CN106053819A - Tetrodotoxin immunochromatography detection card and application thereof - Google Patents
Tetrodotoxin immunochromatography detection card and application thereof Download PDFInfo
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- CN106053819A CN106053819A CN201610420917.6A CN201610420917A CN106053819A CN 106053819 A CN106053819 A CN 106053819A CN 201610420917 A CN201610420917 A CN 201610420917A CN 106053819 A CN106053819 A CN 106053819A
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- fugu ocellatus
- ocellatus toxin
- toxin
- detection card
- fugu
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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 provides a tetrodotoxin immunochromatography detection card and application thereof. An antigen for preparing the tetrodotoxin immunochromatography detection card comprises a tetrodotoxin immunizing antigen and a tetrodotoxin coating antigen. The tetrodotoxin immunizing antigen is obtained by causing tetrodotoxin and KLH proteins to undergo cross-linking reaction through a phthalaldehyde cross-linking agent; and the tetrodotoxin coating antigen is obtained by causing tetrodotoxin and BSA proteins to undergo cross-linking reaction through an adipaldehyde cross-linking agent. By adoption of the technical scheme, the tetrodotoxin immunochromatography detection card is good in tetrodotoxin detection specificity, high in sensitivity, good in matrix interference resistance effect, accurate in detection result and good in reproducibility; the sample pretreatment is simple, the treatment time is short, the tetrodotoxin immunodetection sensitivity is improved, and the tetrodotoxin detection time is reduced. The detection card is compact and easy to carry, and tetrodotoxin can be detected quickly on site.
Description
Technical field
The present invention relates to Fugu ocellatus toxin detection technique field, particularly relate to a kind of Fugu ocellatus toxin immunochromatographydetection detection card and
Application.
Background technology
Fugu ocellatus toxin be (Tetrodotoxin, TTX) be the natural non-protein neurotoxin of a kind of severe toxicity, distribution is wide
General, do not only exist in globe fish, exist in other marine animal bodies, such as basket whelk, goby etc., be that known toxicity is the strongest
One of marine biotoxins.It suppresses the conduction of neural impulse by the blocking effect to sodium-ion channel, makes sensation god
Through paralysis, quadriplegia, dyspnea, the finally death because of respiration inhibition.Its virulence big 1000 times than Cyanogran..People and animals eat by mistake
Rear the most possible lethal.
Owing to Fugu ocellatus meat flavour is delicious, nutritious, therefore it is considered as delicious food by people, especially in Asian countries, people are from ancient times
Just have addicted to the custom eating Fugu ocellatus.The artificial propagation of main globefish kind (such as Puffer fish globefish, Fugurubripes etc.) in recent years
With the success of seed rearing, accelerating to advance the development of China's Fugu ocellatus artificial cultivation, yield and export sales are continuously increased.At present I
The 70% of the Fugu ocellatus Yi Zhan world Fugu ocellatus yield of state's cultivation, the state such as annual a large amount of export trade Japan, Korea S, Fugu ocellatus has become China one
Plant the organism production with higher economic worth and development potentiality.The chemical property of the Fugu ocellatus toxin owing to containing in Fugu ocellatus body
Stable, general cooking means are difficult to destroy, and also lack and effectively save measure after poisoning, the edible peace of serious threat to consumer
Entirely, therefore Fugu ocellatus adds toxicity enrichment positions such as must be completely removed internal organs, eyeball, epidermis man-hour.Although Fugu ocellatus has sternly in processing
The regulation of lattice, the case of Fugu ocellatus toxin poisoning still happens occasionally, and over the nearlyest 3 years, in the Fugu ocellatus toxin of media report, drug case arranges
More than 50.Solution to this problem is exactly to set up the front end pre-alarming system of Fugu ocellatus toxin, right before consumer is edible
The Fugu ocellatus processed carries out toxicity speed survey.But the most all without the on-the-spot speed survey product of Fugu ocellatus toxin, also have no Fugu ocellatus
Toxin scene fast determining method report.Trace it to its cause and be the antibody that Fugu ocellatus toxin molecule is not likely to produce high sensitivity identification, commodity
The detection limit of the Fugu ocellatus toxin ELISA kit changed also can only achieve the level of 100ng/g, therefore uses traditional Fugu ocellatus toxin
Preparation method for antibody, it is impossible to meet the needs of risk control.
Summary of the invention
For above technical problem, the invention discloses a kind of Fugu ocellatus toxin immunochromatographydetection detection card and application thereof, detection
Specificity is good, highly sensitive, and anti-matrix interference is effective, and testing result is accurate, reproducible, can field quick detection tetrodotoxin, TTX
Element.
