CN105906522A - Amantadine artificial antigen and preparation method and application thereof - Google Patents
Amantadine artificial antigen and preparation method and application thereof Download PDFInfo
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- CN105906522A CN105906522A CN201610280030.1A CN201610280030A CN105906522A CN 105906522 A CN105906522 A CN 105906522A CN 201610280030 A CN201610280030 A CN 201610280030A CN 105906522 A CN105906522 A CN 105906522A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C231/00—Preparation of carboxylic acid amides
- C07C231/02—Preparation of carboxylic acid amides from carboxylic acids or from esters, anhydrides, or halides thereof by reaction with ammonia or amines
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C233/00—Carboxylic acid amides
- C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
- C07C233/02—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
- C07C233/09—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with carbon atoms of carboxamide groups bound to carbon atoms of an acyclic unsaturated carbon skeleton
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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
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
Abstract
The invention discloses an amantadine hapten, a corresponding artificial antigen and a preparation method and application of the amantadine hapten and the corresponding artificial antigen. The amantadine hapten is a product as shown in formula 1, and the amantadine antigen can be obtained by connecting the product with carrier protein. The amantadine antigen can be used for preparing an amantadine specific antibody. The preparation method is simple and practicable, low in cost and high in hapten yield. The amantadine artificial antigen has the advantages that the specific antibody aiming at amantadine can be produced by immunizing animals, the amantadine artificial antigen can be used for preparing an enzyme-linked immunosorbent assay kit for detecting amantadine residues, and the amantadine artificial antigen is simple, fast, large in sample processing amount, high in sensitivity, high in specificity, and the like.
Description
Technical field
The invention belongs to technical field of food safety detection, be specifically related to a kind of amantadine haptens, antigen preparation side
Method and application thereof.
Background technology
Amantadine (Amantadine, 1-adamantanamine) is synthesized in nineteen sixty, also known as amantadine, three ring last of the ten Heavenly stems
Amine, it is 1-amantadine or the organic compound of 1-aminoadamantan, it is meant that its structure composition is an adamantane main chain
With four methine positions have a substituted-amino.For white crystalline powder, bitter, soluble in water or ethanol.It is mainly used in
By the prevention of the respiratory tract infection caused by influenza A virus and for the treatment of influenza a virus infection patient, the U.S. in
Asia flu within popular 1966, ratify its as preventive medicine, and confirmed as on the basis of preventive medicine in 1976 treatment
Medicine, is also used for parkinson's syndrome in addition；Its antiviral spectrum is narrower, has obvious inhibiting effect to influenza A, can after medication
Substantially reduce the death rate.2005, report, Chinese poultry resource peasant household used amantadine to protect bird anti-avian influenza,
Rising at that time, regulator of Chinese Government has forbidden that it uses as veterinary drug.
Curative effect and the security of adult patients are the most extensively admitted by amantadine.But therapeutic dose with have side effects
Dosage very close to, to People and have chronic heart and lung diseases or the dosage of kidney trouble person and drug dosage schedule to be difficult to determine, because of
These popularization and application the most clinically.In Japan, amantadine is always as Parkinsonian curative, until ability quilt in 1998
Approval is for the treatment of influenza A virus infectious diseases.Common adverse reactions has central nervous system and gastrointestinal reaction, table
Now for having a dizzy spell, absent minded, have a headache, have a sleepless night, anxiety and anorexia, feel sick, after drug withdrawal, bad reaction is immediately
Disappearing, rare bad reaction has constipation, mouth and nose to be dried.The Ministry of Agriculture sends " disease-resistant about examination amantadine etc. for 2005
The emergency notice of cytotoxic drug ", it is distinctly claimed amantadine, Rimantadine etc. and stops immediately producing, managing and use, offender is by raw
Produce, manage false veterinary drug and use disabling veterinary drug to process.Therefore, for guaranteeing the safety of animal derived food and export abroad trade
Development, sets up accurately and reliably, and highly sensitive qualitative-and-quantitative method is the most necessary.
