesis, the specificity of Cyproheptadine antibody that prior art obtains and discernment not, lack the immunity of Cyproheptadine
Learn the correlation technique report of analysis method.
Content of the invention
It is an object of the invention to filling up the deficiency of the existing detection technique of Cyproheptadine, provide a kind of Cyproheptadine hapten,
Artificial antigen and specific antibody.
It is a further object to provide the preparation side of described Cyproheptadine hapten, artificial antigen and specific antibody
Method.
It is a still further object of the present invention to provide the application of described Cyproheptadine hapten, artificial antigen and specific antibody.
The purpose of the present invention is achieved by the following technical programs：
There is provided a kind of Cyproheptadine hapten, artificial antigen and specific antibody, described Cyproheptadine hapten has formula（Ⅰ）Institute
Show molecular structure：
Described Cyproheptadine artificial antigen, has formula（Ⅱ）Shown molecular structure：
The invention provides the haptenic preparation method of described Cyproheptadine, it is that demethyl Cyproheptadine and pyridine are codissolved in two
Methyl sulfoxide, instills bromo-acetic acid tert-butyl and is reacted at 40 DEG C, and by solvent is evaporated off, column chromatography for separation obtain Cyproheptadine half
Antigen intermediate；Then with formic acid in room temperature reaction, and by solvent being evaporated off, being recrystallized to give described match in ethanol-water system
Pyridine in heptan hapten（Ⅰ）, i.e. carboxyl Cyproheptadine.
Said method is realized by following steps：
（1）Demethyl Cyproheptadine and pyridine are codissolved in dimethyl sulfoxide, bromo-acetic acid tert-butyl is dissolved in dimethyl sulfoxide；
（2）At 40 DEG C, bromo-acetic acid tert-butyl solution is slowly dropped in demethyl Cyproheptadine solution, after continuing reaction 4 hours
Solvent is evaporated off, Cyproheptadine hapten intermediate is obtained by column chromatography for separation；
（3）In step（2）Dimethyl sulfoxide and formic acid is added, room temperature is anti-in the Cyproheptadine hapten intermediate preparing
Solvent is evaporated off after answering 20 hours, in ethanol-water system, is recrystallized to give target hapten（Ⅰ）.
The preparation of Cyproheptadine artificial antigen of the present invention has following technical scheme：
Described Cyproheptadine hapten is dissolved in DMF, is added dropwise over carbodiimides and N- hydroxyl amber
Amber acid imide water fully dissolve after solution, be added dropwise to carrier protein-CB after priming reaction（pH 9.6）In solution, room
Overnight rear 4 DEG C of dialysis obtain purpose product to temperature, -20 DEG C of preservations after subpackage.
Described carrier protein can be bovine serum albumin（BSA）, human albumin（HSA）, hemocyanin（KLH）Or egg white
Albumen（OVA）, preferably bovine serum albumin（BSA）.
Present invention simultaneously provides described Cyproheptadine monoclonal antibody specific, and described Cyproheptadine antigen and antibody are in match
The application of pyridine in heptan immune detection aspect.
The invention has the beneficial effects as follows：
The present invention, to retain the space structure of Cyproheptadine to greatest extent, redesigns and synthesizes new hapten and resist
Former, compared with prior art, the antigen of the present invention obtains more high-quality, efficient antibody after animal immune.
Long term experiments demonstrate that in a large number, the potency of the monoclonal antibody being obtained using the antigen-immunized animal of the present invention up to
1:300000,503nhibiting concentration is 0.20 μ g/L, and it is significantly excellent that described antibody has that specificity is high, sensitivity is high, accuracy is high
More property, therefore, antigen and antibody that the present invention provides, can be used for setting up Cyproheptadine competitive enzyme-linked immune adsorption analyses technology and glue
Body gold immunochromatography technique, thus for the Cyproheptadine residual in quick detection food, have broad application prospects.