To this, the technical solution used in the present invention is:
A kind of Fugu ocellatus toxin immunochromatographydetection detection card, prepares the antigen bag used by described Fugu ocellatus toxin immunochromatographydetection detection card
Including Fugu ocellatus toxin immunizing antigen and Fugu ocellatus toxin envelope antigen, described Fugu ocellatus toxin immunizing antigen is Fugu ocellatus toxin and KLH albumen
Use terephthalic aldehyde cross-linking agent to carry out cross-linking reaction to obtain；Described Fugu ocellatus toxin envelope antigen is Fugu ocellatus toxin and BSA albumen is adopted
Carry out cross-linking reaction with hexandial cross-linking agent to obtain.
As a further improvement on the present invention, described Fugu ocellatus toxin immunizing antigen uses following steps to prepare:
Step S101: be dissolved in methanol by terephthalic aldehyde, obtains the methanol solution of terephthalic aldehyde；
Step S102: by Fugu ocellatus toxin with KLH protein dissolution to distilled water, the methanol being charged with terephthalic aldehyde is molten
Liquid, after reacting 24 hours, adds sodium cyanoborohydride solution at 20～28 DEG C；Use normal saline dialysis 3 days at 4 DEG C, obtain
Fugu ocellatus toxin immunizing antigen.
Preferably, the concentration of sodium cyanoborohydride solution is 10mg/mL.
Preferably, during dialysis, change 3 dialysis solution every day, the holoantigen obtained is sub-packed in the concentration of 1mg/mL
In 0.5mL centrifuge tube, frozen in-20 DEG C of refrigerators.
As a further improvement on the present invention, described Fugu ocellatus toxin, KLH albumen, the mass ratio of terephthalic aldehyde be 1:(5～
15): (5～15).
As a further improvement on the present invention, described Fugu ocellatus toxin, KLH albumen, the mass ratio of terephthalic aldehyde are 1:10:
10。
As a further improvement on the present invention, described Fugu ocellatus toxin envelope antigen uses following steps to prepare:
Step S201: be dissolved in methanol by hexandial, obtains the methanol solution of hexandial；
Step S202: by Fugu ocellatus toxin with BSA protein dissolution to distilled water, the methanol being charged with hexandial is molten
Liquid, after reacting 24 hours, adds sodium cyanoborohydride solution at 20～28 DEG C；Use normal saline dialysis 3 days at 4 DEG C, obtain
Fugu ocellatus toxin envelope antigen.
As a further improvement on the present invention, described Fugu ocellatus toxin, BSA albumen, the mass ratio of hexandial be 1:(5～
15): (5～15).
As a further improvement on the present invention, described Fugu ocellatus toxin, BSA albumen, the mass ratio of hexandial are 1:10:10.
As a further improvement on the present invention, described Fugu ocellatus toxin immunochromatographydetection detection card uses following steps to be prepared into
Arrive:
Step A: synthesize described Fugu ocellatus toxin immunizing antigen；
Step B: synthesize described Fugu ocellatus toxin envelope antigen；
Step C: using described Fugu ocellatus toxin immunizing antigen to merge through cell with immunity Balb/c mice, screening is secreted
The hybridoma of Fugu ocellatus toxin monoclonal antibody, produces ascites with the cell induction mice obtained, obtains tetrodotoxin, TTX after purification
Element monoclonal antibody；
Step D: prepare gold colloidal with trisodium citrate and gold chloride reaction；By gold colloidal and described Fugu ocellatus toxin monoclonal
Antibody is mixed to form gold labeling antibody, obtains the Fugu ocellatus toxin monoclonal antibody complex of colloid gold label after centrifugal redissolution；
Step E: the Fugu ocellatus toxin monoclonal antibody complex of described colloid gold label is dipped on glass fibre element film,
To sample pad；
Step F: be coated on nitrocellulose filter by described Fugu ocellatus toxin envelope antigen, obtains detecting line, and goat-anti is little
Mus IgG is coated on described nitrocellulose filter, obtains nature controlling line；
Preferably, take Fugu ocellatus toxin envelope antigen 2mg/ml 0.01mol/L, pH value is that the PB buffer of 7.2 is with 250 μ
G/ml is spaced, through the BIO-DOT linear observed result being coated in nitrocellulose filter of type XYZ3010 point sample instrument dispenser
Test reaction district, obtains detecting line；
Preferably, first with dispenser, linear to be coated the goat-anti that concentration is 2mg/ml little in the distance detection remote Quality Control of band 5mm
Mus IgG obtains.
