CN107247141A - A kind of Immunofluorescence test card for determining malachite green and preparation method and application - Google Patents
A kind of Immunofluorescence test card for determining malachite green and preparation method and application Download PDFInfo
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- CN107247141A CN107247141A CN201710351734.8A CN201710351734A CN107247141A CN 107247141 A CN107247141 A CN 107247141A CN 201710351734 A CN201710351734 A CN 201710351734A CN 107247141 A CN107247141 A CN 107247141A
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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
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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Abstract
The invention discloses a kind of Immunofluorescence test card for determining malachite green and preparation method and application, detection card includes backing plate, invests sample pad closely coupled on the backing plate and successively, coupling matter pad, reaction film and adsorptive pads, the malachite green antibody that fluorescent microsphere is marked on coupling matter pad, reaction film is provided with detection line and nature controlling line, detection line is close to coupling matter pad, nature controlling line is close to adsorptive pads, the secondary antibody that identification malachite green antibody is coated with malachite green antigen, nature controlling line is coated with detection line；Its preparation method includes preparing sample pad, prepares coupling matter pad, prepare reaction film, prepare the step of adsorptive pads, assembling；Sample to be determined is added in sample pad, immune response is carried out, then the Immunofluorescence test for determining malachite green is placed on fluorescence chart scanner, the intensity to fluorescence signal is read out, you can obtain testing result；It is quick and convenient that the detection card detects that malachite green is used, and sensitivity is very high, can reach 0.1ng/mL.
Description
Technical field
The present invention relates to immunofluorescence analysis technical field, and in particular to a kind of Immunofluorescence test of measure malachite green
Card and preparation method and application.
Background technology
Malachite green (Malachite Green, MG) is a kind of artificial synthesized organic dyestuff, is once widely used in prevention
With saprolegniasis, branchiomycosis and the ich for treating all kinds of aquatic livestocks etc..But because malachite green and its metabolite are colourless
Malachite green can produce high residue in aquatic products body, easily cause the side effects such as carcinogenic, teratogenesis and mutagenesis after people is edible.Europe
The country such as alliance, U.S. has forbidden malachite green being used as the antimicrobial of fishing class, and China also arranges malachite green in May, 2002
Enter《The veterinary drug and its compound inventory of food animal disabling》In.Being presently used for the main method of malachite green vestigial detection is
Instrumental method, because its sample pretreatment process is cumbersome time-consuming, testing cost is high, is unfavorable for extensive use.
Immunofluorescence analysis technology is that the one kind gradually grown up in immunolabelling technique is used as mark using fluorescence
Thing is applied to the analytical technology of antigen-antibody reaction.Can be for detection antigen using immunofluorescence analysis technology, it is also possible to come
Detect antibody.Wherein it is used to detect that the method for antigen is more commonly used, commonly referred to as fluorescence anti-body method.
Thousands of fluorescence molecules can be wrapped up with fluorescent microsphere as label, in each microballoon, so as to carry
The efficiency of height mark, effectively raises detection sensitivity；There is the carboxyl of Suitable Density in fluorescent microsphere surface modification simultaneously, use
In the covalent coupling with albumen or antibody, the stability of label is improved.It is different from traditional Collaurum marking, using fluorescence
When immuno analytical method is detected, its signal specificity is strong, sensitivity is high, is particularly suitable for field quick detection.
Immunochromatography technique is also known as lateral flow technology, is the Ag-Ab immunoassay using solid phase test strips as carrier
Technology.General test strips are made up of sample pad, conjugate pad, nitrocellulose filter and adsorptive pads.Sample pad therein and combination
Pad is glass fiber paper, and sample pad is that testing sample originates flow region, and conjugate pad is label adsorption zone, nitrocellulose filter one
As be made up of detection line and nature controlling line, be the reaction zone of immunoassay, adsorptive pads provide siphon power for the flowing of chromatographic solution.Exempt from
Epidemic disease chromatographic technique biggest advantage is exactly that detection speed is fast, general detection used time 10-15min, it is not necessary to large-scale instrument and equipment, can
For Site Detection.
But at present there is the problem of sensitivity is inadequate in the detection card and detection method of detection malachite green, it is desirable to provide
A kind of higher detection card of sensitivity and detection method.
The content of the invention
The technical problems to be solved by the invention are to provide a kind of immunofluorescence inspection of the high measure malachite green of sensitivity
Survey card and preparation method and application.