Brief description
Fig. 1 Cyproheptadine hapten synthesis route map
Fig. 2 Cyproheptadine hapten hydrogen nuclear magnetic resonance spectrogram
Fig. 3 Cyproheptadine ELISA competition test curve chart
Fig. 4 Cyproheptadine colloidal gold strip structural representation
Fig. 5 a, 5b, 5c Cyproheptadine colloidal gold strip result judgement figure
Specific embodiment
Further describe the present invention with specific embodiment below in conjunction with the accompanying drawings.Experiment used in following embodiments
Method if no special instructions, is conventional method；The material that used, reagent etc., if no special instructions, for commercially
The reagent obtaining and material.
Embodiment 1：Hapten synthesis and identification
First, the haptenic synthesis of Cyproheptadine
The haptenic synthetic route of Cyproheptadine is as shown in Figure 1.
（1）Weigh 0.55g demethyl Cyproheptadine and 1mL pyridine is dissolved in 10mL dimethyl sulfoxide（DMSO）In；
（2）It is slowly added dropwise into step at weighing 40 DEG C after 0.39g bromo-acetic acid tert-butyl is dissolved in 5mL DMSO（1）Described solution
In, solvent is evaporated off after continuing reaction 4 hours, Cyproheptadine hapten intermediate is obtained by column chromatography for separation；
（3）In step（2）20mL DMSO and 5mL formic acid, room temperature is added in the Cyproheptadine hapten intermediate preparing
Reaction was evaporated off solvent after 20 hours, was recrystallized to give target hapten in ethanol-water system（Ⅰ）, two-step reaction total yield is
62%.
2nd, the haptenic identification of Cyproheptadine
Above-mentioned purpose product is taken to identify through proton nmr spectra, as shown in accompanying drawing 2.In collection of illustrative plates 13.0ppm about newly increase
Carboxyl signal peak and 3.3ppm about the methylene signals peak newly increasing, illustrate Cyproheptadine hapten synthesis success.
Embodiment 2：Artificial antigen's synthesis and identification
First, Cyproheptadine antigen（Ⅱ）Synthesis
Cyproheptadine antigen（Ⅱ）Active ester method synthesis：
（1）The Cyproheptadine hapten weighing 20mg is dissolved in 1mL N,N-dimethylformamide（DMF）In；
（2）Take 30mg carbodiimides（EDC）And N-hydroxy-succinamide（NHS）Add after fully being dissolved with 0.2mL water
Enter in hapten solution, stirring reaction 24 hours under room temperature；
（3）Weigh 50mg BSA and be dissolved in 3.8mL CB（pH 9.6）In solution, by step（2）Reacted solution dropwise delays
Slowly it is added in BSA-CB solution, and stirring reaction 24 hours at room temperature；
（4）Will be through step（3）Reacted solution is loaded on bag filter, in 4 DEG C of PBS solution dialysis 72 with 0.01mol/L
Hour, period changes dialysis solution 3 times daily, that is, obtain purpose product, -20 DEG C of preservations after subpackage.
Can also be using human albumin, hemocyanin or ovalbumin, with reference to the match of the present embodiment method in said method
Ibid, simply used carrier albumen is different for pyridine in heptan envelope antigen preparation method.
2nd, Cyproheptadine antigen（Ⅱ）Identification
Cyproheptadine hapten, carrier protein BSA and Cyproheptadine antigen is taken to carry out ultraviolet respectively（200nm-400nm）Scanning,
Identify whether hapten is coupled with carrier protein by comparing the highest light absorption value of three.It was found that Cyproheptadine antigen
Light absorption value significantly different with Cyproheptadine hapten and BSA, illustrate hapten be successfully coupled with BSA prepared Cyproheptadine resist
Former.It is computed, Cyproheptadine hapten is 21 with the combination mol ratio of BSA:1.
Embodiment 3：Prepared by monoclonal antibody
（1）Animal immune：Immunogen is injected in Balb/c mice body, immunizing dose is 150 μ g/ only so as to produce
Antiserum.
（2）Cell fusion and cloning：Take the Balb/c mouse boosting cell producing specific antibody and myeloma cell
SP20 merges, and measures cell supernatant using indirect competitive enzyme-linked immunosorbent analysis method, the positive hole of screening.Using limiting dilution assay
Cloning is carried out to positive hole, obtains and set up the hybridoma cell strain producing monoclonal antibody.