Preferably, the nitrocellulose filter obtaining being coated is preserved in 37 DEG C of dry 2h, 4 DEG C of sealings.
Step G: with PVB as backing, be stacked nitrocellulose filter at the middle part of PVC liner plate, two ends stacked water suction respectively
Pad and sample pad, assemble and obtain described Fugu ocellatus toxin immunochromatographydetection detection card, wherein nitrocellulose filter respectively with adsorptive pads, sample
Product pad connects mutually.
Preferably, in step C, the preparation of described Fugu ocellatus toxin monoclonal antibody comprises the following steps: take ascites about 3mL,
Add 0.06mol/L, pH 4.5 sodium-acetate buffer of 2 times of volumes.Caprylic acid (33 μ g/mL ascites) is dropwise slowly added into sample
In product, stirring while adding, after adding continue stirring 30min, at 4 DEG C, 10000r/min is centrifuged 30min, go precipitation (albumin and
Other non-IgG albumen).Take supernatant through 0.45 μm micro-pore-film filtration, mix with 1/10 volume 10 × PBS (10 × PBS:
80gNaCl, 2g KCl, 11.5g Na2HPO4,2g KH2PO4,0.5845g EDTA, 1000mL distilled water, pH 7.4), use
1mol/L NaOH solution adjusts pH value to 7.4.Supernatant is cooled to 4 DEG C, and (0.277g/mL makes the final saturation be to add ammonium sulfate
45%).Stirring 30min, at 4 DEG C, 10000r/min is centrifuged 30min, abandons supernatant.With a small amount of PBS solution dissolution precipitation, with 50～
The PBS of 100 times of volumes overnight, changes liquid 3 times.Solution PEG-6000 after dialysis suitably concentrates, and preserves standby at 4 DEG C.
Preferably, the preparation of described gold colloidal comprises the following steps: take 1% chlorauric acid solution 1ml, adds 99ml ultra-pure water and becomes eventually
The chlorauric acid solution of concentration 0.01%, after ebuillition of heated, takes 1% trisodium citrate 1.6ml and is disposably rapidly added the chlorine boiled
In auric acid solution, continue to be heated to solution and transferred to the black-and-blue shiny red that eventually becomes by faint yellow, after colour stable, continue heating
5min, room temperature cools down, and supplements dehydration to original volume.
Preferably, the preparation of described colloid gold label Fugu ocellatus toxin monoclonal antibody complex comprises the following steps:
By 120% total amount calculating required protein to be marked that minimum steady is quantitative.Under magnetic stirring, by protein
Solution adds in colloidal gold solution (pH is modulated to 5.9～6.2), should be added dropwise over when adding protein, and the protein of 1mg is about
5min adds.Take 1ml gold colloidal-staphylococcal protein A conjugate liquid (experimental group) and 1ml gold colloidal stock solution (matched group) respectively
Adding 10% sodium chloride solution 0.1ml in test tube, room temperature stands 1h, observed result: if matched group test tube solution is turned by redness
For blueness, it might even be possible to see that polymer precipitates, and experimental group solution still keeps red, without precipitation, can continue next step real
Test, otherwise need to add labelling albumen staphylococcal protein A.It is eventually adding the Polyethylene Glycol (PEG of final concentration of 0.2%
MW20000), stirring 30min is continued.
The application of the Fugu ocellatus toxin immunochromatographydetection detection card described in a kind of as above any one, for Fugu ocellatus soup or Fugu ocellatus group
In the Fugu ocellatus toxin detection knitted.
As a further improvement on the present invention, when the Fugu ocellatus toxin carrying out Fugu ocellatus soup detects, Fugu ocellatus soup lower floor liquid is taken
As sample liquid；Or when the Fugu ocellatus toxin carrying out Fugu ocellatus tissue detects, every gram of homogenizing Fugu ocellatus meat is added 1mL methanol and takes out
Carry, filter, filtrate dilution 5 times is obtained sample liquid；
With sample pad pair on use 1mL pasteur pipet 3 sample liquid of dropping extremely described Fugu ocellatus toxin immunochromatographydetection detection card
At the sample well answered, observed result in placing 5-10 minute, as occurred red stripes at nature controlling line, does not develops the color or aobvious at detection line
The shallow then sample of color ratio nature controlling line is positive；As deeper than nature controlling line red stripes in detected line, then sample is negative；As nature controlling line does not shows
Color then testing result is invalid.
