CN109187961A - One kind is for detecting aflatoxin B1Qualitative, quantitative immuno-chromatographic test paper strip and its preparation method and application - Google Patents
One kind is for detecting aflatoxin B1Qualitative, quantitative immuno-chromatographic test paper strip and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to technical field of food safety detection, in particular to a kind of for detecting aflatoxin B1Qualitative, quantitative immuno-chromatographic test paper strip and its preparation method and application.The qualitative, quantitative immuno-chromatographic test paper strip, successively overlap joint is provided with filter paper, sample pad, glass fibre membrane, nitrocellulose filter and blotting paper on bottom plate；The aflatoxin B of colloid gold label is attached on glass fibre membrane1Monoclonal antibody；It is provided with detection line and nature controlling line on nitrocellulose filter, the aflatoxin B of fluorescent microsphere label is coated in detection line1Artificial antigen is coated with secondary antibody on nature controlling line.Spetrophotometry of the present invention is to the aflatoxin B in detection sample1Qualitative and quantitative detection is realized simultaneously, sample results obtain Dual system verifying in the detection, and it is as a result relatively reliable, save detection time.
Description
Technical field
The present invention relates to technical field of food safety detection, in particular to a kind of for detecting aflatoxin B1It is qualitative
Quantitative immunochromatographic test strips and its preparation method and application.
Background technique
Aflatoxin (aflatoxin B1, claim AFB1) be fungi secondary metabolite, mainly by aspergillus flavus, post
Raw aspergillus and the mould generation in Tequ.It is easily naturally present in the crops such as peanut, cottonseed, corn, wheat and paddy, has strong
Toxicity and carcinogenicity.It is strongest a kind of carcinogenic mutagens in current chemical carcinogen, and toxicity is 10 times stronger than potassium cyanide,
Its carcinogenicity is 75 times stronger than N-nitrosodimethylamine.1993, aflatoxin was drawn by the Agency for Research on Cancer of the World Health Organization
It is set to I class carcinogenic substance, the target organ of effect is mainly liver.
For Grain and its product, aflatoxin B in corn, maize flour (slag, piece) and corn product1Limit standard
For 20 μ g/kg, paddy, brown rice, rice are 10 μ g/kg, and wheat, barley, other cereal are 5.0 μ g/kg, wheat flour, oatmeal, its
His decladding cereal is 5.0 μ g/kg；For beans and its product, aflatoxin B in fermented bean products1Limit standard be 5.0
μg/kg；For nut and its seed class, aflatoxin B in peanut and its product1Limit standard be 20 μ g/kg, other shortenings
Nut and its seed class are 5.0 μ g/kg；For grease and its product, aspergillus flavus is malicious in vegetable fat (except peanut oil, corn oil)
Plain B1Limit standard be 10 μ g/kg, peanut oil, corn oil be 5.0 μ g/kg；For flavouring, soy sauce, vinegar, make vinegar (with
Grain is primary raw material) in aflatoxin B1Limit standard be 5.0 μ g/kg.
Currently, the first method is isotopic dilution liquid chromatography-tandem mass spectrometry in existing national food safety standard food,
Suitable for Grain and its product, beans and its product, nut and seed class, grease and its product, flavouring, dispensed food for baby
With AFB in supplementary food for infants1、AFB2、AFG1And AFG2Measurement.Second method is high performance liquid chromatography-pre-column derivatization,
Suitable for Grain and its product, beans and its product, nut and seed class, grease and its product, flavouring, dispensed food for baby
With AFB in supplementary food for infants1、AFB2、AFG1And AFG2Measurement.Third method is high performance liquid chromatography-post-column derivation method,
Suitable for Grain and its product, beans and its product, nut and seed class, grease and its product, flavouring, dispensed food for baby
With AFB in supplementary food for infants1、AFB2、AFG1And AFG2Measurement.4th method is Enzyme-linked Immunosorbent Assay screening method, is suitable for
Grain and its product, beans and its product, nut and seed class, grease and its product, flavouring, dispensed food for baby and baby children
AFB in youngster's accesary foods1Measurement.5th method is thin-layered chromatography, is suitable for Grain and its product, beans and its product, heavily fortified point
AFB in fruit and seed class, grease and its product, flavouring1Measurement.
