CN107688091A - A kind of immune quantitative test paper bar of quick detection cyproheptadine and preparation method thereof - Google Patents
A kind of immune quantitative test paper bar of quick detection cyproheptadine and preparation method thereof Download PDFInfo
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- CN107688091A CN107688091A CN201710797243.6A CN201710797243A CN107688091A CN 107688091 A CN107688091 A CN 107688091A CN 201710797243 A CN201710797243 A CN 201710797243A CN 107688091 A CN107688091 A CN 107688091A
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- G—PHYSICS
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- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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Abstract
Immune quantitative test paper bar of a kind of quick detection cyproheptadine provided by the invention and preparation method thereof, belongs to vitro detection technical field.Immune quantitative test paper bar provided by the invention is according to Immune competition reaction principle, establish a kind of rapid fluorescence immunologic function test reagent that cyproheptadine residual is detected suitable for animals urine, tissue, by fluorescent value in fluorescence detector quantitative test agent in detection process, to measure the residual of this medicine in detection sample.This kit has the advantages that simple to operate, high sensitivity, quantitative, specificity is good and detection time is short, is adapted to basic unit and field quick detection, and testing result preserved by detecting instrument, print and internet transmits superintendent office, to strengthen to on-site supervision.
Description
Technical field
The invention belongs to the immune quantitative examination of external diagnosis reagent technical field, more particularly to a kind of quick detection cyproheptadine
Paper slip and preparation method thereof.
Background technology
Cyproheptadine (Cyproheptadine, CHD) is a kind of antihistamine drug, and one kind makes H1 receptor antagonists, i.e.,
CNS stimulant, it is used clinically for the treatment of the diseases such as nettle rash, anaphylaxis and bronchial astehma.Cyproheptadine has anti-5- hydroxyls
The effect of tryptamines, the satiety center of hypothalamus can be suppressed and stimulate appetite and put on weight, Cyproheptadine is contained in livestock and poultry cultivation
Feed can promote fowl poultry kind growth and lean meat percentage.The Ministry of Agriculture has issued No. 1519 bulletins for 2010, discloses and forbids to society
The medicine and list of substances used in feed, animal drinking water and beasts, birds and aquatic products breeding process, cyproheptadine are clearly put into
Wherein.
In recent years because country continues to increase to the supervision of clenbuterol hydrochloride class medicine, criminal stares under desire for gain driving
Go up not only cheap and had a cyproheptadine for promoting growth function, so it is used as the substitute of clenbuterol hydrochloride, in animal feed and animal
This medicine is illegally added in drinking-water, because accumulation is caused containing its residual in animal derived food, people eat these foods
Product, weak, sleepy, dizzy, calm, respiration inhibition etc. can be produced, thus will produce very big harm to the health of human body.Reinforcement pair
Feed, the cyproheptadine residue detection of economic animal urine sample are the important means that ensures food safety, and detect cyproheptadine in food at present
The method of residual, the mainly tandem mass spectrometry of liquid chromatogram one (LC/MS/MS), this method sensitivity is higher, but detection time
It is long, operate that cumbersome, testing cost is high, this method is suitable only for conditional laboratory and carried out, and is not suitable for aquaculture and scene is fast
Fast examination.
Although ELISA detections can also reach the purpose accurately detected, ELISA is time-consuming and needs professional and is testing
Carried out in room, it is impossible to meet the requirement of quick detection.In recent years, the immunologic detection method for detecting cyproheptadine label has golden mark
Method, although methods described has, fast and accurately feature, detection sensitivity can not meet the requirement of high-sensitivity detection.
The content of the invention
In view of this, it is an object of the invention to provide a kind of immune detection quantitative test paper bar of quick detection cyproheptadine,
Make the quantitative test paper bar that there is higher sensitivity and accurate testing result, while qualitative and quantitative detection can be completed.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of immune quantitative test paper bar of quick detection cyproheptadine, including bottom plate, the bottom plate is under
Sample uptake zone, solid phase labelling antibody district, reaction zone and suction zones are disposed with upwards, the reaction zone is provided with detection line T
With nature controlling line C；The detection line T is coated with cyproheptadine comlete antigen, and the nature controlling line C is coated with sheep anti-mouse igg；The solid phase
Labelled antibody area is fixed with the anti-cyproheptadine antibody of fluorescent latex particles mark；
The coated mass concentration of cyproheptadine comlete antigen is 0.2~0.5mg/ml；
The coated mass concentration 0.1~0.3% of anti-cyproheptadine antibody of fluorescent latex particles mark.
