CN106940373A - 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 application.It includes the example reaction bottle of immunochromatography time-resolved fluorescence test strips and each monoclonal antibody dried frozen aquatic products marked containing europium；Wherein：The fluorescent test paper strip includes PVC substrates, the one side of substrate pastes adsorptive pads, detecting pad and sample pad successively from top to bottom, adjacent each pad is in the overlapping connection in junction, detecting pad is using nitrocellulose filter as base wad, horizontal nature controlling line and four detection lines are set from top to bottom on nitrocellulose filter, the bovine serum albumin(BSA) conjugate of each toxin is coated with respectively, and the fumonisin B1 monoclonal antibodies are to be secreted to produce by the hybridoma cell strain Fm7A11 that deposit number is CCTCC NO.C201636.Available for aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 contents synchronous detection, with it is simple to operate, quick, sensitivity is high the characteristics of.
Description
Technical field
The present invention relates to mycotoxin immunochromatography time-resolved fluorescence kit, and in particular to a kind of synchronous detection is yellow bent
Mould toxin B1, ochratoxin A, zearalenone, the immunochromatography time-resolved fluorescence examination of fumonisin B1 composite pollutions
Agent box, preparation method and applications.
Background technology
Mycotoxin is a class toxicity very strong chemical substance, is having of being produced under suitable environmental condition of filamentous fungi
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 be by the poisonous secondary metabolite of a class of the generations such as Aspergillus flavus and aspergillus parasiticus,
It is very harmful to human and animal with high toxicity and height be carcinogenic, the characteristic of teratogenesis, mutagenicity.Ochratoxin A is main
Aspergillus and Penicillium some toxigenic bacteriums strain are resulted from, its toxicity is occupied first of 7 kinds of ochratoxins (7 kinds of A, B, C, D etc.),
Distributed in nature is most extensive, is one of mycotoxin of serious harm human health to the pollution of agricultural product also most serious；Reddish brown song
Mould toxin A relatively stablizes and is not easily decomposed in food, most common in the cereal that goes mouldy, feed etc., has been proved to mutagenesis
Property, immunotoxicity, can cause immunosupress, and with carcinogenic, teratogenesis, mutagenesis.Zearalenone is by a variety of sickles
A kind of mycotoxin for on-steroidal structure that knife bacterium produces, is exceeding standard rate and detection water in China's feedstuff and complete feed
One of flat highest mycotoxin, to the toxic effect of reproductive capability, also with 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, with strong neurotoxicity, hepatotoxicity wind agitation and carcinogenic teratogenesis etc., its toxicity mechanism mainly comes
Come from fumonisin B1 and destruction and oxidative stress are synthesized to sphingolipid.And the planting patterns of China's smallholder's decentralized causes
Mycotoxin incidence is high in agricultural product, and the possibility of same agricultural product mycotoxin composite pollution is big, therefore in the urgent need to
The detection technique of mycotoxin composite pollution can quick, be synchronously detected, it is dirty to mycotoxin mixing in grain and feed to realize
Synchronous, the fast slowdown monitoring of dye.
The existing detection method to these mycotoxins mainly includes TLC, precision instrument analytic approach and immunology
Analytic approach.TLC detection mycotoxin is the pathogenic eukaryotes method that earliest research is set up, and is mainly used in fungi poison
Plain qualitative analysis, it is difficult to which quantitative, detection reagent consumption is big, cumbersome and because other component serious interferences cause accuracy
Difference, it is impossible to accurate quantitative analysis, it is larger to experimenter and ambient contamination harm.Precision instrument analytic approach includes high-efficient liquid phase color
The method such as spectrometry, liquid chromatogram and mass spectrum and tandem mass spectrum combination method, has the advantages that accurate, sensitive, but sample pre-treatments mistake
Journey is complicated, and detection time is long, used expensive equipment, requires high to experimental situation and testing staff, it is difficult to realize quick inspection
Survey.Immunoassay method overcomes the shortcoming of TLC and instrumental method, due to its high specificity, sensitivity height, sample
Product pre-treatment is simple, low cost, it is small to the contamination hazard of experimenter and surrounding environment, suitable for live batch detection the advantages of
Fast development is obtained in recent years.Immuno-chromatographic test paper strip based on colloidal gold labeled monoclonal antibody and antigentic specificity association reaction by
It is visible in its testing result naked eyes, it is not necessary to which that large-scale instrument and equipment, testing cost is low, and analysis time is short, in recent years in fungi poison
It is widely applied on the qualitative, online of the minimal residue thing such as element, quick detection.But it is due to that sensitivity is low, can not carries out
Quantitative detection, can not meet the demand of Quantitative detection at the scene in quick detection.And the plantation of China's smallholder's decentralized
Mode causes mycotoxin incidence in agricultural product high, and the risk of composite pollution is bigger, therefore in the urgent need to energy is quick, same
The detection technique of a variety of mycotoxin composite pollutions of step detection, with realize in grain and feed mycotoxin composite pollution it is same
Step, fast slowdown monitoring.
The content of the invention
Problem to be solved by this invention, which is to provide one kind, can synchronously detect aflatoxin B1, ochratoxin A, corn
Zeranol, the immunochromatography time-resolved fluorescence kit of fumonisin B1 composite pollutions, 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 contents, with it is simple to operate, quick, sensitivity is high the characteristics of.