Compared with prior art, the invention has the beneficial effects as follows:
Using technical scheme, detection Fugu ocellatus toxin specificity is good, and highly sensitive, anti-matrix interference is effective,
Testing result is accurate, reproducible；The pre-treatment of sample is simple, detects simple to operate, it is not necessary to use analytical tool.Improve
The sensitivity of Fugu ocellatus toxin immune detection, decreases the Fugu ocellatus toxin detection time.And this card is small and exquisite, it is easy to carry, can show
Fugu ocellatus toxin is quickly detected in field.
Accompanying drawing explanation
Fig. 1 is the package assembly schematic diagram of the present invention a kind of Fugu ocellatus toxin immunochromatographydetection detection card.
Fig. 2 is the structural representation after the present invention a kind of Fugu ocellatus toxin immunochromatographydetection detection card assembles.
Fig. 3 is the schematic diagram that the detection of the present invention a kind of Fugu ocellatus toxin immunochromatographydetection detection card is positive.
Fig. 4 is the schematic diagram that the detection of the present invention a kind of Fugu ocellatus toxin immunochromatographydetection detection card is negative.
Fig. 5 is the schematic diagram that the detection of the present invention a kind of Fugu ocellatus toxin immunochromatographydetection detection card is invalid.
Detailed description of the invention
Below the preferably embodiment of the present invention is described in further detail.
Embodiment 1
Prepare Fugu ocellatus toxin immunochromatographydetection detection card to comprise the following steps:
Step A: the synthesis of Fugu ocellatus toxin immunizing antigen comprises the following steps:
Step S101: weigh terephthalic aldehyde 10mg, is dissolved in 1mL methanol, obtains the methanol solution of terephthalic aldehyde.
Step S102: weigh Fugu ocellatus toxin 1mg, KLH 10mg, be dissolved in 5mL distilled water.It is slowly added dropwise above-mentioned to benzene
The methanol solution of dialdehyde.After room temperature reaction 24 hours, add the sodium cyanoborohydride solution of 1mL, 10mg/mL.Physiology is used at 4 DEG C
Dialysis against saline 3d, changes 3 dialysis solution every day.The holoantigen obtained is sub-packed in 0.5mL centrifuge tube with the concentration of 1mg/mL, freezes
It is stored in-20 DEG C of refrigerators.
Step B: the synthesis of Fugu ocellatus toxin envelope antigen comprises the following steps:
Step S201: weigh hexandial 10mg, is dissolved in 1mL methanol, obtains the methanol solution of hexandial.
Step S202: weigh Fugu ocellatus toxin 1mg, BSA 10mg, be dissolved in 5mL distilled water.Be slowly added dropwise above-mentioned oneself two
The methanol solution of aldehyde.After room temperature reaction 24 hours, add the sodium cyanoborohydride solution of 1mL, 10mg/mL.Physiology salt is used at 4 DEG C
Water dialysis 3d, changes 3 dialysis solution every day.The holoantigen obtained is sub-packed in 0.5mL centrifuge tube with the concentration of 1mg/mL, frozen
In-20 DEG C of refrigerators.
Step C: the preparation of Fugu ocellatus toxin monoclonal antibody comprises the following steps:
(1) experimental animal immune
Employing hapten Fugu ocellatus toxin immunizing antigen merges through cell with immunity Balb/c mice, and the most immune 6 weeks female
Balb/c Mus, often group 3.During first immunisation injection, the immunizing antigen 100 μ L of 100 μ g/mL, helps completely with equivalent Freund respectively
Agent is fully emulsified, abdominal cavity direct injection.After being spaced two weeks, take the antigen of sample, with 100 μ L Freund's incomplete adjuvant emulsifyings, same to sample prescription
Method is injected.
(2) cell merges:
Cell merge before 1d or drew the same day neck put to death mice, be immersed in 70% ethanol, body surface sterilization.Solid with pin
Determine mice on stencil plate, abdominal cut on superclean bench, provoke peritoneum with pincet, inject 5mL RPMI-1640 and train completely
Nutrient solution, rubs abdominal cavity gently with hands, is moved into by its internal liquid aseptic straw in 75mL HAT complete culture solution, described 75mL
HAT complete culture solution is addition 0.75mL100 × HAT liquid in 75mL RPMI-1640 complete culture solution, 2～3 times repeatedly；With suction
Pipe mixes, and spreads 24 orifice plates, and every hole adds 0.5mL, is placed in the 5%CO of 37 DEG C2In incubator.
Mouse orbit blood-letting, collects serum, draws neck to put to death, 70% alcohol-pickled sterilization body surface, and aseptic taking-up spleen is put into
In RPMI-1640 basic culture solution, and carefully reject fascia and fat, shred, be placed in the stainless steel sift of 100 mesh, aseptic grind
Mill, discharges single splenocyte, draws the liquid containing splenocyte and is placed in 50mL sterile centrifugation tube, centrifugal.