Immunochromatography technique is a kind of unique immunoassay formats for coming across the initial stage eighties, it is usually with strip fibre
Dimension chromatographic material is solid phase, makes sample solution swimming on chromatography strip through capillary action, since what antigen-antibody combined is immunized
It reacts, immune complex is enriched with or is trapped in the certain area (detection line) of chromatographic material in chromatography process, passes through enzyme reaction
Or intuitive experimental result directly is obtained (as different colours occurs in detection line with the marker (such as colloidal gold) that can be estimated
Band)；And free label then crosses detection line, reaches the purpose being automatically separated with binding label.Immunochromatography technique is normal
The visual marking carrier seen has colloidal gold, latex, electroselenium etc., wherein being colloidal gold with most successful marker.But glue
Body gold immuno-chromatographic test paper strip has the following deficiencies:
(1) general test strips are and observe by the naked eye result to carry out qualitative analysis, cannot achieve accurately quantitative detection.
(2) different material matrix effects is obvious, and sample background interference is larger, is also easy to produce false positive results.
(3) the positive findings detected can not save, as a result no longer accurate and reliable after usually more than judging the time.
Market in order to meet to sample carry out quantitative detection the needs of, the various quantitative testing test papers based on different markers
Item also emerges one after another, and general current quantitative testing test paper item, is all to pass through miniature instrument after single marker labelled antibody
Color signal value is read, then carries out quantitative detection by drawing standard curve.Fluorescence currently based on different rubidium marking objects is exempted from
Epidemic disease chromatography method also has already appeared, and obtains high sensitivity, but there is also following defects for the quantitative detecting method:
(1) in actually detected, ratio shared by negative findings is very high, but all test strips of this method are required to be read with instrument,
Otherwise it cannot obtain as a result, being taken a long time when doing high-volume pattern detection；
(2) in quantitative detection, there is still a need for a threshold values for detection department to distinguish the negative and positive, so data processing amount
It is larger.
Summary of the invention
In view of this, the present invention provides one kind for detecting aflatoxin B1Qualitative, quantitative immuno-chromatographic test paper strip
And its preparation method and application.The qualitative, quantitative immuno-chromatographic test paper strip high sensitivity, detection time are short, easy to operate, and energy
Aflatoxin B in qualitative and quantitative detection detection sample is realized simultaneously1。
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides one kind for detecting aflatoxin B1Qualitative, quantitative immuno-chromatographic test paper strip, on bottom plate
Successively overlap joint is provided with filter paper, sample pad, glass fibre membrane, nitrocellulose filter and blotting paper；
The aflatoxin B of colloid gold label is attached on glass fibre membrane1Monoclonal antibody；
It is provided with detection line and nature controlling line on nitrocellulose filter, the aspergillus flavus of fluorescent microsphere label is coated in detection line
Toxin B1Artificial antigen is coated with secondary antibody on nature controlling line.
In the present invention, the aflatoxin B of fluorescent microsphere label1Artificial antigen is that coupling has high molecular weight protein and fluorescence
The aflatoxin B of microballoon1。
Preferably, high molecular weight protein is bovine serum albumin(BSA) or ovalbumin.
Preferably, the package fluorescence that the rare earth ion that fluorescent microsphere is 0.01~10 μm of diameter is prepared as marker
The microballoon of substance.
It preferably, fluorescent material is the fluorescent material of organic or inorganic, or is the dopant of multiple fluorescent substance, or
Person is quantum dot.
Preferably, colloid gold particle is the gold particle of 10~100nm of diameter.
Preferably, it is being prepared using trisodium citrate reduction gold chloride for 10~100nm that colloid gold particle, which is diameter,
Gold particle, surface have negative electrical charge, can be coupled with protein.
The present invention also provides the preparation methods of the qualitative, quantitative immuno-chromatographic test paper strip, include the following steps:
By aflatoxin B1It is coupled with macromolecular, obtains aflatoxin B1Then artificial antigen uses fluorescent microsphere pair
Aflatoxin B1Artificial antigen is marked, and obtains the aflatoxin B of fluorescent microsphere label1Artificial antigen；
In the aflatoxin B of the detection line position coating fluorescent microsphere label of nitrocellulose filter1Artificial antigen, in nitre
The Quality Control line position of acid cellulose film is coated with secondary antibody；
Using colloid gold particle to aflatoxin B1Monoclonal antibody is marked, and obtains the aspergillus flavus of colloid gold label
Toxin B1Monoclonal antibody, by the aflatoxin B of colloid gold label1Monoclonal antibody is attached on glass fibre membrane；
Successively overlap joint pastes filter paper, sample pad, glass fibre membrane, nitrocellulose filter, blotting paper on bottom plate, is used
In detection aflatoxin B1Qualitative, quantitative immuno-chromatographic test paper strip.
