CN106940373B - The immunochromatography time-resolved fluorescence kit of four kinds of mycotoxin composite pollutions such as synchronous detection fumonisin B1 and its application - Google Patents
The immunochromatography time-resolved fluorescence kit of four kinds of mycotoxin composite pollutions such as synchronous detection fumonisin B1 and its application Download PDFInfo
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Abstract
The present invention relates to the immunochromatography time-resolved fluorescence kit of four kinds of mycotoxin composite pollutions such as synchronous detection fumonisin B1 and its applications.It includes the example reaction bottle of immunochromatography time-resolved fluorescence test strips and each monoclonal antibody dried frozen aquatic products containing europium label；Wherein: the fluorescent test paper strip includes PVC substrate, the one side of substrate successively pastes water absorption pad, detecting pad and sample pad from top to bottom, adjacent each pad is in the overlapping connection in junction, detecting pad is using nitrocellulose filter as base wad, lateral nature controlling line and four detection lines are set from top to bottom on nitrocellulose filter, it is coated with the bovine serum albumin(BSA) conjugate of each toxin respectively, the fumonisin B1 monoclonal antibody is to be secreted to generate by the hybridoma cell strain Fm7A11 that deposit number is CCTCC NO.C201636.The synchronous detection that can be used for aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 content, has the characteristics that easy to operate, quick, high sensitivity.
Description
Technical field
The present invention relates to mycotoxin immunochromatography time-resolved fluorescence kits, and in particular to a kind of synchronous detection is yellow bent
Mould toxin B1, ochratoxin A, zearalenone, fumonisin B1 composite pollution immunochromatography time-resolved fluorescence examination
Agent box, preparation method and applications.
Background technique
Mycotoxin is the very strong chemical substance of a kind of toxicity, and what to be filamentous fungi generated under suitable environmental condition has
Malicious metabolite.It is a variety of that mycotoxin pollutes the cereal crops such as corn, wheat, rice, peanut and oil crops, feed etc. extensively
Agricultural product food.Aflatoxin B1 is the secondary metabolite toxic by one kind of the generations such as Aspergillus flavus and aspergillus parasiticus,
With high toxicity and height, the characteristic of carcinogenic, teratogenesis, mutagenicity, very harmful to human and animal.Ochratoxin A is main
Aspergillus and some toxigenic bacterium strains of Penicillium are resulted from, toxicity occupies first of 7 kinds of ochratoxins (7 kinds of A, B, C, D etc.),
Distributed in nature is most extensive, is one of the mycotoxin for seriously endangering human health to the pollution of agricultural product also most serious；Reddish brown song
Mould toxin A is more stable in food and is not easily decomposed, most common in mildew cereal, feed etc., has been proved to mutagenesis
Property, immunotoxicity, immunosupress can be caused, and there is carcinogenic, teratogenesis, mutagenesis.Zearalenone is by a variety of sickles
A kind of mycotoxin for on-steroidal structure that knife bacterium generates is exceeding standard rate and detection water in China's feedstuff and complete feed
One of highest mycotoxin is put down, to the toxic effect of reproductive capability, also there is hepatotoxicity wind agitation, renal toxicity, immunotoxicity, cell toxicant
Property and genetoxic, and have significant correlation to tumour.Fumonisin B1 occurs mainly with Fusorium moniliforme Sheldon, many births sickle
Spore, colyliform fusarium and other Fusariumsps, have strong neurotoxicity, hepatotoxicity wind agitation and carcinogenic teratogenesis etc., and toxicity mechanism mainly comes
Destruction and oxidative stress are synthesized to sphingolipid derived from fumonisin B1.And the planting patterns of China smallholder dispersing type causes
Mycotoxin incidence is high in agricultural product, and a possibility that same agricultural product mycotoxin composite pollution is big, therefore there is an urgent need to
The detection technique of quick, the synchronous detection mycotoxin composite pollution of energy, to realize to mycotoxin mixing dirt in grain and feed
The synchronization of dye, fast slowdown monitoring.
The existing detection method to these mycotoxins mainly includes thin layer chromatography, precision instrument analytic approach and immunology
Analytic approach.Thin layer chromatography detection mycotoxin is the pathogenic eukaryotes method that earliest research is established, and is mainly used for fungi poison
Plain qualitative analysis, it is difficult to which quantitative, detection reagent dosage is big, cumbersome and since other component serious interferences lead to accuracy
Difference is unable to accurate quantitative analysis, larger to experimenter and ambient contamination harm.Precision instrument analytic approach includes high-efficient liquid phase color
The methods of spectrometry, liquid chromatogram and mass spectrum and tandem mass spectrum combination method have many advantages, such as accurate, sensitive, but sample pre-treatments mistake
Journey is complicated, and detection time is long, used expensive equipment, requires experimental situation and testing staff high, it is difficult to realize quickly inspection
It surveys.Immunoassay method overcomes the shortcomings that thin layer chromatography and instrumental method, due to its high specificity, high sensitivity, sample
Product pre-treatment is simple, it is at low cost, small to the contamination hazard of experimenter and ambient enviroment, be suitable for the advantages that live batch detection and exist
Fast development is obtained in recent years.Based on the immuno-chromatographic test paper strip of colloidal gold labeled monoclonal antibody and antigentic specificity association reaction by
In its testing result naked eyes as it can be seen that not needing large-scale instrument and equipment, testing cost is low, and analysis time is short, in recent years in fungi poison
It is widely applied in qualitative, online, the quick detection of the minimal residues object such as element.But since sensitivity is low, can not carry out
Quantitative detection is quickly unable to satisfy the demand of rapid quantitative detection at the scene in detection.And the plantation of China smallholder dispersing type
Mode causes mycotoxin incidence in agricultural product high, and the risk of composite pollution is bigger, therefore there is an urgent need to can quickly, together
Step detects the detection technique of a variety of mycotoxin composite pollutions, with realize in grain and feed mycotoxin composite pollution it is same
Step, fast slowdown monitoring.
Summary of the invention
Synchronous aflatoxin B1, ochratoxin A, corn can be detected the problem to be solved by the invention is to provide a kind of
Zeranol, the immunochromatography time-resolved fluorescence kit of fumonisin B1 composite pollution, preparation method and applications.This is exempted from
Epidemic disease chromatography time-resolved fluorescence kit can be used for aflatoxin B1, ochratoxin A, zearalenone, fumonisin
The synchronous detection of B1 content, has the characteristics that easy to operate, quick, high sensitivity.
In order to solve the above technical problems, the technical scheme adopted by the invention is as follows:
Synchronous detection aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 composite pollution are exempted from
Epidemic disease chromatographs time-resolved fluorescence kit, it is characterised in that: it includes immunochromatography time-resolved fluorescence test strips and contains europium
The Gibberella zeae alkene that the aflatoxin B1 monoclonal antibody of label, the ochratoxin A monoclonal antibody of europium label, europium mark
The example reaction bottle for the fumonisin B1 monoclonal antibody dried frozen aquatic products that ketone monoclonal antibody, europium mark；Wherein: the immune layer
Analysis time-resolved fluorescence test strips include PVC substrate, and the one side of substrate successively pastes water absorption pad, detecting pad and sample from top to bottom
Pad, adjacent each pad is in the overlapping connection in junction, and the detecting pad is using nitrocellulose filter as base wad, from upper on nitrocellulose filter
And the lateral nature controlling line of lower setting and four detection lines, the nature controlling line are coated with rabbit-anti mouse polyclonal antibody, four detections
Aflatoxin B1-bovine serum albumin(BSA) conjugate, ochratoxin A-bovine serum albumin(BSA) conjugate, jade are coated on line respectively
Zearlenone-bovine serum albumin(BSA) conjugate, fumonisin B1- bovine serum albumin(BSA) conjugate, the fumonisin B1
Monoclonal antibody is the monoclonal generated by the hybridoma cell strain Fm7A11 secretion that deposit number is CCTCC NO.C201636
Antibody；The hybridoma cell strain Fm7A11 is preserved in China typical culture collection center on March 29th, 2016, protects
Hiding address is China, Wuhan, Wuhan University, and deposit number is CCTCC NO.C201636.