The technical solution used in the present invention is：
A kind of Immunofluorescence test card for determining malachite green, including backing plate, in addition to invest on the backing plate and successively
The malachite green that fluorescent microsphere is marked on closely coupled sample pad, coupling matter pad, reaction film and adsorptive pads, the coupling matter pad
Antibody, the reaction film is provided with detection line and nature controlling line, and the detection line is close to the coupling matter pad, and the nature controlling line is close
It is coated with the adsorptive pads, the detection line on malachite green antigen, the nature controlling line and is coated with identification malachite green antibody
Secondary antibody.
Some preferred embodiment in, the Immunofluorescence test card also include upper shell and lower casing, it is described on
Shell is placed on above the sample pad, the coupling matter pad, the reaction film and the adsorptive pads, and the lower casing is located at described
The lower section of backing plate, the upper shell and the lower casing are mutually covered, and the upper shell is opened in the position of the correspondence sample pad
Provided with well, the upper shell is provided with observation window in the position of the correspondence reaction film.
In addition, present invention also offers a kind of preparation side of the Immunofluorescence test card as described above for determining malachite green
Method, comprises the following steps：
Prepare sample pad；
Prepare coupling matter pad：Coupling matter pad film is cut, the malachite green antibody for taking fluorescent microsphere to mark is sprayed on the film
On, dry；
Prepare reaction film：Reaction film is taken, malachite green antigen solution is sprayed on the reaction film, detection line is formed,
The two corresponding anti-solution of spraying identification malachite green antibody, forms nature controlling line on the reaction film；
Assembling detection card：Adsorptive pads and backing plate are taken, overlap joint is pasted the sample pad, described coupled successively on the backing plate
Thing pad, the reaction film and the adsorptive pads.
Some preferred embodiment in, the preparation method of the malachite green antibody of fluorescent microsphere mark include with
Lower step：
(1) fluorescent microsphere is activated：Fluorescent microsphere to be activated is added in buffer solution, washing, separation of solid and liquid, supernatant discarding
Liquid, collects solid matter and redissolves in buffer solution, n-hydroxysuccinimide and 1- (3- diformazan ammonia are added into the buffer solution
Base propyl group) -3- ethyl-carbodiimide hydrochlorides, oscillating reactions, separation of solid and liquid collects solid matter and is simultaneously dissolved in PB buffer solutions
In, obtain fluorescent microsphere solution；
(2) antibody labeling：Take malachite green antibody to be dissolved in PBS, and be added into the fluorescent microsphere solution
In, oscillating reactions for a period of time, adds monoethanolamine oscillating reactions for a period of time, adds bovine serum albumin solution, and closing is anti-
Should for a period of time, separation of solid and liquid is collected solid matter and suspended again with PB buffer solutions, obtains the malachite green of fluorescent microsphere mark
Antibody.
In further preferred embodiment, the n-hydroxysuccinimide added into the buffer solution and 1- (3-
Dimethylamino-propyl) -3- ethyl-carbodiimide hydrochlorides mass ratio be 1：1.
In further preferred embodiment, the malachite green antibody is with malachite green hapten and carrier protein
Conjugate be antigen obtain.
In further preferred embodiment, the preparation of the malachite green hapten comprises the following steps：Weigh 3-
Nitrobenzaldehyde and DMA, mixing add the concentrated sulfuric acid thereto, and for a period of time, reaction solution is turned for heating response
Enter in sodium carbonate liquor, be extracted with ethyl acetate, take organic phase saturated common salt water washing, then remove what is dissolved in organic phase
Water, suction filtration obtains solid matter, crosses silica gel column chromatography, using ethyl acetate/n-hexane mixed liquor as solvent, obtains in the middle of first
Product, weighs first intermediate product, and tetrahydrofuran, methanol and palladium carbon are added thereto, and decompression removes air, and room temperature hydrogenation is put
Put, obtain the second intermediate product, weigh second intermediate product, add it in anhydrous pyridine, add glutaric anhydride,
It is stirred at room temperature, concentrates dry pyridine, add ethyl acetate and water, aqueous phase is extracted with ethyl acetate, merges organic phase, take organic phase to use
Saturated common salt water washing, then the water dissolved in organic phase is removed, suction filtration obtains malachite green hapten.
In further preferred embodiment, the carrier protein is bovine serum albumin(BSA).