Myeloma cell and the above-mentioned splenocyte prepared are added the centrifuge tube of same 50mL with the ratio of number 5:1
In, adding the RPMI-1640 incomplete culture fluid 20mL of 37 DEG C of temperature baths, mix homogeneously, 1500r/min is centrifuged 6min.Except supernatant,
Bottom finger tapping down centrifuge tube, precipitation is made to mix such as pasty state.Take the PEG 1mL of 37 DEG C of preheatings with pipet, instill centrifuge tube,
After standing 1min, in 37 DEG C of water-baths, in 2min, drip RPMI-1640 complete culture solution 10mL.1000r/min is centrifuged 6min,
Supernatant discarded, adds 75mL HAT culture fluid, mixes gently, is sub-packed in 24 orifice plates of feeder cells by mixing suspension, every hole
0.5mL, in 37 DEG C, 5%CO2The incubator of saturated humidity is hatched.
(3) cultivation of hybridoma and screening
After fusion 6～9d, change liquid 1 time by HT culture fluid half amount, use instead according to proliferative conditions behind 12～14d and cultivate completely
Liquid.Until cell attachment to when accounting for plate hole 1/3, count hole count and the total cellular score of Growth of Hybridoma Cell.Take supernatant, indirectly
ELISA selects titer high and indirect competitive ELISA selects the positive hybridoma cell that Drug inhibition is strong.
Using indirect ELISA and indirect competitive ELISA method to carry out positive hybridoma cell screening, display is positive and occurs competing
The hole striving suppression reaction is the hole producing Fugu ocellatus toxin antibody, and can be used for further sub-clone.
Under aseptic condition, the cell in eluting positive hole, it is transferred to spread with feeder cells in advance by cell with elbow straw
In 96 well culture plates of plate, each archioporus clones into 8 holes, at the bottom of cell attachment covers with 1/2～1/3 hole after, take supernatant,
Connect ELISA detection.Take the strong sub-clone that is positive, 2～5 times the most repeatedly, wait antibody positive in the supernatant of hole, cloned 8
When rate is 100%, picking single cell clone, it is detected as full positive and is transferred to 24 porocyte culture plates or 25mL Tissue Culture Flask
Amplification culture, builds strain and with subpackage, frozen.Carry and inject 0.5mL norphytane the last week to Balb/c mouse peritoneal.Take freeze-stored cell
Strain, after recovery, breeds through mass propgation, collects cell, after full culture medium of cannoing be used up washing secondary, more not exclusively cultivates with 10mL
Base suspends, counting.By cell (every mice 1mL, containing 3.1 × 107 cells) intraperitoneal injection of mice abdominal part, after 10～15d, treat
By No. 16 syringe aseptic collection ascites when mouse web portion substantially expands.2000r/min is centrifuged 10min, remove upper-layer fat and
Lowermost fibre albumen and cell, collect middle level, take part ELISA method and measure its titer, and remaining subpackage-70 DEG C is frozen standby.
(4) preparation and purification of Fugu ocellatus toxin monoclonal antibody
Take ascites about 3mL, add 0.06mol/L, pH 4.5 sodium-acetate buffer of 2 times of volumes.By caprylic acid (33 μ g/
ML ascites) dropwise it is slowly added in sample, stirring while adding, continue stirring 30min after adding, at 4 DEG C, 10000r/min is centrifuged
30min, goes precipitation (albumin and other non-IgG albumen).Take supernatant through 0.45 μm micro-pore-film filtration, with 1/10 volume 10 ×
PBS mix, described 10 × PBS be 80gNaCl, 2g KCl, 11.5g Na2HPO4,2g KH2PO4,0.5845g EDTA,
1000mL distilled water, pH 7.4；Then adjust pH value to 7.4 by 1mol/L NaOH solution.Supernatant is cooled to 4 DEG C, adds concentration and is
0.277g/mL, final saturation are the ammonium sulfate of 45%.Stirring 30min, at 4 DEG C, 10000r/min is centrifuged 30min, abandons supernatant.
With a small amount of PBS solution dissolution precipitation, with the PBS of 50～100 times of volumes overnight, change liquid 3 times.Solution PEG-after dialysis
6000 suitably concentrate, and preserve standby at 4 DEG C, obtain Fugu ocellatus toxin monoclonal antibody.