（3）Cell cryopreservation and recovery：The hybridoma being in exponential phase is taken to make cell suspension with frozen stock solution, point
It is loaded on cryopreservation tube, preserve for a long time in liquid nitrogen.Take out cryopreservation tube during recovery, be immediately placed in 37 DEG C of water-bath middling speeds and melt, centrifugation removes
After frozen stock solution, move into culture culture in glassware.
（4）The preparation of monoclonal antibody and purification：Using inducing method in vivo, by Balb/c mice（8 week old）Intraperitoneal injection
Sterilizing paraffin oil, pneumoretroperitoneum injects hybridoma within 7 ~ 14 days, gathers ascites after 7 ~ 10 days.Enter through octanoic acid-saturated ammonium sulfate method
Row ascites purification, purity is through SDS-PAGE electroresis appraisal, bottle subpackage, -20 DEG C of preservations.
Embodiment 4：The enzyme linked immunological kit of detection Cyproheptadine and its preparation and application
First, the composition of enzyme linked immunological kit
（1）It is coated the ELISA Plate of Cyproheptadine antigen；
（2）Enzyme mark Cyproheptadine antibody：Monoclonal antibody described in embodiment 3 and horseradish peroxidase（HRP）Using
Over-voltage protection carries out coupling and prepares；
（3）Cyproheptadine standard substance：Standard solution concentration be respectively 0 μ g/L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L,
1.35μg/L、4.05μg/L；
（4）Substrate nitrite ion：It is made up of A liquid and B liquid, A liquid is the aqueous solution of 2% urea peroxide, B liquid is 1% tetramethyl connection
The aqueous solution of aniline；
（5）Terminate liquid：2mol/L aqueous sulfuric acid；
（6）Cleaning mixture：Every 1L cleaning mixture is prepared as follows：By 10mL tween 20,5g Hydrazoic acid,sodium salt and 990mL
Phosphate buffer mixes；Wherein, the concentration of phosphate buffer is 0.02mol/L, and pH value is 7.4；
（7）Redissolve liquid：Every 1L redissolves liquid and prepares as follows：12g casein phosphate buffer is dissolved simultaneously
It is settled to 1000mL；Wherein, the concentration of phosphate buffer is 0.02mol/L, and pH value is 7.4.
2nd, the preparation of reagent constituents
（One）The preparation of coating antigen
Using the purpose product described in embodiment 1 as Cyproheptadine hapten, carrier protein is ovalbumin（OVA）, system
Preparation Method is with reference to embodiment 2.Through UV scanning identification, hapten and OVA are coupled successfully, and both combination mol ratios are 13:1.
（Two）It is coated with the preparation of the ELISA Plate of coating antigen
With being coated buffer, above-mentioned coating antigen is diluted to 0.2 μ g/mL, is coated 96 hole polystyrene ELISA Plate, every hole 100
μ L, 37 DEG C of incubation 2h, incline and are coated liquid, washed with cleaning mixture 3 times, each 30s, pat dry, and then add 200 μ L closings in every hole
Liquid, 37 DEG C of incubation 2h, in the hole of inclining liquid, preserved with aluminium film vacuum sealing after being dried.
It is coated buffer：The carbonate buffer solution of pH9.6,0.05mol/L；
Confining liquid：Every 1L confining liquid is prepared as follows：By 5mL horse serum, 1g Hydrazoic acid,sodium salt, the mixing of 30g casein,
Dissolved with phosphate buffer and be settled to 1000mL；Wherein, the concentration of phosphate buffer is 0.02mol/L, and pH value is 7.2.
3rd, kit test method
（One）Sample pre-treatments
1st, pig urine sample
The limpid urine sample of 50 μ L is taken directly to measure（As urine sample muddiness must be by filtration or more than 3000g, room temperature（20~25
℃/68~77℉）10min is until limpid for centrifugation）, the sample that wouldn't use answers freezen protective（Extension rate：1 times）.