In order to solve the above technical problems, the technical solution adopted in the present invention is：
Synchronous detection aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 composite pollutions 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 aflatoxin B1 monoclonal antibody of mark, the ochratoxin A monoclonal antibody of europium mark, the Gibberella zeae alkene of europium mark
The example reaction bottle of ketone monoclonal antibody, the fumonisin B1 monoclonal antibody dried frozen aquatic productses of europium mark；Wherein：Described immune layer
Analysing time-resolved fluorescence test strips includes PVC substrates, and the one side of substrate pastes adsorptive pads, detecting pad and sample successively 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 it is lower horizontal nature controlling line and four detection lines are set, the nature controlling line is 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 with line respectively
Zearlenone-bovine serum albumin(BSA) conjugate, fumonisin B1- bovine serum albumin(BSA) conjugates, described fumonisin B1
Monoclonal antibody is to secrete the monoclonal produced by the hybridoma cell strain Fm7A11 that deposit number is CCTCC NO.C201636
Antibody；Described 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.
By such scheme, the monoclonal antibody of the europium mark is to prepare in accordance with the following methods：
The aflatoxin B1 monoclonal antibody of europium mark：By the europium mark after aflatoxin B1 monoclonal antibody and activation
Note reagent is mixed in borate buffer, oscillating reactions, then obtains target product europium mark through centrifugation, redissolution, closing step
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 mark：By the europium mark after ochratoxin A monoclonal antibody and activation
Reagent is mixed in borate buffer, oscillating reactions, then obtains target product europium mark through centrifugation, redissolution, closing step
Ochratoxin A monoclonal antibody；Europium labelled reagent after 1mL activation can be coupled ochratoxin A monoclonal antibody：40μg-
90μg。
The zearalenone monoclonal antibody of europium mark：By the europium mark after zearalenone monoclonal antibody and activation
Note reagent is mixed in borate buffer, oscillating reactions, then obtains target product europium mark 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 antibodies of europium mark：Europium after fumonisin B1 monoclonal antibodies and activation is marked into examination
Agent is mixed in borate buffer, oscillating reactions, and the volt of target product europium mark is then obtained through centrifugation, redissolution, closing step
Horse toxin B1 monoclonal antibodies；Europium labelled reagent after 1mL activation can be coupled fumonisin B1 monoclonal antibodies：30μg-80μg.
By such scheme, described activation method is takes europium labelled reagent, with the 0.2mol/L of pH 8.2 borate buffer
Middle ultrasonic disperse, is then slowly added into Carbodiimide solution, and shaken at room temperature activation centrifuges and removes supernatant, with pH 8.2
0.2mol/L borate buffer redissolves, standby, and described soak time is 15-30min.
The monoclonal antibody of the above-mentioned europium mark prepared is redissolved in containing 1.5% (m/v) trehalose, 2% (m/v) cow's serum
It is standby in the 0.01mol/L pH 7.4 of albumin phosphate buffer, in use, taking the monoclonal of each europium mark of certain consumption
Antibody is put into example reaction bottle, is placed in freeze drier and is freezed, and obtains the monoclonal antibody dried frozen aquatic products of each europium mark, standby
With.
By such scheme, adsorptive pads 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；Examined in the fluorescent test paper strip
The spacing on edge is 5-10mm in the upper detection line and nitrocellulose filter close to nature controlling line of survey pad, 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；Described example reaction bottle is
1-2.5mL cillin bottle.
By such scheme, institute per cm in the detection line of the coating aflatoxin B1-bovine serum albumin(BSA) conjugate
The package amount of the aflatoxin B1 needed-bovine serum albumin(BSA) conjugate is 50-250ng；It is coated with ochratoxin A-ox blood
The package amount of required ochratoxin A-bovine serum albumin(BSA) conjugate per cm in the detection line of pure protein conjugate
For 100-300ng；It is coated with required corn per cm in the detection line of zearalenone-bovine serum albumin(BSA) conjugate red
The package amount of mould ketenes-bovine serum albumin(BSA) conjugate is 100-300ng；It is coated with the coupling of fumonisin B1- bovine serum albumin(BSA)s
The package amount of fumonisin B1- bovine serum albumin(BSA) conjugates required in the detection line of thing is 50-150ng, matter per cm
The package amount of rabbit-anti mouse polyclonal antibody needed for control line is 50-150ng；
The content for the aflatoxin B1 monoclonal antibody dried frozen aquatic products that europium is marked is 0.1-0.3 μ in the example reaction bottle
G, the content of the ochratoxin A monoclonal antibody dried frozen aquatic products of europium mark is 0.1-0.3 μ g, the zearalenone list of europium mark
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 productses of europium mark is
0.1-0.3μg。
By such scheme, described aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 are immunized
Chromatographing time-resolved fluorescence quick testing reagent box also includes sample diluting liquid and sample diluting liquid suction pipe, and described sample diluting liquid is
Volume fraction is the 0.01%-0.30% Tween-20 aqueous solution.