Step D: the preparation of gold colloidal comprises the following steps:
Take 1% chlorauric acid solution 1ml, add 99ml ultra-pure water and become the chlorauric acid solution of final concentration 0.01%, after ebuillition of heated,
Take 1% trisodium citrate 1.6ml to be disposably rapidly added in the chlorauric acid solution boiled, continue to be heated to solution by faint yellow turn
For the black-and-blue shiny red that eventually becomes, continuing heating 5min after colour stable, room temperature cools down, and supplements dehydration to original volume.
Step E: the preparation of colloid gold label Fugu ocellatus toxin monoclonal antibody complex comprises the following steps:
By 120% total amount calculating required protein to be marked that minimum steady is quantitative.Under magnetic stirring, by protein
Solution adds in colloidal gold solution (pH is modulated to 5.9～6.2), should be added dropwise over when adding protein, and the protein of 1mg is about
5min adds.Take 1ml gold colloidal-staphylococcal protein A conjugate liquid (experimental group) and 1ml gold colloidal stock solution (matched group) respectively
Adding 10% sodium chloride solution 0.1ml in test tube, room temperature stands 1h, observed result: if matched group test tube solution is turned by redness
For blueness, it might even be possible to see that polymer precipitates, and experimental group solution still keeps red, without precipitation, can continue next step real
Test, otherwise need to add labelling albumen staphylococcal protein A.It is eventually adding the Polyethylene Glycol (PEG of final concentration of 0.2%
MW20000), continue stirring 30min, obtain colloid gold label Fugu ocellatus toxin monoclonal antibody complex.
Step F: the preparation of gold mark pad and sample pad comprises the following steps:
The colloid gold label Fugu ocellatus toxin monoclonal antibody complex stock solution that preservation liquid dilution embodiment 5 obtains is dense to working
Degree, is the most uniformly dipped in glass fibre element film, obtains gold mark pad, makees with the glass fibre element film of 1mg/mL BSA sealing treatment
For sample pad, described gold mark pad and sample are padded on-20 DEG C frozen, after lyophilisation, seal and preserve.
Step G: the preparation of Fugu ocellatus toxin envelope antigen solid phase nitrocellulose filter comprises the following steps:
The PB buffer (pH 7.2) taking Fugu ocellatus toxin envelope antigen 2mg/ml 0.01mol/L is spaced with 250 μ g/ml,
Test reaction through the BIO-DOT linear observed result being coated in nitrocellulose filter of type XYZ3010 point sample instrument dispenser
District, is defined as detecting line, and distance detection line 5mm is far coated goat anti-mouse igg (2mg/ml), as matter with dispenser is linear
Control line.37 DEG C of dry 2h, 4 DEG C seal preservation.
Step H: test strips assembles and comprises the following steps:
As depicted in figs. 1 and 2, being backing by PVC board 1, be stacked nitrocellulose filter 8 at the middle part of PVC board 1, nitric acid is fine
Being coated with detection line 4 and nature controlling line 3 according to step G on dimension element film 8, the two ends of nitrocellulose filter 8 are stacked adsorptive pads 7 He respectively
Sample pad 5, nitrocellulose filter 8 connects mutually with adsorptive pads 7, sample pad 5, then assembles, and sample pad 5 is provided with well
2 mark pad 6 with gold, and the Fugu ocellatus toxin immunochromatographydetection detection card after completing is as shown in Figure 2.
Embodiment 2
The application of detection card.
(1) when Fugu ocellatus soup is detected by needs, take Fugu ocellatus soup, take off the less liquid of layer fat content as sample
Liquid, uses 1mL pasteur pipet dropping 3 to drop to sample well.
When Fugu ocellatus tissue is detected by needs, take 1g homogenizing Fugu ocellatus meat and add the extracting of 1mL methanol, filter, by filtrate
Dilute 5 times and become sample liquid, use 1mL pasteur pipet 3 sample liquid of dropping to sample well.
(2) result judges
The Fugu ocellatus toxin immunochromatographydetection detection card having dripped sample places observed result in 5-10 minute.
As it is shown on figure 3, as red stripes occurs at nature controlling line location of C, do not develop the color or develop the color at detection line T location and compare Quality Control
The shallow then sample of line is positive；
As shown in Figure 4, if at nature controlling line location of C, developed the color at detection line T location, but detection line is more red than nature controlling line
Band is deep, then sample is negative；
As it is shown in figure 5, it is as invalid in the then testing result that do not develops the color at nature controlling line location of C.
