CN108241060A - Mycotoxin detects colloidal gold quick measuring card and kit and the method being detected to mycotoxin - Google Patents
Mycotoxin detects colloidal gold quick measuring card and kit and the method being detected to mycotoxin Download PDFInfo
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The present invention relates to field of biological detection, disclose mycotoxin detection colloidal gold quick measuring card and kit and the method being detected to mycotoxin, specifically, the colloidal gold quick measuring card detected for mycotoxin is disclosed, which includes the sample application zone, conjugate area, the area of observation coverage and the suction zones that are sequentially arranged on bottom plate；The method being detected to mycotoxin is also disclosed, the method includes：Pre-treatment is carried out to sample to be tested, obtains mycotoxin extract, and using the colloidal gold quick measuring card mycotoxin extract is detected and interpretation of result.Also disclose a kind of detection mycotoxin kit, which is characterized in that the kit includes the colloidal gold quick measuring card, the bottle equipped with pure water and the bottle equipped with alcohol.The colloidal gold quick measuring card of the present invention can quickly and effectively improve sensitivity, specificity and the precision of detection.
Description
Technical field
The present invention relates to field of biological detection, and in particular, to a kind of colloidal gold quick measuring card detected for mycotoxin,
A kind of method being detected to mycotoxin and a kind of detection mycotoxin kit.
Background technology
Mycotoxin is metabolite caused by fungi grows in food or feed, all harmful to human and animal.
Mycotoxin causes the ergot poisoning that the earliest record of poisoning is 11 Century Europeans, and the clinical symptoms of this poisoning are once in Middle Ages
Icon painting in described.
Common mycotoxin is aflatoxin (B1), vomitoxin, zearalenone, fumonisin B1, T-2 poison
Element, ochratoxin A, wherein, it is most commonly seen with aflatoxin B1.
Aflatoxin is mainly that the one kind generated by aspergillus flavus and aspergillus parasiticus has serious carcinogenic, teratogenesis and mutagenesis
The similar secondary metabolite general name of chemical constitution.Wherein, aflatoxin B1 (AFB1) often coexists with other mycotoxins,
After being eaten can collective effect in human body, bigger is threatened to human health.At present, aflatoxin is primarily present in grain and oil crop
In feed.AFB1 poisoning symptoms show as the hepatitis symptoms such as vomiting, apocleisis, fever, jaundice, ascites, can induce deformity, cancer
Generation, prove that the experiment of fish, birds, domestic animal and primate the effect of its tumor inducing is very big, and can induce a variety of
The generation of cancer.Therefore, the detection reagent of the quick detection AFB1 of exploitation is to ensureing that food security and citizen's health have important meaning
Justice.
At present, the focus studied both at home and abroad is concentrated mainly on thin layer chromatography, liquid chromatography, radiommunoassay side
The inspection criterion of comparative maturity including method, enzyme-linked immunization, immunochromatographic method etc..However, there is also for example all for these methods
Phase is long, program is complicated, many shortcomings such as of high cost.Rarely has report especially for the detection technique of aflatoxin in itself
Road far lags behind the research and application of associated toxin detection technique.So far, it is main to producing the detection of aflatoxin fungi
Continue to use traditional culture identification method, that is, on the basis of isolated pathogen single bacterium colony, pass through morphological observation and kock
Rule detects to be verified, but this conventional method is real to judge the type of pathogen using pathogen Toxin producing C
It applies relatively difficult.For example, entire Testing and appraisal process usually needs expensive equipment, and operator is required to have profession
Pathogenicbacteria separation technology, Morphological Identification knowledge and rich experience, time-consuming and laborious, generally requiring several days could complete.This
Outside, traditional form identification method due to time-consuming and efficiency is low, and is susceptible to false positive or false negative result, it is difficult to it is full
Foot is to the actual needs of production aflatoxin fungi rapid sensitive detection.In addition, sensitivity and the spy of the method for the prior art
The opposite sex is relatively low.
Invention content
The purpose of the invention is to overcome in the prior art to mycotoxin detection time length, sensitivity, specificity and
The defects of precision is low and of high cost provides the detection fungi of a kind of quick energy, highly sensitive, high specific and high accurancy and precision
The colloidal gold quick measuring card and kit of toxin and a kind of method being detected to mycotoxin.
To achieve these goals, in a first aspect, the present invention provides a kind of colloidal gold speed for mycotoxin detection
Card is surveyed, which includes the sample application zone, conjugate area, the area of observation coverage and the suction zones that are sequentially arranged on bottom plate；
Wherein, the conjugate area includes antibody gold label pad, and the list of antimycotic toxin is fixed on the antibody gold label pad
Clonal antibody；
The sample application zone is included in the sample pad soak containing BSA, sucrose and/or trehalose and tween after dipping
And dry sample pad；
The area of observation coverage includes chromatographic film, and detection line and nature controlling line are fixed in the chromatographic film, wherein, the detection line
Mycotoxin to be detected and the conjugate of BSA are coated with, the nature controlling line is coated with the monoclonal antibody with antimycotic toxin
The secondary antibody of specific binding.
Second aspect, the present invention provides a kind of method being detected to mycotoxin, the method includes：To be measured
Sample carries out pre-treatment, obtains mycotoxin extract, and using colloidal gold quick measuring card as described above to the mycotoxin
Extract is detected and interpretation of result.
The third aspect, the present invention also provides a kind of detection mycotoxin kit, wherein, which is included as above
Colloidal gold quick measuring card, the bottle equipped with pure water and the bottle equipped with alcohol.