By such scheme, the preparation method of described time-resolved fluorescence test strips is as follows：
(1) blotting paper is cut out into obtain adsorptive pads；
(2) preparation of detecting pad：
By aflatoxin B1-bovine serum albumin(BSA) conjugate, ochratoxin A-bovine serum albumin(BSA) conjugate, corn
Zeranol-bovine serum albumin(BSA) conjugate, fumonisin B1- bovine serum albumin(BSA) conjugates are configured to concentration for 0.10-
0.50mg/mL coating buffer, mode is sprayed with line by it in carrying out being respectively separated coating on nitrocellulose filter, 4 detections are obtained
Line, is then dried 60-120 minutes 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, spraying mode with line laterally wraps it
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 soaked, is taken out, is dried 4-6 hours under the conditions of 37-40 DEG C, obtains sample
Pad, then puts room temperature preservation in drier；
(4) assembling of immunochromatography time-resolved fluorescence test strips：
Adsorptive pads, detecting pad, sample pad are pasted successively from top to bottom in the one side of cardboard, and adjacent each pad is overlapping in junction
Connection, produces fluorescent test paper strip；
By such 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 bag
It is by the coating buffer solution used in liquid, fumonisin B1- bovine serum albumin(BSA) conjugate coating buffers：Contain ox in per 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；
Preparing the coating buffer solution used in rabbit-anti mouse polyclonal antibody coating buffer is：Contain sodium azide in per 10mL
0.002g, sodium chloride 0.08g, disodium hydrogen phosphate 0.029g, potassium chloride 0.002g, potassium dihydrogen phosphate 0.002g；
The confining liquid used in the preparation of the fluorescent test paper strip is：Contain ovalbumin 0.5-2g, sucrose in per 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 detections：After testing sample is obtained into testing sample solution through pre-treatment, sample is added
In reaction bulb, mix, insert time-resolved fluorescence test strips, after 37 DEG C are reacted 10 minutes, entered with time-resolved fluorescence tester
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 obtained and nature controlling line fluorescence intensity is malicious with aspergillus flavus respectively
Plain B1, ochratoxin A, zearalenone, the relation curve of fumonisin B1 concentration, obtain yellow bent in testing sample solution
Mould toxin B1, ochratoxin A, zearalenone, fumonisin B1 content, most produce Huang in testing sample through conversion afterwards
Aspertoxin B1, ochratoxin A, zearalenone, fumonisin B1 content；
By such scheme, described immunochromatography time-resolved fluorescence ELISA test strip 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 liquid；
(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, is mixed, and inserts fluorescent test paper strip, and 37 DEG C are reacted 6-10 minutes, use the time
Resolved fluorometric immunity analysis instrument detects the fluorescence intensity level for obtaining 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 and nature controlling line fluorescence intensity；
(3) immunochromatography time-resolved fluorescence ELISA test strip line fluorescence intensity and nature controlling line fluorescence intensity are obtained through fitting
Ratio (T/C) and aflatoxin B1, fumonisin B1, ochratoxin A, the relation curve of zearalenone.
Beneficial effects of the present invention：
(1) quick, synchronous quantitatively detection aflatoxin B1, ochratoxin A, zearalenone and fumonisin
B1.The immunochromatography time-resolved fluorescence kit that the present invention is provided can be realized in a test strips to aflatoxin B1,
Ochratoxin A, the synchronization of zearalenone and fumonisin B1, Quantitative detection, the antibody used are monoclonal
Antibody, specific good, sensitivity is high, noiseless between the detection of each mycotoxin, simple, quick.
(2) sensitivity is high.The immunochromatography time-resolved fluorescence kit that the present invention is provided is to aspergillus flavus in detection solution
Toxin B1 lowest detection 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, fumonisin B1 lowest detection is limited to 0.2ng/mL, the test limit can meet European Union to food
The limitation requirement of 4 kinds of mycotoxins of this in product.
Brief description of the drawings
Fig. 1 is immunized for the aflatoxin B1 of the invention provided, ochratoxin A, zearalenone, fumonisin B1
Chromatograph the structural representation 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.
Embodiment
The acquisition of the aflatoxin B1 monoclonal antibody of embodiment 1
Aflatoxin B1 monoclonal antibody is by hybridoma cell strain 3G1 point of the deposit number for CCTCC NO.C201014
Generation is secreted, is made in advance for the method reported in ZL201210117614.9 patent with specific reference to grant number, preparation method is：
The BALB/c mouse that the hybridoma cell strain 3G1 injections of acquisition are treated with freund 's incomplete adjuvant in advance, collects mouse
Aflatoxin B1 monoclonal antibody is obtained after ascites, purification process.Wherein, purification process is caprylic acid-ammonium, specific behaviour
As：Mouse ascites are filtered with double-layer filter paper, the ascites after filtering is in 4 DEG C, and 12000r/min is centrifuged in more than 15min, absorption
Clearly, supernatant is mixed with the acetate buffer of 4 times of volumes, be slowly added to while stirring needed for caprylic acid, every milliliter of ascites
Caprylic acid volume is 30-35 μ L, mixed at room temperature 30-60min, 4 DEG C of standing more than 2h, then 4 DEG C, 12000r/min centrifugations
More than 30min, abandons precipitation, after obtained supernatant is filtered with double-layer filter paper, and the molar concentration for adding 1/10 filtrate volume is
0.1mol/L and pH value 7.4 phosphate buffer, adjusted with 2mol/L sodium hydroxide solution the pH value of the mixed liquor to
7.4,4 DEG C of precoolings, are slowly added to ammonium sulfate to the final concentration of 0.277g/mL of ammonium sulfate, 4 DEG C of standing more than 2h, then 4 DEG C,
12000r/min centrifuges more than 30min, abandons supernatant, and the 0.01mol/L phosphate of the former ascites volume 1/10 of gained precipitation is delayed
Fliud flushing is resuspended, loads bag filter, is dialysed with pure water, the protein solution fully dialysed is put into -70 DEG C of refrigerator freezings, then with cold
Freeze vacuum drier to freeze, collect freeze-dried powder, produce the aflatoxin B1 monoclonal antibody purified, antibody is put -20 DEG C