According to the above scheme, the monoclonal antibody of the europium label is to be prepared in accordance with the following methods:
The aflatoxin B1 monoclonal antibody of europium label: by the europium mark after aflatoxin B1 monoclonal antibody and activation
Note reagent mix in borate buffer, oscillating reactions, then through centrifugation, redissolution, closing step obtain target product europium mark
Aflatoxin B1 monoclonal antibody；Europium labelled reagent after 1mL activation can be coupled aflatoxin B1 monoclonal antibody: 30
μg-80μg。
The ochratoxin A monoclonal antibody of europium label: the europium after ochratoxin A monoclonal antibody and activation is marked
Reagent mixes in borate buffer, oscillating reactions, then obtains target product europium label through centrifugation, redissolution, closing step
Ochratoxin A monoclonal antibody；Europium labelled reagent after 1mL activation can be coupled ochratoxin A monoclonal antibody: 40 μ g-
90μg。
The zearalenone monoclonal antibody of europium label: by the europium mark after zearalenone monoclonal antibody and activation
Note reagent mixes in borate buffer, oscillating reactions, then obtains target product europium label through centrifugation, redissolution, closing step
Zearalenone monoclonal antibody；Europium labelled reagent after 1mL activation can be coupled zearalenone monoclonal antibody: 30
μg-90μg。
The fumonisin B1 monoclonal antibody of europium label: the europium after fumonisin B1 monoclonal antibody and activation is marked into examination
Agent mixes in borate buffer, oscillating reactions, then obtains the volt of target product europium label through centrifugation, redissolution, closing step
Horse toxin B1 monoclonal antibody；Europium labelled reagent after 1mL activation can be coupled fumonisin B1 monoclonal antibody: 30 μ g-80 μ g.
According to the above scheme, the activation method is to take europium labelled reagent, with the borate buffer of 8.2 0.2mol/L of pH
Middle ultrasonic disperse is then slowly added into Carbodiimide solution, and shaken at room temperature activation, supernatant is removed in centrifugation, with pH 8.2
The borate buffer of 0.2mol/L redissolves, spare, and the activation time is 15-30min.
The monoclonal antibody of above-mentioned prepared europium label is redissolved in containing 1.5% (m/v) trehalose, 2% (m/v) cow's serum
It is spare in the phosphate buffer of the 0.01mol/L pH 7.4 of albumin, in use, taking the monoclonal of each europium label of certain dosage
Antibody is put into example reaction bottle, is placed in freeze drier and is lyophilized, and the monoclonal antibody dried frozen aquatic products of each europium label are obtained, standby
With.
According to the above scheme, the water absorption pad long 10-16mm, wide 3-5mm in the fluorescent test paper strip；The long 25-30mm of detecting pad,
Wide 3-5mm；Sample pad long 12-18mm, wide 3-5mm, the adjacent overlapping length respectively padded are 1-3mm；It is examined in the fluorescent test paper strip
The spacing on the upper edge in the detection line of nature controlling line and nitrocellulose filter of survey pad is 5-10mm, between every adjacent two detection lines
Spacing be 1.5-4.5mm, close to nature controlling line detection line and nature controlling line spacing be 3-5mm；The example reaction bottle is
The cillin bottle of 1-2.5mL.
According to the above scheme, institute per cm in coating aflatoxin B1-bovine serum albumin(BSA) conjugate detection line
The aflatoxin B1 needed-bovine serum albumin(BSA) conjugate package amount is 50-250ng；It is coated with ochratoxin A-ox blood
In the detection line of pure protein conjugate it is per cm required for ochratoxin A-bovine serum albumin(BSA) conjugate package amount
For 100-300ng；It is red to be coated with required corn per cm in zearalenone-bovine serum albumin(BSA) conjugate detection line
Mould ketenes-bovine serum albumin(BSA) conjugate package amount is 100-300ng；It is coated with the coupling of fumonisin B1- bovine serum albumin(BSA)
The package amount of required fumonisin B1- bovine serum albumin(BSA) conjugate is 50-150ng, matter per cm in the detection line of object
The package amount of rabbit-anti mouse polyclonal antibody needed for controlling line is 50-150ng；
The content for the aflatoxin B1 monoclonal antibody dried frozen aquatic products that europium marks in the example reaction bottle is 0.1-0.3 μ
G, the content of the ochratoxin A monoclonal antibody dried frozen aquatic products of europium label are 0.1-0.3 μ g, the zearalenone list of europium label
The content of clonal antibody dried frozen aquatic products is 0.1-0.3 μ g, and the content of the fumonisin B1 monoclonal antibody dried frozen aquatic products of europium label is
0.1-0.3μg。
According to the above scheme, the aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 are immune
Chromatographing time-resolved fluorescence quick testing reagent box further includes sample diluting liquid and sample diluting liquid suction pipe, and the sample diluting liquid is
Volume fraction is the Tween-20 aqueous solution of 0.01%-0.30%.
According to the above scheme, the time-resolved fluorescence test strips the preparation method is as follows:
(1) blotting paper is cut out into obtain water absorption pad；
(2) preparation of detecting pad:
By aflatoxin B1-bovine serum albumin(BSA) conjugate, ochratoxin A-bovine serum albumin(BSA) conjugate, corn
It is 0.10- that zeranol-bovine serum albumin(BSA) conjugate, fumonisin B1- bovine serum albumin(BSA) conjugate, which are configured to concentration,
The coating buffer of 0.50mg/mL, with line spray mode by it in carrying out being respectively separated coating on nitrocellulose filter, obtain 4 and detect
Line, it is then 60-120 minutes dry under the conditions of 37-40 DEG C；
Rabbit-anti mouse polyclonal antibody is made into the coating buffer that concentration is 0.1-0.5mg/mL, is laterally wrapped it with line spray mode
By on nitrocellulose filter, obtaining nature controlling line, then dried 60-120 minutes under the conditions of 37-40 DEG C；
(3) preparation of sample pad:
Glass fibre membrane is put into confining liquid and is soaked, is taken out, it is 4-6 hours dry under the conditions of 37-40 DEG C, obtain sample
Pad, then sets room temperature preservation in drier；
(4) assembling of immunochromatography time-resolved fluorescence test strips:
Water absorption pad, detecting pad, sample pad are successively pasted from top to bottom in the one side of cardboard, and adjacent each pad is overlapping in junction
Connection is to get fluorescent test paper strip；
According to the above scheme, aflatoxin B1-bovine serum albumin(BSA) conjugate is prepared in the preparation of the fluorescent test paper strip
Coating buffer, ochratoxin A-bovine serum albumin(BSA) conjugate coating buffer, zearalenone-bovine serum albumin(BSA) conjugate packet
The coating buffer used in liquid, fumonisin B1- bovine serum albumin(BSA) conjugate coating buffer are as follows: contain ox in every 10mL
Seralbumin 0.1g, sodium azide 0.002g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride
0.002g, potassium dihydrogen phosphate 0.002g；
Prepare coating buffer used in rabbit-anti mouse polyclonal antibody coating buffer are as follows: contain sodium azide in every 10mL
0.002g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g；
Confining liquid used in the preparation of the fluorescent test paper strip are as follows: contain ovalbumin 0.5-2g, sucrose in every 100mL
2g, sodium azide 0.02g, sodium chloride 0.8g, disodium hydrogen phosphate 0.29g, potassium chloride 0.02g, potassium dihydrogen phosphate
0.02g, 0.5g Tween-20 (Tween 20%)；
Above-mentioned immunochromatography time-resolved fluorescence quick testing reagent box is in aflatoxin B1, ochratoxin A, Gibberella zeae
Application in ketenes, fumonisin B1 content detection: by sample to be tested after pre-treatment obtains testing sample solution, sample is added
In reaction flask, mix, be inserted into time-resolved fluorescence test strips, 37 DEG C reaction after ten minutes, with time-resolved fluorescence tester into
Row detection, obtains the ratio of detection line (T) fluorescence intensity and nature controlling line (C) fluorescence intensity on fluorescent test paper strip；Based on obtaining in advance
The ratio (T/C) of the fluorescent test paper strip detection line time-resolved fluorescence intensity and nature controlling line fluorescence intensity that obtain is malicious with aspergillus flavus respectively
Plain B1, ochratoxin A, zearalenone, fumonisin B1 concentration relation curve, obtain yellow bent in testing sample solution
The content of mould toxin B1, ochratoxin A, zearalenone, fumonisin B1 are most converted afterwards up to yellow in sample to be tested
The content of aspertoxin B1, ochratoxin A, zearalenone, fumonisin B1；
According to the above scheme, the immunochromatography time-resolved fluorescence test strips detection line fluorescence intensity and nature controlling line fluorescence
The ratio (T/C) of intensity respectively with aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 concentration pass
It is that curve is obtained using following methods:
(1) prepare obtain graded series concentration aflatoxin B1, fumonisin B1, ochratoxin A and corn it is red
Mould ketenes standard solution；
(2) by the aflatoxin B1 of appropriate above-mentioned each gradient, ochratoxin A, zearalenone, fumonisin B1
Standard solution is added separately in example reaction bottle, mix, be inserted into fluorescent test paper strip, 37 DEG C reaction 6-10 minutes, use the time
Resolved fluorometric immunity analysis instrument detects to obtain the fluorescence intensity level of detection line (T) and nature controlling line (C) on each fluorescent test paper strip, thus
Obtain the ratio (T/C) of each fluorescent test paper strip detection line time-resolved fluorescence intensity Yu nature controlling line fluorescence intensity；
(3) immunochromatography time-resolved fluorescence test strips detection line fluorescence intensity and nature controlling line fluorescence intensity are obtained through fitting
Ratio (T/C) and aflatoxin B1, fumonisin B1, ochratoxin A, zearalenone relation curve.