2nd, serum, Carnis Sus domestica, beef, Hepar Sus domestica sample
Weigh the tissue after 2.0g ± 0.05g homogenizing, liver samples/absorption 2.0mL serum sample to 50mL polystyrene
In centrifuge tube；It is separately added into 0.3mL 2mol/L sulfuric acid solution and 3.7mL acetonitrile, vibrates 5min with agitator, mix, 3000g
More than, room temperature（20~25℃/68~77℉）Centrifugation 5min；Pipette 2mL upper organic phase to 50mL polystyrene centrifuge tube, plus
Enter 0.2mL 2mol/L sodium hydroxide solution, gently vibrate 20s, then be separately added into 2mL chloroform, 4mL normal hexane, with vibration
Device vibrates 5min, mixes, more than 3000g, room temperature（20~25℃/68~77℉）Centrifugation 5min；Pipette 2mL upper organic phase extremely
In the clean teat glass of 10mL, in 50 ~ 60 DEG C（122~140℉）Dry up under water-bath nitrogen stream；1mL is added to redissolve liquid, with being vortexed
Instrument whirling motion 30s, takes 50 μ L to be used for analyzing（Extension rate：6 times）.
（Two）Detected with test kit
1st, the making of standard curve
Add Cyproheptadine standard solution 50 μ L in the ELISA Plate micropore be coated with coating antigen, add enzyme mark Cyproheptadine and resist
Body running liquid 50 μ L, gently vibration mixes, and with reacting 40min in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate, pours out in the hole liquid,
Every hole adds cleaning mixture 250 μ L, pours out in the hole liquid after 30s, repeats operation and washes plate 5 times altogether, is patted dry with absorbent paper；Every hole
Add substrate nitrite ion A liquid and each 50 μ L of B liquid, gently vibration mixes, with reacting in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate
15min；Every hole adds terminate liquid 50 μ L, and gently vibration mixes, and sets microplate reader at 450nm, measures the absorbance in every hole.
Absorbance values with the standard solution of each concentration（B）Divided by first standard solution（0 standard）'s
Absorbance values（B0）It is multiplied by 100% again, obtain percentage absorbance.With the logarithm value of Cyproheptadine standard concentration as X-axis,
Percentage absorbance is Y-axis, draws canonical plotting.The standard curve obtaining is as shown in Figure 3.
Percentage absorbance（%）=（B/B0）×100%
2nd, in sample Cyproheptadine concentration mensure
Add sample to be tested solution 50 μ L in the ELISA Plate micropore be coated with coating antigen, add enzyme mark Cyproheptadine antibody
Working solution 50 μ L, gently vibration mixes, and with reacting 40min in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate, pours out in the hole liquid,
Every hole adds cleaning mixture 250 μ L, pours out in the hole liquid after 30s, repeats operation and washes plate 5 times altogether, is patted dry with absorbent paper；Every hole
Add substrate nitrite ion A liquid and each 50 μ L of B liquid, gently vibration mixes, with reacting in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate
15min；Every hole adds terminate liquid 50 μ L, and gently vibration mixes, and sets microplate reader at 450nm, measures the absorbance in every hole.
Result judges：Absorbance values with each test sample solution（B）Divided by first standard solution（0 mark
Accurate）Absorbance values（B0）It is multiplied by 100% again, obtain percentage absorbance.The hundred of each test sample solution corresponding
Divide absorbance, then the concentration corresponding to test sample solution can be read from standard curve, then be multiplied by its corresponding dilution times
Number is the practical residue limit of Cyproheptadine in sample solution.
4th, test kit Detection results are evaluated
（One）Lowest detectable limit
Take each 20 parts of the sample without Cyproheptadine, carry out after pre-treatment, it being examined according to method described in experiment three
Survey, represent test limit with the meansigma methodss of 20 parts of sample detection concentration plus 3 times of standard deviations.
Result shows, the method is limited to 0.5 μ g/L to the lowest detection of pig urine sample, to serum, Carnis Sus domestica, beef, Hepar Sus domestica
The lowest detection of sample is limited to 0.3 μ g/kg.