It is quick the present invention provides one kind, mycotoxin is accurately detected, preferably aflatoxin B1 colloidal gold is quickly examined
Test agent.This detection method does not need to any supplementary instrument and is handled and interpretation, and testing cost is low, easy to operate, and
Experimental result can be quickly obtained, is very suitable for carrying out primary dcreening operation detection in production line.For the colloidal gold speed of mycotoxin detection
It is the quick detection reagent prepared based on immunology principle to survey card.Matrix for detection is mainly the samples such as grain and oil crop and feed
Product, this method pre-treatment is simple, and when detection only needs to be detected using methanol solution as extracting solution, and detection process is
Detection sample is instilled in well, result is judged by the colour developing situation of detection line and control line.This method is compared
Large-scale instrument method has many advantages, such as that sensitivity, specificity and precision are high, applied widely, while can generate certain warp
Benefit of helping and social benefit.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Specific embodiment
The specific embodiment of the present invention is described in detail below.It is it should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood to comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It between the endpoint value of a range and individual point value and can be individually combined with each other between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
On the one hand, the present invention provides a kind of colloidal gold quick measuring card for mycotoxin detection, the colloidal gold quick measuring cards
Including sample application zone, conjugate area, the area of observation coverage and the suction zones being sequentially arranged on bottom plate；
Wherein, the conjugate area includes antibody gold label pad, and the list of antimycotic toxin is fixed on the antibody gold label pad
Clonal antibody；
The sample application zone is included in the sample pad soak containing BSA, sucrose and/or trehalose and tween after dipping
And dry sample pad；
The area of observation coverage includes chromatographic film, and detection line and nature controlling line are fixed in the chromatographic film, wherein, the detection line
Mycotoxin to be detected and the conjugate of BSA are coated with, the nature controlling line is coated with the monoclonal antibody with antimycotic toxin
The secondary antibody of specific binding.
, according to the invention it is preferred to, the sample pad soak is containing BSA, sucrose and/or trehalose and tween
PH value is the PBS buffer solution of 7-9.5 or PB buffer solutions, and pH value is preferably 8.0-9.0.Wherein, the concentration of tween can be
The concentration of 0.005-0.02 volumes %, BSA can be 1-10 weight %, and the concentration of sucrose and/or trehalose can be 2-10 weights
Measure %.Still more preferably, the tween is Tween 80.Herein it should be noted that when containing in sucrose and trehalose
When a kind of, concentration as above refers to the concentration of one of which, when containing sucrose and during two kinds of trehalose, what concentration as above referred to
It is the concentration of both.
According to the present invention, the sample pad soak can also with the difference of the mycotoxin type of detection and not
Together, for example, the sample pad soak can also according to the type of the mycotoxin of the detection optionally contain cholic acid, poly-
Vinylpyrrolidone, RHODASURF ON-870, STANDAPOLES-1, wire tray 60 and one kind or more in Triton X-100
Plant the detection to adapt to different type mycotoxin.Wherein, it is further preferred that a concentration of 0.01-2 weights of the cholic acid
Measure %, preferably 0.1-1 weight %；A concentration of 0.005-0.03 weight %, preferably 0.008- of polyvinylpyrrolidone
0.015 weight %；(addition polymerization product of oleyl alcohol and ethylene oxide, the structural formula of product are RHODASURF ON-870
C18H35O(CH2CH2O)n- H), STANDAPOLES-1 (laureth sulfuric ester sodium salt), (sorbitol anhydride list is stearic for wire tray 60
Acid esters) and Triton X-100 (Triton X-100) total content be 0.03-3 weight %, more preferably 0.05-
0.4 weight %.
Wherein, the PBS buffer solution and PB buffer solutions are known in the art common buffer solution, herein no longer in detail
It repeats.
Wherein, sample pad in sample pad soak is as above impregnated, subsequent detection process can be significantly reduced
Cross reacting rate in middle antigen-antibody cohesive process.And sample pad the present invention sample pad soak in have significantly improve
Hydrophily.
According to the present invention, the time that the sample pad is impregnated in sample pad soak can carry out in a wider scope
Selection, as long as the ingredient in soak is enabled adequately to combine in sample pad, for example, 20-40min.
According to the present invention, the method that the sample pad of the immersion is dried can be the selection of this field routine, example
Such as, the temperature of the drying can be 35-40 DEG C, and the dry time can be 40-80min.
According to the present invention, the sample pad is preferably the high-absorbent materials such as glass fibre membrane, blotting paper.
According to the present invention, the mycotoxin can be the various mycotoxins of this field routine, for example, can be yellow bent
Mould toxin (preferably AFB1), vomitoxin, zearalenone, fumonisin B1, T-2 toxin or ochratoxin A.It is excellent
Choosing, the mycotoxin is aflatoxin, preferably aflatoxin B1.
, according to the invention it is preferred to, the antibody gold label pad of the monoclonal antibody for being fixed with antimycotic toxin passes through such as
Under method prepared：
(1) monoclonal antibody for resisting mycotoxin to be measured is added in into the colloidal gold solution of pH7.5-9, contacts 40-
80min obtains being marked with the colloidal gold solution of the monoclonal antibody；
(2) there are addition BSA solution in the colloidal gold solution of monoclonal antibody, contact to the label that step (1) obtains
40-80min；
(3) mixed material obtained step (2) carries out separation of solid and liquid, and sediment is redissolved in containing sucrose/or seaweed
In the boric acid solution of sugar, BSA and Sodium azide, gold labeling antibody solution is obtained；
(4) by the gold labeling antibody solution spraying prepared by step (3) in the gold-labelled pad soak containing tween and BSA
After immersion and through in drying to the gold-labelled pad of water content 60-80 weight %, drying to water content be less than 10 weight % later.