Preferably, the anti-cyproheptadine antibody of the fluorescent latex particles mark is to be wrapped in every milliliter 0.1% of fluorescent latex
Quality containing antibody is 40~60 μ g.
Preferably, fluorescer is rare earth elements europium element in fluorescent latex particles.
Preferably, the anti-cyproheptadine antibody of fluorescent latex particles mark is to lead to fluorescent latex particles and anti-cyproheptadine antibody
Superamide key connection.
Preferably, the coated mass concentration of cyproheptadine comlete antigen is 0.3~0.4mg/ml on the detection line T.
Preferably, cyproheptadine comlete antigen is coupled connection by coupling agent by cyproheptadine and coupling protein and obtained；The idol
Connection albumen includes bovine serum albumin(BSA) or ovalbumin；The coupling agent is that 1- ethyls-(3- dimethylaminopropyls) carbon two is sub-
Amine.
Present invention also offers a kind of preparation method of the immune quantitative test paper bar of the quick detection cyproheptadine, including under
Row step：
1) the anti-cyproheptadine antibody that fluorescent latex particles mark is dissolved in buffer solution, the labelled antibody diluted,
The labelled antibody of the dilution is sprayed on solid phase labelling antibody district, the labelled antibody area after spraying carries out being placed on relative humidity small
Dried 7~9 hours in 20% and 30~38 DEG C of environment；
2) cyproheptadine comlete antigen solution is lined into reaction zone and forms detection line T lines, sheep anti-mouse igg solution is lined
The quality control region of reaction zone forms nature controlling line C line, and the reaction zone of line is placed on into the wet ring for being less than 20% and 30~38 DEG C of relative degree
Dried 7~9 hours in border；
3) reaction zone for obtaining the step 2) pastes fixed bottom plate center, and one end of reaction zone is pasted solid phase labelling and resisted successively
Body area and sample uptake zone, the other end of reaction zone stick suction zones, cut into bar again, obtain immune quantitative test paper bar；
The wherein limitation of step 1) and step 2) without time sequencing.
Preferably, the molar concentration that it is 1%BSA containing mass concentration that the buffer solution, which is, is 10mM, the PBS of pH value 7.2 is molten
Liquid.
A kind of immune quantitative test paper bar of quick detection cyproheptadine provided by the invention, detected using Immune competition method principle
Cyproheptadine, by the fluorescence labeling of cyproheptadine antibody, fluorescent value is quantitatively detected by luminoscope in detection process, to measure detection
The residual of cyproheptadine in sample.The immune quantitative test paper bar is easy to carry and preserved；Simple to operate, method is stable, can be single
Test, and test is completed within 10min.The concentration of immune quantitative test paper bar detection cyproheptadine of the present invention, the results showed that
The sensitivity of the immune quantitative test paper bar is 0.2ppb, and the range of linearity is 0~8ppb, and the current detection sensitivity of Gold standard exists
In the range of 5~10ppb, ELISA can accomplish 0.2ppb or so, but ELISA is time-consuming and needs professional and enters in the lab
OK, it is impossible to meet the requirement of quick detection.The present invention is applicable single detection simultaneously, applies also for high-volume examination and scene is examined
Survey.
Brief description of the drawings
Fig. 1 is the structural representation of immune quantitative test paper card of the present invention；
Fig. 2 is the standard curve for drawing to obtain in embodiment 3.
Embodiment
The invention provides a kind of immune quantitative test paper bar of quick detection cyproheptadine, including bottom plate, in the bottom plate
Surface is disposed with sample uptake zone, solid phase labelling antibody district, reaction zone and suction zones from bottom to top, and the reaction zone is provided with
Detection line T and nature controlling line C；The detection line T is coated with cyproheptadine comlete antigen, and the nature controlling line C is coated with sheep anti-mouse igg；
The solid phase labelling antibody district is fixed with the anti-cyproheptadine antibody of fluorescent latex particles mark；
The coated mass concentration of cyproheptadine comlete antigen is 0.2~0.5mg/ml；
The anti-coated mass concentration of cyproheptadine antibody of fluorescent latex particles mark is 0.1~0.3%.
