CN103217528A - Non-labeled immunization analysis method for detecting content of aflatoxin B1 - Google Patents
Non-labeled immunization analysis method for detecting content of aflatoxin B1 Download PDFInfo
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Abstract
The invention relates to a non-labeled immunization analysis method for detecting a content of aflatoxin B1. The method comprises the following steps of: detecting fluorescence intensity values of a sample solution to be measured at 440nm part before and after fluorescence of aflatoxin B1 is fully quenched by a monoclonal antibody 1C11 generally for resisting aflatoxin, wherein the sample solution to be measured is excited under 365nm of excitation wavelength, and the monoclonal antibody 1C11 generally for resisting aflatoxin is generated by a hybridomas cell strain 1C11 with an accession number CCTCCNP.C201013; and based on a relative curve of fluorescence quenching intensity by the monoclonal antibody 1C11 generally for resisting aflatoxin to concentration of aflatoxin B1, the content of the aflatoxin B1 in the sample solution to be measured is obtained by the fluorescence quenching intensity of the sample solution to be measured by the antibody. The method can be used for directly and quantitatively determining aflatoxin B1, and has advantages of simple and rapid operation, no label, low detection cost, and small pollution harm.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to a kind of non-marked immune analysis method that detects aflatoxin B1 content.
Background technology
Aflatoxin is a class formation similar compounds, and the aflatoxin of occurring in nature mainly is the secondary metabolite that is produced by Aspergillus flavus and aspergillus parasiticus.Kind surplus the aflatoxin of finding has 20 at present, wherein common with aflatoxin B1, B2, G1, G2 and M1.Aflatoxin can produce destruction to the liver organization of people and animal, can cause liver cancer even death when serious.Aflatoxin delimited by the World Health Organization (WHO) and be I class carcinogenic substance, and the strongest with aflatoxin B1 toxicity, its toxicity is 68 times of arsenic.
The agricultural product that aflatoxin can occur in the torrid zone or subtropical zone are produced in the process, also may introduce the pollution of aflatoxin simultaneously in links such as storage, transportations.Peanut is the agricultural product that the easiest discovery has aflatoxin, and other all might be subjected to aflatoxin contamination as corn, fig, kernel and cereal.Because the cropping pattern of China smallholder decentralized, agricultural product each link from the farmland to the dining table all might be subjected to the pollution of aflatoxin.And aflatoxin toxicity is big, and harm is serious, and therefore, strict limit standard has been formulated to the content of aflatoxin in the agricultural product in countries in the world, becomes the key link of control agricultural product quality and safety.
In order more effectively to monitor aflatoxin content, the technology of analyzing and testing aflatoxin increases gradually in recent years, and sensitivity also improves gradually.Mainly contain instrument analytical method and immune analysis method.Immunological method has overcome instrumental method expense height, operating conditions and has required shortcomings such as high, big for environment pollution, have quick, easy, specificity is good, to advantages such as operating personnel and environmental hazard are little.Test strips chromatographic technique and enzyme linked immunological kit based on immunization method have been widely used in on-the-spot qualitative and detection by quantitative.The test strips chromatographic technique can be realized detecting in 15min, but can only carry out qualitative analysis, can't realize detection by quantitative; Enzyme linked immunological kit can carry out detection by quantitative, but the several hrs that needs consuming time, and operation steps is many.Therefore, but research set up a kind of simple, fast, the method for sensitive detection by quantitative aflatoxin B1 is for fast, accurately, aflatoxin has great importance in the on-line monitoring agricultural product.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of non-marked immune analysis method that detects aflatoxin B1 content at the deficiency of above-mentioned prior art existence.This method can be used for aflatoxin B1 detection in the agricultural product, compares with the enzyme labeled immunoassay analytical approach with conventional fluorescence to have the advantages that to need not mark.
The present invention solves the problems of the technologies described above the technical scheme that is adopted to be:
A kind of non-marked immune analysis method that detects aflatoxin B1 content is characterized in that: may further comprise the steps:
(1) provides testing sample solution, detecting testing sample solution excites down in the 365nm excitation wavelength, adding aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 makes the fluorescence intensity level at the 440nm place (wavelength of fluorescence intensity maximum correspondence in the fluorescence emission spectrum) before and after the abundant cancellation of aflatoxin B1 fluorescence, and described aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 is the hybridoma cell strain 1C11 generation of CCTCC NO:C201013 by preserving number;
(2) based on the fluorescence that obtains in advance by the relation curve of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration, promptly added the difference of two fluorescence intensities at the 440nm place before and after the aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation by the fluorescence of this testing sample solution by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity level, try to achieve the content of aflatoxin B1 in the testing sample solution.