According to the present invention, in step (1), colloidal gold solution that can be by conventional pH adjusting agent to preparing as described above
PH value be adjusted, for example, the organic or inorganic acids such as boric acid, phosphoric acid, hydrochloric acid, acetic acid, ethanedioic acid.The present invention preferably passes through boron
Its pH is adjusted in acid.
According to the present invention, the addition of the monoclonal antibody of the anti-mycotoxin to be measured is preferably so that the antibody can
It is adequately combined with colloid gold particle, it is preferred that the addition of the monoclonal antibody causes the monoclonal antibody
A concentration of 1-3mg/ml.Wherein, the type of the antibody can be selected according to the type of toxin to be detected, the antibody
The commercially available monoclonal antibody for particular toxin that can be known in the art can also pass through well known monoclonal antibody
Preparation method is voluntarily prepared, and the present invention is not particularly limited this.
According to the present invention, the contact is preferably dynamic Contact, for example, being connect under conditions of vibrating or overturning mixing
It touches, the temperature of the contact is preferably room temperature, for example, 15-35 DEG C.
According to the present invention, in step (2), in order to stablize and close the colloid for being combined with antibody obtained in step (1)
Gold particle, the present invention need to add in BSA solution into the product of step (1), wherein, the addition of the BSA solution is not
Special limitation, as long as can fully the product of step (1) be stablized and be closed, it is preferred that its addition causes
A concentration of 0.5-1.5 weight % of BSA.Wherein, the contact with BSA is preferably dynamic Contact, for example, mixed vibrating or overturning
It is contacted under conditions of conjunction, the temperature of the contact is preferably room temperature, for example, 15-35 DEG C.
According to the present invention, in step (3), can be by the method that the mixed material that step (2) obtains carries out separation of solid and liquid
The selection of this field routine, the present invention is not particularly limited this, for example, can pass through the method (10000- of centrifugation
15000rpm, 30-50min, 2-6 DEG C) or filtering method.
In addition, the present inventor also found in the course of the study, by using containing sucrose/or trehalose, BSA
The solid phase after as above separation of solid and liquid is redissolved with the boric acid solution of Sodium azide, to obtain gold labeling antibody solution, uses the gold
Labeling antibody solution coats gold-labelled pad, and the sensitivity to Mycotoxin identification, specificity and precision is enabled to obtain further
It is promoted.In the case of preferred, in the boric acid solution, a concentration of 0.5-4 weight % of sucrose and/or trehalose, preferably
1-3 weight %；A concentration of 0.5-1.5 weight % of BSA, preferably 0.8-1.2 weight %；A concentration of 0.01-1 weights of Sodium azide
Measure %, preferably 0.03-0.08 weight %；The pH value of the boric acid solution is 7-9, preferably 7.5-8.5.It needs to illustrate herein
, when containing a kind of in sucrose and trehalose, " a concentration of 0.5-4 weight % of sucrose and/or trehalose, preferably
1-3 weight % " refers to the concentration of one of which, when containing sucrose and during two kinds of trehalose, " sucrose and/or trehalose it is dense
It spends and refers to the concentration of both for 0.5-4 weight %, preferably 1-3 weight % ".
In step (4), it was found by the inventors of the present invention that as above specifically preparing the preparation of antibody gold label pad in the present invention
Specific gold-labelled pad soak is combined in the process, the colloidal gold for including the antibody gold label pad speed for enabling to finally prepare
Card is surveyed to greatly improve the sensitivity of Mycotoxin identification, specificity and precision.Wherein, it is preferred that when the fungi
When toxin is aflatoxin, preferably aflatoxin B1, the gold-labelled pad soak can be to contain only polysorbas20 and BSA
PBS buffer solution；Preferably, a concentration of 1-5 weights of a concentration of 0.005-0.01 volumes % of the polysorbas20, the BSA
Measure %.It is preferred in the case that this, method of the invention the detection of aflatoxin can be realized maximum detection sensitivity,
Specificity and precision.
According to the present invention, the soak can also containing one kind in sucrose, trehalose, polyethylene glycol and casein or
It is a variety of.Wherein, the polyethylene glycol is preferably polyethylene glycol 2000.It is further preferred that the sucrose and/or trehalose is dense
It spends for 2-10 weight %, a concentration of 0.01-1 weight % of polyethylene glycol, a concentration of 0.1-2 weight % of casein.It needs herein
It is noted that when containing a kind of in sucrose and trehalose, " a concentration of 2-10 of the sucrose and/or trehalose weights
Amount % " refers to the concentration of one of which, when containing sucrose and during two kinds of trehalose, " the concentration of the sucrose and/or trehalose
The concentration of both is referred to for 2-10 weight % ".
According to the present invention, the time that the gold-labelled pad is impregnated in gold-labelled pad soak can carry out in a wider scope
Selection, as long as the ingredient in soak is enabled adequately to combine in gold-labelled pad, for example, 20-40min.
According to the present invention, the method that the gold-labelled pad of the immersion is dried can be the selection of this field routine, example
Such as, the temperature of the drying can be 35-40 DEG C, and the dry time can be 40-80min.
According to the present invention, in order to further increase the colloidal gold quick measuring card pair prepared by antibody gold label pad prepared by the present invention
Sensitivity, specificity and the precision of mycotoxin detection, method of the invention further include：Coating the gold mark particle solution
The preceding gold-labelled pad by dry immersion gold labeling antibody solution is more than 80% in relative humidity, preferably 80-90% and 2-6 DEG C
Under the conditions of place 20-360min.
Can be that this field is conventional to the method that the gold-labelled pad after contact gold-labelled pad soak is dried according to the present invention
Selection, for example, the temperature of the drying can be 35-40 DEG C, the dry time can be 40-80min.
According to the present invention, the antibody gold label pad is preferably glass fibre membrane or polyester film.