It is standby in refrigerator；
Described acetate buffer is 0.29g sodium acetates, and 0.141mL acetic acid, which adds water, to be settled to obtained by 100mL；Described
0.1mol/L phosphate buffer is 0.8g sodium chloride, 0.29g disodium hydrogen phosphates, 0.02g potassium chloride, biphosphate
Potassium 0.02g, adds water and is settled to obtained by 100mL；
The acquisition of the ochratoxin A monoclonal antibody of embodiment 2；
Ochratoxin A monoclonal antibody is by hybridoma cell strain 1H2 point of the deposit number for CCTCC NO.C201329
Generation is secreted, is made in advance with specific reference to the method reported in the patent of Application No. 201310115921.8, preparation method is：Will
Hybridoma cell strain 1H2 is expelled to the belly of the BALB/c mouse treated in advance with freund 's incomplete adjuvant, collects the mouse
Ascites, purifying produce ochratoxin A monoclonal antibody.Described purification process is caprylic acid-ammonium, specific steps
For：Mouse ascites are filtered with double-layer filter paper, 4 DEG C, 12000r/min centrifugation more than 15min draw supernatant, by gained ascites supernatant
Mixed with the acetate buffer of 4 times of volumes, caprylic acid is slowly added under stirring, the 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 more than 2h, then 4 DEG C, 12000r/min centrifugation more than 30min abandon heavy
Form sediment, after obtained supernatant is filtered with double-layer filter paper, the molar concentration for adding 1/10 filtrate volume is 0.1mol/L and pH value
For 7.4 phosphate buffer, the pH value of the mixed liquor is adjusted with 2mol/L sodium hydroxide solution to 7.4,4 DEG C of precoolings, is delayed
The slow ammonium sulfate that adds is to the final concentration of 0.277g/mL of ammonium sulfate, 4 DEG C of standing more than 2h, then 4 DEG C, 12000r/min centrifugations
More than 30min, abandons supernatant, by the 0.01mol/L of the former ascites volume 1/10 of gained precipitation, the phosphate-buffered that pH value is 7.4
Liquid is resuspended, loads bag filter, is dialysed with pure water, and the protein solution fully dialysed is put into -70 DEG C of refrigerator freezings, afterwards with freezing
Drying machine is freezed, and is collected freeze-dried powder, is produced the ochratoxin A monoclonal antibody purified, antibody is put into standby in -20 DEG C of refrigerators
With；
Described acetate buffer is 0.29g sodium acetates, and 0.141mL acetic acid adds water obtained by constant volume to 100mL；Described
0.01mol/L phosphate buffer is 0.8g sodium chloride, 0.29g disodium hydrogen phosphates, 0.02g potassium chloride, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.02g, adds water obtained by constant volume to 100mL；Described 0.1mol/L phosphate buffer is 8g sodium chloride, 2.9g 12
Water disodium hydrogen phosphate, 0.2g potassium chloride, potassium dihydrogen phosphate 0.2g adds water obtained by constant volume to 100mL；
The acquisition of the zearalenone monoclonal antibody of embodiment 3
Zearalenone monoclonal antibody is by hybridoma cell strain 2D3 point of the deposit number for CCTCC NO.C201328
Generation is secreted, is made in advance with specific reference to the method reported in the patent of Application No. 201310115825.3, preparation method is：Will
Hybridoma cell strain 2D3 is expelled to the belly of the BALB/c mouse treated in advance with freund 's incomplete adjuvant, collects the mouse
Ascites, purifying produce zearalenone monoclonal antibody；
Described purification process is caprylic acid-ammonium, is concretely comprised the following steps：With double-layer filter paper filter mouse ascites, 4 DEG C,
12000r/min centrifuges more than 15min, draws supernatant, gained ascites supernatant is mixed with the acetate buffer of 4 times of volumes, stirred
Mix down and be slowly added to caprylic acid, the caprylic acid volume needed for every milliliter of ascites is 30-35 μ L, mixed at room temperature 30-60min, and 4 DEG C quiet
More than 2h is put, then 4 DEG C, 12000r/min centrifugation more than 30min abandon precipitation, obtained supernatant is filtered with double-layer filter paper
Afterwards, the molar concentration for adding 1/10 filtrate volume is the phosphate buffer that 0.1mol/L, pH value are 7.4, with 2mol/L hydrogen
Sodium hydroxide solution adjusts the pH value of the mixed liquor to 7.4,4 DEG C of precoolings, is slowly added to ammonium sulfate final concentration of to ammonium sulfate
0.277g/mL, 4 DEG C of standing more than 2h, then 4 DEG C, 12000r/min centrifugation more than 30min abandon supernatant, and gained precipitation is former
The 0.01mol/L of ascites volume 1/10, pH value are resuspended for 7.4 phosphate buffer, load bag filter, are dialysed with pure water, will
The protein solution fully dialysed puts -70 DEG C of refrigerator freezings, lyophilized with freeze drier afterwards, collects freeze-dried powder, produces purifying
Good zearalenone monoclonal antibody, puts standby in -20 DEG C of refrigerators by antibody；
Described acetate buffer is 0.29g sodium acetates, and 0.141mL acetic acid adds water obtained by constant volume to 100mL；Described
0.01mol/L phosphate buffer is 0.8g sodium chloride, 0.29g disodium hydrogen phosphates, 0.02g potassium chloride, di(2-ethylhexyl)phosphate
Hydrogen potassium 0.02g, adds water obtained by constant volume to 100mL；Described 0.1mol/L phosphate buffer is 8g sodium chloride, 2.9g 12
Water disodium hydrogen phosphate, 0.2g potassium chloride, potassium dihydrogen phosphate 0.2g adds water obtained by constant volume to 100mL；
The acquisition of the fumonisin B1 monoclonal antibodies of embodiment 4
Hybridoma cell strain Fm7A11 screening
1. antigen is synthesized and animal immune
The commercially available fumonisin B of purchase1Standard items carry out comlete antigen synthesis, and specific synthesis step is as follows:By 2mg FB1
Standard items powder and 2mg EDC are dissolved in 500 μ L 0.01mol/LPBS solution respectively, obtain EDC solution and FB1Solution, will
The FB dissolved is added dropwise in 4mg/mL (solution is 0.01mol/LPBS) EDC solution1In solution, 10 are gently mixed at room temperature
Minute.5mg/mL (solution is 0.01mol/LPBS) BSA solution is added dropwise in above-mentioned mixed liquor, reaction is stirred at room temperature
4 hours.Dialysis 3 days.Conventional UV scanning method identification is finally carried out, qualification result shows FB1- BSA comlete antigens are 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 fumonisin comlete antigen is emulsified with isometric Freund's complete adjuvant, in the subcutaneous multi-point injection of mouse nape part.