Some preferred embodiment in, the preparation of the malachite green antibody comprises the following steps：With malachite green
The conjugate of haptens and carrier protein is antigen, the mice immunized with antigen is used, by the spleen cell of immunized mice and mouse source bone
Myeloma cells are merged, screening, obtain secreting the hybridoma cell strain of malachite green monoclonal antibody, hole is obtained by preparing ascites
Sparrow malachite green antibody.
In addition, present invention also offers a kind of using the Immunofluorescence test card progress as described above for determining malachite green
The method of malachite green detection, comprises the following steps：
(1) sample to be determined is added in the sample pad, carries out immune response；
(2) Immunofluorescence test for determining malachite green is placed on fluorescence chart scanner, the intensity to fluorescence signal is entered
Row is read；
(3) judgement of result：When the detection line and the nature controlling line show orange when 345nm excites light irradiation similarly hereinafter
Fluorescence, and deep or both the color of the detection line nature controlling line is same or like, that is, it is feminine gender, explanation to represent testing result
Malachite green is not contained in detectable substance or contained malachite green concentration is less than 0.1ng/mL；When the detection line and the nature controlling line
In the case where 345nm excites light irradiation, the nature controlling line shows fluorescent orange, and the detection line does not show fluorescence or the nature controlling line
Colour developing is shallow, that is, represents that testing result is the positive, illustrates to contain malachite green in detectable substance and concentration is more than 0.1ng/mL.
The beneficial effects of the invention are as follows：
The present invention devises a kind of new Immunofluorescence test card for being used to determine malachite green, by malachite green antigen
It is coated on reaction film and forms detection line, the secondary antibody of malachite green antibody is coated on reaction film and forms nature controlling line, by fluorescence
The malachite green antibody of microballoon mark is coated on coupling matter pad, obtained Immunofluorescence test card, the inspection for malachite green
Survey very easy to use, as long as sample extracting solution to be determined is put into sample pad, fluorescence signal is then read after immune response
Whether intensity, the process of immune response can also obtain as a result, it is possible to contain in quick detection sample quickly, within 10min
There is malachite green, and the detection sensitivity of malachite green is very high, can reach 0.1ng/mL, disclosure satisfy that food security device to hole
Sparrow malachite green remains the demand of quick detection, is particularly suitable for supervision department's Site Detection and uses, the detection card is easy to use, quick,
Detection sensitivity is high.
Brief description of the drawings
Fig. 1 is the structural representation of the Immunofluorescence test card of the measure malachite green of embodiment 1.
Fig. 2 is the structural representation of the Immunofluorescence test card of the measure malachite green of embodiment 2.
Embodiment
Embodiment 1：
Reference picture 1, Fig. 1 is the structural representation of the Immunofluorescence test card of the measure malachite green of embodiment 1, this implementation
Example provides a kind of Immunofluorescence test card for determining malachite green, including backing plate 1, in addition to invest on the backing plate 1 and according to
Fluorescent microsphere is marked on secondary closely coupled sample pad 2, coupling matter pad 3, reaction film 4 and adsorptive pads 5, the coupling matter pad 3
Malachite green antibody, the reaction film 4 be provided with detection line 6 and nature controlling line 7, the detection line 6 close to the coupling matter pad 3,
The nature controlling line 7 is coated with malachite green antigen, the nature controlling line 7 on the adsorptive pads 5, the detection line 6 and is coated with
There is the secondary antibody of identification malachite green antibody.
The as described above Immunofluorescence test card for determining malachite green is through the following steps that prepare：(1) make
Standby sample pad 2：1.1cm × 10cm plain the film of glass fibre is cut, it is uniformly soaked in 10min in confining liquid, room temperature is clean
Condition is dried in vacuo, and obtains sample pad 2 standby；(2) coupling matter pad 3 is prepared：20mm × 4mm glass fibre membrane is cut, is taken suitable
The malachite green antibody of the fluorescent microsphere mark of amount is sprayed on glass fibre membrane with BIODOT specking instruments by 4ul/cm, and 37 DEG C are done
Dry 1h, obtains coupling matter pad film 3；(3) reaction film 4 is taken, in the market has purchasing for given size：Take reaction film 4, reaction film 4
It is the region that Immunofluorescence test card carries out immunoassay, malachite green antigen solution is sprayed on the reaction film 4, forms inspection
Survey line 6, the two corresponding anti-solution of spraying identification malachite green antibody, forms nature controlling line 7 on the reaction film 4；(4) adsorptive pads 5 are taken,
Can market directly buy；(5) assembling detection card：The backing plate 1 for taking market to buy, overlap joint pastes the sample successively on backing plate 1
Pad 2, the coupling matter pad 3, the reaction film 4 and the adsorptive pads 5.