According to the present invention, quantity for spray gold labeling antibody produced above being sprayed in the gold-labelled pad can be wider
In the range of be changed.Preferably, quantity for spray is 0.5-1.5 μ L/cm.
The present inventor has found in the course of the study, by under given conditions by the gold chloride of special ratios
Redox reaction is carried out, and reaction product is made to cool down in specific rate with sodium citrate, it is big particle can be obtained
Colloid gold particle of the small uniform and grain size in the range of 10-35nm, preferably 10-30nm.It is prepared when with this colloid gold particle
Colloidal gold quick measuring card for mycotoxin detection when, sensitivity, specificity and the precision of detection can be effectively improved.
Therefore, the preparation method of the colloid gold particle in colloidal gold solution of the invention preferably includes：
(i) under conditions of stirring, sodium citrate is added in into the chlorauric acid solution of boiling, makes gold chloride and sodium citrate
Haptoreaction 5-15min, obtains reaction product；
(ii) reaction product that step (1) obtains is cooled to 20-35 DEG C under 15-20 DEG C/min cooling rates, obtained
Grain size is the colloid gold particle of 10-35nm；
Wherein, the addition of sodium citrate so that the weight ratio of gold chloride and sodium citrate is 1：2-5, preferably 1：3-4.
The chlorauric acid solution is preferably the aqueous solution of gold chloride, and it is further preferred that in the chlorauric acid solution, the chlorine
A concentration of 1-2 weight % of auric acid.
According to the present invention, after sodium citrate solution is added in the chlorauric acid solution, in ebuillition of heated and stirring
Under the conditions of, the color of solution can show variation of light blue, the blue, purple to claret, after claret is showed, solution face
Color no longer changes, and keeps 3-10min under conditions of ebuillition of heated and stirring again at this time, you can obtain reaction product.It should
Course probably needs the time of 5-15min in total.
Can be the selection of this field routine by the method that the reaction product cools down, for example, can according to the present invention
With by cooling down with cooling media contact, details are not described herein by the present invention.
According to the present invention, the sodium citrate can be trisodium citrate.
According to the present invention, the chromatographic film is preferably prepared by following steps：By the idol of the vomitoxin and BSA
Connection object and the secondary antibody are fixed in the chromatographic film, are then dried and are assembled；The chromatography water content of membrane is 30-80 before drying
Weight %；The chromatography water content of membrane is less than 10 weight % after drying.Wherein, the fixation preferably by way of spraying into
Row.The method of the drying has hereinbefore carried out specific description, is no longer described in detail herein.
According to the present invention, the secondary antibody is preferably sheep anti mouse secondary antibody.
Wherein, the fixed mode can be the selection of this field routine, for example, for fixed coating buffer solution
For the PB buffer solutions of pH7.4, Seal treatment is carried out to T lines and C lines using BSA later.Wherein, in buffer solution is coated with, vomiting
The concentration of toxin and BSA conjugate can be 0.1-0.5mg/mL, a concentration of 0.1-0.5mg/mL of secondary antibody, and spraying package amount is
0.3-1μL/cm.Further, the chromatographic film being coated with is dried according to any way as above.
In addition, in assembling, the detection line can be 0.5-1mm apart from conjugate area, the nature controlling line distance detection
Line can be 0.5-1mm.Wherein, it is further preferred that the sample pad and gold-labelled pad, gold-labelled pad and chromatographic film, chromatographic film with
The overlapped cover width of suction zones is 1-2mm.
According to the present invention, the chromatographic film can be nitrocellulose filter.
According to the present invention, the substrate of the suction zones is blotting paper.
Second aspect, the present invention provides a kind of method being detected to mycotoxin, the method includes：To be measured
Sample carries out pre-treatment, obtains mycotoxin extract, and using colloidal gold quick measuring card as described above to the mycotoxin
Extract is detected and interpretation of result.
According to the present invention, to the method that the toxin in sample to be tested extracts, it is preferable to use methanol solutions, preferably 75-85
The methanol solution of volume % handles sample to be tested.Wherein, under preferable case, relative to the sample to be tested of 1g, the first
The addition of alcoholic solution is 0.5-1mL.The use of the method that methanol solution handles sample to be tested can be conventional selection,
For example, concussion mixing or the mixing that turns upside down manually, the present invention is not particularly limited this.The time of processing is preferred
For 5-30min.After processing contact, preferably solid-liquid is carried out by centrifuging the method for (4000-6000rpm, 4-8min, 20-35 DEG C)
Separation obtains supernatant.
It is to be detected when containing in sample to be tested when the colloidal gold quick measuring card of the present invention is used to be detected toxin to be measured
Toxin when, after sample to be tested is added into sample application zone, under the action of suction zones, sample to be tested is transported from sample application zone to suction zones
Dynamic, when moving to conjugate area, toxin to be detected is combined with the specific antibody of conjugate, is continued thereafter with and is travelled forward,
Since the toxin in the specific antibody and sample in conjugate area is combined in advance, during (T lines) to be exercised to detection line, just
It will not be combined again with the conjugate in T lines, so as to which T lines do not develop the color, when moving to nature controlling line (C lines), the antibody in C lines is with treating
The specific antibody specific binding of the toxin of detection, so as to develop the color.That is, when T lines do not develop the color, and during the colour developing of C lines, knot
Fruit is the positive.Conversely, when not containing toxin to be detected in sample, the toxin specific antibody in conjugate area can be with the idol
Join object to combine, so as to develop the color, that is, when T lines and C lines develop the color, result is feminine gender.If C lines do not develop the color, then may be used
Directly judge that testing result is invalid.