The external enzyme linked immunological kit having developed detection amantadine, but the kit of domestic production is accurately
Property, sensitivity, the aspect such as specific can't be fully achieved the requirement of detection.Amantadine haptens disclosed by the invention, anti-
Originally it was that development amantadine antibody and amantadine enzyme linked immunological kit provide raw material further.Amantadine enzyme linked immunological
Reaction kit utilizes competitiveness enzyme-linked immune response principle, firm to tissue (beef, chicken and pork) and other Gold Samples
The residual of alkanamine quantitatively detects.
Summary of the invention
It is an object of the invention to provide a kind of amantadine haptens, antigen preparation procedure and application thereof.
The amantadine haptens that the present invention provides, is compound shown in formula 1:
Formula 1.
The invention also discloses the preparation method of product shown in formula 1, comprise the steps:
0.94g adamantanamine hydrochloride is dissolved in 30ml anhydrous pyridine, adds maleic anhydride 0.49g, after dissolving, adds 4-diformazan ammonia
Yl pyridines 10mg, 70 DEG C of back flow reaction 3h, rotation is evaporated off pyridine, adds deionized water 10ml, and pH5.0 adjusted by glacial acetic acid, separates out a large amount of
Precipitation, filters, water washing and precipitating, dried haptens.
The amantadine antigen that the present invention provides, is conjugate product shown in formula 1 and carrier protein couplet obtained.
The present invention also protects the preparation method of described amantadine antigen, comprises the steps:
Weigh 5.57mg haptens and be dissolved in 1ml DMF, add EDC 4.3mg, NHS 5.2mg, reaction 2h is stirred at room temperature；50mg
BSA is dissolved in 5ml 0.1M sodium bicarbonate buffer liquid, is added dropwise in protein solution by above-mentioned pharmacological activation, was stirred at room temperature
At night, PBS 4 degree dialyses 72 hours, and period changes dislysate 6 times.Dislysate is aseptically crossed the filter membrane in 0.22 μm aperture,
Being sub-packed in ampere bottle ,-20 degree preserve.
Coating antigen is prepared with method.
Common carrier albumen all can use, including bovine serum albumin(BSA) (BSA), ovalbumin (OVA), human serum albumins
(HSA), mouse serum albumin (MSA), thyroprotein (TG) or hemocyanin (KLH) etc..
Described amantadine antigen can prepare amantadine specific antibody as immunogen immune animal, it is also possible to makees
ELISA Plate is prepared for coating antigen.
Described antibody specific can be monoclonal antibody.
Product shown in formula 1, described amantadine antigen, described antibody all can be applicable to detect amantadine.
The present invention also disclosed the enzyme linked immunological applying amantadine antigen and amantadine monoclonal antibody to prepare
Kit.
Described enzyme-linked immunologic detecting kit, be by be coated with the ELISA Plate of amantadine antigen, enzyme labelled antibody working solution,
Amantadine serial standards, substrate nitrite ion, stop buffer, concentration redissolution liquid, concentrated cleaning solution.
The present invention relies on immunology, immunochemistry general principle and retention analysis technological means, design, synthesized micromolecule mesh
Mark analyte haptens, and and carrier protein couplet, prepare effective artificial antigen.Preparation method of the present invention is simple and feasible, cost
Relatively low, yield of hapten is higher.The amantadine artificial antigen of the present invention, can be created for amantadine by immune animal
Specific antibody, the amantadine residual in quickly detection food.
Accompanying drawing explanation
Fig. 1 is amantadine haptenic Mass Spectrometer Method result.
Fig. 2 is the MALDI-TOF-MAS figure of BSA.
Fig. 3 is the MALDI-TOF-MAS figure of amantadine antigen " amantadine haptens+BSA ".
Fig. 4 is amantadine enzyme-linked immunologic detecting kit calibration curve.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is certainly
Routine biochemistry reagent shop is commercially available.