Beneficial effects of the present invention:
(1) quick, synchronous quantitative detection aflatoxin B1, ochratoxin A, zearalenone and fumonisin
B1.Immunochromatography time-resolved fluorescence kit provided by the invention can be realized in a test strips to aflatoxin B1,
Ochratoxin A, zearalenone be synchronous with fumonisin B1's, rapid quantitative detection, and the antibody used is monoclonal
Antibody, specific good, high sensitivity is noiseless between the detection of each mycotoxin, simple, quickly.
(2) high sensitivity.Immunochromatography time-resolved fluorescence kit provided by the invention is to aspergillus flavus in detection solution
The lowest detection of toxin B1 is limited to 0.06ng/mL, and the lowest detection of ochratoxin A is limited to 0.5ng/mL, zearalenone
Lowest detection be limited to 1ng/mL, the lowest detection of fumonisin B1 is limited to 0.2ng/mL, the detection limit be able to satisfy European Union to food
The limitation requirement of 4 kinds of mycotoxins of this in product.
Detailed description of the invention
Fig. 1 is aflatoxin B1 provided by the invention, ochratoxin A, zearalenone, fumonisin B1 are immune
Chromatograph the structural schematic diagram of immunochromatography time-resolved fluorescence test strips in time-resolved fluorescence quick testing reagent box.In figure: 1 water suction
Pad, 2 detecting pads, 3 sample pads, 4 nature controlling lines, 5 aflatoxin B1 detection lines, 6 ochratoxin A detection lines, 7 Gibberella zeae alkene
Ketone detection line, 8 fumonisin B1 detection lines.
Specific embodiment
The acquisition of 1 aflatoxin B1 monoclonal antibody of embodiment
Aflatoxin B1 monoclonal antibody is by hybridoma cell strain 3G1 points that deposit number is CCTCC NO.C201014
Generation is secreted, is made in advance with specific reference to the method reported in the patent that grant number is ZL201210117614.9, the preparation method comprises the following steps:
The processed BALB/c mouse of freund 's incomplete adjuvant is used into the hybridoma cell strain 3G1 injection of acquisition in advance, collects mouse
Ascites obtains aflatoxin B1 monoclonal antibody after purification process.Wherein, purification process is caprylic acid-ammonium, specific to grasp
As: mouse ascites are filtered with double-layer filter paper, filtered ascites is on 4 DEG C, 12000r/min centrifugation 15min or more, absorption
Clearly, supernatant is mixed with the acetate buffer of 4 times of volumes, is slowly added to caprylic acid while stirring, needed for every milliliter of ascites
Caprylic acid volume is 30-35 μ L, and mixed at room temperature 30-60min, 4 DEG C of standing 2h or more, then 4 DEG C, 12000r/min is centrifuged
30min or more abandons precipitating, and after obtained supernatant is filtered with double-layer filter paper, the molar concentration that 1/10 filtrate volume is added is
The phosphate buffer of 0.1mol/L and pH value 7.4, with the sodium hydroxide solution of 2mol/L adjust the pH value of the mixed liquor to
7.4,4 DEG C of pre-coolings, are slowly added to ammonium sulfate to the final concentration of 0.277g/mL of ammonium sulfate, 4 DEG C of standing 2h or more, and then 4 DEG C,
12000r/min is centrifuged 30min or more, abandons supernatant, and the gained precipitating 0.01mol/L phosphate of former ascites volume 1/10 is delayed
Fliud flushing is resuspended, and is packed into bag filter, is dialysed with pure water, the protein solution sufficiently dialysed is set -70 DEG C of refrigerator freezings, then with cold
Freeze vacuum drier freeze-drying, collects freeze-dried powder to get purified aflatoxin B1 monoclonal antibody, antibody is set -20 DEG C
It is spare in refrigerator；
The acetate buffer is 0.29g sodium acetate, and 0.141mL acetic acid adds water to be settled to obtained by 100mL；Described
The phosphate buffer of 0.1mol/L is 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, biphosphate
Potassium 0.02g adds water to be settled to obtained by 100mL；
The acquisition of 2 ochratoxin A monoclonal antibody of embodiment；
Ochratoxin A monoclonal antibody is by hybridoma cell strain 1H2 points that deposit number is CCTCC NO.C201329
Generation is secreted, is made in advance with specific reference to the method reported in the patent application No. is 201310115921.8, the preparation method comprises the following steps: will
Hybridoma cell strain 1H2 is injected into the abdomen for using the processed BALB/c mouse of freund 's incomplete adjuvant in advance, collects the mouse
Ascites, purify up to ochratoxin A monoclonal antibody.The purification process is caprylic acid-ammonium, specific steps
Are as follows: mouse ascites are filtered with double-layer filter paper, 4 DEG C, 12000r/min is centrifuged 15min or more, supernatant is drawn, by gained ascites supernatant
It is mixed with the acetate buffer of 4 times of volumes, caprylic acid is slowly added under stirring, caprylic acid volume needed for every milliliter of ascites is
30-35 μ L, mixed at room temperature 30-60min, 4 DEG C of standing 2h or more, then 4 DEG C, 12000r/min is centrifuged 30min or more, and it is heavy to abandon
It forms sediment, after obtained supernatant is filtered with double-layer filter paper, the molar concentration that 1/10 filtrate volume is added is 0.1mol/L and pH value
For 7.4 phosphate buffer, the pH value for adjusting the mixed liquor with the sodium hydroxide solution of 2mol/L is delayed to 7.4,4 DEG C of pre-coolings
The slow ammonium sulfate that is added is to the final concentration of 0.277g/mL of ammonium sulfate, and 4 DEG C of standing 2h or more, then 4 DEG C, 12000r/min is centrifuged
30min or more abandons supernatant, by the phosphate-buffered that gained precipitates the 0.01mol/L with former ascites volume 1/10, pH value is 7.4
Liquid is resuspended, and is packed into bag filter, is dialysed with pure water, and the protein solution sufficiently dialysed is set -70 DEG C of refrigerator freezings, later with freezing
Drying machine freeze-drying is collected freeze-dried powder to get purified ochratoxin A monoclonal antibody, antibody is set standby in -20 DEG C of refrigerators
With；
The acetate buffer is 0.29g sodium acetate, and 0.141mL acetic acid adds obtained by water constant volume to 100mL；Described
The phosphate buffer of 0.01mol/L is 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.02g adds obtained by water constant volume to 100mL；The phosphate buffer of the 0.1mol/L is 8g sodium chloride, 2.9g 12
Water disodium hydrogen phosphate, 0.2g potassium chloride, potassium dihydrogen phosphate 0.2g add obtained by water constant volume to 100mL；
The acquisition of 3 zearalenone monoclonal antibody of embodiment
Zearalenone monoclonal antibody is by hybridoma cell strain 2D3 points that deposit number is CCTCC NO.C201328
Generation is secreted, is made in advance with specific reference to the method reported in the patent application No. is 201310115825.3, the preparation method comprises the following steps: will
Hybridoma cell strain 2D3 is injected into the abdomen for using the processed BALB/c mouse of freund 's incomplete adjuvant in advance, collects the mouse
Ascites, purify up to zearalenone monoclonal antibody；
The purification process be caprylic acid-ammonium, specific steps are as follows: with double-layer filter paper filter mouse ascites, 4 DEG C,
12000r/min is centrifuged 15min or more, draws supernatant, gained ascites supernatant is mixed with the acetate buffer of 4 times of volumes, is stirred
It mixes down and is slowly added to caprylic acid, caprylic acid volume needed for every milliliter of ascites is 30-35 μ L, and mixed at room temperature 30-60min, 4 DEG C quiet
2h or more is set, then 4 DEG C, 12000r/min is centrifuged 30min or more, abandons precipitating, obtained supernatant is filtered with double-layer filter paper
Afterwards, be added 1/10 filtrate volume molar concentration be 0.1mol/L, the phosphate buffer that pH value is 7.4, with the hydrogen of 2mol/L
Sodium hydroxide solution adjusts the pH value of the mixed liquor to 7.4,4 DEG C of pre-coolings, and it is final concentration of to ammonium sulfate to be slowly added to ammonium sulfate