（Two）Accuracy and precision
Using the response rate as accuracy estimating index, the testing result relative standard deviation of a certain concentration samples of replication
（RSD%）As precision evaluation index.Computing formula is：The response rate（%）=actual measured value/theoretical value × 100%, wherein
The interpolation concentration of theoretical value sample；Relative standard deviation RSD%=SD/X × 100%, wherein SD are standard deviation, and X is to survey
The meansigma methodss of fixed number evidence.
Add Cyproheptadine standard substance in the sample without Cyproheptadine, make end in pig urine samples for the Cyproheptadine standard substance dense
Spend for 0.5,1.0,2.0 μ g/L, final concentration of 0.3,0.6,1.2 μ g/L (kg) in serum, Carnis Sus domestica, beef, Pig Liver；
Sample after adding is carried out pre-treatment according to method described in experiment three respectively, obtains test sample solution.
Respectively extract 3 test kits to be detected, institute in detection method such as experiment three from the test kit of three different batches
State, each experiment is repeated 5 times, calculate relative standard deviation respectively.The results are shown in Table 1.
Table 1 accuracy and Precision Experiment result
Result shows, the TIANZHU XINGNAO Capsul of all samples 83.7% ~ 114.3%, in batch relative standard deviation 4.8% ~
9.5%, between batch, relative standard deviation is 7.4% ~ 13.8%.
（Three）Test kit storage life
Test kit preservation condition is 2-8 DEG C, through the mensure of 12 months, the maximum absorbance value of test kit（Zero standard）、
50% inhibition concentration, Cyproheptadine TIANZHU XINGNAO Capsul are all within normal range.Have improper guarantor in view of transport with during using
Condition of depositing occurs, and test kit is placed 8 days at 37 DEG C and carries out accelerated aging tests, the indices of this test kit of result are complete
Meet the requirements；Occur in view of test kit freezing situation, test kit is placed 8 days in -20 DEG C of refrigerators, this test kit of result
Indices are also completely normal.Can show that test kit at least can preserve more than 12 months at 2-8 DEG C from result above.
Embodiment 5：The colloidal gold strip of detection Cyproheptadine and its preparation and application
First, the structure of test strips（Fig. 4）
Described test strips are made up of base plate, sample absorption pad, conjugate release pad, reaction film and adsorptive pads；
Described sample absorption pad 1, conjugate release pad 2, reaction film 3 and adsorptive pads 4 are pasted onto base plate 7 successively in order
On, conjugate release pad has 1/3 region to be absorbed by the sample pad covering, the end of conjugate release pad and reaction film from initiating terminal
Top connects, and the end of reaction film is connected with the top of adsorptive pads；
The Cyproheptadine monoclonal antibody of colloid gold label is coated with described conjugate release pad；
Detection line 5 and nature controlling line 6, detection line are had on described reaction film（C line）And nature controlling line（T line）It is and described reagent paper
The perpendicular ribbon of the length of bar；Detection line is located at the side of the end near conjugate release pad；Nature controlling line is located remotely from knot
The side of the end of compound release pad；Detection line is coated with coating antigen, and nature controlling line is coated with sheep anti mouse anti antibody.
2nd, the preparation of test strips
（One）The preparation of colloidal gold labeled monoclonal antibody
The preparation of gold colloidal：With double distilled deionized water, 1% gold chloride is diluted to 0.01%（Mass fraction）, take 100mL to put
In conical flask, it is heated to seething with excitement with thermostatic electromagnetic agitator, in continuous high temperature, continuously stirred lower addition 1.5mL 1% citric acid
Trisodium, continuing at the uniform velocity to be heated with stirring to solution is in stop during bright claret, and after being cooled to room temperature, deionized water returns to
Original volume, 4 DEG C of preservations.It is limpid transparent for prepare good gold colloidal detecting by an unaided eye, and does not have muddiness, and liquid surface no floats
Thing, the color of observing colloid gold is claret in the sunlight.