It is immune to after 3 weeks and carries out for the second time, is emulsified using incomplete Freund's adjuvant with isometric fumonisin comlete antigen, in mouse
The subcutaneous multi-point injection of nape part.Third time with the 4th time it is immune respectively with last immunization interval two weeks, immunization wayses 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 mice serum sensitivity, selection is determined with indirect competitive ELISA method
The corresponding mouse of the of a relatively high serum of potency, sensitivity carries out last time booster immunization, and immunizing dose is 2 measured before
Times.
2. cell fusion
In after last time booster immunization 3 days, use 50% (percetage by weight) polyethylene glycol i.e. PEG (molecular weight for
1450) make fusion agent, cell fusion, specific steps are carried out according to a conventional method：Neck is taken off under aseptic condition and puts to death mouse to be fused, point
From splenocyte, with mouse source myeloma cell SP2/0 with 5：1 number washes mixing with RPMI-1640 basic culture solutions thin than mixing
Born of the same parents, 1200rpm centrifuges 5min.Supernatant discarded, is drained, and adds 1mLPEG, is merged 1 minute, is slowly added to RPMI-1640 bases
Nutrient solution, centrifugation abandons supernatant, precipitation is fused cell, resuspended with 20mL complete mediums, and the cell hanged is added to
It is added in 80mL semisolid culturemediums, after mixing in 6 porocyte culture plates, 2mL/ holes, is placed in 37 DEG C of CO2gas incubator trainings
Support.
The described cell culture complete medium containing 1%HAT contains 20% (percentage by volume) hyclone, 75% (volume
Percentage) RPMI-1640 basic culture solutions, 1% (percetage by weight) Glu, 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% (percetage by weight) hypoxanthine-amino butterfly ridge-thymidine be HAT and methyl fibre
Dimension element is purchased from sigma-Aldrich companies.
3. screening and the clone of cell line
After 2-3 weeks after cell fusion, cell colony length to naked eyes it is visible when, will be cloned with micropipettor from culture medium
Choose, be transferred to 96 porocyte culture plates using HAT Liquid Cultures, when cell length to 2/3 bottom hole, draw 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；The positive hole that second step is filtered out using indirect competitive ELISA method to the first step is detected, uses fumonisin B1
Former as competition, (light absorption value is higher to refer to the hole i.e. Positive control wells that competition was 0 originally in selection light absorption value and sensitivity higher hole
Final tested volume it is higher, the higher competition original content also IC referred to when inhibiting rate is 50% of sensitivity50Value is smaller), using limited
Dilution method is subcloned, and is detected after subclone using same two-step method, is so repeated after being subcloned 4-5 times, is obtained
Hybridoma cell strain Fm7A11.
Anti- fumonisin B1Monoclonal antibody hybridoma cell strain Fm7A11 antibody variable sequences are determined
(1) total serum IgE is extracted：Using Tiangeng company total RNA extraction reagent box and to specifications extract can produce hybridization
Tumor cell strain Fm7A11 total serum IgE；
(2) cDNA is synthesized：Total serum IgE using step 1 acquisition is template, and oligo (dT) 15 is primer, according to
SuperScriptTM- 2II reverse transcriptase specification carries out reverse transcription, synthesizes the chains of cDNA first；Primer oligo (dT) 15 by
Invitrogen is bought；
(3) PCR methods clone variable region gene：Drawn according to the conserved positions design of mouse antibody gene sequence in GENBANK
Thing, heavy chain of antibody, chain variable region gene are expanded by masterplate of CDNA.PCR programs are：94℃30s、58℃45s、72℃
1min, expands 30 circulations, last 72 DEG C of extensions 10min.Ago-Gel electricity of the PCR primer Jing Guo 1% (percetage by weight)
After swimming separation, DNA fragmentation is reclaimed 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 is delivered to Shanghai Sani bio tech ltd and is sequenced.The sequence of wherein primer is respectively：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 merger base, M=A/C, R=A/G, S=G/C, W
=A/T, light chain variable district primer is 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 weight chain variable district coding gene sequence, sequence such as SEQ ID NO:1 institute
Show, the weight chain variable district according to coded by the gene order obtained derives the gene order is made up of 126 amino acid, sequence
Row such as SEQ ID NO:Shown in 3.The long 348bp of light chain variable district coding gene sequence, sequence such as SEQ ID NO:Shown in 2, according to
The gene order obtained derives that the light chain variable district coded by the gene order is made up of 116 amino acid, sequence such as SEQ
ID NO:Shown in 4.