This hybridoma cell strain 1C11 has been preserved in Chinese typical culture collection center (CCTCC) on July 13rd, 2010, the preservation address is, China, and Wuhan, Wuhan University, deposit number is CCTCC NO. C201013.
Press such scheme, the fluorescence intensity level at the 440nm place in the described step (1) after the abundant cancellation of aflatoxin B1 fluorescence is to add an amount of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 in testing sample solution, make the concentration of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 in the testing sample system that adds behind the aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 reach the Cmin of the abundant cancellation of fluorescence that makes aflatoxin B1 in the system at least, mixing then, place 0.5h at least in 37 ± 5 ℃, excite test to obtain recording through the 365nm excitation wavelength again.
Press such scheme, described fluorescence is adopted following method to obtain by the relation curve of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration:
1. preparation obtains the aflatoxin B1 standard solution of a series of concentration, excites the fluorescence emission spectrum that obtains the aflatoxin B1 standard solution of each concentration down in the 365nm excitation wavelength, the fluorescence intensity level at record 440nm place;
2. the aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 that in the aflatoxin B1 standard solution of above-mentioned each concentration, adds equivalent, described aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 is the hybridoma cell strain 1C11 generation of CCTCC NO:C201013 by preserving number, make the concentration of aspergillus flavus resisting toxin general purpose single clonal antibody in the aflatoxin B1 standard solution system that adds each concentration behind the aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 reach the Cmin of the abundant cancellation of fluorescence that makes the aflatoxin B1 in the Cmax aflatoxin B1 standard solution at least, and make the fully cancellation under the effect of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 of aflatoxin B1 fluorescence, excite the fluorescence emission spectrum that obtains the aflatoxin B1 standard solution that respectively adds aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 down in the 365nm excitation wavelength then, the fluorescence intensity level at record 440nm place;
3. obtain fluorescence by the relation curve c(x of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration through match, y)---x aflatoxin B1 concentration, y is fluorescence is promptly added 440nm place, aspergillus flavus resisting toxin general purpose single clonal antibody cancellation front and back fluorescence intensity by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity a difference.
Press such scheme, described step 1. in the aflatoxin B1 standard solution of a series of concentration be that methyl alcohol storing solution dilution preparation with aflatoxin B1 obtains, concentration is respectively 10,7.5,5,3.5,2,1.5,1,0.75,0.5,0.3,0.1 ng/mL; The concentration of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 is 〉=20 μ g/mL in the solution system in the aflatoxin B1 standard solution of described each solution behind the adding aspergillus flavus resisting toxin general purpose single clonal antibody 1C11, is preferably 20 μ g/mL.
Press such scheme, the dilution of described step in 1. can be phosphate buffer, as follows preparation: 0.8g sodium chloride, and the 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to 1000mL.
Press such scheme, described step is to add aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 to add the back mixing in the aflatoxin B1 standard solution of each concentration in 2., and place at least 0.5h so that aflatoxin B1 fluorescence by fully cancellation, carries out subsequent step then in 37 ± 5 ℃.
Press such scheme, 3. described step is piecewise fitting, and fluorescence is by the relation curve c of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration when obtaining small concentration respectively
1(x
1, y
1)---x
1Aflatoxin B1 concentration, 0.1-1 ng/ml, y
1For fluorescence by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity promptly add the difference of the 440nm place fluorescence intensity before and after the aspergillus flavus resisting toxin general purpose single clonal antibody and greatly during concentration fluorescence by the relation curve c of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration
2(x
2, y
2)---x
2Aflatoxin B1 concentration, 1-10 ng/ml, y
2The difference that is promptly added the 440nm place fluorescence intensity of aspergillus flavus resisting toxin general purpose single clonal antibody front and back for fluorescence by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity;
Fluorescence was by the relation curve c of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration when correspondingly, the fluorescence of aflatoxin B1 was determined to select small concentration for use by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity in asking when calculating aflatoxin B1 concentration in the testing sample according to testing sample solution
1(x
1, y
1), fluorescence is by the relation curve c of aspergillus flavus resisting toxin general purpose single clonal antibody cancellation intensity and aflatoxin B1 concentration during still big concentration
2(x
2, y
2), the fluorescence in conjunction with this testing sample solution is calculated aflatoxin B1 concentration in the testing sample by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity level then.