The third aspect, the present invention also provides a kind of detection mycotoxin kits, which includes as described above
Colloidal gold quick measuring card, the bottle equipped with pure water and the bottle equipped with alcohol.Wherein, the pure water and alcohol can be used for extracting sample
Toxin in product, the method for the extraction can be preferably methanol by simple soak extraction, the alcohol.Further, institute
State the specification of application method and Toxic extraction method that the colloidal gold quick measuring card is further included in kit.
The present invention will be described in detail by way of examples below.In following embodiment,
Embodiment 1
The present embodiment is used for the preparation method for illustrating colloidal gold quick measuring card provided by the invention
(1) preparation of colloidal gold solution
The chlorauric acid solution of a concentration of 1.3 weight % is positioned on heating magnetic stirring apparatus, is heated to boiling, it is quick to add
Entering 2.5ml citric acid three sodium solutions, (molar ratio of gold chloride and trisodium citrate is 1：3.5), solution starts to change colour, by it is light blue,
Blue, purple, claret change colour successively, when solution becomes claret, continue to heat 5min, then with the speed of 15 DEG C/min
Be cooled to room temperature, be put in 4 DEG C it is spare.Through transmission electron microscope microscopy, colloid gold particle is in the same size, between 20-25nm, established practice
Spherical shape then；
(2) preparation of gold labeling antibody
1) pH value for the colloidal gold solution for being prepared step (1) using borate buffer is adjusted to 8.5；
2) under conditions of stirring, aspergillus flavus resisting toxin B1 monoclonal antibody (potency is rapidly added into colloidal gold solution
It is 1:500000), antibody concentration reaches 5.0mg/mL, and room temperature slowly overturns mixing 1h；
3) the BSA solution of 10 weight % is added in into the colloidal gold solution for be marked with antibody, makes final concentration of 1 weight of BSA
% is measured, closes colloidal gold solution to stablize, room temperature slowly overturns mixing 1h；
4) supernatant is abandoned after centrifuging (12000rpm, 40min, about 4 DEG C), with containing 2 weight % sucrose, 1 weight %BSA and 0.05
The borate buffer of the pH8.0 of the Sodium azide of weight % redissolves sediment, is in store at about 4 DEG C；
(3) preparation of antibody gold label pad
1) 0.01mL polysorbas20s and 5g BSA processing are weighed or measure, with the PBS buffer solution (pH 9.0) of 0.01 weight %
Constant volume continues mixing 30min at a slow speed, obtains gold-labelled pad soak to 100mL after being completely dissolved；
2) glass fibre gold-labelled pad is completely soaked the 30min in prepared gold-labelled pad soak；Later with clean tweezer
Son takes out gold-labelled pad, is positioned in 37 DEG C of clean environment, dries 1h；
4) dried gold-labelled pad is positioned over 30min in the environment that relative humidity is about 4 DEG C of 80% temperature, to increase gold
The hydrophily of pad is marked, the gold labeling antibody prepared in spraying process (2) later is positioned over 37 DEG C of clean rings immediately after spraying
In border, 1h is dried.
(4) preparation of sample pad
1) weigh or measure 0.01mL Tween 80s and 5g BSA processing, 5g sucrose, with the PBS buffer solution of 0.01 weight %
(pH 8.5) constant volume continues mixing 30min at a slow speed to 100mL after being completely dissolved；
2) double glazing fiber sample pad is completely soaked the 30min in prepared sample pad soak；Later with clean
Net tweezers take out gold-labelled pad, are positioned in 37 DEG C of clean environment, dry 1h.
(5) preparation of nitrocellulose filter (NC films)
Aflatoxin B1 and BSA are coupled, and are coated on NC films as T lines, sheep anti mouse secondary antibody is sprayed at NC films
It is upper to be used as C lines, Seal treatment is carried out to T lines and C lines using BSA later.
(6) assembling of colloidal gold quick measuring card
Sample pad, gold-labelled pad, NC films and water absorption pad are affixed on successively on PVC bottom plates, wherein, NC films be affixed on gold-labelled pad it
Under, it is overlapped 2mm, and T linear distance gold-labelled pad 0.8mm, C linear distance T lines 0.8mm；Blotting paper 2mm Chong Die with NC films.In addition, sample
Pad and gold-labelled pad also be overlapped 2mm.
Embodiment 2
The present embodiment is used for the preparation method for illustrating colloidal gold quick measuring card provided by the invention
(1) preparation of colloidal gold solution
The chlorauric acid solution of a concentration of 1 weight % is positioned on heating magnetic stirring apparatus, is heated to boiling, rapidly join
(molar ratio of gold chloride and trisodium citrate is 1 to 2.5ml citric acid three sodium solutions：4), solution starts to change colour, by light blue, blue
Color, purple, claret change colour successively, when solution becomes claret, continue to heat 8min, then be dropped with the speed of 18 DEG C/min
Warm to room temperature, be put in 4 DEG C it is spare.Through transmission electron microscope microscopy, colloid gold particle is in the same size, between 15-20nm, into rule
Spherical shape；
(2) preparation of gold labeling antibody
1) pH value for the colloidal gold solution for being prepared step (1) using borate buffer is adjusted to 7.5；
2) under conditions of stirring, aspergillus flavus resisting toxin B1 monoclonal antibody (potency is rapidly added into colloidal gold solution
It is 1:500000), antibody concentration reaches 3.0mg/mL, and room temperature slowly overturns mixing 1h；
3) the BSA solution of 10 weight % is added in into the colloidal gold solution for be marked with antibody, makes final concentration of the 0.8 of BSA
Weight % closes colloidal gold solution to stablize, and room temperature slowly overturns mixing 1h；
4) centrifuge (15000rpm, 30min, about 4 DEG C) after abandon supernatant, with containing 1 weight % sucrose, 0.8 weight %BSA and
The borate buffer of the pH8.5 of the Sodium azide of 0.08 weight % redissolves sediment, is in store at about 4 DEG C；
(3) preparation of antibody gold label pad
1) 0.005mL polysorbas20s and 3g BSA processing are weighed or measure, with the PBS buffer solution (pH 8.5) of 0.01 weight %
Constant volume continues mixing 30min at a slow speed to 100mL after being completely dissolved；
2) glass fibre gold-labelled pad is completely soaked the 30min in prepared gold-labelled pad soak；Later with clean tweezer
Son takes out gold-labelled pad, is positioned in 35 DEG C of clean environment, dries 80min；
4) dried gold-labelled pad is positioned over 30min in the environment that relative humidity is about 4 DEG C of 85% temperature, to increase gold
The hydrophily of pad is marked, the gold labeling antibody prepared in spraying process (2) later is positioned over 40 DEG C of clean rings immediately after spraying
In border, 40min is dried.