5. anti-fumonisin B1Preparation purifying, hypotype and the CHARACTERISTICS IDENTIFICATION of monoclonal antibody
The anti-fumonisin B that embodiment 1 is obtained1Monoclonal antibody hybridoma cell strain Fm7A11 injections use Freund in advance
The treated BALB/c mouse of Freund's incomplete adjuvant, collects the ascites of the mouse, using caprylic acid-ammonium antibody purification, specifically
Operate and be：Mouse ascites are filtered with double-layer filter paper, 4 DEG C, 12000r/min centrifugation more than 15min draw supernatant, by gained ascites
Supernatant is mixed with the acetate buffer of 4 times of volumes, and caprylic acid is slowly added under stirring, the 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 more than 2h.12000r/min, 4 DEG C of centrifugation more than 30min, abandons heavy
Form sediment, after obtained supernatant is filtered with double-layer filter paper, the molar concentration for adding 1/10 filtrate volume is 0.1mol/L and pH is
7.4 phosphate buffer, is adjusted with 2mol/L sodium hydroxide solution and is slowly added in the pH to 7.4 of the mixed liquor, ice bath
Ammonium sulfate is to the final concentration of 0.277g/mL of ammonium sulfate, 4 DEG C of standing more than 2h, then 12000r/min, 4 DEG C of centrifugation 30min with
On, supernatant is abandoned, it is phosphate that 0.01mol/L, pH are 7.4 that gained, which is precipitated with the molar concentration of original ascites volume 1/10 volume,
Buffer solution is resuspended, loads bag filter, is dialysed two days with 0.01mol/LPBS, then uses PB dialysis two days instead, by albumen in bag filter
Solution takes out, centrifugation, collects supernatant, abandons precipitation, be put into after -70 DEG C of pre-freezes be put into freeze dryer it is lyophilized.Freeze-dried powder is collected, is
The anti-fumonisin B purified1Monoclonal antibody；
Described acetate buffer is 0.29g sodium acetates, and 0.141mL acetic acid adds water obtained by constant volume to 100mL；Described
0.01mol/L phosphate buffer is 0.8g sodium chloride, 0.29g disodium hydrogen phosphates, 0.02g potassium chloride, 0.02g phosphorus
Acid dihydride potassium, adds water obtained by constant volume to 100mL；Described 0.1mol/L phosphate buffer is 8g sodium chloride, 2.9g 12
Water disodium hydrogen phosphate, 0.2g potassium chloride, 0.2g potassium dihydrogen phosphates add water obtained by constant volume to 100mL.
The anti-fumonisin B1 monoclonals for identifying hybridoma cell strain Fm7A11 secretions with commercially available hypotype identification kit resist
The hypotype of body is IgG2b.
Mouse ascites are measured with conventional non-competing enzyme linked immunosorbent assay (ELISA) purify obtained antibody titer and 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 determined with conventional indirect competitive ELISA
Fumonisin B1Sensitivity is 0.32ng/mL.To fumonisin B2、B3Cross reacting rate be 4.3% and 12.8%.It is bent with Huang
Mould toxin, zearalenone, T-2 toxin, ochratoxin, the cross reacting rate of vomitoxin are respectively less than 0.1%.
Embodiment 5
200 μ l europium mark fluorescent microballoons are taken, in the borate buffer for adding 1mL 0.2mol/L pH8.2, at 300w ultrasounds
Reason 40 seconds, is then slowly added into after 40uL 15mg/mL carbodiimide, shaken at room temperature 20min, and 17000g is centrifuged 15 minutes, is received
Collection precipitation, is redissolved with 0.2mol/L pH 8.2 borate buffer, the fluorescent microsphere of activation is redissolved in 5mL 0.01mol/L
In pH8.2 borate buffer, 1mg/ml antibody (aflatoxin B1 monoclonal antibody 35ul, ochratoxin A Dan Ke are 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 centrifugation 10min collect precipitation, redissolve in containing 1.5% (m/v) trehalose, 2% (m/v) bovine serum albumin(BSA)
In 0.2mol/L pH8.2 borate buffer, the mycotoxin antibody of europium mark is produced, 4 DEG C is placed in and saves backup.