The malachite green antibody of the fluorescent microsphere mark is prepared by following steps：(1) fluorescent microsphere is activated：Will
Fluorescent microsphere to be activated is added in buffer solution, and the buffer solution is 2- (N- morpholines) ethyl sulfonic acid solution that pH is 6.0, ultrasound
Wash microballoon several minutes, high speed refrigerated centrifuge 15min abandons supernatant, repeat washing step, collect solid matter and add buffering
In liquid, the buffer solution is 2- (N- morpholines) ethyl sulfonic acid solution that pH is 6.0, and N- hydroxyls are successively added into the buffer solution
Succinimide (NHS) and 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDS), N- hydroxysuccinimidyls acyl are sub-
The mass ratio of amine (NHS) and 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDS) is 6:1, shaken at room temperature is anti-
30min is answered, above-mentioned reaction solution is centrifuged into 15min, supernatant is discarded, solid matter is collected and is dissolved in PB buffer solutions
(0.05mol/L, pH be 7.4) in, obtain fluorescent microsphere solution；(2) antibody labeling：Malachite green antibody is taken to be dissolved in PBS bufferings
In liquid (pH is 7.4), antibody concentration is diluted to 1mg/mL, and is added into the fluorescent microsphere solution, shaken at room temperature is anti-
60min is answered, the μ L of monoethanolamine 10, shaken at room temperature reaction 60min is added, adds bovine serum albumin solution (BSA solution), close
60min is reacted, above-mentioned reaction system is centrifuged into 15min, supernatant is abandoned, solid matter PB buffer solutions (0.05mol/L, pH is collected
7.4) to be resuspended, the malachite green antibody of fluorescent microsphere mark, 4 DEG C of preservations are obtained.
Above-mentioned malachite green antibody is obtained by antigen of the conjugate of malachite green hapten and carrier protein, specifically
Step is：Conjugate using malachite green hapten and bovine serum albumin(BSA) (BSA) uses the mice immunized with antigen as antigen,
The spleen cell of immunized mice is merged with mouse source myeloma cell, screened, obtains secreting the hybridization of malachite green monoclonal antibody
Tumor cell strain, substantial amounts of malachite green antibody is can obtain by preparing ascites.And malachite green hapten is by following step
Suddenly prepare：1) in 100mL single port bottle, 3- nitrobenzaldehydes (12.81g, 84.77mmol) and N, N- dimethyl are added
Aniline (24.76g, 204.3mmol), adds the concentrated sulfuric acid (5mL), is heated to 120 DEG C and reacts 48 hours, reaction solution is transferred into matter
Fraction is measured in 20% sodium carbonate liquor, to be extracted with ethyl acetate twice, organic phase saturated common salt water washing is taken, then with nothing
Aqueous sodium persulfate dries the water for removing and being dissolved in organic phase, and suction filtration obtains solid matter, then using dry method loading column chromatography again, crosses silicon
Glue post (silica gel 300-400 mesh), solvent is ethyl acetate：N-hexane=1:5 (volume ratios), obtain 3.2g single-points yellow and consolidate
Body, is the first intermediate product, weighs first intermediate product, in the single port bottle for adding 100mL, and 70mL tetrahydrochysenes are added thereto
Furans, 70mL methanol and 280mg palladium carbons, decompression remove air, and room temperature hydrogenation is placed, and is obtained the second intermediate product, is weighed described the
Two intermediate product 200mg (0.579mmol), are added it in 5mL anhydrous pyridines, add glutaric anhydride (80mg,
0.701mmol), it is stirred at room temperature, concentrates dry pyridine, add ethyl acetate and water, aqueous phase is extracted with ethyl acetate, merges organic
Phase, takes organic phase saturated common salt water washing, then anhydrous sodium sulfate drying to remove the water dissolved in organic phase, suction filtration obtains green
Color solid 170mg, as malachite green hapten, obtain the haptens of solid forms, more easy to maintain, are also convenient for next step coupling
Reaction, because reagent associated with idol is different from the reagent of this synthetic reaction.Though hapten synthesis yield is low, raw material it is sensitive
Degree is higher.