The present invention has testing result accurate using Immune competition method principle detection cyproheptadine, the immune quantitative test paper bar
Really, the characteristics of high sensitivity, and it is easy to carry and preserves；Simple to operate, method is stable, single can test, and within 10min
Complete test.The advantages of with quantitative detection, testing result preserved by detecting instrument, is printed and internet transmission supervision portion
Door, to strengthen to on-site supervision and in-situ processing.
The structural representation of immune quantitative test paper card of the present invention is as shown in Figure 1.Wherein 1, sample pad is treated；2nd, it is solid
Phase labelled antibody area；3rd, detection line T；4th, nature controlling line C；5th, nitrocellulose membrane；6th, blotting paper；7th, PVC bottom plates；8th, paper slip examination card；
9th, well；10th, procuratorial work window.
Immune quantitative test paper bar provided by the invention includes bottom plate.Material, size and source of the present invention to the bottom plate
There is no special limitation, using the bottom plate well known to those skilled in the art being used in immune quantitative test paper bar.In this hair
In bright, the material of the bottom plate is preferably plastics or film condensation material.Bottom plate used is PVC board in the embodiment of the present invention, is city
Sell commodity.
Immune quantitative test paper bar provided by the invention includes being arranged on the sample absorption that the backplate surface is located at lowermost end
Area.In the present invention, the material of the sample uptake zone is preferably glass fibre.The present invention does not have to the source of the glass fibre
There are any restrictions, glass fibre well-known to those skilled in the art.The present invention is pasted onto bottom to the sample uptake zone
The lower end of plate, the lower edge of the sample uptake zone is concordant with bottom plate lower edge.Sample uptake zone constitutes about floor length
A quarter.
Immune quantitative test paper bar provided by the invention includes the solid phase labelling antibody district being arranged on bottom plate, the solid phase mark
Note antibody district is located above sample uptake zone, and the solid phase labelling antibody district absorbs area overlapping 1.5mm with sample and set.
In the present invention, the material of the solid phase labelling antibody district is preferably glass fibre or polyester film, more preferably polyester film.The present invention
There is no any restrictions, glass fibre or polyester well-known to those skilled in the art to the source of the glass fibre or polyester film
Film.
In the present invention, the solid phase labelling antibody district is fixed with the anti-cyproheptadine antibody of fluorescent latex particles mark, institute
In the fluorescent latex that the anti-cyproheptadine antibody for stating fluorescent latex particles mark is every milliliter 0.1% quality comprising antibody be 40~
60 μ g, more preferably 50ug.The anti-cyproheptadine antibody of fluorescent latex particles mark is preferably through fluorescent latex particles and anti-match
Pyridine in heptan antibody passes through acid amides key connection.Fluorescer is preferably rare earth elements europium element in fluorescent latex particles.The fluorescent latex
The source of particle is not particularly limited, using fluorescent latex particles well-known to those skilled in the art.
Immune quantitative test paper bar provided by the invention includes being arranged on reaction zone on the bottom plate, the reaction zone close to
The solid phase labelling antibody district, positioned at the top of the solid phase labelling antibody district, and the reaction zone and the solid phase labelling
Antibody area overlapping 1.5mm.In the present invention, the material of the reaction zone is preferably nitrocellulose filter or cellulose acetate film.
The present invention does not have any restrictions to the source of the nitrocellulose filter or cellulose acetate film, well known to those skilled in the art
Nitrocellulose filter or cellulose acetate film.
In the present invention, the reaction zone is provided with detection line T and nature controlling line C.In the present invention, the detection line T and
Nature controlling line C spacing is preferably 5mm, and the detection line T is located at below nature controlling line C, and the detection line T resists close to solid phase labelling
Body area, the nature controlling line C is close to water suction section.The spacing of the nature controlling line C and suction zones is preferably 3mm.On the detection line C
The coating quality concentration of sheep anti-mouse igg is 0.8~1.2mg/ml, more preferably 1.0mg/ml.The solvent of the sheep anti-mouse igg is
0.01M pH7.2 phosphate buffers containing 2% sucrose.