Synchronous detection aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 composite pollutions are synchronous
The immunochromatography time-resolved fluorescence kit of detection and its application
Synchronously detection is immune for aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 composite pollutions
Time-resolved fluorescence quick testing reagent box is chromatographed, it includes immunochromatography time-resolved fluorescence test strips, the Huang song marked containing europium
Mould toxin B1 monoclonal antibodies dried frozen aquatic products, the ochratoxin A monoclonal antibody dried frozen aquatic products of europium mark, the Gibberella zeae of europium mark
Ketenes monoclonal antibody dried frozen aquatic products, the example reaction bottle of the fumonisin B1 monoclonal antibody dried frozen aquatic productses of europium mark, sample dilution
Liquid and sample diluting liquid suction pipe,
Described fluorescent test paper strip includes PVC substrates, and the one side of PVC substrates pastes adsorptive pads, detection successively from top to bottom
Pad and sample pad, adjacent each pad is in the overlapping connection in junction, and overlapping length is 1mm；
(1) preparation of adsorptive pads
Blotting paper is cut out into growth 16mm, wide 4mm specification produces adsorptive pads；
(2) preparation of detecting pad
The coating of detection line：
Aflatoxin B1-bovine serum albumin(BSA) conjugate is used to the solution for being coated with buffer into 0.25mg/mL, in
Away from the position on nitrocellulose filter along 10mm, it is coated in line spray mode on nitrocellulose filter and is obtained 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)s conjugate (OTA-BSA) is with coating buffer of the buffer into 0.35mg/mL is coated with, in away from detection line I
4mm position, is coated on nitrocellulose filter with line spray mode needed for obtaining on detection line II, detection line II per cm
The package amount of ochratoxin A-bovine serum albumin(BSA) conjugate is 160ng；By zearalenone-bovine serum albumin(BSA) coupling
Thing, into 0.4mg/mL coating buffer, in the position away from detection line II 4mm, is wrapped with coating buffer with line spray mode
By zearalenone-bovine serum albumin needed for being obtained on nitrocellulose filter on detection line III, detection line III per cm
The package amount of white conjugate is 200ng；By fumonisin B1- bovine serum albumin(BSA)s conjugate with coating buffer into
0.25mg/mL coating buffer, in the position away from detection line III 4mm, is coated on nitrocellulose filter with line spray mode
The package amount of fumonisin B1- bovine serum albumin(BSA) conjugates is needed for obtaining on detection line IV, detection line IV per cm
150ng；Then dried 120 minutes 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 solution；In away from detection line I
Its transverse direction, is coated on nitrocellulose filter, needed for obtaining nature controlling line, nature controlling line per cm by 5mm position with line spray mode
The package amount of rabbit-anti mouse polyclonal antibody is 90ng, is then dried 120 minutes under the conditions of 40 DEG C；
Described coating buffer solution is：By 1mg rabbit-anti mouse polyclonal antibodies, 0.002g sodium azide, 0.08g sodium chloride,
0.029g disodium hydrogen phosphates, 0.002g potassium chloride, 0.002g potassium dihydrogen phosphates add water and are settled to obtained by 10mL；
Described nitrocellulose filter long 25mm, wide 4mm.
(3) preparation of sample pad：
Glass fibre membrane is cut out into growth 12mm, wide 4mm specification is put into confining liquid and soaked, and takes out, in 40 DEG C of conditions
Lower drying 4 hours, obtains sample pad, then puts room temperature preservation in drier；
Described confining liquid is 1g oralbumins, 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 phosphates add water and are settled to obtained by 100mL；
(4) assembling of fluorescent test paper strip：
Adsorptive pads, detecting pad and sample pad are pasted successively from top to bottom in the one side of PVC substrates, and adjacent each pad is in junction
Overlapping connection, overlapping length is 1mm, is produced；See Fig. 1.
The acquisition of described example reaction bottle：By the above-mentioned redissolution prepared in containing 1.5% (m/v) trehalose, 2% (m/
V) the aflatoxin B1 monoclonal of the europium mark in the 0.01mol/L pH 7.4 of bovine serum albumin(BSA) phosphate buffer resists
Body, the ochratoxin A monoclonal antibody of europium mark, the zearalenone monoclonal antibody of europium mark, the volt horse of europium mark
Toxin B1 monoclonal antibodies, respectively take it is a certain amount of be put into example reaction bottle in, be placed in freeze drier freeze, obtain europium mark
Monoclonal antibody dried frozen aquatic products, it is standby.
The content for the aspergillus flavus resisting toxin B1 monoclonal antibody dried frozen aquatic productses that europium is marked is 0.25 μ g in the example reaction bottle,
The content of the anti-ochratoxin A monoclonal antibody dried frozen aquatic products of europium mark is 0.3 μ g, the anti-zearalenone Dan Ke of europium mark
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 productses of europium mark is 0.2 μ g.
The aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 composite pollutions are synchronously detected
Immunochromatography time-resolved fluorescence detection kit is in sample aflatoxin B1, ochratoxin A, zearalenone, volt
Application in horse toxin B1 detections：
Learn from else's experience confirmation negative Feed Sample 20g add 100mL 70% (volume fraction) methanol solution, 2 points of homogeneous
Clock, stands, is filtered with double-layer filter paper, collect filtrate 1mL, adds 5mL sample diluting liquids dilution filtrate, mixes, obtain blank base
Matter solution；
With vehicle solution configuration aflatoxin B1, ochratoxin A, zearalenone, B1 points of fumonisin
The aflatoxin B1 of following concentration gradient, ochratoxin A, zearalenone, fumonisin B1 mixing marks 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 liquid is repeated 5 times, with poison more
Plain test strip is detected：The μ L of above-mentioned standard product solution 150 are added in example reaction bottle, mixed, immunochromatography is inserted
Time-resolved fluorescence test strips, after 37 DEG C are reacted 10 minutes, sample pad residual liquid are blotted with blotting paper, time resolution is used immediately
Fluorescence immunity analyzer detects (excitation wavelength：365nm, determines wavelength：615nm), the fluorescence signal intensity of detection zone is read