The preparation of colloidal gold labeled monoclonal antibody：Under magnetic stirring, the pH to 7.2 of gold colloidal is adjusted with 0.2mol/L potassium carbonate
（The pH label range of different antibodies, between 7 ~ 8, can change）, by addition antibody 20 ~ 50 g in every milliliter of colloidal gold solution
Standard add Cyproheptadine monoclonal antibody in colloidal gold solution, stirring and evenly mixing, after room temperature standing 10min, add 10%BSA
Make its final concentration of 1% in colloidal gold solution, stand 10min.12000r/min, 4 DEG C of centrifugation 40min, abandon supernatant, use
Volume is that the redissolution buffer of initial colloid gold volume 1/10 will precipitate resuspended, puts standby in 4 DEG C of environment.
Redissolve buffer：Every 1L redissolves buffer and prepares as follows：By 5mL tween 80,1g casein and
995mL phosphate buffer mixes；Wherein, the concentration of phosphate buffer is 0.02mol/L, and pH value is 7.2.
（Two）The preparation of conjugate release pad
By conjugate release pad be soaked in containing 0.5% BSA, pH be 7.2, in the phosphate buffer of 0.5mol/L, uniformly
Soak 1h, 37 DEG C of baking 3h are standby.With Isoflow spray film instrument, the colloidal gold labeled monoclonal antibody preparing even application is released in conjugate
Put on pad, after every 1cm conjugate release pad spraying 0.01mL colloidal gold labeled monoclonal antibody, be placed in 60min in 37 DEG C of environment and take out, put
Save backup in dry environment.
（Three）The preparation of reaction film
With the phosphate buffer of 0.01mol/L, pH7.4, coating antigen is diluted to 10mg/mL, will with Isoflow point film instrument
It is coated in the detection line on nitrocellulose filter（T line）, package amount is 0.8 μ L/cm；Sheep anti mouse anti antibody is diluted to 200 μ
G/mL, is coated in the nature controlling line on nitrocellulose filter with Isoflow point film instrument（C line）, package amount is 1.0 μ L/cm.Will
The reaction film being coated is dried 2h under the conditions of being placed in 37 DEG C, standby.
（Four）The preparation of sample absorption pad
Sample absorption pad is placed in the phosphate buffer containing 0.5% BSA, pH7.2,0.1mol/L and soaks 2h, 37 DEG C
Dry 2h standby.
（Five）The assembling of test strips
Base plate, sample absorption pad, conjugate release pad, reaction film, adsorptive pads are assembled according to a conventional method, Ran Houyong
Machine is cut into the wide little bar of 3mm, is contained in special plastics fabrication, forms test card.
3rd, detected with test card
（One）Sample pre-treatments and detection
Limpid urine sample is taken directly to measure（As urine sample muddiness must be by filtration or more than 3000g, room temperature（20~25℃/68~
77℉）10min is until limpid for centrifugation）, the sample that wouldn't use answers freezen protective.
Take out test card and lie against desktop, draw the vertical Deca of measuring samples solution 2 ~ 3 in well with suction pipe, liquid
Start timing during body flowing, react 5 ~ 10min, result of determination.
（Two）Result judges
Negative（-）：T line and C line all develop the color, and represent that in sample, Cyproheptadine concentration is less than test limit, such as Fig. 5 a.
Positive（+）：T line does not develop the color the colour developing of C line, represents that in sample, Cyproheptadine concentration is equal to or higher than test limit, such as Fig. 5 b.
Invalid：C line does not occur, shows incorrect operating process or test strips deterioration failure, such as Fig. 5 c.In this situation
Under, description should be read over again, and retested with new test strips.
4th, test card Detection results are evaluated
（One）Test limit
To in the pig urine samples without Cyproheptadine add Cyproheptadine standard substance so as in pig urine samples final concentration of 5,
10、20μg/L；Sample after adding is carried out being detected with test card after pre-treatment according to method described in experiment three respectively,
Each sample is repeated three times.
During with test card detection pig urine samples, when wherein Cyproheptadine adds concentration for 5 μ g/L, test card shows meat
The visible two red lines of eye, are negative；When wherein Cyproheptadine adds concentration for 10,20 μ g/L, test card nature controlling line shows
Color, detection line does not develop the color, and is positive, and shows that this test card is limited to 10 μ g/L to the detection of Cyproheptadine in pig urine samples.