Press such scheme, the concentration of aflatoxin B1 should be 0.1-10 ng/mL in by the valid analysing range of the relation curve of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration at the fluorescence in this method step (2) in the described testing sample solution, as exceed, can to testing sample solution carry out corresponding concentrate or dilution process after detect again; Concentration 〉=20 μ g/mL of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 in the system are preferably 20 μ g/mL behind the described testing sample solution adding aspergillus flavus resisting toxin general purpose single clonal antibody 1C11.
[0014]The implication of the aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 Cmin of the abundant cancellation of fluorescence that makes aflatoxin B1 in the system of the present invention is: when aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 is a certain concentration in system, increase along with this concentration, the fluorescent quenching rate of aflatoxin B1 does not increase thereupon yet in the system, and this concentration is the aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 Cmin of the abundant cancellation of fluorescence that makes aflatoxin B1 in the system.
The aflatoxin B1 fluorescent quenching reaches the principle of work based on its application in aflatoxin B1 detects: the aflatoxin B1 under the state of nature is being subjected to ultraviolet light (365nm) can send fluorescence when exciting, the wavelength of fluorescence intensity maximum correspondence is 440nm in the fluorescence emission spectrum, and after this aflatoxin B1 aspergillus flavus resisting toxin specific general purpose single clonal antibody 1C11 reaction, being subjected to ultraviolet light (365nm) again when exciting with it, the fluorescence overwhelming majority of aflatoxin B1 is by cancellation.This method directly utilize aflatoxin fluorescence can by the characteristic of its aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation can realize to aflatoxin B1 fast, detection by quantitative, the multistep reaction step that does not need similar enzyme linked immunosorbent assay, do not need to add extra signal probe, as antibody of probe mark etc. yet.
Beneficial effect of the present invention is:
(1) direct quantitative detects aflatoxin B1.Utilize the fluorescence of aflatoxin B1 can be, can realize detection by quantitative aflatoxin B1 in conjunction with the fluorescence intensity level before and after aflatoxin B1 solution and its antibody response by the characteristic of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation.
(2) simple to operate fast, do not need the multistep reaction step of similar enzyme linked immunosorbent assay.
(3) need not mark, detect that cost is low, contamination hazard is little.Only need in this detection method that adding aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 can detect in testing sample solution (testing sample get through the sample pre-treatments of routine and aflatoxin B1 immune affinity column purified treatment), do not need to add in addition harmful chemical reagent such as fluorescent material, the antibody or the carrier that also do not need probe mark, to the harm of operator and environment with pollute little, with low cost.
Description of drawings
Fig. 1 is the aflatoxin B1 standard solution of series concentration and the comparison of its aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 reaction front and back fluorescence intensity, among the figure: in two row of each concentration value correspondence: left column is the fluorescence intensity of each concentration aflatoxin B1 standard solution, and the fluorescence intensity that adds after aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 reacts cancellation is classified on the right side as;
Fig. 2 is aflatoxin B1 concentration standard curve c
1(x
1, y
1), concentration range is 0.1～1ng/mL;
Fig. 3 is aflatoxin B1 concentration standard curve c
2(x
2, y
2), concentration range is 1～10ng/mL.