Embodiment 3
On the basis of embodiment 1, in this example, when preparing Fugu ocellatus toxin immunizing antigen, the consumption of terephthalic aldehyde is
The consumption of 5mg, KLH is 15mg.When preparing Fugu ocellatus toxin envelope antigen, the consumption of hexandial is that the consumption of 15mg, BSA is
5mg.Other are with embodiment 1.
Embodiment 4
On the basis of embodiment 1, in this example, when preparing Fugu ocellatus toxin immunizing antigen, the consumption of terephthalic aldehyde is
The consumption of 15mg, KLH is 5mg.When preparing Fugu ocellatus toxin envelope antigen, the consumption of hexandial is that the consumption of 5mg, BSA is
15mg.Other are with embodiment 1.
Embodiment 5
Use the present invention Fugu ocellatus toxin immunochromatographydetection detection card carry out Fugu ocellatus toxin detection limit in sensitivity, Fugu ocellatus soup,
In Fugu ocellatus tissue, Fugu ocellatus toxin detection limit is tested, and using immunizing antigen, envelope antigen all use formaldehyde crosslinking method as
Control sample 1, all uses Euplotes woodruffi for control sample 2 with immunizing antigen, envelope antigen, and result is as shown in table 1.
Table 1 embodiment 1,3 and 4 and control sample 1, the sensitivity technique result table of control sample 2
| Project | Control sample 1 | Control sample 2 | Embodiment 1 | Embodiment 3 | Embodiment 4 |
| Sensitivity | 200μg/L | 400μg/L | 20μg/L | 50μg/L | 60μg/L |
| Fugu ocellatus toxin detection limit in Fugu ocellatus soup | 500μg/L | 1000μg/L | 50μg/L | 80μg/L | 100μg/L |
| Fugu ocellatus toxin detection limit in Fugu ocellatus tissue | 2000μg/kg | 4000μg/kg | 100μg/kg | 120μg/L | 150μg/L |
From table 1, using the embodiment 1,3 and 4 of technical scheme, detection Fugu ocellatus toxin specificity is good, spirit
Sensitivity is high, exceeds far away control sample 1 and control sample 2.
Simultaneously by the Fugu ocellatus toxin immunochromatographydetection detection card of the present invention and control sample 1 and the control sample 2 pre-treatment to sample
Time compares, and result is as shown in table 2.Wherein, the sample pre-treatments of control sample 1 uses the method for acetic acid extracting, comparison
The sample pre-treatments of sample 2 uses the method for trichloroacetic acid extracting.
Table 2 present invention and control sample 1, the time for sample pretreatment contrast table of control sample 2
| Sample | Control sample 1 | Control sample 2 | The present invention |
| Fugu ocellatus soup | —— | —— | Without pre-treatment |
| Fugu ocellatus tissue | About 1 hour | About 1.5 hours | 10 minutes |
Contrast by table 2 is visible, uses the Fugu ocellatus toxin immunochromatographydetection detection card of technical solution of the present invention to carry out Fugu ocellatus
During Mycotoxin identification, the pre-treatment of sample is simple, processes the time short, decreases the Fugu ocellatus toxin detection time.
Above content is to combine concrete preferred implementation further description made for the present invention, it is impossible to assert
Being embodied as of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of present inventive concept, it is also possible to make some simple deduction or replace, all should be considered as belonging to the present invention's
Protection domain.
Claims (10)
1. a Fugu ocellatus toxin immunochromatographydetection detection card, it is characterised in that: prepare described Fugu ocellatus toxin immunochromatographydetection detection card institute
Antigen include that Fugu ocellatus toxin immunizing antigen and Fugu ocellatus toxin envelope antigen, described Fugu ocellatus toxin immunizing antigen are Fugu ocellatus toxin
Use terephthalic aldehyde cross-linking agent to carry out cross-linking reaction with KLH albumen to obtain；Described Fugu ocellatus toxin envelope antigen be Fugu ocellatus toxin and
BSA albumen employing hexandial cross-linking agent carries out cross-linking reaction and obtains.
Fugu ocellatus toxin immunochromatographydetection detection card the most according to claim 1, it is characterised in that: described Fugu ocellatus toxin immunity resists
Former employing following steps prepare:
Step S101: be dissolved in methanol by terephthalic aldehyde, obtains the methanol solution of terephthalic aldehyde；
Step S102: by Fugu ocellatus toxin with KLH protein dissolution to distilled water, be charged with the methanol solution of terephthalic aldehyde,
After reacting 24 hours at 20 ~ 28 DEG C, add sodium cyanoborohydride solution；Use normal saline dialysis 3 days at 4 DEG C, obtain Fugu ocellatus
Toxin immunity antigen.