0.277g/mL, 4 DEG C of standing 2h or more, then 4 DEG C, 12000r/min is centrifuged 30min or more, abandons supernatant, and gained is precipitated with former
The 0.01mol/L of ascites volume 1/10, the phosphate buffer that pH value is 7.4 are resuspended, and are packed into bag filter, are dialysed with pure water, will
The protein solution sufficiently dialysed sets -70 DEG C of refrigerator freezings, is lyophilized later with freeze drier, collects freeze-dried powder to get purifying
Good zearalenone monoclonal antibody, antibody is set spare in -20 DEG C of refrigerators；
The acetate buffer is 0.29g sodium acetate, and 0.141mL acetic acid adds obtained by water constant volume to 100mL；Described
The phosphate buffer of 0.01mol/L is 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.02g adds obtained by water constant volume to 100mL；The phosphate buffer of the 0.1mol/L is 8g sodium chloride, 2.9g 12
Water disodium hydrogen phosphate, 0.2g potassium chloride, potassium dihydrogen phosphate 0.2g add obtained by water constant volume to 100mL；
The acquisition of 4 fumonisin B1 monoclonal antibody of embodiment
The screening of hybridoma cell strain Fm7A11
1. antigen synthesis and animal immune
Buy commercially available fumonisin B1Standard items carry out comlete antigen synthesis, and specific synthesis step is as follows: by the FB of 2mg1
The EDC of standard items powder and 2mg are dissolved in the 0.01mol/LPBS solution of 500 μ L respectively, obtain EDC solution and FB1Solution, will
The FB dissolved is added dropwise in the EDC solution of 4mg/mL (solution 0.01mol/LPBS)1In solution, it is gently mixed 10 at room temperature
Minute.The BSA solution of 5mg/mL (solution 0.01mol/LPBS) is added dropwise in above-mentioned mixed liquor, reaction is stirred at room temperature
4 hours.Dialysis 3 days.The identification of Conventional UV scanning method is finally carried out, qualification result shows FB1- BSA comlete antigen is successfully prepared.
Buy 6 week old BALB/c mouse 6, the fumonisin comlete antigen FB of immunization experiment room synthesis1-BSA.For the first time
It is immunized after emulsifying fumonisin comlete antigen and isometric Freund's complete adjuvant, in the subcutaneous multi-point injection of mouse the nape of the neck.
It carries out, is emulsified using incomplete Freund's adjuvant and isometric fumonisin comlete antigen, in mouse after being immune to for the second time 3 weeks
The subcutaneous multi-point injection of the nape of the neck.Third time with the 4th time it is immune respectively with last immunization interval two weeks, immunization ways and second
It is secondary identical.Four times immunizing dose is identical, and only 100 μ g/ are only.7th day after third time is immune, mouse tail vein blood sampling separates blood
Clearly, mice serum potency is monitored using indirect elisa method, and measures mice serum sensitivity, selection with indirect competitive ELISA method
The corresponding mouse of the relatively higher serum of potency, sensitivity carries out last time booster immunization, and immunizing dose is 2 measured before
Times.
2. cell fusion
After last time booster immunization 3 days, use 50% (weight percent) polyethylene glycol i.e. PEG (molecular weight for
1450) make fusion agent, carry out cell fusion according to a conventional method, specific steps: taking off neck under aseptic condition and put to death mouse to be fused, point
It from splenocyte, is mixed with source of mouse myeloma cell SP2/0 with the number ratio of 5:1, it is thin to wash mixing with RPMI-1640 basic culture solution
Born of the same parents, 1200rpm are centrifuged 5min.It discards supernatant, drains, 1mLPEG is added, merge 1 minute, be slowly added to the basis RPMI-1640
Supernatant is abandoned in culture solution, centrifugation, and precipitating is fused cell, is resuspended with 20mL complete medium, the cell hanged is added to
It is added in 80mL semisolid culturemedium, after mixing in 6 porocyte culture plates, the hole 2mL/, is placed in 37 DEG C of carbon dioxide incubator trainings
It supports.
The cell culture complete medium containing 1%HAT contains 20% (percentage by volume) fetal calf serum, 75% (volume
Percentage) RPMI-1640 basic culture solution, 1% (weight percent) L-Glutamine, 1% (percentage by volume) HEPES, 1%
(percentage by volume) is dual anti-(10000 units per ml penicillin and 10000 micrograms per millilitre streptomysins), 2% (volume basis
Number) growth factor (HFCS) and 1% (weight percent) hypoxanthine-amino butterfly ridge-thymidine, that is, HAT and methyl fibre
Dimension element is purchased from sigma-Aldrich company.
3. screening and the clone of cell strain
2-3 weeks after cell fusion, when cell colony is long visible to naked eyes, it will be cloned from culture medium with micropipettor
Choose, is transferred to 96 porocyte culture plates using HAT Liquid Culture, when cell length to 2/3 bottom hole, draws culture supernatant and carry out
Detection.Using two step screening method, the first step uses indirect ELISA method, filters out anti-fumonisin without anti-carrier protein BSA
Positive hole；Second step detects the positive hole that the first step filters out using indirect competitive ELISA method, with fumonisin B1
Former as competition, selecting the higher hole of light absorption value and sensitivity, (higher refer to of light absorption value competes the hole i.e. Positive control wells for 0 originally
Final tested volume it is higher, the higher competition original content also IC referred to when inhibiting rate is 50% of sensitivity50It is worth smaller), use is limited
Dilution method is subcloned, and is detected after subclone using same two-step method, after so repeating subclone 4-5 times, is obtained
Hybridoma cell strain Fm7A11.
Anti- fumonisin B1The measurement of the monoclonal antibody hybridoma cell antibody variable sequences strain Fm7A11
(1) extract total serum IgE: using Tiangeng company total RNA extraction reagent box and to specifications extraction can produce hybridization
The total serum IgE of tumor cell strain Fm7A11；
(2) synthesize cDNA: for the total serum IgE obtained using step 1 as template, oligo (dT) 15 is primer, according to
SuperScriptTM- 2II reverse transcriptase specification carries out reverse transcription, synthesizes the first chain of cDNA；Primer oligo (dT) 15 by
Invitrogen is bought；
(3) PCR method clones variable region gene: being drawn according to the conserved positions design of mouse antibody gene sequence in GENBANK
Object expands heavy chain of antibody, light-chain variable region gene by template of CDNA.PCR program are as follows: 94 DEG C of 30s, 58 DEG C of 45s, 72 DEG C
1min expands 30 circulations, last 72 DEG C of extensions 10min.Ago-Gel electricity of the PCR product Jing Guo 1% (weight percent)
After swimming separation, DNA fragmentation is recycled with kits, is connected in carrier pMD18-T, conversion bacillus coli DH 5 alpha competence is thin
Born of the same parents, picking positive colony send to Shanghai Sani Biotechnology Co., Ltd and are sequenced.Wherein the sequence of primer is respectively as follows: weight
Chain variable region primers are 5 '-CAG GTS MAR CTG MAG GAG TCW G-3 ' (22mer) and 5 '-CAG GGG CCA GTG
GAT AGA CAG ATG GGG G-3 ' (28mer), wherein S, M, R and W are to annex base, M=A/C, R=A/G, S=G/C, W
=A/T, light chain variable region primer are 5 '-GAC ATC AAG ATG ACC CAG TCT CCA-3 ' (24mer) and 5 '-CCG
TTT TAT TTC CAG CTT GGT CCC-3’(24mer)。
Obtained gene order result: the long 379bp of heavy chain variable region coding gene sequence, sequence such as SEQ ID NO:1 institute
Show, derives that the encoded heavy chain variable region of the gene order is made of 126 amino acid according to gene order obtained, sequence
Column are as shown in SEQ ID NO:3.The long 348bp of light chain variable region coding gene sequence, sequence as shown in SEQ ID NO:2, according to
Gene order obtained derives that the encoded light chain variable region of the gene order is made of 116 amino acid, sequence such as SEQ
Shown in ID NO:4.