Embodiment
Following used aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 is that to adopt deposit number be that the hybridoma cell strain 1C11 of CCTCC NO:C201013 is that reported method makes in advance in the patent of CN201010245095.5 according to number of patent application, and concrete preparation method is as follows:
With deposit number is the BALB/c mouse that the hybridoma cell strain 1C11 injection of CCTCC NO. C201013 was handled with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, adopt sad-ammonium sulfate method antibody purification, concrete operations are: with double-deck filter paper filtering mouse ascites, 4 ℃, the centrifugal 15min of 12000r/min, draw supernatant, gained ascites supernatant is mixed with the acetate buffer of 4 times of volumes, stir and slowly add caprylic acid down, every milliliter of required caprylic acid volume of ascites is 33 μ L, mixed at room temperature 30min, 4 ℃ leave standstill 2h, 4 ℃ then, the centrifugal 30min of 12000r/min, abandon precipitation, with the supernatant that obtains with double-deck filter paper filtering after, the volumetric molar concentration that adds 1/10 filtrate volume is that 0.1mol/L and pH value are 7.4 phosphate buffer, regulate the pH value to 7.4 of this mixed liquor with the sodium hydroxide solution of 2 mol/L, 4 ℃ of precoolings, slowly adding ammonium sulfate to ammonium sulfate final concentration is 0.277g/mL, 4 ℃ leave standstill 2h, 4 ℃ then, the centrifugal 30min of 12000r/min abandons supernatant, the gained precipitation is resuspended with the 0.01mol/L phosphate buffer of former ascites volume 1/10, the bag filter of packing into, to the pure water dialysis, it is freezing that the protein solution that fully dialysis is good is put-70 ℃ of refrigerators, use the freeze drier freeze-drying afterwards, collect freeze-dried powder, promptly get the good aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 of purifying, antibody is put in-20 ℃ of refrigerators standby;
Described acetate buffer is the 0.29g sodium acetate, and 0.141mL acetic acid adds water and is settled to the 100mL gained; The phosphate buffer of described 0.1mol/L is a 0.8g sodium chloride, the 0.29g disodium hydrogen phosphate, and 0.02g potassium chloride, potassium dihydrogen phosphate 0.02g adds water and is settled to the 1000mL gained.
Embodiment 1: the interpolation of aflatoxin B1 detects application experiment in the peanut
I fluorescence is by the relation curve c(x of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration, foundation y)
(1) with concentration is the aflatoxin B1 methyl alcohol storing solution dilution phosphate buffer dilution of 500ng/mL, preparation obtains the aflatoxin B1 standard solution (concentration is respectively 10,7.5,5,3.5,2,1.5,1,0.75,0.5,0.3,0.1 and 0 ng/mL) of series concentration, described phosphate-buffered formula of liquid is as follows: 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate adds water and is settled to 1000mL.The aflatoxin B1 standard solution 2mL that gets each series concentration excites under the 365nm excitation wavelength, obtains the fluorescence emission spectrum of the aflatoxin B1 standard solution of each series concentration, and the fluorescence intensity level at recording wavelength 440nm place is seen Fig. 1.
(2) in the aflatoxin B1 standard solution of above-mentioned 10ng/mL, add the different aspergillus flavus-resistance toxin general purpose single clonal antibody 1C11 that measure respectively, (concentration of aspergillus flavus resisting toxin general purpose single clonal antibody is respectively 0.1 to the system of acquisition variable concentrations aspergillus flavus resisting poison general purpose single clonal antibody 1C11 in the system, 0.5,5,10,20,30,40ng/mL), adopt as above condition to carry out fluorescent quenching, fluorescence is compared with the fluorescence intensity level before the adding aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity level, obtained the fluorescent quenching rate of the aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 of variable concentrations.Concrete outcome: when aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 concentration is 0.1ng/mL, the fluorescent quenching rate 31% of aflatoxin B1; When aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 concentration was 0.5 ng/mL, the cancellation rate was 37%; When aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 concentration was 5ng/mL, the cancellation rate was 49%; When aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 concentration was 10ng/mL, the cancellation rate was 78%; When aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 concentration is 20ng/mL, the cancellation rate is 91%, and continue to increase up to antibody concentration be 30 or during 40ng/mL to aspergillus flavus resisting toxin general purpose single clonal antibody content in the system, the cancellation rate still is 91%, when aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 concentration is 20ng/mL in the explanation system, can make the abundant cancellation of fluorescence of aflatoxin B1.So in conjunction with cost, selecting the concentration of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 in the following system for use is 20ng/mL.
(3) in each aflatoxin B1 standard solution of step (1), add aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 respectively, make the final concentration of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 in each system be 20 μ g/mL, mixing, place 1h, get each system solution 2mL, excite under the 365nm excitation wavelength, obtaining respectively is the fluorescence emission spectrum of system, get the fluorescence intensity level at maximum wavelength 440nm place, see Fig. 1.