Preferably, the aflatoxin B of colloid gold label1The quantity for spray of monoclonal antibody is 1.5~2.5 μ L/cm.
Preferably, the aflatoxin B of colloid gold label1The quantity for spray of monoclonal antibody is 2.0 μ L/cm.
In the present invention, the aflatoxin B of colloid gold label1Monoclonal antibody the preparation method is as follows:
Colloidal gold solution 40mL is taken, adjusting pH with the potassium carbonate of 0.02M is 8.0, and aflatoxin B is added1Monoclonal is anti-
Body makes its final concentration reach 5~50 μ g/mL, and after mixing, 1~4h of reaction is stirred at room temperature, 10% bovine serum albumin(BSA) is then added
(BSA), 0.5h~1h is reacted at room temperature, is centrifuged 5~15min, the precipitating phosphoric acid of 0.02M pH 8.0 in (2000~5000) × g
Salt buffer redissolves, and multiple liquor capacity is 1/10th of initial volume, saves in 4 DEG C.
In specific embodiment provided by the invention, the aflatoxin B of colloid gold label1The preparation side of monoclonal antibody
Method is as follows:
Colloidal gold solution 40mL is taken, adjusting pH with the potassium carbonate of 0.02M is 8.0, and the aflatoxin B of 200 μ g is added1It is single
Clonal antibody after mixing, is stirred at room temperature reaction 1h, is then added 10% bovine serum albumin(BSA) (BSA), react at room temperature 0.5h,
5000 × g is centrifuged 5~15min, and the precipitating phosphate buffer of 0.02M pH 8.0 redissolves, and multiple liquor capacity is initial volume
1/10th, in 4 DEG C save.
In the present invention, by the aflatoxin B of colloid gold label1Monoclonal antibody is attached on glass fibre membrane specifically
Are as follows: above-mentioned redissolution liquid is sprayed on glass fibre membrane, 25 DEG C of 1~2h of vacuum drying, quantity for spray is 2~4 μ L/cm.
Preferably, the aflatoxin B of fluorescent microsphere label1The peridium concentration of artificial antigen is 0.2~3.0mg/mL,
Quantity for spray is 0.7~0.8 μ L/cm；The peridium concentration of secondary antibody is 0.5~5.0mg/mL, and quantity for spray is 0.7~0.8 μ L/
cm。
Preferably, the aflatoxin B of fluorescent microsphere label1The peridium concentration of artificial antigen is 0.2~3.0mg/mL, spray
Painting amount is 0.74 μ L/cm；The peridium concentration of secondary antibody is 0.5~5.0mg/mL, and quantity for spray is 0.74 μ L/cm.
Preferably, the aflatoxin B of fluorescent microsphere label1The peridium concentration of artificial antigen is 0.2~3.0mg/mL,
Quantity for spray is 0.74 μ L/cm；The peridium concentration of secondary antibody is 0.5~5.0mg/mL, and quantity for spray is 0.74 μ L/cm.
In embodiment provided by the invention, the aflatoxin B of fluorescent microsphere label1The peridium concentration of artificial antigen is
0.5mg/mL, quantity for spray are 0.74 μ L/cm；The peridium concentration of secondary antibody is 1mg/mL, and quantity for spray is 0.74 μ L/cm.
Preferably, the aflatoxin B that fluorescent microsphere is marked1Artificial antigen and secondary antibody are coated with to cellulose nitrate
On plain film by the way of spraying, the spraying process of detection line and nature controlling line on nitrocellulose filter are as follows:
The aflatoxin B of fluorescent microsphere label is adjusted with the phosphate buffer of 0.01~0.5M pH7.01It is artificial anti-
Original content is 0.2~3.0mg/mL, is sprayed at detection line position, and quantity for spray is 0.7~0.8 μ L/cm；
Adjusting secondary antibody concentration with the phosphate buffer of 0.01~0.5M pH7.0 is 0.5~5.0mg/mL, by it
It is sprayed at Quality Control line position, quantity for spray is 0.7~0.8 μ L/cm；
By nitrocellulose filter under the conditions of 37 DEG C 8~12h of drying and processing.
Preferably, the aflatoxin B of fluorescent microsphere label is adjusted with the phosphate buffer of 0.02M pH7.01Manually
Antigen concentration is 0.5mg/mL.
Preferably, adjusting secondary antibody concentration with the phosphate buffer of 0.02M pH7.0 is 1.0mg/mL.