The present invention is one to the bar number of the detection line T.The coated matter of cyproheptadine comlete antigen on the detection line T
Amount concentration is 0.2~0.5mg/ml, preferably 0.3~0.4mg/ml.Cyproheptadine comlete antigen is led to by cyproheptadine and coupling protein
Coupling agent coupling connection is crossed to obtain；The coupling protein includes bovine serum albumin(BSA) or ovalbumin；The coupling agent is 1- second
Base-(3- dimethylaminopropyls) carbodiimide.The solvent of the cyproheptadine comlete antigen solution is mole dense containing 2% sucrose
It is 7.2 phosphate buffer to spend for 0.01mol/L, pH value.
Immune quantitative test paper bar provided by the invention includes being arranged on suction zones on the bottom plate, the suction zones close to
Reaction zone, the top of the reaction zone is arranged on, has overlapping 1.5mm, and the top of the reaction zone and bottom plate with reaction zone
Edge is concordant.In the present invention, the material of the suction zones is preferably blotting paper or filter paper.Source of the present invention to the blotting paper
There is no any restrictions, blotting paper well-known to those skilled in the art.
The present invention provides the plastic housing that immune quantitative test paper bar preferably also includes containing detector bar.In the present invention, it is described
Plastic housing includes bottom and the top cover coordinated with the bottom, and the bottom has width groove corresponding with quantitative test paper bar, recessed
The size of groove and the length and width of quantitative test paper bar are coincide, and are provided with the top cover and reaction zone and sample addition area's size
Identical hole, procuratorial work window and well are formed, so that operator passes through luminoscope test result.
The preparation method of immune quantitative test paper bar, comprises the following steps described in above-mentioned technical proposal provided by the invention：
1) the anti-cyproheptadine antibody that fluorescent latex particles mark is dissolved in buffer solution, the labelled antibody diluted,
The labelled antibody of the dilution is sprayed on solid phase labelling antibody district, the labelled antibody area after spraying is placed on that relative degree is wet to be less than
20% and 30~38 DEG C of environment in dry 7~9 hours；
2) detection zone that cyproheptadine comlete antigen solution is lined to nitrocellulose membrane forms detection line T lines, by sheep anti mouse
The quality control region that IgG solution lines nitrocellulose membrane forms nature controlling line C line, and it is wet small that the nitrocellulose membrane of line is placed on into relative degree
Dried 7~9 hours in 20% and 30~38 DEG C of environment；
3) nitrocellulose membrane of line is pasted into fixed bottom plate center, solid phase labelling antibody is pasted in one end of nitrocellulose membrane successively
Area and sample uptake zone, the reaction zone other end stick suction zones, cut into bar again, obtain immune quantitative test paper bar；
The wherein limitation of step 1) and step 2) without time sequencing.
The anti-cyproheptadine antibody of fluorescent latex particles mark is dissolved in buffer solution by the present invention, by the mark of obtained dilution
Note antibody is sprayed on solid phase labelling antibody district, and the labelled antibody area after spraying is dried.
In the present invention, the buffer solution including mass concentration is 0.5%BSA, mass concentration is 4% sucrose and volumetric concentration
Molar concentration for 0.5% Tween-20 is 10mmol/L, and pH value is 7.4 phosphate buffer solution.Marked after the dilution anti-
The mass concentration of body is preferably 0.1%~0.3%, and more preferably 0.2%.The concentration of antibody is 0.5~1% before dilution.
In the present invention, the preparation method of the anti-cyproheptadine antibody of fluorescent latex particles mark preferably includes following steps：
1. surface is carried to the europium latex beads and MES (MES) solution mixing, washing of carboxyl, separation of solid and liquid,
Surface after being washed carries the europium latex beads of carboxyl；
2. europium latex beads of the surface after washing with carboxyl is mixed into resuspension with MES solution, weight is obtained
Suspension；
3. by the re-suspension liquid and N- hydroxysuccinimides, 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine
Hydrochloride (EDC.HCl) admixture activation 50min~1.5h, second of separation of solid and liquid, obtains solid phase components；
4. the solid phase components are resuspended with MES solution, obtained re-suspension liquid mixes instead with cyproheptadine antibody
1.5~2.5h is answered, third time separation of solid and liquid, obtains the antibody of mark europium latex beads.