(4) to aflatoxin B1 standard items concentration be the scope of the scope of 0.1-1ng/mL and 1-10ng/mL carry out respectively match be piecewise fitting when obtaining small concentration fluorescence by the relation curve c of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration
1(x
1, y
1), curvilinear equation is: y
1=10
6x
1+ 8859, wherein: x
1Aflatoxin B1 concentration, 0.1-1ng/mL, y
1The difference that is promptly added the 440nm place fluorescence intensity of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 front and back for fluorescence by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity, fluorescence is by the relation curve c of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration when seeing Fig. 2 and obtaining big concentration
2(x
2, y
2), curvilinear equation is: y
2=71535x
2+ 10
6, wherein: x
2Aflatoxin B1 concentration, 1-10ng/mL, y
2For fluorescence is promptly added the difference of the 440nm place fluorescence intensity before and after the aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity, see Fig. 3.
The interpolation of aflatoxin B1 detects in the II peanut:
Take by weighing peanut blank sample 5 grams that ground, the methyl alcohol storing solution that adds 100 μ L aflatoxin B1, the interpolation concentration of aflatoxin B1 is 10 ng/g peanut blank samples, place half an hour, treating to be evaporated completely the back by methyl alcohol, to add the 15mL volumetric concentration be 80% methanol-water (contain massfraction be 4% sodium chloride), under 50～60 ℃ of water-baths ultrasonic 10 minutes, quantitative filter paper filters, get filtrate 4mL and add the 2mL sherwood oil, vortex 1 minute, standing demix, take off a layer methanol-water 3mL, add 9 mL water, mixing, with the sample extracting solution of this dilution is the mixing membrane filtration of 0.45 μ m with the aperture, get the 10mL filtrate flow and cross the aflatoxin B1 immune affinity column, with 1mL methanol-eluted fractions affinity column, collect meoh eluate, under 60 ℃, dry up with nitrogen, with the above-mentioned dilution of 2mL aflatoxin is hanged again, be sample solution, the fluorescence emission spectrum of scanning samples solution under the 365nm excitation wavelength excites, in sample solution, add again aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 to aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 final concentration be 20 μ g/mL, mixing, place after 1 hour, excite in the 365nm excitation wavelength to obtain its fluorescence emission spectrum down, the fluorescence intensity level at record 440nm place.
Testing result: by antibody cancellation intensity level, fluorescence was by the relation curve c of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration when testing sample solution fluorescence was promptly added the big concentration of difference substitution of the 440nm place fluorescence intensity before and after the aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 by antibody cancellation intensity level according to system fluorescence after adding aspergillus flavus resisting toxin general purpose single clonal antibody 1C11
2(x
2, y
2) in (as Fig. 3), obtain the aflatoxin B1 concentration in the sample solution, again in conjunction with the dilution situation in the sample preparation process, the interpolation concentration that the variation relation of concentration calculates the aflatoxin B1 in the actual peanut sample in the processing procedure is 9.4 ng/g per sample.
Embodiment 2: the detection application experiment of aflatoxin B1 content in the peanut sample
I fluorescence is by the relation curve c(x of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration, and foundation y) is as described in the above-mentioned embodiment 1.
The detection of aflatoxin B1 content in the II peanut:
Take by weighing the peanut sample 1# to be measured that has ground, 2# or 3# 5 grams, add the 15mL volumetric concentration and be 80% methanol-water (contain massfraction be 4% sodium chloride), under 50～60 ℃ of water-baths ultrasonic 10 minutes, quantitative filter paper filters, get filtrate 4m L and add the 2mL sherwood oil, vortex 1 minute, standing demix, take off a layer methanol-water 3mL, add 9mL water, mixing, with the sample extracting solution of this dilution is the mixing membrane filtration of 0.45 μ m with the aperture, get the 10mL filtrate flow and cross the immune affinity column of aflatoxin B1, with 1mL methanol-eluted fractions affinity column, collect meoh eluate, under 60 ℃, dry up, with the above-mentioned dilution of 2mL aflatoxin is hanged again, be sample solution with nitrogen, the fluorescence emission spectrum of scanning samples solution under the 365nm excitation wavelength excites, the fluorescence intensity level at record 440nm place, adding aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 in sample solution again, to aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 final concentration be 20 μ g/mL, mixing, place after 1 hour, excite in the 365nm excitation wavelength to obtain its fluorescence emission spectrum down, the fluorescence intensity level at record 440nm place.