Preferably, detection line and nature controlling line are separated by 5~7mm.
In specific embodiment provided by the invention, detection line and nature controlling line are separated by 5mm.
In the present invention, it is separated by 10 with the film top margin of glass fibre membrane overlapped nitrocellulose filter one end and detection line~
12mm。
Preferably, it is separated by 10mm with the film top margin of glass fibre membrane overlapped nitrocellulose filter one end and detection line.
In the present invention, it is separated by 15 with the film top margin of glass fibre membrane overlapped nitrocellulose filter one end and nature controlling line~
19mm。
Preferably, it is separated by 15mm with the film top margin of glass fibre membrane overlapped nitrocellulose filter one end and nature controlling line.
In specific embodiment provided by the invention, secondary antibody is sheep anti-mouse antibody.
The present invention also provides a kind of qualitative and quantitative detection aflatoxin Bs1Method, include the following steps:
(1) qualitative detection
Detection sample is added in the sample pad of qualitative, quantitative immuno-chromatographic test paper strip, after reacting 10~12min, observation examination
The red stripes situation that paper slip detection line and nature controlling line occur:
Detection line and nature controlling line are all displayed in red band, then detect aflatoxin B in sample1Content less than 5 μ g/
Kg qualitatively judges as feminine gender；
Detection line is not displayed in red band, and nature controlling line is displayed in red band, then detects aflatoxin B in sample1Content
More than or equal to 5 μ g/kg, qualitatively judge as the positive；
If faint pale red band occurs in qualitative detection result detection line when judging, prompt to contain in sample
AFB1, but concentration is lower than 5 μ g/kg, then quantitative approach can be used and detected.
(2) quantitative detection
Quantitative detection is that aflatoxin B in detection sample is obtained according to standard curve1Content.
In the present invention, detection sample is cassia seed.
The present invention provides one kind for detecting aflatoxin B1Qualitative, quantitative immuno-chromatographic test paper strip and its preparation
Methods and applications.The qualitative, quantitative immuno-chromatographic test paper strip, successively overlap joint is provided with filter paper, sample pad, glass fibers on bottom plate
Tie up film, nitrocellulose filter and blotting paper；The aflatoxin B of colloid gold label is attached on glass fibre membrane1Monoclonal is anti-
Body；It is provided with detection line and nature controlling line on nitrocellulose filter, the aflatoxin of fluorescent microsphere label is coated in detection line
B1Artificial antigen is coated with secondary antibody on nature controlling line.The beneficial effects of the present invention are:
1, spetrophotometry is to the aflatoxin B in detection sample1Realize qualitative and quantitative detection simultaneously: this method exists
In one test strips, colloidal gold Color Appearance System is first passed through, range estimation the presence or absence of detection line and nature controlling line red stripes is qualitative to carry out
Detection.If colloidal gold is shown as positive sample, then instrument is read by fluorescent microsphere, fluorescence developing system is read out, obtained
To the actual concentrations of positive sample, quantitative detection is realized.One test strips realizes that two kinds of detection functions, sample results are being examined simultaneously
Dual system verifying is obtained in survey, it is as a result relatively reliable.
2, in practical applications, the possession ratio of negative sample is the largest, and when detecting high-volume sample, first passes through range estimation
Negative findings are excluded, detection time can be greatlyd save, positive findings can carry out accurate quantification again again, realize result data
Change and facilitate preservation.
Detailed description of the invention
Fig. 1 is aflatoxin B1The structural schematic diagram of qualitative, quantitative immuno-chromatographic test paper strip；Wherein, filter paper 1, sample pad
2, glass fibre membrane (colloidal gold antibody compound pad) 3, nitrocellulose filter (NC film) 4, detection line 5, nature controlling line 6, blotting paper
7, PVC bottom plates 8；
Fig. 2 is aflatoxin B1The detection principle diagram of qualitative, quantitative immuno-chromatographic test paper strip, whereinShow aspergillus flavus poison
Plain B1(AFB1),Show colloidal gold-AFB1 antibody complex,Show fluorescent microsphere-AFB1 coupled antigen,Show anti-mouse two
It is anti-.
Specific embodiment
The invention discloses one kind for detecting aflatoxin B1Qualitative, quantitative immuno-chromatographic test paper strip and its preparation
Methods and applications, those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular
It is that all similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this
Invention.Method and application of the invention is described by preferred embodiment, and related personnel can obviously not depart from this
Method described herein and application are modified or appropriate changes and combinations in summary of the invention, spirit and scope, realizing and
Using the technology of the present invention.