(4) preparation of sample pad
1) weigh or measure 0.02mL Tween 80s and 3g BSA processing, 8g sucrose, with the PBS buffer solution of 0.01 weight %
(pH 9.0) constant volume continues mixing 30min at a slow speed to 100mL after being completely dissolved；
2) double glazing fiber sample pad is completely soaked the 30min in prepared sample pad soak；Later with clean
Net tweezers take out gold-labelled pad, are positioned in 37 DEG C of clean environment, dry 1h.
(5) preparation of nitrocellulose filter (NC films)
Aflatoxin B1 and BSA are coupled, and are coated on NC films as T lines, sheep anti mouse secondary antibody is sprayed at NC films
It is upper to be used as C lines, Seal treatment is carried out to T lines and C lines using BSA later.
(6) assembling of colloidal gold quick measuring card
Sample pad, gold-labelled pad, NC films and water absorption pad are affixed on successively on PVC bottom plates, wherein, NC films be affixed on gold-labelled pad it
Under, it is overlapped 2mm, and T linear distance gold-labelled pad 0.8mm, C linear distance T lines 0.8mm；Blotting paper 2mm Chong Die with NC films.
Embodiment 3
The present embodiment is used for the preparation method for illustrating colloidal gold quick measuring card provided by the invention
(1) preparation of colloidal gold solution
The chlorauric acid solution of a concentration of 2 weight % is positioned on heating magnetic stirring apparatus, is heated to boiling, rapidly join
(molar ratio of gold chloride and trisodium citrate is 1 to 2.5ml citric acid three sodium solutions：2.5), solution starts to change colour, by light blue, blue
Color, purple, claret change colour successively, when solution becomes claret, continue to heat 10min, then with the speed of 20 DEG C/min
Be cooled to room temperature, be put in 4 DEG C it is spare.Through transmission electron microscope microscopy, colloid gold particle is in the same size, between 25-30nm, established practice
Spherical shape then；
(2) preparation of gold labeling antibody
1) pH value for the colloidal gold solution for being prepared step (1) using borate buffer is adjusted to 9.0；
2) under conditions of stirring, aspergillus flavus resisting toxin B1 monoclonal antibody (potency is rapidly added into colloidal gold solution
It is 1:500000), antibody concentration reaches 1.0mg/mL, and room temperature slowly overturns mixing 1h；
3) 10% BSA solution is added in into the colloidal gold solution for be marked with antibody, makes final concentration of 1.5 weight of BSA
% is measured, closes colloidal gold solution to stablize, room temperature slowly overturns mixing 1h；
4) centrifuge (10000rpm, 50min, about 4 DEG C) after abandon supernatant, with containing 3 weight % sucrose, 1.2 weight %BSA and
The borate buffer of the pH 7.5 of the Sodium azide of 0.03 weight % redissolves sediment, is in store at about 4 DEG C；
(3) preparation of antibody gold label pad
1) 0.007mL polysorbas20s and 1g BSA processing are weighed or measure, with the PBS buffer solution (pH 9.0) of 0.01 weight %
Constant volume continues mixing 30min at a slow speed to 100mL after being completely dissolved；
2) glass fibre gold-labelled pad is completely soaked the 30min in prepared gold-labelled pad soak；Later with clean tweezer
Son takes out gold-labelled pad, is positioned in 37 DEG C of clean environment, dries 1h；
4) dried gold-labelled pad is positioned over 30min in the environment that relative humidity is about 4 DEG C of 90% temperature, to increase gold
The hydrophily of pad is marked, the gold labeling antibody prepared in spraying process (2) later is positioned over 37 DEG C of clean rings immediately after spraying
In border, 1h is dried.
(4) preparation of sample pad
1) weigh or measure 0.005mL Tween 80s and 8g BSA processing, 2g sucrose, with the PBS buffer solution of 0.01 weight %
(pH 8.0) constant volume continues mixing 30min at a slow speed to 100mL after being completely dissolved；
2) double glazing fiber sample pad is completely soaked the 30min in prepared sample pad soak；Later with clean
Net tweezers take out gold-labelled pad, are positioned in 37 DEG C of clean environment, dry 1h.
(5) preparation of nitrocellulose filter (NC films)
Aflatoxin B1 and BSA are coupled, and are coated on NC films as T lines, sheep anti mouse secondary antibody is sprayed at NC films
It is upper to be used as C lines, Seal treatment is carried out to T lines and C lines using BSA later.
(6) assembling of colloidal gold quick measuring card
Sample pad, gold-labelled pad, NC films and water absorption pad are affixed on successively on PVC bottom plates, wherein, NC films be affixed on gold-labelled pad it
Under, it is overlapped 2mm, and T linear distance gold-labelled pad 0.8mm, C linear distance T lines 0.8mm；Blotting paper 2mm Chong Die with NC films.