Testing result: by the value of antibody cancellation, fluorescence is by the relation curve c of aspergillus flavus resisting toxin general purpose single clonal antibody cancellation intensity and aflatoxin B1 concentration when determining that the fluorescence of each sample solution promptly added the difference substitution small concentration of the 440nm fluorescence intensity before and after the antibody by the value of antibody cancellation according to system fluorescence after adding aflatoxin general purpose single clonal antibody
1(x
1, y
1) or during big concentration fluorescence by the relation curve c of aspergillus flavus resisting toxin general purpose single clonal antibody cancellation intensity and aflatoxin B1 concentration
2(x
2, y
2), obtain the aflatoxin B1 concentration in the sample solution, again in conjunction with the dilution situation in the sample preparation process, the variation relation of concentration calculates the concentration of the aflatoxin B1 in the peanut sample in the processing procedure per sample, obtain that aflatoxin B1 concentration is below the detection limit 0.1ng/mL in the solution to be measured of peanut sample 1, promptly among the sample 1# aflatoxin B1 content for not detecting; Aflatoxin B1 concentration is 0.6ng/mL in the solution to be measured of peanut sample 2, and promptly aflatoxin B1 content is 2.16 μ g/kg among the sample 2#; Aflatoxin B1 concentration is 3.2ng/m L in the solution to be measured of peanut sample 3, and promptly aflatoxin B1 content is 11.52 μ g/kg among the sample 3#.Adopt national standard method GB/T 18979-2003 to detect above-mentioned peanut sample 1# simultaneously, 2# or 3#, its testing result is followed successively by: aflatoxin B1 does not detect among the peanut sample 1#, aflatoxin B1 content is 2.3 μ g/kg among the peanut sample 2#, and aflatoxin B1 content is 12.2 μ g/kg among the peanut sample 3#.Can find out in conjunction with above-mentioned detection: the testing result relative differences of above-mentioned two kinds of detection methods is all in 10%.
Claims (8)
1. non-marked immune analysis method that detects aflatoxin B1 content is characterized in that: may further comprise the steps:
(1) provides testing sample solution, detecting testing sample solution excites down in the 365nm excitation wavelength, adding aspergillus flavus resisting toxin general purpose single clonal antibody aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 makes the fluorescence intensity level at the 440nm place before and after the abundant cancellation of aflatoxin B1 fluorescence, and described aspergillus flavus resisting toxin general purpose single clonal antibody aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 is the hybridoma cell strain 1C11 generation of CCTCC NO:C201013 by preserving number;
(2) based on the fluorescence that obtains in advance by the relation curve of aspergillus flavus resisting toxin general purpose single clonal antibody aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration, promptly added the difference of two fluorescence intensities at the 440nm place before and after the aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation by the fluorescence of this testing sample solution by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity level, try to achieve the content of aflatoxin B1 in the testing sample solution.
2. according to the non-marked immune analysis method of the detection aflatoxin B1 content described in the claim 1, it is characterized in that: the fluorescence intensity level at the 440nm place in the described step (1) after the abundant cancellation of aflatoxin B1 fluorescence is to add an amount of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 in testing sample solution, make the concentration of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 in the testing sample system that adds behind the aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 reach the Cmin of the abundant cancellation of fluorescence that makes aflatoxin B1 in the system at least, mixing then, place 0.5h at least in 37 ± 5 ℃, excite test to obtain recording through the 365nm excitation wavelength again.