The haptenic preparation of embodiment 1, amantadine
One, the haptenic preparation of amantadine
0.94g adamantanamine hydrochloride is dissolved in 30ml anhydrous pyridine, adds maleic anhydride 0.49g, after dissolving, adds 4-diformazan ammonia
Yl pyridines 10mg, 70 DEG C of back flow reaction 3h, rotation is evaporated off pyridine, adds deionized water 10ml, and pH5.0 adjusted by glacial acetic acid, separates out a large amount of
Precipitation, filters, water washing and precipitating, dried haptens.
Two, the haptenic qualification of amantadine
Products obtained therefrom is carried out Mass Spectrometric Identification ,-MS:M-1=248.7(Fig. 1).
Result shows its chemical structural formula as shown in Equation 1, is amantadine haptens.
Formula 1.
Embodiment 2, the preparation of amantadine artificial antigen and qualification
One, the synthesis of amantadine immunizing antigen
Weigh 5.57mg haptens and be dissolved in 1ml DMF, add EDC 4.3mg, NHS 5.2mg, reaction 2h is stirred at room temperature； 50mg
BSA is dissolved in 5ml 0.1M sodium bicarbonate buffer liquid, is added dropwise in protein solution by above-mentioned pharmacological activation, was stirred at room temperature
At night, PBS 4 degree dialyses 72 hours, and period changes dislysate 6 times.Dislysate is aseptically crossed the filter membrane in 0.22 μm aperture,
Being sub-packed in ampere bottle ,-20 degree preserve.
Coating antigen is prepared with method.
Two, the qualification of amantadine artificial antigen
Immunogene MALDI-TOF-MS qualification result display coupling ratio is: R=(AMD-MH-BSA)-(BSA)/249.7=
(73828.009-67485.901)/249.7=25.40(Fig. 2 and Fig. 3).I.e. in immunogene, described amantadine haptens (formula
I) mol ratio with bovine serum albumin(BSA) (BSA) coupling is 25.40:1.
Embodiment 3, the preparation of enzyme mark monoclonal antibody and specificity identification
One, the preparation of amantadine monoclonal antibody
1, by the above-mentioned immunogene prepared by 100 μ g/ only, with physiological saline solution immunogene and Freund's complete adjuvant equal-volume
Mixing, the female mouse of neck dorsal sc injection immunity 6 ~ 8 week old Balb/c, after initial immunity the 7th, 14,28 days with immunogene and Freund
Freund's incomplete adjuvant equal-volume mixes, each supplementary immunization once, merge first 3 days with immune complex 100 μ g/ only, be not added with Freund assistant
Agent supplementary immunization again is once.
2, carry out according to a conventional method, take the splenocyte of immune mouse and be in the murine myeloma cell of exponential phase
(SP2/0) mixing, the fusion agent (PEG4000) being then slowly added to preheating in 45s merges, and suspends with HAT culture medium
Uniformly, add appropriate feeder cells, be incubated at 96 well culture plates, in 37 DEG C, 5%CO2Incubator is cultivated, uses after 5 days
HT culture medium partly changes liquid, entirely changes liquid when 9 days.
3, after cell merges, when cell grows to the 1/4 of culture hole area, use substep screening method screening hybridoma thin
Born of the same parents.Primary election uses indirect ELISA method, and with envelope antigen, (with square formation method conventional titration, it is most preferably coated concentration and the positive in advance
Serum dilution) coated elisa plate, add measured hole culture supernatant, hatch, after cleaning, add sheep anti-mouse igg-HRP and IgM-
HRP, OPD carry out chromogenic reaction.The positive Kong Zaiyong indirect competitive ELISA method screening filtered out, first by cell conditioned medium and 100
The amantadine equal-volume mixing of μ g/mL, 37 DEG C of water-bath effect 30min, it is then added in the ELISA Plate being coated.Use simultaneously
PBS replaces amantadine and compares, and remaining step is ibid.If the OD after amantadine blocks450Nm value drops to control wells
Less than 50%, then it is judged to the positive, is all positive hole through 2 ~ 3 detections, carries out subcloning with limiting dilution assay immediately.