The present invention provides a kind of detection aflatoxin B1Qualitative, quantitative immuno-chromatographic test paper strip.It includes bottom plate, with
And the filter paper pasted with successively being overlapped on bottom plate, sample pad, fiberglass packing, nitrocellulose filter and blotting paper, wherein institute
The aflatoxin B of colloid gold label is coated on the fiberglass packing stated1Monoclonal antibody compound, on the nitrocellulose filter
It is coated with the aflatoxin B of fluorescent microsphere label1Artificial antigen is as detection line and is coated with anti-mouse antibody as nature controlling line.
The fluorescent microsphere is the rare earth ion for the use longer fluorescence half-life period that diameter is 0.01~10 μm as label
For object come the special microballoon of the package fluorescent material prepared, surface is connected with active group；The fluorescent material include it is organic or
The dopant and quantum dot of inorganic fluorescent material or multiple fluorescent substance.
The colloid gold particle is the gold for restoring gold chloride using trisodium citrate to prepare that diameter is 10~100nm
Particle, surface have negative electrical charge, can be coupled with protein.
Another technical solution that the present invention takes is to provide a kind of method for preparing above-mentioned test strips, it includes following
Step:
(1) the preparation of nitrocellulose filter
1. preparing aflatoxin B1Artificial antigen；
In order to prepare nitrocellulose filter, first by aflatoxin B1Standard items and protein macromolecule pass through covalent coupling
Method preparation detects trivial required artificial antigen, coupling method are as follows: EDC-NHS oximate method.Coupling protein is optional: bovine serum albumin
White and ovalbumin after being coupled macro-molecular protein, then by EDC-NHS oximate method is marked fluorescent microsphere and holoantigen
Note.
2. the preparation of detection line and nature controlling line.
The aflatoxin B that fluorescent microsphere is marked respectively1Artificial antigen and anti-mouse antibody are coated with to nitrocellulose filter
On, detection line and nature controlling line is made.Coating object concentration is adjusted respectively with the PBS (phosphate buffer solution) of 0.1~0.5MpH7.0
For 0.2~3.0mg/mL, spray film amount is 0.74 μ L/cm, detects wire spraying aflatoxin B1Artificial antigen, Quality Control wire spraying are anti-
Mouse antibody, twoth area are separated by 5mm；After 37 DEG C of drying processing overnight, saved backup in the environment of drying at room temperature.
(2) the preparation of colloidal gold antibody compound pad
1. colloid gold particle marks aflatoxin B by charge effect and Van der Waals force1Monoclonal antibody: colloidal gold solution is taken to use
It is 8.0 that the potassium carbonate of 0.02M, which adjusts pH, and the aflatoxin B of 5~50 μ g is added1Monoclonal antibody after being sufficiently mixed, is stirred at room temperature anti-
1~4h is answered, 1% bovine serum albumin(BSA) is then added, reacts at room temperature 1h, is centrifuged 5~15min in 2000 × g, precipitating uses 0.02M
The phosphate buffer of pH 8.0 redissolves, and redissolves 1/10th that volume is initial volume, saves in 4 DEG C stand-by.
2. gold labeling antibody compound is sprayed on glass fibre membrane with BIODOTDispensing System, 25 DEG C of vacuum
Dry 1~2h is placed in spare in the environment of drying at room temperature.
(3) the assembling of test strips
Paste materials described below with successively overlapping on bottom plate:
1. filter paper；
2. sample pad；
3. being coated with colloidal gold-aflatoxin B1The fiberglass packing of monoclonal antibody complex；
4. the aflatoxin B of fluorescent microsphere label1Artificial antigen is as detection line and anti-mouse antibody as nature controlling line
Nitrocellulose filter；
5. blotting paper.
It is assembled into immuno-chromatographic test paper strip of the invention.
(4) detection method
With aflatoxin B in above-mentioned immuno-chromatographic test paper strip detection Cassia subsample1Method, including following step
It is rapid:
1. weighing cassia seed 2g, 1min is shaken after 2mL extracting solution is added, supernatant is taken to detect；
2. 110 μ L cassia seed extracting solution samples are added in test paper well, 10min is reacted, estimates test strips detection line
The red stripes situation occurred with nature controlling line；
3. the red stripes if detection line and nature controlling line develop the color, aflatoxin B in sample1Content less than 5 μ g/
Kg qualitatively judges as feminine gender, whereas if detection line does not develop the color, only nature controlling line develops the color, then aflatoxin B in sample1Contain
Amount is greater than 5 μ g/kg, qualitatively judges as the positive.