The method that malachite green detection is carried out using the Immunofluorescence test card of said determination malachite green, including following step
Suddenly：
(1) sample to be determined is added in the sample pad, carries out immune response；
(2) Immunofluorescence test for determining malachite green is placed on fluorescence chart scanner, the intensity to fluorescence signal is entered
Row is read；
(3) judgement of result：When the detection line and the nature controlling line show orange when 345nm excites light irradiation similarly hereinafter
Fluorescence, and deep or both the color of the detection line nature controlling line is same or like, that is, it is feminine gender, explanation to represent testing result
Malachite green is not contained in detectable substance or contained malachite green concentration is less than 0.1ng/mL；When the detection line and the nature controlling line
In the case where 345nm excites light irradiation, the nature controlling line shows fluorescent orange, and the detection line does not show fluorescence or the nature controlling line
Colour developing is shallow, that is, represents that testing result is the positive, illustrates to contain malachite green in detectable substance and concentration is more than 0.1ng/mL.The detection
The sensitivity of card detection malachite green is very high, can reach 0.1ng/mL.
Embodiment 2：
The Immunofluorescence test card for the measure malachite green that the present embodiment is provided is substantially the same manner as Example 1, difference
Be in：The Immunofluorescence test card also includes upper shell 9 and lower casing 8, and the upper shell 9 is placed on the sample pad 2, institute
Coupling matter pad 3, the reaction film 4 and the top of the adsorptive pads 5 are stated, the lower casing 8 is located at the lower section of the backing plate 1, described
Upper shell 9 and the lower casing 8 are mutually covered, and the periphery of the lower casing 8 is provided with latch 11, and the periphery of the upper shell 9 is set
There is the hole clipping 10 coordinated with the latch 11, the upper shell 9 and the lower casing 8 pass through the latch 11 and the hole clipping 10
Connection.The upper shell 9 offers well 12 in the position of the correspondence sample pad 2, and sample to be determined is from the well
Add, flow to the sample pad 2, then spread successively along sample pad 2, coupling matter pad 3, reaction film 4 and adsorptive pads 5, it is described
Upper shell 9 is provided with observation window 13, detection line 6 and nature controlling line 7 on observing response film 4 in the position of the correspondence reaction film 4
Fluorescence signal.
Claims (10)
1. a kind of Immunofluorescence test card for determining malachite green, including backing plate, it is characterised in that also including investing the backing plate
Fluorescent microsphere mark on upper and sample pad closely coupled successively, coupling matter pad, reaction film and adsorptive pads, the coupling matter pad
Malachite green antibody, the reaction film is provided with detection line and nature controlling line, and the detection line is close to the coupling matter pad, the matter
Control line, which is coated with the adsorptive pads, the detection line on malachite green antigen, the nature controlling line, is coated with identification peacock
The secondary antibody of malachite green antibody.
2. the Immunofluorescence test card according to claim 1 for determining malachite green, it is characterised in that the immunofluorescence
Detection card also include upper shell and lower casing, the upper shell be placed on the sample pad, the coupling matter pad, the reaction film and
Above the adsorptive pads, the lower casing is located at the lower section of the backing plate, and the upper shell and the lower casing are mutually covered, institute
State shell and offer well in the position of the correspondence sample pad, the upper shell is set in the position of the correspondence reaction film
There is observation window.
3. a kind of preparation method of the Immunofluorescence test card of measure malachite green as described in any one of claim 1 or 2, its
It is characterised by, comprises the following steps：
Prepare sample pad；
Prepare coupling matter pad：Coupling matter pad film is cut, the malachite green antibody spraying that fluorescent microsphere is marked is taken on the membrane, does
It is dry；
Prepare reaction film：Reaction film is taken, malachite green antigen solution is sprayed on the reaction film, detection line is formed, described
The two corresponding anti-solution of spraying identification malachite green antibody, forms nature controlling line on reaction film；
Assembling detection card：Adsorptive pads and backing plate are taken, overlap joint pastes the sample pad, the coupling matter successively on the backing plate
Pad, the reaction film and the adsorptive pads.