4,2 ~ 3 subclones are built the hybridoma after strain and expands cultivation, collect supernatant indirect ELISA and measure effect
Valency, frozen；And only take 8 ~ 10 week old Balb/c mouse peritoneal injecting fluid paraffin 0.5mL/, lumbar injection hybridoma after 7 ~ 10 days
Cell 1 ~ 2 × 106/ only, and extract mouse ascites, centrifuging and taking supernatant after 7 ~ 10 days, measure titer, and frozen standby.
Two, the preparation of enzyme labelled antibody
1, weigh horseradish peroxidase (HRP) 2 mg to be dissolved in 0.5 mL water, add 0.5 mL 0.06 mol/L NaIO4
Solution, 4 DEG C of lucifuge effect 30 min；
2, the ethylene glycol 0.5mL of 160 mmol/L, room temperature effect 30 min are added；
3, add amantadine monoclonal antibody 2 mg of step one preparation, in the bag filter that after mixing, loading processed, put 1000 mL's
Dialysing in 0.05 mmol/L sodium carbonate buffer, 4 DEG C overnight；
4, during dislysate is drawn to the centrifuge tube of 10 mL, the NaBH of 0.25mL 5g/L is added4Solution, mixes rearmounted 4 DEG C of 2 h；
5, adding isopyknic saturated ammonium sulfate solution, after 4 DEG C of effect 30 min, at 4 DEG C, 3000 r/min are centrifuged 25 min, abandon
Supernatant；
6, precipitation is dissolved in the PBS of 1.5 mL0.02 mol/L pH 7.4, sucks in bag filter, at 0.02mol/L pH
7.4 PBS, 4 DEG C overnight (PBS is changed 3 times in midway)；
7, by during in bag filter, liquid is drawn to microcentrifugal tube, at 4 DEG C, 10000r/min is centrifuged 30min, by supernatant sucking-off, adds
Equivalent glycerine, mixing ,-20 DEG C save backup.
Three, the mensuration of enzyme mark amantadine antibody titer
Amantadine standard items are purchased from Sigma company.
The working concentration of the monoclonal antibody of amantadine envelope antigen and step one preparation, amantadine is determined by square formation titration
The working concentration of envelope antigen is 2.5 μ g/mL, and the working concentration of monoclonal antibody is 1:60000.
Doing experimental solutions with the amantadine standard solution of variable concentrations, its concentration is as follows: 0,0.5,1.5,4.5,
13.5、40.5μg/L.Use 8 groups of parallel tests (n=8).
Competitive ELISA method:
(1) with the antigen coated ELISA Plate of amantadine of above-mentioned working concentration, by amantadine standard items experimental solutions and enzyme mark
Antibody-solutions is simultaneously introduced in ELISA Plate micropore, and arrange blank well (changing the antibody-solutions of interpolation into high purity water, other one simultaneously
Cause) and negative control hole (standard items experimental solutions PBS solution being replaced, other is consistent), 25 DEG C of light protected environment react
30min；
(2) pour out liquid in hole, wash 3 ~ 5 times with cleaning solution, ELISA Plate is upside down on blotting paper and pats dry；
(3) add substrate chromophoric solution in ELISA Plate micropore, 25 DEG C of light protected environment react 15min；
(4) add stop buffer, mixing of vibrating gently, at wavelength 450nm, measure OD value with ELIASA.
With OD value as ordinate, with the log10 value of amantadine experimental solutions concentration as abscissa, draw semilog standard
Curve map.Calibration curve has complete reverse-s shape shape, and has upper mounting plate and lower platform, the parallel determination number of times 8 of calibration curve
Secondary, experimental repeatability is good, and relative standard deviation (coefficient of variation) is all within 10%.