（Two）False positive rate and false negative rate
The negative pig urine of confirmation of learning from else's experience（＜ containing Cyproheptadine 10 μ g/L）With positive pig urine（Containing Cyproheptadine >=10 μ g/L）Sample
Each 50 parts, sample is detected with test card respectively according to after method process described in experiment three, is calculated false positive rate and vacation
Negative rate.
It was found that in 50 parts of negative pig urine samples measure, test card detects totally 1 part of positive, false positive rate
For 2%；In 50 parts of positive pig urine samples measure, test card detects 0 part of negative sample, and false negative rate is 0%.
（Three）Test card storage life
Stability test result shows, this test card cool place place of drying under the conditions of 4-30 DEG C can preserve 12 months.
Claims (6)
1. a kind of Cyproheptadine artificial antigen is it is characterised in that be by Cyproheptadine hapten and carrier protein couplet using active ester method
Prepare, molecular structure is：
Wherein, the haptenic preparation method of described Cyproheptadine comprises the following steps：
(1) demethyl Cyproheptadine and pyridine are codissolved in dimethyl sulfoxide, bromo-acetic acid tert-butyl is dissolved in dimethyl sulfoxide；
At (2) 40 DEG C, bromo-acetic acid tert-butyl solution is slowly dropped in demethyl Cyproheptadine solution, continues reaction, after completion of the reaction
Solvent is evaporated off, Cyproheptadine hapten intermediate is obtained by column chromatography for separation；
(3) addition dimethyl sulfoxide and formic acid in the Cyproheptadine hapten intermediate that step (2) prepares, room temperature reaction,
Solvent is evaporated off after completion of the reaction, in ethanol-water system, is recrystallized to give target hapten, its molecular structure is：
2. a kind of preparation method of Cyproheptadine artificial antigen described in claim 1 is it is characterised in that comprise the following steps：
(1) weigh Cyproheptadine hapten and be dissolved in N,N-dimethylformamide；
(2) take carbodiimides and N-hydroxy-succinamide water fully dissolve after be added to step (1) Cyproheptadine hapten
In solution, priming reaction；
(3) carrier protein is dissolved in CB solution, the solution after step (2) priming reaction is added dropwise to carrier protein solution
In, overnight；
(4) purpose product will be obtained through the dialysis of step (3) reacted solution, -20 DEG C of preservations after subpackage.
3. preparation method as claimed in claim 2 is it is characterised in that described carrier protein is bovine serum albumin, human serum egg
In vain, hemocyanin or ovalbumin.
4. Cyproheptadine artificial antigen described in a kind of claim 1 is preparing Cyproheptadine antibody or answering in terms of detection Cyproheptadine
With.
5. a kind of Cyproheptadine monoclonal antibody being prepared using Cyproheptadine artificial antigen described in claim 1.
6. application in detection Cyproheptadine for the Cyproheptadine monoclonal antibody described in a kind of claim 5.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310138339.3A CN104109112B (en) | 2013-04-19 | 2013-04-19 | Cyproheptadine semiantigen, artificial antigen, antibody, preparation methods of cyproheptadine semiantigen and artificial antigen, and application of artificial antigen and antibody |
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| CN106243096B (en) * | 2016-07-29 | 2019-11-29 | 上海璃道医药科技有限公司 | The new application of tricyclic drugs |
| CN107688091A (en) * | 2017-09-06 | 2018-02-13 | 上海容晖生物科技有限公司 | A kind of immune quantitative test paper bar of quick detection cyproheptadine and preparation method thereof |
| CN108424880A (en) * | 2018-03-16 | 2018-08-21 | 江南大学 | A kind of hybridoma cell strain and preparation method of secretion cyproheptadine monoclonal antibody |
| CN114456224B (en) * | 2022-04-12 | 2022-08-12 | 北京纳百生物科技有限公司 | Synthesis and application of estrone sulfate hapten |
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