Fugu ocellatus toxin immunochromatographydetection detection card the most according to claim 2, it is characterised in that: described Fugu ocellatus toxin, KLH egg
In vain, the mass ratio of terephthalic aldehyde is 1:(5 ~ 15): (5 ~ 15).
Fugu ocellatus toxin immunochromatographydetection detection card the most according to claim 3, it is characterised in that: described Fugu ocellatus toxin, KLH egg
In vain, the mass ratio of terephthalic aldehyde is 1:10:10.
Fugu ocellatus toxin immunochromatographydetection detection card the most according to claim 1, it is characterised in that: described Fugu ocellatus toxin is coated anti-
Former employing following steps prepare:
Step S201: be dissolved in methanol by hexandial, obtains the methanol solution of hexandial；
Step S202: by Fugu ocellatus toxin with BSA protein dissolution to distilled water, be charged with the methanol solution of hexandial,
After reacting 24 hours at 20 ~ 28 DEG C, add sodium cyanoborohydride solution；Use normal saline dialysis 3 days at 4 DEG C, obtain tetrodotoxin, TTX
Element envelope antigen.
Fugu ocellatus toxin immunochromatographydetection detection card the most according to claim 5, it is characterised in that: described Fugu ocellatus toxin, BSA egg
In vain, the mass ratio of hexandial is 1:(5 ~ 15): (5 ~ 15).
Fugu ocellatus toxin immunochromatographydetection detection card the most according to claim 6, it is characterised in that: described Fugu ocellatus toxin, BSA egg
In vain, the mass ratio of hexandial is 1:10:10.
8. according to the Fugu ocellatus toxin immunochromatographydetection detection card described in claim 1 ~ 7 any one, it is characterised in that: described Fugu ocellatus
Toxin immunity chromatography detection card uses following steps to prepare:
Step A: synthesize described Fugu ocellatus toxin immunizing antigen；
Step B: synthesize described Fugu ocellatus toxin envelope antigen；
Step C: using described Fugu ocellatus toxin immunizing antigen to merge through cell with immunity Balb/c mice, screening obtains secreting Fugu ocellatus
The hybridoma of toxin monoclone antibody, produces ascites with the cell induction mice obtained, obtains Fugu ocellatus toxin list after purification
Clonal antibody；
Step D: prepare gold colloidal with trisodium citrate and gold chloride reaction；By gold colloidal and described Fugu ocellatus toxin monoclonal antibody
It is mixed to form gold labeling antibody, after centrifugal redissolution, obtains the Fugu ocellatus toxin monoclonal antibody complex of colloid gold label；
Step E: the Fugu ocellatus toxin monoclonal antibody complex of described colloid gold label is dipped on glass fibre element film, obtains gold
Mark pad, with the glass fibre element film of 1mg/mL BSA sealing treatment as sample pad；
Step F: be coated on nitrocellulose filter by described Fugu ocellatus toxin envelope antigen, obtains detecting line, by goat anti-mouse igg
It is coated on described nitrocellulose filter, obtains nature controlling line；
Step G: with PVB as backing, be stacked nitrocellulose filter at the middle part of PVC liner plate, two ends be stacked respectively adsorptive pads and
Sample pad, assembles and obtains described Fugu ocellatus toxin immunochromatographydetection detection card, wherein nitrocellulose filter respectively with adsorptive pads, sample pad
Connect mutually.
9. the application of the Fugu ocellatus toxin immunochromatographydetection detection card as described in claim 1 ~ 8 any one, it is characterised in that:
In the Fugu ocellatus toxin of Fugu ocellatus soup or Fugu ocellatus tissue detects.
The application of Fugu ocellatus toxin immunochromatographydetection detection card the most according to claim 9, it is characterised in that:
When the Fugu ocellatus toxin carrying out Fugu ocellatus soup detects, take Fugu ocellatus soup lower floor liquid as sample liquid, or carrying out Fugu ocellatus group
During the Fugu ocellatus toxin detection knitted, every gram of homogenizing Fugu ocellatus meat is added the extracting of 1mL methanol, filters, filtrate dilution 5 times is obtained sample
Liquid；
1mL pasteur pipet is used to drip 3 sample liquid corresponding with sample pad to described Fugu ocellatus toxin immunochromatographydetection detection card
At sample well, observed result in placing 5-10 minute, as occurred red stripes at nature controlling line, do not develop the color or develop the color at detection line ratio
The shallow then sample of nature controlling line is positive；As deeper than nature controlling line red stripes in detected line, then sample is negative；Do not develop the color then such as nature controlling line
Testing result is invalid.
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