5. anti-fumonisin B1Preparation purifying, hypotype and the CHARACTERISTICS IDENTIFICATION of monoclonal antibody
The anti-fumonisin B that embodiment 1 is obtained1Freund is used in monoclonal antibody hybridoma cell strain Fm7A11 injection in advance
The processed BALB/c mouse of Freund's incomplete adjuvant, collects the ascites of the mouse, using caprylic acid-ammonium antibody purification, specifically
Operation are as follows: filter mouse ascites with double-layer filter paper, 4 DEG C, 12000r/min is centrifuged 15min or more, supernatant is drawn, by gained ascites
Supernatant is mixed with the acetate buffer of 4 times of volumes, and caprylic acid is slowly added under stirring, caprylic acid body needed for every milliliter of ascites
Product is 30-35 μ L, mixed at room temperature 30-60min, 4 DEG C of standing 2h or more.12000r/min, 4 DEG C of centrifugation 30min or more, it is heavy to abandon
It forms sediment, after obtained supernatant is filtered with double-layer filter paper, the molar concentration that 1/10 filtrate volume is added is 0.1mol/L and pH is
7.4 phosphate buffer adjusts the pH to 7.4 of the mixed liquor with the sodium hydroxide solution of 2mol/L, is slowly added in ice bath
Ammonium sulfate is to the final concentration of 0.277g/mL of ammonium sulfate, 4 DEG C of standing 2h or more, then 12000r/min, 4 DEG C of centrifugation 30min with
On, supernatant is abandoned, with the molar concentration of original ascites volume 1/10 volume is phosphate that 0.01mol/L, pH are 7.4 by gained precipitating
Buffer is resuspended, and is packed into bag filter, is dialysed two days with 0.01mol/LPBS, then uses PB instead and dialyse two days, by albumen in bag filter
Solution takes out, and supernatant is collected in centrifugation, abandons precipitating, is put into after -70 DEG C of pre-freezes to be put into freeze dryer and is lyophilized.Freeze-dried powder is collected, as
Purified anti-fumonisin B1Monoclonal antibody；
The acetate buffer is 0.29g sodium acetate, and 0.141mL acetic acid adds obtained by water constant volume to 100mL；Described
The phosphate buffer of 0.01mol/L is 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g phosphorus
Acid dihydride potassium adds obtained by water constant volume to 100mL；The phosphate buffer of the 0.1mol/L is 8g sodium chloride, 2.9g 12
Water disodium hydrogen phosphate, 0.2g potassium chloride, 0.2g potassium dihydrogen phosphate add obtained by water constant volume to 100mL.
Anti- fumonisin B1 monoclonal with the identification hybridoma cell strain Fm7A11 secretion of commercially available subtype identification kit is anti-
The hypotype of body is IgG2b.
Measuring the antibody titer that mouse ascites are purified with conventional non-competing enzyme linked immunosorbent assay (ELISA) can reach
3.2×105, i.e. antibody dilution 3.2 × 105Times when solution measurement result be the positive.It is right that its is measured with conventional indirect competitive ELISA
Fumonisin B1Sensitivity is 0.32ng/mL.To fumonisin B2、B3Cross reacting rate be 4.3% and 12.8%.With Huang song
Mould toxin, zearalenone, T-2 toxin, ochratoxin, vomitoxin cross reacting rate be respectively less than 0.1%.
Embodiment 5
200 μ l europium mark fluorescent microballoons are taken, are added in the borate buffer of 1mL 0.2mol/L pH8.2, at 300w ultrasound
Reason 40 seconds, is then slowly added into the carbodiimide of 40uL 15mg/mL, and after shaken at room temperature 20min, 17000g is centrifuged 15 minutes, receives
Collection precipitating is redissolved with the borate buffer of 0.2mol/L pH 8.2, the fluorescent microsphere of activation is redissolved in 5mL 0.01mol/L
In the borate buffer of pH8.2,1mg/ml antibody (aflatoxin B1 monoclonal antibody 35ul, ochratoxin A Dan Ke is added
Grand antibody 45ul, zearalenone monoclonal antibody 55ul, fumonisin B1 monoclonal antibody 40ul), 4 DEG C of oscillating reactions
After 12h, 17000g is centrifuged 10 minutes, and the borate buffer of the 0.2mol/L pH8.2 containing 1%BSA redissolves, 4 DEG C of oscillating reactions
4h, 17000g are centrifuged 10min and collect precipitating, redissolve in containing 1.5% (m/v) trehalose, 2% (m/v) bovine serum albumin(BSA)
To get the mycotoxin antibody of europium label in the borate buffer of 0.2mol/L pH8.2, it is placed in 4 DEG C and saves backup.
Synchronous detection aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 composite pollution are synchronous
The immunochromatography time-resolved fluorescence kit of detection and its application
Aflatoxin B1, ochratoxin A, zearalenone, the synchronous detection of fumonisin B1 composite pollution are immune
Time-resolved fluorescence quick testing reagent box is chromatographed, it includes immunochromatography time-resolved fluorescence test strips, the Huang song containing europium label
The Gibberella zeae that mould toxin B1 monoclonal antibody dried frozen aquatic products, the ochratoxin A monoclonal antibody dried frozen aquatic products of europium label, europium mark
Ketenes monoclonal antibody dried frozen aquatic products, the example reaction bottle of the fumonisin B1 monoclonal antibody dried frozen aquatic products of europium label, sample dilution
Liquid and sample diluting liquid suction pipe,
The fluorescent test paper strip includes PVC substrate, and the one side of PVC substrate successively pastes water absorption pad, detection from top to bottom
Pad and sample pad, adjacent each pad is in the overlapping connection in junction, overlapping length 1mm；
(1) preparation of water absorption pad
Blotting paper is cut out into growth 16mm, the specification of wide 4mm is to get water absorption pad；
(2) preparation of detecting pad
The coating of detection line:
By aflatoxin B1-bovine serum albumin(BSA) conjugate with coating buffer at the solution of 0.25mg/mL, in
Away from the position on nitrocellulose filter along 10mm, it is coated on nitrocellulose filter with line spray mode and obtains detection line I, often
The package amount of aflatoxin B1-bovine serum albumin(BSA) conjugate needed on centimetre detection line I is 100ng；By ochratoxin
A- bovine serum albumin(BSA) conjugate (OTA-BSA) uses coating buffer at the coating buffer of 0.35mg/mL, in away from detection line I
The position of 4mm is coated on nitrocellulose filter with line spray mode and obtains detection line II, required on detection line II per cm
Ochratoxin A-bovine serum albumin(BSA) conjugate package amount is 160ng；By zearalenone-bovine serum albumin(BSA) coupling
Object is wrapped in the position away from detection line II 4mm with line spray mode with coating buffer at the coating buffer of 0.4mg/mL
By in obtaining detection line III on nitrocellulose filter, on detection line III per cm needed for zearalenone-bovine serum albumin
The package amount of white conjugate is 200ng；By fumonisin B1- bovine serum albumin(BSA) conjugate with coating buffer at
The coating buffer of 0.25mg/mL is coated on nitrocellulose filter in the position away from detection line III 4mm with line spray mode
Detection line IV is obtained, the package amount of required fumonisin B1- bovine serum albumin(BSA) conjugate is on detection line IV per cm
150ng；Then 120 minutes dry under the conditions of 37 DEG C；
The coating of nature controlling line:
Rabbit-anti mouse polyclonal antibody is made into the coating buffer that concentration is 0.30mg/mL with coating buffer；In away from detection line I
Its transverse direction is coated on nitrocellulose filter by the position of 5mm with line spray mode, obtains nature controlling line, needed for nature controlling line per cm
The package amount of rabbit-anti mouse polyclonal antibody is 90ng, then 120 minutes dry under the conditions of 40 DEG C；
The coating buffer are as follows: by 1mg rabbit-anti mouse polyclonal antibody, 0.002g sodium azide, 0.08g sodium chloride,
0.029g disodium hydrogen phosphate, 0.002g potassium chloride, 0.002g potassium dihydrogen phosphate add water to be settled to obtained by 10mL；
The nitrocellulose filter long 25mm, wide 4mm.