Half amount of suppression (IC is drawn according to calibration curve50), determine detection sensitivity.
Inhibiting rate is with being calculated as follows:
In formula: ODmax: for being not added with light absorption value during standard items, ODx is light absorption value during standard items x, and ODmin is blank
The light absorption value in hole.
Being calculated amantadine antibody half amount of suppression (IC50) in buffer solution by above-mentioned formula is 1.3 μ g/L.
Embodiment 4, the enzyme linked immunological kit of detection amantadine and preparation thereof
One, enzyme linked immunological kit is made up of following substances:
1, the haptenic ELISA Plate of amantadine it is coated；
2, enzyme mark amantadine antibody working solution: enzyme labelled antibody solution described in embodiment 3；
3, amantadine standard items: amantadine standard solution concentration is respectively 0,0.5,1.5,4.5,13.5,40.5 μ g/L；
4, substrate nitrite ion: be made up of A liquid and B liquid, A liquid is the aqueous solution of 2% urea peroxide, and B liquid is 1% tetramethyl benzidine
(TMB) the aqueous solution；
5, stop buffer: 0.2M aqueous sulfuric acid；
6, concentrated cleaning solution: every 1 liter of described cleaning solution is prepared as follows and obtained: by 10mL Tween-20,5g nitrine
Change sodium and the mixing of 990mL phosphate buffer, obtain described cleaning solution；The concentration of described phosphate buffer is 0.01M pH value
It is 7.4；
Two, ELISA Plate and the preparation thereof of AMD-OVA it are coated with
It is coated the polystyrene ELISA Plate of AMD-OVA: with the carbonate solution of 0.05M by antigen diluent to 2.5 μ g/mL, be coated
96 hole polystyrene ELISA Plates, every hole 100 μ L, 37 DEG C of incubation 2h, incline and be coated liquid, wash 3 times with cleaning solution, each 10s,
Pat dry, in every hole, then add 150 μ L confining liquids, 37 DEG C of incubation 2h, liquid in hole of inclining, seal by aluminium film vacuum after drying
Preserve.
It is coated buffer solution: the sodium carbonate buffer of pH9.6,0.05mo1/L；
Confining liquid: every 1 liter of confining liquid is prepared as follows: by 5mL horse serum, 1g sodium azide, the mixing of 30g casein,
Dissolve with phosphate buffer and be settled to 1000mL, obtaining confining liquid；Wherein, the concentration of phosphate buffer is 0.02M,
PH value is 7.2.
Three, kit test method
(1) sample pre-treatments
Chicken, pork, duck, egg (coefficient of dilution: 2)
1, the sample after 2 ± 0.01 g homogeneous is accurately weighed in 50 mL centrifuge tubes；
2,1 mL 1% acetic acid solution, 7mL acetonitrile, violent whirling motion 3 min are added；
3, under room temperature (25 ± 2 DEG C), 4000 g are centrifuged 5min；
4,2 mL supernatants are taken in clean centrifuge tube；
5,60-70 DEG C of water-bath nitrogen dries up；
6,0.5mL sample diluting liquid, violent whirling motion 30 s are added；
7, take 200 L of supernatant liquid in new centrifuge tube, add 200 L sample diluting liquids, violent whirling motion 30 s；
8, take 50 L to detect.
(2) detect with kit
1, the making of calibration curve
In the ELISA Plate micropore be coated with AMD-OVA, add amantadine standard solution 50 μ L, be subsequently adding enzyme labelled antibody
Working solution 50 μ L/ hole, vibration mixes gently, with reacting 30min in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.Carefully take off
Uncap plate film, liquid in hole is dried, add wash operating solution 250mL/ hole, fully wash 4 ~ 5 times, every minor tick 10s, sprinkle
Cleaning solution in plate hole, pats dry with blotting paper.Addition substrate A liquid 50 μ L/ hole, substrate B liquid 50 μ L/ hole, mixing of vibrating gently, 25 DEG C
Insulating box lucifuge colour developing 15min, every hole adds stop buffer 50 μ L, mixing of vibrating gently, with ELIASA, measures every hole absorbance
Value.