Embodiment 4
The present embodiment is used for the preparation method for illustrating colloidal gold quick measuring card provided by the invention
The preparation of colloidal gold quick measuring card is carried out according to the method for embodiment 1, the difference is that in step (2), the Dan Ke
A concentration of 0.5mg/mL of grand antibody, in the boric acid solution, a concentration of 0.1 weight % of the sucrose, the BSA's is dense
It spends for 3 weight %, without Sodium azide, and low temperature uses after placing for 3 week after being resuspended.
Embodiment 5
The present invention provides the preparation methods of colloidal gold quick measuring card for explanation for the present embodiment
The preparation of colloidal gold quick measuring card is carried out according to the method for embodiment 1, the difference is that in step (3), the gold mark
It pads soak and also contains sucrose and/or trehalose.
Embodiment 6
The present invention provides the preparation methods of colloidal gold quick measuring card for explanation for the present embodiment
The preparation of colloidal gold quick measuring card is carried out according to the method for embodiment 1, the difference is that the gold-labelled pad soak is
The PBS solution of the sucrose and trehalose of the BSA of 5 weight % and 5 weight %.
Embodiment 7
The present invention provides the preparation methods of colloidal gold quick measuring card for explanation for the present embodiment
The preparation of colloidal gold quick measuring card is carried out according to the method for embodiment 1, the difference is that in step (3), is being coated
Before the gold labeling antibody solution, dry gold-labelled pad is not placed under conditions of humidity is more than 80% and about 4 DEG C.
Embodiment 8
The present invention provides the preparation methods of colloidal gold quick measuring card for explanation for the present embodiment
The preparation of colloidal gold quick measuring card is carried out according to the method for embodiment 1, the difference is that the sample pad is 1 layer of glass
Glass fiber.
Embodiment 9
This comparative example is used to illustrate that the method for reference provides the preparation method of colloidal gold quick measuring card
The preparation of colloidal gold quick measuring card is carried out according to the method for embodiment 1, the difference is that in step (1), reaction product
It is warmed to room temperature under conditions of stirring with the decline of the cooling rate of 5 DEG C/min, and the molar ratio of gold chloride and sodium citrate is 1：
1.The size of acquired colloid gold particle is between 30-50nm.
Embodiment 10
The present invention provides the preparation methods of colloidal gold quick measuring card for explanation for the present embodiment
The preparation of colloidal gold quick measuring card is carried out according to the method for embodiment 1, the difference is that the sample pad soak is
The PBS solution of the cyclodextrin solution of polysorbas20 containing 0.01 volume %, the BSA solution of 5 weight % and 5 weight %.
Comparative example 1
The present invention provides the preparation methods of colloidal gold quick measuring card for explanation for the present embodiment
The preparation of colloidal gold quick measuring card is carried out according to the method for embodiment 1, the difference is that the mycotoxin is reddish brown song
Mould toxin, the specific antibody be ochratoxin monoclonal antibody, potency 1:500000.
Test case 1
The colloidal gold quick measuring card of embodiment 1-10 and comparative example 1 is lain on detection platform, is then added dropwise and is voluntarily configured
The solution containing 1ng/ml aflatoxin B1s in well, when sample is just moved to NC films, stop be added dropwise sample
Liquid records result after 15min.Each sample is repeated 10 times, while record positive number.It the results are shown in Table 1.
In addition, the solution containing 10ng/ml ochratoxins is added dropwise to embodiment 1-10 and the well of comparative example 1
In, when sample is just moved to NC films, stop that sample liquid is added dropwise, each sample is repeated 10 times, while records cross reaction sample
Product number and the developing time of record detection line and nature controlling line.It the results are shown in Table 1.
As a result judge：
Negative findings：A Luminescent bands are shown at C lines, Luminescent bands are shown at T lines.
Positive findings：A Luminescent bands are shown at C lines, while do not show colour developing Luminescent bands at T lines.
In vain：After the completion of reaction, if not showing Luminescent bands at C lines, it was demonstrated that the detecting system is invalid.
Table 1
Test case 2
(1) it is formulated as follows the solution of the aflatoxin B1 of concentration respectively：500ng/ml, 250ng/ml, 125ng/ml,
63ng/ml, 32ng/ml, 16ng/ml, 8ng/ml, 4ng/ml, 2ng/ml, 1ng/ml, 0.5ng/ml.
(2) the colloidal gold quick measuring card prepared according to the method for test case 1 using embodiment 1-10 and comparative example 1 is to above-mentioned
Sample is detected.
(3) colour developing situation is observed.And record the minimum effectively colour developing toxin concentration of each colloidal gold quick measuring card.Each concentration
Gradient is repeated 3 times.It the results are shown in Table 2.
Table 2
| Minimum developing concentration (ng/ml) | |
| Embodiment 1 | 0.5 |
| Embodiment 2 | 0.5 |
| Embodiment 3 | 0.5 |
| Embodiment 4 | 2 |
| Embodiment 5 | 2 |
| Embodiment 6 | 4 |
| Embodiment 7 | 8 |
| Embodiment 8 | 4 |
| Embodiment 9 | 8 |
| Embodiment 10 | 4 |
| Comparative example 1 | ---- |
The colloidal gold quick measuring card that it can be seen from the result of more than Tables 1 and 2 prepared by method using the present invention it is accurate
Degree, specificity and sensitivity are higher, and in the case that currently preferred, the detection performance can be carried further
It rises.