(3) preparation of sample pad:
Glass fibre membrane is cut out into growth 12mm, the specification of wide 4mm is put into confining liquid and soaks, and takes out, in 40 DEG C of conditions
Lower drying 4 hours, obtains sample pad, then sets room temperature preservation in drier；
The confining liquid is 1g oralbumin, 2g sucrose, 0.02g sodium azide, 0.8g sodium chloride, 0.29g 12
Water disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate add water to be settled to obtained by 100mL；
(4) assembling of fluorescent test paper strip:
Water absorption pad, detecting pad and sample pad are successively pasted from top to bottom in the one side of PVC substrate, and adjacent each pad is in junction
Overlapping connection, overlapping length be 1mm to get；See Fig. 1.
The acquisition of the example reaction bottle: by above-mentioned prepared redissolution in containing 1.5% (m/v) trehalose, 2% (m/
V) the aflatoxin B1 monoclonal of the europium label in the phosphate buffer of the 0.01mol/L pH 7.4 of bovine serum albumin(BSA) is anti-
The volt horse that body, the ochratoxin A monoclonal antibody of europium label, the zearalenone monoclonal antibody of europium label, europium mark
Toxin B1 monoclonal antibody, respectively take it is a certain amount of be put into example reaction bottle, be placed in freeze drier and be lyophilized, obtain europium label
Monoclonal antibody dried frozen aquatic products, it is spare.
The content for the aspergillus flavus resisting toxin B1 monoclonal antibody dried frozen aquatic products that europium marks in the example reaction bottle is 0.25 μ g,
The content of the anti-ochratoxin A monoclonal antibody dried frozen aquatic products of europium label is 0.3 μ g, the anti-zearalenone Dan Ke of europium label
The content of grand antibody dried frozen aquatic products is 0.2 μ g, and the content of the anti-fumonisin B1 monoclonal antibody dried frozen aquatic products of europium label is 0.2 μ g.
The aflatoxin B1, ochratoxin A, zearalenone, the synchronous detection of fumonisin B1 composite pollution
Immunochromatography time-resolved fluorescence detection kit is in sample aflatoxin B1, ochratoxin A, zearalenone, volt
Application in horse toxin B1 detection:
The methanol solution of 100mL 70% (volume fraction) is added in the negative Feed Sample 20g of confirmation of learning from else's experience, and homogeneous 2 divides
Clock stands, is filtered with double-layer filter paper, collects filtrate 1mL, and 5mL sample diluting liquid is added and dilutes filtrate, mixes, obtains blank base
Matter solution；
With vehicle solution configuration aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 points
Aflatoxin B1, the ochratoxin A, zearalenone, fumonisin B1 mixing mark of following concentration gradient are not corresponded to
Quasi- solution 6, aflatoxin B1 (0ng/mL, 0.05ng/mL, 0.1ng/mL, 0.25ng/mL, 0.5ng/mL, 1.0ng/
ML), fumonisin B1 (0ng/mL, 0.05ng/mL, 0.1ng/mL, 0.25ng/mL, 0.5ng/mL, 1.0ng/mL), Aspergillus ochraceus
Toxin A (0ng/mL, 0.25ng/mL, 0.5ng/mL, 1.0ng/mL, 2ng/mL, 4ng/mL), zearalenone (0ng/mL,
0.5ng/mL,1.0ng/mL,2.5ng/mL,5.0ng/mL,10ng/mL).The each concentration point of standard solution is repeated 5 times, and how malicious use is
Plain test strip is detected: 150 μ L of above-mentioned standard product solution being added in example reaction bottle, is mixed, immunochromatography is inserted into
Time-resolved fluorescence test strips, 37 DEG C of reactions after ten minutes, blot sample pad residual liquid with blotting paper, use time resolution immediately
Fluorescence immunity analyzer detection (excitation wavelength: 365nm measures wavelength: 615nm), reads the fluorescence signal intensity of detection zone
Value, calculates 5 repetition average values.With fluorescence signal intensity value/C line of standard series concentration value natural logrithm (Lnc) and T line
Fluorescence signal intensity value (T/C) draws standard curve, and the standard of calibration curve formula such as Y=a*lnc+b, 5 kinds of test objects are bent
Line parameter is as shown in the table:
| a | b | R2 | Detect limit/ng/mL | |
| Aflatoxin B1 | -1.425 | 3.524 | 0.991 | 0.06 |
| Fumonisin B1 | -1.750 | 3.448 | 0.989 | 0.2 |
| Ochratoxin A | -1.594 | 4.788 | 0.989 | 0.5 |
| Zearalenone | -1.750 | 5.751 | 0.979 | 1 |
It takes Feed Sample 20g to be checked that the methanol solution of 100mL 70% (volume fraction) is added, homogeneous 2 minutes, stands, uses
Double-layer filter paper filtering, collects filtrate 1mL, and 5mL sample diluting liquid is added and dilutes filtrate, mixes；Take Feed Sample detection liquid to be checked
150 μ L are added in example reaction bottle, mix, and are inserted into immunochromatography time-resolved fluorescence test strips, and 37 DEG C of reactions after ten minutes, are used
Blotting paper blots sample pad residual liquid, obtains each fluorescent test paper strip 4 with time-resolved fluorescence immunoassay instrument detection immediately
Then the ratio (T/C) of detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity substitutes into it respectively
The ratio (T/C) of the fluorescent test paper strip detection line time-resolved fluorescence intensity stated and nature controlling line time-resolved fluorescence intensity with
The relation curve of aflatoxin B1 concentration, ochratoxin A concentration, zearalenone concentration, fumonisin B1 concentration, obtains
Aflatoxin B1 content in the sample is 5.4 μ g/kg, ochratoxin content is 0 μ g/kg, zearalenone content
It is 0 μ g/kg for 0 μ g/kg, ochratoxin content.
Embodiment 6
As different from Example 5: the long 14mm of water absorption pad, wide 3mm in immunochromatography time-resolved fluorescence test strips；Detection
Pad long 30mm, wide 3mm；Sample pad long 16mm, wide 3mm, the adjacent overlapping length respectively padded are 2mm；It is examined in the fluorescent test paper strip
The spacing on the upper edge in the detection line of nature controlling line and nitrocellulose filter of survey pad is 10mm, between every adjacent two detection lines
Spacing is 3.5mm.It is per cm required yellow in coating aflatoxin B1-bovine serum albumin(BSA) conjugate detection line
The package amount of aspertoxin B1- bovine serum albumin(BSA) conjugate is 125ng；It is coated with ochratoxin A-bovine serum albumin(BSA) coupling
In the detection line of object it is per cm required for ochratoxin A-bovine serum albumin(BSA) conjugate package amount be 200ng；Coating
Required zearalenone-ox blood per cm is pure in zearalenone-bovine serum albumin(BSA) conjugate detection line
The package amount of protein conjugate is 250ng；Required for being coated in the detection line of fumonisin B1- bovine serum albumin(BSA) conjugate
The package amount of fumonisin B1- bovine serum albumin(BSA) conjugate is 150ng, rabbit-anti mouse Anti-TNF-α needed for nature controlling line per cm
The package amount of body is 75ng；The content for the aspergillus flavus resisting toxin B1 monoclonal antibody dried frozen aquatic products that europium marks in the example reaction bottle
For 0.2 μ g, the content of the anti-ochratoxin A monoclonal antibody dried frozen aquatic products of europium label is 0.25 μ g, and the anti-corn of europium label is red
The content of mould ketenes monoclonal antibody dried frozen aquatic products is 0.2 μ g, and the anti-fumonisin B1 monoclonal antibody dried frozen aquatic products of europium label contain
Amount is 0.23 μ g.The aflatoxin B1, ochratoxin A, zearalenone, the synchronous inspection of fumonisin B1 composite pollution
Survey immunochromatography time-resolved fluorescence detection kit sample aflatoxin B1, ochratoxin A, zearalenone,
Application in fumonisin B1 detection:
(aflatoxin B1 content is 3.5 μ g/ to the corn sample containing more toxin of high performance liquid chromatography of learning from else's experience confirmation
Kg, ochratoxin content are 2.6 μ g/kg, zearalenone content is 42 μ g/kg, fumonisin B1 content is 8.1 μ g/
Kg), take 25g that the methanol solution of 100mL 70% (volume fraction) is added, homogeneous 2 minutes, stand, filtered with double-layer filter paper, received
Collect filtrate 1mL, 4mL sample diluting liquid is added and dilutes filtrate, mixes；Take corn sample detection 150 μ L of liquid to be checked that sample is added anti-
It answers in bottle, mixes, be inserted into immunochromatography time-resolved fluorescence test strips, 37 DEG C of reactions after ten minutes, blot sample with blotting paper
Residual liquid is padded, obtains each 4 detection line time resolutions of fluorescent test paper strip with time-resolved fluorescence immunoassay instrument detection immediately
Then it is substituted into fluorescence examination obtained above by the ratio (T/C) of fluorescence intensity and nature controlling line time-resolved fluorescence intensity respectively
Paper slip detection line time-resolved fluorescence intensity and the ratio (T/C) and aflatoxin B1 of nature controlling line time-resolved fluorescence intensity are dense
The relation curve of degree, ochratoxin A concentration, zearalenone concentration, fumonisin B1 concentration, the Huang obtained in the sample are bent
Mould toxin B1 content is 3.2 μ g/kg, ochratoxin content is 2.5 μ g/kg, zearalenone content is 45 μ g/kg, volt
Horse toxin B1 content is 8.5 μ g/kg.