4. the preparation method of the Immunofluorescence test card according to claim 3 for determining malachite green, it is characterised in that institute
The preparation method for stating the malachite green antibody of fluorescent microsphere mark comprises the following steps：
(1) fluorescent microsphere is activated：Fluorescent microsphere to be activated is added in buffer solution, washing, separation of solid and liquid, abandoning supernatant,
Collect solid matter to redissolve in buffer solution, n-hydroxysuccinimide and 1- (3- dimethylaminos are added into the buffer solution
Propyl group) -3- ethyl-carbodiimide hydrochlorides, oscillating reactions, separation of solid and liquid, collect solid matter simultaneously be dissolved in PB buffer solutions,
Obtain fluorescent microsphere solution；
(2) antibody labeling：Take malachite green antibody to be dissolved in PBS, and be added into the fluorescent microsphere solution, shake
Reaction a period of time is swung, monoethanolamine oscillating reactions is added for a period of time, adds bovine serum albumin solution, one section of capping
Time, separation of solid and liquid is collected solid matter and suspended again with PB buffer solutions, obtains the malachite green antibody of fluorescent microsphere mark.
5. the preparation method of the Immunofluorescence test card according to claim 4 for determining malachite green, it is characterised in that to
The n-hydroxysuccinimide added in the buffer solution and 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides
Mass ratio is 1：1.
6. the preparation method of the Immunofluorescence test card according to claim 4 for determining malachite green, it is characterised in that institute
Stating malachite green antibody is obtained by antigen of the conjugate of malachite green hapten and carrier protein.
7. the preparation method of the Immunofluorescence test card according to claim 6 for determining malachite green, it is characterised in that institute
The preparation for stating malachite green hapten comprises the following steps：3- nitrobenzaldehydes and DMA are weighed, is mixed, Xiang Qi
For a period of time, reaction solution is transferred in sodium carbonate liquor, is extracted with ethyl acetate for the middle addition concentrated sulfuric acid, heating response, is taken organic
Saturated common salt water washing mutually is used, then removes the water dissolved in organic phase, suction filtration obtains solid matter, silica gel column chromatography is crossed, with second
Acetoacetic ester/n-hexane mixed liquor is solvent, obtains the first intermediate product, weighs first intermediate product, add thereto
Tetrahydrofuran, methanol and palladium carbon, decompression remove air, and room temperature hydrogenation is placed, obtains the second intermediate product, weigh in the middle of described second
Product, is added it in anhydrous pyridine, adds glutaric anhydride, is stirred at room temperature, and concentrates dry pyridine, add ethyl acetate and
Water, aqueous phase is extracted with ethyl acetate, and merges organic phase, takes organic phase saturated common salt water washing, then remove dissolving in organic phase
Water, suction filtration obtains malachite green hapten.
8. the preparation method of the Immunofluorescence test card according to claim 6 for determining malachite green, it is characterised in that institute
Carrier protein is stated for bovine serum albumin(BSA).
9. the preparation method of the Immunofluorescence test card of the measure malachite green according to claim any one of 4-8, it is special
Levy and be, the preparation of the malachite green antibody comprises the following steps：With malachite green hapten and the conjugate of carrier protein
For antigen, the mice immunized with antigen is used, the spleen cell of immunized mice is merged with mouse source myeloma cell, screens, is divided
The hybridoma cell strain of malachite green monoclonal antibody is secreted, malachite green antibody is obtained by preparing ascites.
10. a kind of Immunofluorescence test card of the measure malachite green described in use claim 1 or 2 carries out malachite green detection
Method, it is characterised in that comprise the following steps：
(1) sample to be determined is added in the sample pad, carries out immune response；
(2) Immunofluorescence test for determining malachite green is placed on fluorescence chart scanner, the intensity to fluorescence signal is read
Take；
(3) judgement of result：When the detection line and the nature controlling line show orange glimmering when 345nm excites light irradiation similarly hereinafter
Light, and deep or both the color of the detection line nature controlling line is same or like, that is, represents that testing result is feminine gender, illustrate to examine
Survey and malachite green or contained malachite green concentration are not contained in thing less than 0.1ng/mL；When the detection line and the nature controlling line exist
345nm is excited under light irradiation, and the nature controlling line shows fluorescent orange, and the detection line does not show fluorescence or the nature controlling line is aobvious
Color is shallow, that is, represents that testing result is the positive, illustrates to contain malachite green in detectable substance and concentration is more than 0.1ng/mL.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108107202A (en) * | 2017-12-27 | 2018-06-01 | 石家庄市畜产品质量监测中心 | A kind of time-resolved fluoroimmunoassay chromatograph test strip and preparation method thereof |
| CN110954582A (en) * | 2019-12-30 | 2020-04-03 | 佛山职业技术学院 | Malachite green electrochemical sensor |
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