Test case 3
100 batches of corn on the ground such as Hebei, Shandong, Shanxi are chosen, after the niblet is ground to pulverulence, are made
5min is impregnated under conditions of stirring with 80% methanol, is taken in supernatant to centrifuge tube.Implementation is used according to the method for test case 1
The colloidal gold quick measuring card of example 1 is detected the aflatoxin B1 in the supernatant.Positive findings recall rate is 1%.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail, within the scope of the technical concept of the present invention, a variety of simple variants can be carried out to technical scheme of the present invention, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case of shield, it can be combined by any suitable means.In order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (10)
1. a kind of colloidal gold quick measuring card for mycotoxin detection, which includes being sequentially arranged on bottom plate
Sample application zone, conjugate area, the area of observation coverage and suction zones；
Wherein, the conjugate area includes antibody gold label pad, and the monoclonal of antimycotic toxin is fixed on the antibody gold label pad
Antibody；
The sample application zone is included in the sample pad soak containing BSA, sucrose and/or trehalose and tween after dipping and does
Dry sample pad；
The area of observation coverage includes chromatographic film, and detection line and nature controlling line are fixed in the chromatographic film, wherein, the detection line coating
There are mycotoxin to be detected and the conjugate of BSA, the nature controlling line is coated with special with the monoclonal antibody of antimycotic toxin
Property combine secondary antibody.
2. colloidal gold quick measuring card according to claim 1, wherein, it is fixed with antimycotic toxin on the antibody gold label pad
The coupling conjugate of monoclonal antibody and colloid gold particle, the granularity of the colloid gold particle is 10-30nm；
Preferably, the material of the antibody gold label pad is glass fibre.
3. colloidal gold quick measuring card according to claim 1, wherein, the sample pad soak be containing BSA, sucrose and/
Or the pH value of trehalose and tween is the PBS solution of 7-9.5 or PB buffer solutions；
Preferably, the sample pad soak also containing cholic acid, polyvinylpyrrolidone, RHODASURF ON-870,
It is one or more in STANDAPOLES-1, wire tray 60 and Triton X-100；
Preferably, the sample pad is double medium filtration pad.
4. the colloidal gold quick measuring card according to any one in claim 1-3, wherein, the mycotoxin is aspergillus flavus poison
Element, preferably aflatoxin B1.
5. the colloidal gold quick measuring card according to any one in claim 1-3, wherein, the preparation side of the antibody gold label pad
Method includes：
(1) monoclonal antibody for resisting mycotoxin to be measured is added in into the colloidal gold solution of pH7.5-9, contacts 40-80min,
Obtain being marked with the colloidal gold solution of the monoclonal antibody；
(2) there is addition BSA solution in the colloidal gold solution of monoclonal antibody to the label that step (1) obtains, contact 40-
80min；
(3) mixed material obtained step (2) carries out separation of solid and liquid, by sediment redissolve in containing sucrose and/or trehalose,
In the boric acid solution of BSA and Sodium azide, gold labeling antibody solution is obtained；
(4) the gold labeling antibody solution spraying prepared by step (3) is impregnated in the gold-labelled pad soak containing tween and BSA
Gold-labelled pad on, and dry be less than 10 weight % to water content；
Preferably, the gold-labelled pad soak is also containing one or more in sucrose, trehalose, polyethylene glycol and casein.
6. colloidal gold quick measuring card according to claim 5, wherein, this method further includes：Coating, the gold labeling antibody is molten
Before liquid, dry gold-labelled pad is placed into 20-360min under conditions of relative humidity is more than 80% and 2-6 DEG C.
7. colloidal gold quick measuring card according to claim 1, wherein, the chromatographic film is prepared by following steps：It is right
The conjugate and the secondary antibody of the mycotoxin and BSA are fixed, and then dry and assemble；The chromatographic film contains before drying
Water is 30-80 weight %；The chromatography water content of membrane is less than 10 weight % after drying；
Preferably, the detection line is 0.5-1mm apart from conjugate area, and the nature controlling line is 0.5-1mm apart from detection line.
8. a kind of method being detected to mycotoxin, the method includes：Pre-treatment is carried out to sample to be tested, obtains fungi
Toxic extraction object, and using the colloidal gold quick measuring card described in any one in claim 1-7 to the mycotoxin extract
It is detected and interpretation of result.
9. according to the method described in claim 8, wherein,
The pre-treatment includes：Sample to be tested is contacted into 5-30min with methanol solution, separation of solid and liquid takes supernatant later；
The interpretation of result includes：(1) when liquid reaches the area of observation coverage, detection line and nature controlling line are as it can be seen that be determined as feminine gender；
(2) after standing 5-15min, nature controlling line is visible, detection line is invisible, is determined as the positive；(3) stand 5-15min after, detection line with
Nature controlling line is invisible or detection line is visible and nature controlling line is invisible, and judgement detection is invalid.
10. a kind of detection mycotoxin kit, which is characterized in that the kit includes any one in claim 1-7
Colloidal gold quick measuring card, the bottle equipped with pure water and the bottle equipped with alcohol.
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| CN201611203900.1A CN108241060A (en) | 2016-12-23 | 2016-12-23 | Mycotoxin detects colloidal gold quick measuring card and kit and the method being detected to mycotoxin |
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| CN201611203900.1A CN108241060A (en) | 2016-12-23 | 2016-12-23 | Mycotoxin detects colloidal gold quick measuring card and kit and the method being detected to mycotoxin |
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| CN111929446A (en) * | 2020-08-25 | 2020-11-13 | 郑州安图生物工程股份有限公司 | Kit for quantitatively and qualitatively detecting anti-LKM-1 antibody and preparation method thereof |
| CN112684177A (en) * | 2020-12-17 | 2021-04-20 | 北京维德维康生物技术有限公司 | Dairy multi-factor rapid detection kit and detection method thereof |
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