Sequence table
110 > Inst. of Oil Crops, Chinese Academy of Agriculture of <
The immunochromatography time-resolved fluorescence of four kinds of mycotoxin composite pollutions such as the synchronous detection fumonisin B1 of 120 > of <
Kit and its application
160 > 4 of <
210 > 1 of <
211 > 379bp of <
212 > DNA of <
213 > mouse of <
400 > 1 of <
caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag 50
cctgtccatc acttgcactg tctctgggct ttcattaacc agctatggtg 100
tacactgggt tcgtcaggcc ccaggaaagg gtctggagtg gctgggagta 150
atttggggtg gtggaaacac aaattataat tcggctctca tgtccagact 200
gagcatcagc aaagacaact ccaggagcca agttttctta agaatgaaca 250
gtctgcaaat tgatgacaca gccatgtact attgtgccag aggcaggatg 300
gactactggg gtcaaggaac ctcagtcacc gtctcgtcag ccaaaacgac 350
acccccatct gtctatccac tggcccctg 379
210 > 1 of <
211 > 348bp of <
212 > DNA of <
213 > mouse of <
400 > 2 of <
gacatcaaga tgacccagtc tccatcttcc atgtatgcat ctctaggaga 50
aagagtcact atcacttgca aggcgagtca ggacattagt agctatttag 100
gctggttaca gcagaaacca gggaaatctc ctaagaccct gatctatcgt 150
gcaaacacat tggtagaagg ggtcccatcc agattcagtg gcagtggatc 200
tggggaagat tattctctca ccatcagcag cctggagtat gaagatatgg 250
gaatttatta ttgtctacag tatgatgagt ttccgtacac gttcggaggg 300
gggaccaagc tggaaataaa acgggctgat gctgcaccaa ctgtatcc 348
210 > 1 of <
211 > 126 of <
212 > PRT of <
213 > mouse of <
400 > 3 of <
Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser 15
Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Leu Ser Leu Thr 30
Ser Tyr Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 45
Glu Trp Leu Gly Val Ile Trp Gly Gly Gly Asn Thr Asn Tyr Asn 60
Ser Ala Leu Met Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Arg 75
Ser Gln Val Phe Leu Arg Met Asn Ser Leu Gln Ile Asp Asp Thr 90
Ala Met Tyr Tyr Cys Ala Arg Gly Arg Met Asp Tyr Trp Gly Gln 105
Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser 120
Val Tyr Pro Leu Ala Pro 126
210 > 1 of <
211 > 116 of <
212 > PRT of <
213 > mouse of <
400 > 4 of <
Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu 15
Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Ser 30
Ser Tyr Leu Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Pro Lys 45
Thr Leu Ile Tyr Arg Ala Asn Thr Leu Val Glu Gly Val Pro Ser 60
Arg Phe Ser Gly Ser Gly Ser Gly Glu Asp Tyr Ser Leu Thr Ile 75
Ser Ser Leu Glu Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln 90
Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 105
Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser 116
Claims (10)
1. synchronous detection aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 composite pollution it is immune
Chromatograph time-resolved fluorescence kit, it is characterised in that: it includes immunochromatography time-resolved fluorescence test strips and containing europium mark
The zearalenone that the aflatoxin B1 monoclonal antibody of note, the ochratoxin A monoclonal antibody of europium label, europium mark
The example reaction bottle for the fumonisin B1 monoclonal antibody dried frozen aquatic products that monoclonal antibody, europium mark；Wherein: the immunochromatography
Time-resolved fluorescence test strips include PVC substrate, and the one side of substrate successively pastes water absorption pad, detecting pad and sample from top to bottom
Pad, adjacent each pad is in the overlapping connection in junction, and the detecting pad is using nitrocellulose filter as base wad, from upper on nitrocellulose filter
And the lateral nature controlling line of lower setting and four detection lines, the nature controlling line are coated with rabbit-anti mouse polyclonal antibody, four detections
Aflatoxin B1-bovine serum albumin(BSA) conjugate, ochratoxin A-bovine serum albumin(BSA) conjugate, jade are coated on line respectively
Zearlenone-bovine serum albumin(BSA) conjugate, fumonisin B1- bovine serum albumin(BSA) conjugate, the fumonisin B1
Monoclonal antibody is the monoclonal generated by the hybridoma cell strain Fm7A11 secretion that deposit number is CCTCC NO. 201636
Antibody；The hybridoma cell strain Fm7A11 is preserved in China typical culture collection center on March 29th, 2016, protects
Hiding address is China, Wuhan, Wuhan University, and deposit number is CCTCC NO. 201636.
2. immunochromatography time-resolved fluorescence kit according to claim 1, it is characterised in that: the list of the europium label
Clonal antibody is to be prepared in accordance with the following methods:
The aflatoxin B1 monoclonal antibody of europium label: the europium after aflatoxin B1 monoclonal antibody and activation is marked into examination
Agent mixes in borate buffer, oscillating reactions, then obtains the Huang of target product europium label through centrifugation, redissolution, closing step
Aspertoxin B1 monoclonal antibody；Europium labelled reagent after 1mL activation can be coupled aflatoxin B1 monoclonal antibody: 30 μ g-
80μg；
The ochratoxin A monoclonal antibody of europium label: by the europium labelled reagent after ochratoxin A monoclonal antibody and activation
Then mixing, oscillating reactions in borate buffer obtain the reddish brown song of target product europium label through centrifugation, redissolution, closing step
Mould toxin A monoclonal antibody；Europium labelled reagent after 1mL activation can be coupled ochratoxin A monoclonal antibody: 40 μ g-90 μ g；
The zearalenone monoclonal antibody of europium label: the europium after zearalenone monoclonal antibody and activation is marked into examination
Agent mixes in borate buffer, oscillating reactions, then obtains the jade of target product europium label through centrifugation, redissolution, closing step
Zearlenone monoclonal antibody；Europium labelled reagent after 1mL activation can be coupled zearalenone monoclonal antibody: 30 μ g-
90μg；
The fumonisin B1 monoclonal antibody of europium label: the europium labelled reagent after fumonisin B1 monoclonal antibody and activation is existed
It is mixed in borate buffer, oscillating reactions, then obtains the volt horse poison of target product europium label through centrifugation, redissolution, closing step
Plain B1 monoclonal antibody；Europium labelled reagent after 1mL activation can be coupled fumonisin B1 monoclonal antibody: 30 μ g-80 μ g.
3. immunochromatography time-resolved fluorescence kit according to claim 2, it is characterised in that: the work of europium labelled reagent
Change method is ultrasonic disperse in the borate buffer for take 8.2 0.2mol/L of europium labelled reagent pH, is then slowly added into carbon two
Supernatant is removed in imide liquor, shaken at room temperature activation, centrifugation, is redissolved with the borate buffer of 8.2 0.2mol/L of pH, spare, living
The change time is 15-30min.