In the present invention, during the mixing, washing, europium latex beads quality in 10mmol MES solution
Concentration is 0.1~0.2%, more preferably 0.15%.The particle diameter of europium latex beads of the surface with carboxyl be preferably
200nm.The europium latex beads is visually seen as milky under visible light, purchased from Thermo Fisher.
In the present invention, the time of the activation is preferably 1h.In activation process, europium latex beads is sub- with N- maloyls
The mass ratio of amine and 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (EDC.HCl) is 1：0.15：0.15.
In the present invention, the re-suspension liquid and the reaction time of cyproheptadine antibody are preferably 2h.
In the present invention, the separation of solid and liquid, second of separation of solid and liquid and third time separation of solid and liquid are preferably to centrifuge.It is described from
The rotating speed of the heart is preferably 14000rpm.The time of the centrifugation is preferably 10~15min.
In the present invention, the method for resuspension is preferably resuspended using ultrasound.The ultrasonic power is preferably 1800w.It is described super
The sound time is preferably 1min, is spaced 5s.
In the present invention, 4. mixture preferably also includes after third time separation of solid and liquid in step：Europium latex will be marked micro-
The antibody of ball uses diluted to be resuspended into mass concentration for 0.5% solution and ultrasound.
Heretofore described dilution is preferably to include the molar concentration that mass concentration is 0.1~0.2%BSA to be
10mmol/L, pH value are 7.2 PBS.The mass concentration of the BSA is more preferably 0.15%.
In the present invention, after the labelled antibody diluted, the labelled antibody of dilution is fixed on solid phase labelling and resisted by the present invention
In body area, obtain being coated with the solid phase labelling antibody district of labelled antibody.The spraying is preferably to apply paving, hand coatings or instrument spray
Apply.
Fixed method is that the labelled antibody of dilution is sprayed in solid phase labelling antibody district in the embodiment of the present invention, described
Main machine sprays or applied by hand paving.After solid phase labelling antibody district after being sprayed, by the solid phase labelling antibody district after spraying
It is dried under drying room environment, obtains solid phase labelling antibody district.In the present invention, the temperature of the drying is preferably 30~38
DEG C, more preferably 37 DEG C, relative humidity≤20%, drying time is preferably 7~9h.
Cyproheptadine comlete antigen solution is lined formation detection line T lines on reaction zone by the present invention, by sheep anti-mouse igg solution
Line and nature controlling line C line is formed on reaction zone, the reaction zone rule.The present invention is not limited to the mode of the line
System, using labelled antibody spraying method well known to those skilled in the art.Line uses lining instrument in the embodiment of the present invention
Rule, the model HM3230 of the lining instrument, purchased from Harbin De Yuan Science and Technology Development Co., Ltd.s.The coating buffer spray
Painting amount is preferably to spray 24ul on wide, long 30cm nitrocellulose filters bar per 2cm.
In the present invention, the preparation method of cyproheptadine comlete antigen solution comprises the following steps：
Substitution reaction occurs for I, cyproheptadine hydrochlorides and 4- bromo-butyric acids, obtains N- butyric acid cyproheptadine (CHD-COOH)；
The CHD-COOH and carrier protein be coupled under EDC by II, obtain cyproheptadine comlete antigen solution.
In the present invention, the mol ratio of cyproheptadine hydrochloride and the 4- bromo-butyric acid is preferably 1：1.5, flow back in methanol, instead
The temperature answered is 65~70 DEG C, more preferably 68 DEG C；The time of reaction is preferably 4~6h, more preferably 5h.
In the present invention, the coupling carrier is preferably BSA or OVA.The temperature of the coupling is preferably 20~30 DEG C, more excellent
Elect 23~27 DEG C, most preferably 25 DEG C as.