With the absorbance values of the standard solution of each concentration divided by the extinction of first standard solution (0 standard)
Angle value (B0) it is multiplied by 100% again, obtain percentage absorbance.With the semilog value of amantadine standard concentration (μ g/L) as X
Axle, percentage absorbance is Y-axis, draws canonical plotting.The calibration curve obtained is as shown in Figure 4.
Percentage absorbance (%)=(B/B0) × 100%
2, the mensuration of Gold Samples firm alkanamine concentration
With the absorbance values (B) of each test sample solution divided by the absorbance of first standard solution (0 standard)
(B0) it is multiplied by 100% again, obtain percentage absorbance.The percentage absorbance of each test sample solution corresponding, then can be from
Reading the absorbance of test sample solution on calibration curve, the concentration value further according to standard solution converses in sample solution
The residual quantity of amantadine, is multiplied by the extension rate of each sample pretreatment process the most again, can calculate the firm alkane of Gold Samples
The concentration of amine.
Four, kit Detection results is evaluated
(1) degree of accuracy and precision test
To without the chicken meat sample of amantadine adds amantadine standard items, make amantadine standard items ends in the sample
Concentration is respectively 1,2,4 μ g/L；Sample after adding carries out pre-treatment according to method described in experiment three respectively, is detected
Sample solution.
From the kit of three different batches, 3 kits of each extraction detect, institute in detection method such as experiment three
Stating, each experiment is repeated 5 times, and calculates the coefficient of variation respectively.Result is shown in Table 1 respectively.
Table 1 degree of accuracy and Precision test result
Variation within batch coefficient: with the coefficient of variation of each parallel samples in once measuring.
Interassay coefficient of variation: same sample, in the coefficient of variation of different batches measurement result, takes its mean value.
Result shows: the average TIANZHU XINGNAO Capsul of chicken meat sample 86.9 ~ 97.3%, variation within batch coefficient 6.4 ~
13.6%, interassay coefficient of variation is at 9.8 ~ 10.1 %.
(2) kit storage life
Kit preservation condition is 2 ~ 8 DEG C, and through the mensuration of 15 months, the maximum absorbance value (0 standard) of kit, 50% pressed down
Concentration processed, amantadine add practical measurement value all within normal range (NR).During transport and using, have anon-normal
Often preservation condition occurs, places 9 days, be accelerated senile experiment under conditions of being preserved at 37 DEG C by kit, and result shows this
The indices of kit complies fully with requirement.Occur in view of kit freezing situation, kit is put into-20 DEG C of refrigerators cold
Freezing 9 days, measurement result also indicates that kit indices is the most normal.Can show that kit can be at 2 ~ 8 DEG C from result above
At least can preserve more than 12 months.
(3) cross reacting rate test
Select to carry out cross reaction test, by the calibration curve of various medicines with AMD structure or intimate other drug
Respectively obtain its 50% inhibition concentration.The kit cross reacting rate to other analog is calculated with following formula.Friendship with other drug
Fork reactivity is the least, illustrates that amantadine enzyme-linked immunologic detecting kit is the best to the detection of amantadine.Result is shown in
Table 2.
Table 2 amantadine kit cross reacting rate
Result of the test shows, kit of the present invention to the cross reacting rate of amantadine be 100%, Amoxicillin, Cloxacillin,
OXA, the cross reacting rate of Ceftiofur are respectively less than 1%, so the best to amantadine of kit, the i.e. present invention
Kit can detect amantadine.
Claims (10)
1. an amantadine haptens, for product shown in formula 1:
Formula 1.