4. according to colloidal gold red stripes show as a result, if when sample is positive, test strips insertion fluorescence is read
The power of the detection window of instrument, detection line and nature controlling line fluorescence can be shown over the display with the height of numerical value, according in instrument
The standard curve of typing can calculate aflatoxin B in sample1Content, realize the quantitative detection of positive sample
(after colloidal gold colour developing, the fluorescence data read in 20 minutes is effective).
Provided by the present invention for detecting aflatoxin B1Qualitative, quantitative immuno-chromatographic test paper strip and preparation method thereof
It is available on the market with agents useful for same in application or instrument.
Below with reference to embodiment, the present invention is further explained:
Embodiment 1: aflatoxin B is detected in cassia seed1Qualitative, quantitative immuno-chromatographic test paper strip preparation
One, the preparation process of immuno-chromatographic test paper strip
1. the preparation of nitrocellulose filter:
(1) aflatoxin B of fluorescent microsphere label is prepared1(AFB1- BSA) artificial antigen:
It is EDC-NHS oximate method with the coupling method of protein, coupling ratio 1:10-1:100, dialysis purification after coupling,
Obtain AFB1-BSA.Labeling method with fluorescent microsphere is EDC-NHS method, micro- with fluorescence with the calculating of artificial antigen protein concentration
The coupling ratio of ball is 1:1.Ultrafiltration centrifuge washing 3 times after label obtain the aflatoxin B of fluorescent microsphere label1Artificial antigen.
(2) preparation of detection line and nature controlling line:
The AFB of fluorescent microsphere label1In BSA conjugate and anti-mouse antibody coating to nitrocellulose filter: using 0.02M pH
The concentration of 7.0 PBS (phosphate buffer) dilution antigen conjugates is 0.5mg/mL, and resulting solution sprays conduct on film
Detection line；The concentration for diluting anti-mouse antibody is 1mg/mL, and resulting solution is sprayed on film as nature controlling line, the spray membrane body of two lines
Product is 0.74 μ L/cm, detection line and film top margin interval 10mm, and two line midfeather 5mm, 37 DEG C dry 12 hours, are placed in
It is saved backup in drying cupboard.
2. the preparation of colloidal gold composite pad:
Colloid gold label aflatoxin B1The preparation of monoclonal antibody: taking 40mL colloidal gold (25nm or so size), uses
The pH that solution of potassium carbonate adjusts colloidal gold is 8.0, and 200 μ g aflatoxin Bs are added1Monoclonal antibody, after being sufficiently mixed, room temperature
It is stirred to react 1h, 10% bovine serum albumin(BSA) 4mL, capping 30min, 5000 × g centrifugal force 15min is added, is surpassed
Pure water centrifuge washing 3 times, the precipitating PBS (wherein comprising 5% sucrose and 0.05%Tween-20) of 0.02M pH 8.0 redissolves
The spraying that bonding pad is carried out after to the 1/10 of initial volume, is sprayed on the glass fibre membrane of 30 × 0.8cm, 25 DEG C of vacuum drying
1~2h is put in spare in drying cupboard.The semi-finished product and NC film assembling detection sample results are as follows: standard curve concentration under this condition
Are as follows: 0,1,3,9,15,27ppb, R2It is 0.9912, IC50For 5.5ng/mL, linear regression equations are as follows: y=-827.1232x+
917.2319。
3. assembling test strips:
(1) filter paper and sample pad specification are 1 × 30cm；
(2) the glass fibre of colloidal gold antibody compound is coated with, and specification is 0.8 × 30cm；
(3) the nitrocellulose filter of detection line and nature controlling line has been sprayed, and specification is 2.5 × 30cm；
(4) blotting paper, specification are 1.2 × 30cm；
(5) PVC bottom plate, specification are 5.5 × 30cm.
The above material is successively pasted according to each component position in test strips structure schematic diagram, uses cutter after assembling
The test strips for cutting into 4 × 55mm are fitted into during plastics get stuck, aluminium foil bag are packed into after compression, and after desiccant is added, sealing is saved,
The room temperature environment shelf-life is 6 months.
As shown in Figure 1, the composition of the immuno-chromatographic test paper strip are as follows: on PVC bottom plate 8, successively overlap joint ground, which is pasted, is coated with
The NC film 4 of detection line 5 and nature controlling line 6, the gold pad 3 for being coated with colloidal gold antibody compound, sample pad 1, filter paper 2 and blotting paper 7,
The test strips of 4 × 55mm are cut into after pasting by cutting machine, are fitted into during plastics get stuck, an as complete Test paper
Item.