4. immunochromatography time-resolved fluorescence kit according to claim 1, it is characterised in that: the fluorescent test paper strip
In water absorption pad long 10-16mm, wide 3-5mm；Detecting pad long 25-30mm, wide 3-5mm；Sample pad long 12-18mm, wide 3-5mm,
The adjacent overlapping length respectively padded is 1-3mm；It is fine close to the detection line of nature controlling line and nitric acid on detecting pad in the fluorescent test paper strip
The spacing for tieing up edge on plain film is 5-10mm, and the spacing between every adjacent two detection lines is 1.5-4.5mm, close to the inspection of nature controlling line
The spacing of survey line and nature controlling line is 3-5mm；The example reaction bottle is the cillin bottle of 1-2.5mL.
5. immunochromatography time-resolved fluorescence kit according to claim 1, it is characterised in that: the coating aspergillus flavus
Required aflatoxin B1-bovine serum albumin(BSA) per cm is even in the detection line of toxin B1- bovine serum albumin(BSA) conjugate
The package amount for joining object is 50-250ng；It is coated with per cm required in ochratoxin A-bovine serum albumin(BSA) conjugate detection line
The ochratoxin A wanted-bovine serum albumin(BSA) conjugate package amount is 100-300ng；It is coated with zearalenone-cow's serum
In the detection line of albumin conjugate it is per cm required for the package amount of zearalenone-bovine serum albumin(BSA) conjugate be
100-300ng；It is coated with required fumonisin B1- ox blood in the detection line of fumonisin B1- bovine serum albumin(BSA) conjugate
The package amount of pure protein conjugate is 50-150ng, and the package amount of rabbit-anti mouse polyclonal antibody needed for nature controlling line per cm is
50-150ng。
6. immunochromatography time-resolved fluorescence kit according to claim 1, it is characterised in that: the example reaction bottle
The content of the aflatoxin B1 monoclonal antibody dried frozen aquatic products of middle europium label is 0.1-0.3 μ g, the ochratoxin A list of europium label
The content of clonal antibody dried frozen aquatic products is 0.1-0.3 μ g, and the content of the zearalenone monoclonal antibody dried frozen aquatic products of europium label is
0.1-0.3 μ g, the content of the fumonisin B1 monoclonal antibody dried frozen aquatic products of europium label are 0.1-0.3 μ g.
7. immunochromatography time-resolved fluorescence kit according to claim 1, it is characterised in that: the aspergillus flavus poison
Plain B1, ochratoxin A, zearalenone, fumonisin B1 immunochromatography time-resolved fluorescence quick testing reagent box further include
Sample diluting liquid and sample diluting liquid suction pipe, the sample diluting liquid are the Tween-20 that volume fraction is 0.01%-0.30%
Aqueous solution.
8. immunochromatography time-resolved fluorescence kit according to claim 1, it is characterised in that: the time resolution
Fluorescent test paper strip the preparation method is as follows:
(1) blotting paper is cut out into obtain water absorption pad；
(2) preparation of detecting pad:
By aflatoxin B1-bovine serum albumin(BSA) conjugate, ochratoxin A-bovine serum albumin(BSA) conjugate, Gibberella zeae
It is 0.10-0.50mg/ that ketenes-bovine serum albumin(BSA) conjugate, fumonisin B1- bovine serum albumin(BSA) conjugate, which are configured to concentration,
The coating buffer of mL, with line spray mode by it in carrying out being respectively separated coating on nitrocellulose filter, obtain 4 detection lines, then
It is 60-120 minutes dry under the conditions of 37-40 DEG C；
Rabbit-anti mouse polyclonal antibody is made into the coating buffer that concentration is 0.1-0.5mg/mL, is coated in its transverse direction with line spray mode
On nitrocellulose filter, nature controlling line is obtained, it is then 60-120 minutes dry under the conditions of 37-40 DEG C；
(3) preparation of sample pad:
Glass fibre membrane is put into confining liquid and is soaked, is taken out, it is 4-6 hours dry under the conditions of 37-40 DEG C, sample pad is obtained, so
Room temperature preservation in postposition drier；
(4) assembling of immunochromatography time-resolved fluorescence test strips:
Water absorption pad, detecting pad, sample pad are successively pasted from top to bottom in the one side of cardboard, and adjacent each pad is in the overlapping company in junction
It connects to get fluorescent test paper strip.
9. immunochromatography time-resolved fluorescence quick testing reagent box described in claim 1 is in aflatoxin B1, ochratoxin
A, zearalenone, the application in fumonisin B1 content detection, it is characterised in that: by sample to be tested through pre-treatment obtain to
It after sample solution, is added in example reaction bottle, mixes, be inserted into time-resolved fluorescence test strips, 37 DEG C of reactions after ten minutes, are used
Time-resolved fluorescence tester is detected, and the ratio of detection line fluorescence intensity and nature controlling line fluorescence intensity on fluorescent test paper strip is obtained
Value；Ratio based on the fluorescent test paper strip detection line time-resolved fluorescence intensity that is obtained ahead of time and nature controlling line fluorescence intensity respectively with
Aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 concentration relation curve, obtain sample to be tested it is molten
Aflatoxin B1, the content of ochratoxin A, zearalenone, fumonisin B1 in liquid are most converted afterwards up to be measured
Aflatoxin B1, the content of ochratoxin A, zearalenone, fumonisin B1 in sample.
10. application according to claim 9, it is characterised in that: the immunochromatography time-resolved fluorescence test strips inspection
The ratio of survey line fluorescence intensity and nature controlling line fluorescence intensity respectively with aflatoxin B1, ochratoxin A, Gibberella zeae alkene
Ketone, fumonisin B1 concentration relation curve obtained using following methods:
(1) it prepares and obtains aflatoxin B1, fumonisin B1, ochratoxin A and the Gibberella zeae alkene of graded series concentration
Ketone standard solution；
(2) by aflatoxin B1, ochratoxin A, zearalenone, the fumonisin B1 standard of appropriate above-mentioned each gradient
Product solution is added separately in example reaction bottle, mix, be inserted into fluorescent test paper strip, 37 DEG C reaction 6-10 minutes, use time resolution
Fluorescence immunity analyzer detects to obtain the fluorescence intensity level of detection line and nature controlling line on each fluorescent test paper strip, thus to obtain each fluorescence
The ratio of test strips detection line time-resolved fluorescence intensity and nature controlling line fluorescence intensity；
(3) ratio of immunochromatography time-resolved fluorescence test strips detection line fluorescence intensity and nature controlling line fluorescence intensity is obtained through fitting
Value with aflatoxin B1, fumonisin B1, ochratoxin A, zearalenone relation curve.
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| CN110873794A (en) * | 2018-08-29 | 2020-03-10 | 中国农业大学 | High-flux ultrasensitive double-label time-resolved immunochromatographic test strip and application thereof |
| CN114660293B (en) * | 2020-12-22 | 2023-06-27 | 中国农业大学 | SERS immunoassay kit for detecting mycotoxin exposure markers in blood plasma or urine |
| CN114660294B (en) * | 2020-12-23 | 2023-07-04 | 中国农业大学 | Kit for synchronously detecting six mycotoxins and application thereof |
| CN112684180A (en) * | 2021-01-26 | 2021-04-20 | 无锡精检生物技术有限公司 | Fluorescence immunoassay quantitative POCT detection method for mycotoxin in agricultural products |
| CN113092766A (en) * | 2021-03-30 | 2021-07-09 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Detection kit for various mycotoxins, preparation method, detection method and application thereof |
| CN113552359B (en) * | 2021-07-16 | 2024-05-28 | 江南大学 | Duplex fluorescent immunity quantitative test strip for simultaneously detecting AFB1 and Cd in grains |
| CN113588962B (en) * | 2021-07-28 | 2024-04-30 | 江南大学 | Method for detecting fumonisin B1 based on fluorescent immunity quantitative test strip |
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| CN114814205A (en) * | 2022-04-28 | 2022-07-29 | 江南大学 | Quintuplet fluorescence immunoassay test strip for simultaneously detecting various mycotoxins and heavy metal Cd in grains |
| CN115267176A (en) * | 2022-07-18 | 2022-11-01 | 中国农业科学院油料作物研究所 | Iron-based porous carbon immunochromatographic test strip and application thereof |
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