The preparation method of product shown in formula 1 the most according to claim 1, comprises the steps: 0.94g amantadine hydrochloric acid
Salt is dissolved in 30ml anhydrous pyridine, adds maleic anhydride 0.49g, after dissolving, adds DMAP 10mg, and 70 DEG C of backflows are anti-
Answering 3h, rotation that pyridine is evaporated off, add deionized water 10ml, pH5.0 adjusted by glacial acetic acid, separates out a large amount of precipitation, filters, water washing and precipitating, dry
Haptens is obtained after dry.
3. an amantadine antigen, is conjugate product shown in formula 1 and carrier protein couplet obtained.
Amantadine antigen the most according to claim 3, it is characterised in that described carrier protein includes bovine serum albumin
In vain, ovalbumin, human serum albumins, mouse serum albumin, thyroprotein or hemocyanin.
5. the preparation method of amantadine antigen described in claim 3, comprises the steps: that weighing 5.57mg haptens is dissolved in
1ml DMF, adds EDC 4.3mg, NHS 5.2mg, and reaction 2h is stirred at room temperature；50mg BSA is dissolved in 5ml 0.1M sodium acid carbonate and delays
Rushing liquid, be added dropwise in protein solution by above-mentioned pharmacological activation, stirred overnight at room temperature, PBS 4 DEG C dialyses 72 hours, and period changes
Dislysate 6 times；Dislysate is aseptically crossed the filter membrane in 0.22 μm aperture, is sub-packed in ampere bottle ,-20 DEG C of preservations, with
Method prepares coating antigen.
6. amantadine antigen application in preparing amantadine specific antibody described in claim 3.
7. the specific antibody that amantadine antigen described in application claim 3 prepares.
8. antibody described in amantadine antigen, claim 7 described in product, claim 3 described in claim 1 is at detection Buddha's warrior attendant
Application in alkanamine.
9. specific antibody described in amantadine antigen, claim 7 described in product, claim 3 described in application claim 1
The enzyme-linked immunologic detecting kit prepared.
10. enzyme-linked immunologic detecting kit described in claim 9, it is characterised in that it includes: be coated with amantadine antigen
ELISA Plate, enzyme labelled antibody working solution, amantadine serial standards, substrate nitrite ion, stop buffer, concentration redissolution liquid, concentration are washed
Wash liquid.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610280030.1A CN105906522A (en) | 2016-04-29 | 2016-04-29 | Amantadine artificial antigen and preparation method and application thereof |
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|---|---|---|---|
| CN201610280030.1A CN105906522A (en) | 2016-04-29 | 2016-04-29 | Amantadine artificial antigen and preparation method and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106565846A (en) * | 2016-10-13 | 2017-04-19 | 华中农业大学 | Monoclonal antibody and enzyme-linked immunosorbent assay kit for detecting amantadine and rimantadine |
| CN108152499A (en) * | 2017-12-06 | 2018-06-12 | 华南农业大学 | A kind of antigen of amantadine, antibody and its enzyme-linked immunologic detecting kit |
| CN111533665A (en) * | 2020-05-29 | 2020-08-14 | 深圳市金阅科技有限责任公司 | Rimantadine hapten, rimantadine complete antigen, preparation method thereof and test strip |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6042991A (en) * | 1997-02-18 | 2000-03-28 | Fuji Photo Film Co., Ltd. | Positive working photosensitive composition |
| CN101643454A (en) * | 2009-05-27 | 2010-02-10 | 北京维德维康生物技术有限公司 | Melamine hapten and antigen as well as preparation method and application thereof |