As shown in Fig. 2, testing principle is as follows:
After test strips are added in sample, sample is if it is negative (aflatoxin B1Content is less than 5 μ g/kg), sample is with layer
Direction chromatography is analysed, the colloidal gold antibody compound on bonding pad springs up the fluorescent microsphere mark on detection line position, with detection line
The aflatoxin B of note1Coupled antigen occur based on antigen-antibody in conjunction with immunological response and form antigen antibody complex,
Colloidal gold, which is assembled and shown in detection line, there are red stripes；Colloidal gold antibody compound of the part not in conjunction with coupled antigen
Quality Control line position is sprung up, anti-mouse secondary antibody can occur to combine with mouse monoclonal antibody to lead to which nature controlling line also shows red stripes
Negative findings can be directly judged as by crossing naked eyes.Sample is if it is the positive, then the aflatoxin B in sample1Elder generation and colloidal gold
Antibody complex combines, and combines aflatoxin B1Colloidal gold antibody compound cannot again with the fluorescent microsphere in detection line
The aflatoxin B of label1Coupled antigen combines, then detection line colloidal gold colour developing disappears, then is visually judged as positive.Because of glue
The colour developing of body gold can carry out optical quenching to the fluorescent microsphere in detection line, so the negative findings of colour developing, fluorescent value is lower, and
Colour developing as a result, being quenched because of no colloidal gold, fluorescent value is stronger, utilizes yellow in this fluorescent microsphere fluorescent value and sample to be positive
Aspertoxin B1The positive correlation of presentation reads instrument with fluorescent microsphere and reads fluorescence intensity of the fluorescent microsphere in detection line, leads to
Data analysis is crossed, obtains aflatoxin B in Cassia subsample1Specific concentration.In this detection architecture, colloidal gold color development system
For carrying out qualitative detection, fluorescent microsphere color development system is used to carry out quantitative detection.
Two, the aflatoxin B in qualitative and quantitative detection sample1
1. Cassia subsample are crushed, 18 meshes are crossed, 1g is accurately weighed, is extracted with the methanol solution of 80% concentration,
3min is vibrated, 4 DEG C of 4000r/min are centrifuged 10min, take supernatant with 0.01MPBS (pH7.4) solution to be diluted to methanol final concentration of
20%, 0.22 μm of organic filter is crossed, takes 110 μ L samples to be added dropwise in test strips sample pad, reacts 10min；
2. estimating the red stripes situation of test strips detection line and nature controlling line appearance；
3. if detection line and nature controlling line are all displayed in red band, aflatoxin B in cassia seed1Content less than 5 μ
G/kg qualitatively judges as feminine gender, whereas if detection line does not develop the color, only nature controlling line is displayed in red band, then yellow bent in sample
Mould toxin B1Content is greater than 5 μ g/kg, qualitatively judges as the positive.
4. according to colloidal gold red stripes show as a result, if when sample is positive, test strips insertion fluorescence is read
The power of the detection window of instrument, detection line and nature controlling line fluorescence can be shown over the display with the height of numerical value, according in instrument
The standard curve of typing can calculate aflatoxin B in sample1Content, realize the quantitative detection of positive sample
(after colloidal gold colour developing, the fluorescence data read in 20 minutes is effective).
5. sample is verified: in experimentation, with aflatoxin B1Standard items prepare the sample of known series of concentrations, measure
Then the standard curve established according to this series of values and corresponding concentration is stored in instrument by the numerical value of its corresponding fluorescence intensity
In.5 known concentration Cassia subsamples are detected (known sample through GC-MS standard measure is 0,3,5.3,9.8,
15ppb), it is detected with this method, complies fully with confirmation sample results, specific data are shown in Table 1.
Table 1: this detection method and instrument confirmation methods and results compare n=3
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. one kind is for detecting aflatoxin B1Qualitative, quantitative immuno-chromatographic test paper strip, which is characterized in that on bottom plate successively
Overlap joint is provided with filter paper, sample pad, glass fibre membrane, nitrocellulose filter and blotting paper；
The aflatoxin B of colloid gold label is attached on the glass fibre membrane1Monoclonal antibody；
It is provided with detection line and nature controlling line on the nitrocellulose filter, the Huang of fluorescent microsphere label is coated in the detection line
Aspertoxin B1Artificial antigen is coated with secondary antibody on the nature controlling line.