It is of the invention by sample uptake zone, solid phase labelling antibody district, reaction zone after obtaining solid phase labelling antibody district and reaction zone
It is fixed on after being cut with suction zones on bottom plate, the bottom plate assembled.
The present invention is preferably first from bottom to top to paste sample uptake zone, solid phase labelling antibody successively in bottom plate to the assembling
Area, reaction zone, suction zones, it is described to paste the overlapping 1.5~2mm for making each area.
After the bottom plate assembled, the present invention is cut the bottom plate of assembling, obtains immune quantitative test paper bar.The present invention
The method of the slitting is not particularly limited, using slitting method well-known to those skilled in the art.It is of the invention real
Apply slitting in example to cut using cutting machine, the cutting machine model ZQ4200, biological Co., Ltd is marked purchased from Shanghai gold.
In the present invention, the width of the slitting is preferably 2.5~5mm, more preferably 3mm.
In the present invention, the slitting places bottom, then covers on bottom the top cover that the bottom coordinates, is detected
Card.
In the present invention, the test strips are in use, the data that the colour developing situation of the detection line T is determined with fluorescence detector
Obtain quantitative testing result.The quantitative values of detection sample are obtained according to default standard curve control.
In the present invention, the detected sample that the sample uptake zone absorbs is preferably blood, serum, blood plasma or urine.Institute
The source for stating blood does not limit, using blood well-known to those skilled in the art or serum, blood plasma.The urine
Source does not limit, using urine well-known to those skilled in the art.
In the present invention, the application method of the immune quantitative test paper bar is not particularly limited, using those skilled in the art
Known detection method.In the embodiment of the present invention, detection sample is added drop-wise to the sample uptake zone of test strips, stands 5
After~10min, detection examination card insertion is entered into Fluorescent reader and measures its fluorescent value, obtains measurement result.
With reference to embodiment to quick detection cyproheptadine immune quantitative test paper bar provided by the invention and preparation method thereof
It is described in detail, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
It is prepared by antibody antigen
1st, the preparation of cyproheptadine comlete antigen
Cyproheptadine hydrochloride (CHDNHCl) and 4- bromo-butyric acids are combined into CHD-COOH, CHD-COOH carrier protein couplet shapes
Into comlete antigen.CHDNHCl 30mg add 2ml methanol and add 200ul pyridines, be heated to reflux that 23mg is added dropwise in three mouthfuls of anti-bottles
4- bromo-butyric acids, flow back 4-5 hours, add 3ml deionized waters to be extracted with benzene, rotary evaporation removes benzene liquid, obtains 30mg white solids and connects
The haptens CHD-COOH of arm.10mg haptens CHD-COOH, 8mgNHS and 10mg EDC are dissolved in DMF, 4 DEG C stirred
Night.Next day, first 30mg BSA (or OVA) are dissolved in 3ml 0.02mol/L disodium phosphate soln, and by upper reaction solution by
It is added dropwise to, stirs 6h.Load bag filter, with 10mM PBS, and change liquid 2 times, obtain cyproheptadine comlete antigen.
2nd, the preparation of cyproheptadine antibody
By immunizing antigen by the dosage of 50ug/ml with waiting subcutaneous multi-point injection of the fully emulsified postabdomen of body Freund's adjuvant to exempt from
4 6-8 week old male BALB/C mices of epidemic disease, it is immunized 7-10 days, tail vein blood, indirect ELISA detection serum titer.Will be immune
The preferable mouse spleen of effect passes through PEG mediated fusions, indirect ELISA screening positive clone with myeloma cell SP2/0；It is positive
The monoclonal cell strain that inheritance stability is obtained after limiting dilution is subcloned is cloned, after cell line expands culture, is inoculated with and is planted through drop
The ascites of alkane sensitization BALB/C mice abdominal cavity induction production monoclonal antibody.Ascites slightly purifies through thiamines, then the purifying of sad thiamines,
Obtain corresponding monoclonal antibody.
Embodiment 2
Cyproheptadine preparative chromatography detection reagent card
1st, Eu is wrapped up3+Fluorescent latex mark cyproheptadine antibody：The particle diameter for taking 100ul mass concentrations to be 1% is 200nm tables
Face carries the europium latex beads of carboxyl, adds 900ul 0.01M MES pH 6.1 to wash, and 14000rpm centrifugation 10min, abandons supernatant.