| CN102952782A (en) * | 2011-08-31 | 2013-03-06 | 北京勤邦生物技术有限公司 | Vomitoxin hybridoma, monoclonal antibody, and preparation method and application of monoclonal antibody |
| CN103524427A (en) * | 2012-07-03 | 2014-01-22 | 北京勤邦生物技术有限公司 | Preparation method as well as application of trimethoprem hapten |
| CN104402753A (en) * | 2014-11-17 | 2015-03-11 | 浙江省农业科学院 | Amantadine artificial hapten and artificial antigen as well as preparation method and application of amantadine artificial hapten and artificial antigen |
| CN104480072A (en) * | 2014-11-17 | 2015-04-01 | 浙江省农业科学院 | Hybridoma cell strain secreting anti-amantadine monoclonal antibody and application of hybridoma cell strain |
| CN104897864A (en) * | 2015-06-18 | 2015-09-09 | 北京勤邦生物技术有限公司 | ELISA (enzyme linked immunosorbent assay) kit for detecting amantadine and application of ELISA kit |
| CN104892445A (en) * | 2015-05-14 | 2015-09-09 | 江南大学 | Preparation and application of highly specific amantadine artificial antigen |
| CN105112376A (en) * | 2015-09-07 | 2015-12-02 | 江南大学 | Amantadine monoclonal antibody hybridoma cell strain and application thereof |
| CN105319351A (en) * | 2014-07-24 | 2016-02-10 | 江苏维赛科技生物发展有限公司 | Adamantanamine enzyme-linked immunosorbent assay detection special-purpose detection kit |
-
2016
- 2016-04-29 CN CN201610280030.1A patent/CN105906522A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6042991A (en) * | 1997-02-18 | 2000-03-28 | Fuji Photo Film Co., Ltd. | Positive working photosensitive composition |
| CN101643454A (en) * | 2009-05-27 | 2010-02-10 | 北京维德维康生物技术有限公司 | Melamine hapten and antigen as well as preparation method and application thereof |
| CN102952782A (en) * | 2011-08-31 | 2013-03-06 | 北京勤邦生物技术有限公司 | Vomitoxin hybridoma, monoclonal antibody, and preparation method and application of monoclonal antibody |
| CN103524427A (en) * | 2012-07-03 | 2014-01-22 | 北京勤邦生物技术有限公司 | Preparation method as well as application of trimethoprem hapten |
| CN105319351A (en) * | 2014-07-24 | 2016-02-10 | 江苏维赛科技生物发展有限公司 | Adamantanamine enzyme-linked immunosorbent assay detection special-purpose detection kit |
| CN104402753A (en) * | 2014-11-17 | 2015-03-11 | 浙江省农业科学院 | Amantadine artificial hapten and artificial antigen as well as preparation method and application of amantadine artificial hapten and artificial antigen |
| CN104480072A (en) * | 2014-11-17 | 2015-04-01 | 浙江省农业科学院 | Hybridoma cell strain secreting anti-amantadine monoclonal antibody and application of hybridoma cell strain |
| CN104892445A (en) * | 2015-05-14 | 2015-09-09 | 江南大学 | Preparation and application of highly specific amantadine artificial antigen |
| CN104897864A (en) * | 2015-06-18 | 2015-09-09 | 北京勤邦生物技术有限公司 | ELISA (enzyme linked immunosorbent assay) kit for detecting amantadine and application of ELISA kit |
| CN105112376A (en) * | 2015-09-07 | 2015-12-02 | 江南大学 | Amantadine monoclonal antibody hybridoma cell strain and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 李杰: "《药物应用基础(上)》", 28 February 2011 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106565846A (en) * | 2016-10-13 | 2017-04-19 | 华中农业大学 | Monoclonal antibody and enzyme-linked immunosorbent assay kit for detecting amantadine and rimantadine |
| CN106565846B (en) * | 2016-10-13 | 2019-11-01 | 华中农业大学 | For detecting the monoclonal antibody and enzyme linked immunological kit of amantadine and Rimantadine |
| CN108152499A (en) * | 2017-12-06 | 2018-06-12 | 华南农业大学 | A kind of antigen of amantadine, antibody and its enzyme-linked immunologic detecting kit |
| CN111533665A (en) * | 2020-05-29 | 2020-08-14 | 深圳市金阅科技有限责任公司 | Rimantadine hapten, rimantadine complete antigen, preparation method thereof and test strip |
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