2. qualitative, quantitative immuno-chromatographic test paper strip according to claim 1, which is characterized in that the fluorescent microsphere label
Aflatoxin B1Artificial antigen is the aflatoxin B that coupling has high molecular weight protein and fluorescent microsphere1。
3. qualitative, quantitative immuno-chromatographic test paper strip according to claim 2, which is characterized in that the high molecular weight protein is ox
Seralbumin or ovalbumin.
4. qualitative, quantitative immuno-chromatographic test paper strip according to claim 2, which is characterized in that the fluorescent microsphere is diameter
The microballoon for the package fluorescent material that 0.01~10 μm of rare earth ion is prepared as marker.
5. qualitative, quantitative immuno-chromatographic test paper strip according to claim 4, which is characterized in that the fluorescent material is organic
Or inorganic fluorescent material, perhaps for the dopant of multiple fluorescent substance or be quantum dot.
6. qualitative, quantitative immuno-chromatographic test paper strip according to claim 1, which is characterized in that the colloid gold particle is straight
The gold particle of 10~100nm of diameter.
7. the preparation method of qualitative, quantitative immuno-chromatographic test paper strip as described in any one of claims 1 to 6, which is characterized in that
Include the following steps:
By aflatoxin B1It is coupled with macromolecular, obtains aflatoxin B1Artificial antigen, then using fluorescent microsphere to yellow bent
Mould toxin B1Artificial antigen is marked, and obtains the aflatoxin B of fluorescent microsphere label1Artificial antigen；
In the aflatoxin B of the detection line position coating fluorescent microsphere label of nitrocellulose filter1Artificial antigen, in nitric acid fibre
The Quality Control line position for tieing up plain film is coated with secondary antibody；
Using colloid gold particle to aflatoxin B1Monoclonal antibody is marked, and obtains the aflatoxin of colloid gold label
B1Monoclonal antibody, by the aflatoxin B of colloid gold label1Monoclonal antibody is attached on glass fibre membrane；
Successively overlap joint pastes filter paper, sample pad, glass fibre membrane, nitrocellulose filter, blotting paper on bottom plate, obtains for examining
Survey aflatoxin B1Qualitative, quantitative immuno-chromatographic test paper strip.
8. preparation method according to claim 7, which is characterized in that the aflatoxin B of the colloid gold label1Dan Ke
The quantity for spray of grand antibody is 1.5~2.5 μ L/cm.
9. preparation method according to claim 7, which is characterized in that the aflatoxin B of the fluorescent microsphere label1People
The peridium concentration of work antigen is 0.2~3.0mg/mL, and quantity for spray is 0.7~0.8 μ L/cm；The peridium concentration of the secondary antibody
For 0.5~5.0mg/mL, quantity for spray is 0.7~0.8 μ L/cm.
10. a kind of qualitative and quantitative detection aflatoxin B1Method, which comprises the steps of:
(1) qualitative detection
Detection sample is added in the sample pad of qualitative, quantitative immuno-chromatographic test paper strip described in any one of claims 1 to 6, instead
After answering 10~12min, the red stripes situation of test strips detection line and nature controlling line appearance is observed:
Detection line and nature controlling line are all displayed in red band, then detect aflatoxin B in sample1Content less than 5 μ g/kg, it is qualitative
It is judged as negative；
Detection line is not displayed in red band, and nature controlling line is displayed in red band, then detects aflatoxin B in sample1Content be greater than etc.
In 5 μ g/kg, qualitatively judge as the positive；
(2) quantitative detection
Detection qualitatively judges the fluorescence intensity of the test strips for the positive, obtains aflatoxin in detection sample according to standard curve
B1Content.
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| CN110095595A (en) * | 2019-05-27 | 2019-08-06 | 武汉上成生物科技有限公司 | A kind of fluorescence immune chromatography test paper detecting aflatoxin B1 |
| CN110220891A (en) * | 2019-05-24 | 2019-09-10 | 华南农业大学 | A kind of portable mycotoxin immunochromatography quantitative testing device and detection method |
| CN112180081A (en) * | 2020-09-07 | 2021-01-05 | 天津浩泰科技有限公司 | Small molecule immunochromatography detection method, test strip and kit |
| CN112505032A (en) * | 2020-12-10 | 2021-03-16 | 厦门禾堂餐饮企业管理有限公司 | Food intellectual detection system device |
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Application publication date: 20190111 |