Add 1ml 0.01M MES pH6.1 ultrasounds to be resuspended, add 15ul10mg/ml NHS (N- hydroxysuccinimides) and 15ul
10mg/ml EDC.HCl (1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate), priming reaction 1 is small at room temperature
When, 14000rpm centrifugation 10min, abandon supernatant.It is resuspended with 1ml 0.01M MES pH6.1 ultrasounds, adds cyproheptadine antibody
0.06mg is mixed, (25-30 DEG C) reaction 2h of room temperature.Latex after mark centrifuges 30min with 14000rpm, abandons supernatant.Add 1ml
MES ultrasounds of the 0.01M pH6.1 containing 0.2%BSA is resuspended, and closes 2 hours, 14000rpm centrifugations, 10 minutes, abandons at room temperature
Clearly, emulsion particle is resuspended with 0.2ml 10mM pH7.2 PBS dilutions containing 0.1%BSA ultrasound, i.e. concentration is that 0.5% mark is glimmering
Light latex.
2nd, the preparation of latex fluorescence labeling pad：
By the mark fluorescent latex of preparation with containing 0.5%BSA, 4% sucrose and 0.5% Tween-20 surfactant
PH7.4 10mM phosphate buffer solutions dilution, is made into 0.1% concentration, is equably coated on polyester film, the mark being coated with
At 37 DEG C, relative humidity is less than 20% hothouse and dried 8 hours pad.
3rd, the preparation of nitrocellulose filter detection zone and check plot
0.01M pH7.2 phosphate of the concentration 0.4mg/ml containing 2% sucrose is coated with the detection zone of nitrocellulose membrane to delay
CHD-BSA (cyproheptadine-bovine serum albumin(BSA)) conjugate in fliud flushing, while contain in check plot coating concentration for 0.8mg/ml
The sheep anti-mouse igg of the 0.01M pH7.2 phosphate-buffereds of 2% sucrose, package amount are that the nitrocellulose membrane of the length per 30cm sprays 24 μ l,
At 37 DEG C, relative humidity is less than 20% hothouse and dried 9 hours.
4th, the preparation of sample pad
According to the form below 1, which is formulated and (calculated by mass percentage), to be prepared, and is uniformly layered on sample treatment pad and is maintained 37 DEG C, phase
It is less than 20% to humidity to dry.
The sample pad prescription for the treatment of liquid of table 1
| Title | Content |
| Sodium taurocholate | 0.5% |
| S17 | 1% |
| S9 | 0.3% |
S17 (Rhodasurf ON-870, RHODIA, INC), S9 (Tetronic-1037, RHODIA, INC).
5th, test card assembles
It is coated with nitrocellulose filter, blotting paper, fluorescent latex label pad, the sample of CHD-BSA conjugates and sheep anti-mouse igg
Pad is attached in PVC board forms test-strips successively, and is loaded, as shown in figure 1, examination is snapped fits into aluminium foil bag, to seal composition complete
Test card.
Embodiment 3
Sample detection
A. standard curve is established
The 0.01M pH7.2 for the cyproheptadine that 1.0ml concentration is 8ng/ml PBS solution is prepared, takes 500ul to be carried out with PBS
The cyproheptadine solution of doubling dilution acquisition various concentrations, (8.00,4.00,2.00,1.00,0.5,0.25,0.125,0.00ng/
Ml) using PBS as blank control.Take 100ul standard items to be fed directly in strip or the sample pad of examination card, react 8 minutes at room temperature,
The fluorescent value for card insertion will be detected entering Fluorescent reader again and detect T and C areas.
B. data processing
After the fluorescent value for obtaining T lines and C lines, T/C ratios (being shown in Table 2) are calculated, using sample concentration as X-axis, T/C ratios are
Y-axis makees curve map.