3. according to the non-marked immune analysis method of the detection aflatoxin B1 content described in the claim 1, it is characterized in that: the described fluorescence of step (2) is adopted following method to obtain by the relation curve of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration:
1. preparation obtains the aflatoxin B1 standard solution of a series of concentration, excites the fluorescence emission spectrum that obtains the aflatoxin B1 standard solution of each concentration down in the 365nm excitation wavelength, the fluorescence intensity level at record 440nm place;
2. the aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 that in the aflatoxin B1 standard solution of above-mentioned each concentration, adds equivalent, described aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 is the hybridoma cell strain 1C11 generation of CCTCC NO:C201013 by preserving number, make the concentration of aspergillus flavus resisting toxin general purpose single clonal antibody in the aflatoxin B1 standard solution system that adds each concentration behind the aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 reach the Cmin of the abundant cancellation of fluorescence that makes the aflatoxin B1 in the Cmax aflatoxin B1 standard solution at least, and make the fully cancellation under the effect of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 of aflatoxin B1 fluorescence, excite the fluorescence emission spectrum that obtains the aflatoxin B1 standard solution that respectively adds aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 down in the 365nm excitation wavelength then, the fluorescence intensity level at record 440nm place;
3. obtain fluorescence by the relation curve c(x of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration through match, y)---x aflatoxin B1 concentration, y is fluorescence is promptly added 440nm place, aspergillus flavus resisting toxin general purpose single clonal antibody cancellation front and back fluorescence intensity by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity a difference.
4. according to the non-marked immune analysis method of the detection aflatoxin B1 content described in the claim 3, it is characterized in that: described step 1. in the aflatoxin B1 standard solution of a series of concentration be that methyl alcohol storing solution dilution preparation with aflatoxin B1 obtains, concentration is respectively 10,7.5,5,3.5,2,1.5,1,0.75,0.5,0.3,0.1 ng/mL; The concentration of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 is 〉=20 μ g/mL in the solution system in the aflatoxin B1 standard solution of described each solution behind the adding aspergillus flavus resisting toxin general purpose single clonal antibody 1C11.
5. according to the non-marked immune analysis method of the detection aflatoxin B1 content described in the claim 4, it is characterized in that: the dilution of described step in 1. is phosphate buffer, preparation as follows: 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate adds water and is settled to 1000mL.
6. according to the non-marked immune analysis method of the detection aflatoxin B1 content described in the claim 3, it is characterized in that: described step adds the back mixing for add aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 in the aflatoxin B1 standard solution of each concentration in 2., and place at least 0.5h so that aflatoxin B1 fluorescence by fully cancellation, carries out subsequent step then in 37 ± 5 ℃.
7. according to the non-marked immune analysis method of the detection aflatoxin B1 content described in the claim 3, it is characterized in that: 3. described step is piecewise fitting, and fluorescence is by the relation curve c of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration when obtaining small concentration respectively
1(x
1, y
1)---x
1Aflatoxin B1 concentration, 0.1-1 ng/ml, y
1For fluorescence by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity promptly add the difference of the 440nm place fluorescence intensity before and after the aspergillus flavus resisting toxin general purpose single clonal antibody and greatly during concentration fluorescence by the relation curve c of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration
2(x
2, y
2)---x
2Aflatoxin B1 concentration, 1-10 ng/ml, y
2The difference that is promptly added the 440nm place fluorescence intensity of aspergillus flavus resisting toxin general purpose single clonal antibody front and back for fluorescence by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity;
Fluorescence was by the relation curve c of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration when correspondingly, the fluorescence of aflatoxin B1 was determined to select small concentration for use by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity in asking when calculating aflatoxin B1 concentration in the testing sample according to testing sample solution
1(x
1, y
1), fluorescence is by the relation curve c of aspergillus flavus resisting toxin general purpose single clonal antibody cancellation intensity and aflatoxin B1 concentration during still big concentration
2(x
2, y
2), the fluorescence in conjunction with this testing sample solution is calculated aflatoxin B1 concentration in the testing sample by aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity level then.
8. according to the non-marked immune analysis method of the detection aflatoxin B1 content described in the claim 3, it is characterized in that: the concentration of aflatoxin B1 should be 0.1-10 ng/mL in by the valid analysing range of the relation curve of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 cancellation intensity and aflatoxin B1 concentration at the fluorescence in this method step (2) in the described testing sample solution, as exceed, can to testing sample solution carry out corresponding concentrate or dilution process after detect again; Concentration 〉=20 μ g/mL of aspergillus flavus resisting toxin general purpose single clonal antibody 1C11 in the system behind the described testing sample solution adding aspergillus flavus resisting toxin general purpose single clonal antibody 1C11.
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