T/C ratio statistical results under 2 different sample concentrations of table
| Concentration (ng/ml) | 0 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 |
| T/C | 1.51 | 1.46 | 1.33 | 1.1 | 0.91 | 0.65 | 0.36 | 0.15 |
Using T/C ratios as ordinate, the logarithm of cyproheptadine mass concentration is the linear relationship that abscissa is mapped well, line
Property regression equation is Y=-0.754X+0.854, R2=0.99.
C. detecting step：
Reagent is taken out, is kept flat on the table.
100ul measuring samples are drawn with liquid-transfering gun, are dripped in well, and start timing；
In room temperature reaction 8min, then detection examination card insertion is entered into Fluorescent reader and measures its fluorescent value, obtain measure knot
Fruit.
D. detection sensitivity：
0.2ppb can be detected, the range of linearity is 0~8ppb.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (8)
1. a kind of immune quantitative test paper bar of quick detection cyproheptadine, including bottom plate, the bottom plate are disposed with from bottom to top
Sample uptake zone, solid phase labelling antibody district, reaction zone and suction zones, it is characterised in that the reaction zone is provided with detection line T and matter
Control line C；Cyproheptadine comlete antigen is coated with the detection line T, sheep anti-mouse igg is coated with the nature controlling line C；The solid phase
Labelled antibody area is fixed with the anti-cyproheptadine antibody of fluorescent latex particles mark；
The coated mass concentration of cyproheptadine comlete antigen is 0.2~0.5mg/ml；
The coating quality concentration of the anti-cyproheptadine antibody of fluorescent latex particles mark is 0.1%~0.3%.
2. immune quantitative test paper bar according to claim 1, it is characterised in that the anti-match of the fluorescent latex particles mark
The quality comprising antibody is 40~60 μ g in the fluorescent latex that pyridine in heptan antibody is every milliliter 0.1%.
3. immune quantitative test paper bar according to claim 1, it is characterised in that fluorescent latex particles include fluorescer and breast
Glue particle；The fluorescer is rare earth elements europium element.
4. the immune quantitative test paper bar according to claims 1 to 3 any one, it is characterised in that the fluorescent latex
The anti-cyproheptadine antibody of grain mark is to obtain fluorescent latex particles and anti-cyproheptadine antibody by acid amides key connection.
5. immune quantitative test paper bar according to claim 1, it is characterised in that cyproheptadine is completely anti-at the detection line T
Former mass concentration is 0.3~0.4mg/ml.
6. immune quantitative test paper bar according to claim 5, it is characterised in that the cyproheptadine comlete antigen is by cyproheptadine
Connection is coupled with coupling protein by coupling agent to obtain；The coupling protein includes bovine serum albumin(BSA) or ovalbumin；It is described
Coupling agent is 1- ethyls-(3- dimethylaminopropyls) carbodiimide.
7. the preparation method of the immune quantitative test paper bar of quick detection cyproheptadine described in claim 1~6 any one, its feature
It is, comprises the following steps：
1) the anti-cyproheptadine antibody that fluorescent latex particles mark is dissolved in buffer solution, the labelled antibody diluted, by institute
The labelled antibody for stating dilution is coated in solid phase labelling antibody district, and the solid phase labelling antibody district after spraying is placed on relative humidity less than 20%
With dry 7~9h in 30~38 DEG C of environment；
2) cyproheptadine comlete antigen solution is lined into reaction zone and forms detection line T lines, sheep anti-mouse igg solution is lined into reaction
Area forms nature controlling line C line, by the reaction zone of line be seated in it is relatively wet be less than in 20% and 30~38 DEG C of environment dry 7~9
Hour；
3) reaction zone for obtaining the step 2) fixes bottom plate center, and solid phase labelling antibody is pasted in one end of nitrocellulose membrane successively
Area and sample uptake zone, the other end patch suction zones of reaction zone, cut into bar, obtain immune quantitative test paper bar；
The wherein limitation of step 1) and step 2) without time sequencing.
8. preparation method according to claim 7, it is characterised in that the BSA that it is 1% containing mass concentration that the buffer solution, which is,
The PBS solution that molar concentration is 10mmol/L, pH value is 7.2.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110646616A (en) * | 2019-09-05 | 2020-01-03 | 桂林理工大学 | Hypersensitivity fluorescence quenching immunosensor for detecting human cTnI in serum and detection method |
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