Value, calculates 5 repetition average values.With fluorescence signal intensity value/C lines of standard series concentration value natural logrithm (Lnc) and T lines
Fluorescence signal intensity value (T/C) draws standard curve, and calibration curve formula such as Y=a*lnc+b, the standard of 5 kinds of detection objects is bent
Line parameter is as shown in the table：
| a | b | R2 | Test 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 |
Feed Sample 20g to be checked is taken to add 100mL 70% (volume fraction) methanol solution, homogeneous 2 minutes stands, used
Double-layer filter paper is filtered, and collects filtrate 1mL, is added 5mL sample diluting liquids dilution filtrate, is mixed；Take Feed Sample detection liquid to be checked
150 μ L are added in example reaction bottle, are mixed, and insert immunochromatography time-resolved fluorescence test strips, after 37 DEG C are reacted 10 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
The ratio (T/C) of detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity, then substitutes into it respectively
State the ratio (T/C) of obtained fluorescent test paper strip detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity with
Aflatoxin B1 concentration, ochratoxin A concentration, zearalenone concentration, the relation curve of fumonisin B1 concentration, are obtained
Aflatoxin B1 content in the sample is that 5.4 μ g/kg, ochratoxin content are 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 adsorptive pads, 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；Examined in the fluorescent test paper strip
The spacing on edge is 10mm in the upper detection line and nitrocellulose filter close to nature controlling line of survey pad, between every adjacent two detection lines
Spacing is 3.5mm.Required Huang per cm in the detection line of the coating aflatoxin B1-bovine serum albumin(BSA) conjugate
The package amount of aspertoxin B1- bovine serum albumin(BSA) conjugates is 125ng；It is coated with ochratoxin A-bovine serum albumin(BSA) coupling
The package amount of required ochratoxin A-bovine serum albumin(BSA) conjugate per cm is 200ng in the detection line of thing；Coating
Required zearalenone-ox blood per cm is pure in the detection line of zearalenone-bovine serum albumin(BSA) conjugate
The package amount of protein conjugate is 250ng；It is coated with required in the detection line of fumonisin B1- bovine serum albumin(BSA) conjugates
The package amount of fumonisin B1- bovine serum albumin(BSA) conjugates is 150ng, the 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 productses that europium is marked 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 mark is 0.25 μ g, and the anti-corn of europium mark 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 productses of europium mark contain
Measure as 0.23 μ g.The aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 composite pollutions are synchronously examined
Survey immunochromatography time-resolved fluorescence detection kit sample aflatoxin B1, ochratoxin A, zearalenone,
Application in fumonisin B1 detections：
(aflatoxin B1 content is 3.5 μ g/ to the corn sample containing many toxin that high performance liquid chromatography of learning from else's experience confirms
Kg, ochratoxin content are that 2.6 μ g/kg, zearalenone content are that 42 μ g/kg, fumonisin B1 contents are 8.1 μ g/
Kg), 25g is taken to add 100mL 70% (volume fraction) methanol solution, homogeneous 2 minutes stands, filtered with double-layer filter paper, received
Collect filtrate 1mL, add 4mL sample diluting liquids dilution filtrate, mix；Corn sample detection liquid 150 μ L to be checked are taken to add sample anti-
Answer in bottle, mix, insert immunochromatography time-resolved fluorescence test strips, after 37 DEG C are reacted 10 minutes, sample is blotted with blotting paper
Residual liquid is padded, each 4 detection line time resolutions of fluorescent test paper strip are obtained with time-resolved fluorescence immunoassay instrument detection immediately
The ratio (T/C) of fluorescence intensity and nature controlling line time-resolved fluorescence intensity, then substitutes into it fluorescence examination obtained above respectively
The ratio (T/C) and aflatoxin B1 of paper slip detection line time-resolved fluorescence intensity and nature controlling line time-resolved fluorescence intensity are dense
Degree, ochratoxin A concentration, zearalenone concentration, the relation curve of fumonisin B1 concentration, the Huang obtained in the sample are bent
Mould toxin B1 contents are that 3.2 μ g/kg, ochratoxin content are that 2.5 μ g/kg, zearalenone content are 45 μ g/kg, volt
Horse toxin B1 contents are 8.5 μ g/kg.
Sequence table
The ＞ Inst. of Oil Crops, Chinese Academy of Agriculture of ＜ 110
The ＞ of ＜ 120 synchronously detect the immunochromatography time-resolved fluorescence reagent of four kinds of mycotoxin composite pollutions such as fumonisin B1
Box and its application
The ＞ 4 of ＜ 160
The ＞ 1 of ＜ 210
The ＞ 379bp of ＜ 211
The ＞ DNA of ＜ 212
The ＞ mouse of ＜ 213
The ＞ 1 of ＜ 400
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
The ＞ 1 of ＜ 210
The ＞ 348bp of ＜ 211
The ＞ DNA of ＜ 212
The ＞ mouse of ＜ 213
The ＞ 2 of ＜ 400
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
The ＞ 1 of ＜ 210
The ＞ 126 of ＜ 211
The ＞ PRT of ＜ 212
The ＞ mouse of ＜ 213
The ＞ 3 of ＜ 400
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
The ＞ 1 of ＜ 210
The ＞ 116 of ＜ 211
The ＞ PRT of ＜ 212
The ＞ mouse of ＜ 213
The ＞ 4 of ＜ 400
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 pollutions it is immune
Chromatograph time-resolved fluorescence kit, it is characterised in that：It includes immunochromatography time-resolved fluorescence test strips and contains europium mark
The aflatoxin B1 monoclonal antibody of note, the ochratoxin A monoclonal antibody of europium mark, the zearalenone of europium mark
The example reaction bottle of monoclonal antibody, the fumonisin B1 monoclonal antibody dried frozen aquatic productses of europium mark；Wherein：Described immunochromatography
Time-resolved fluorescence test strips include PVC substrates, and the one side of substrate pastes adsorptive pads, detecting pad and sample successively 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 it is lower horizontal nature controlling line and four detection lines are set, the nature controlling line is 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 with line respectively
Zearlenone-bovine serum albumin(BSA) conjugate, fumonisin B1- bovine serum albumin(BSA) conjugates, described fumonisin B1
Monoclonal antibody is to secrete the monoclonal produced by the hybridoma cell strain Fm7A11 that deposit number is CCTCC NO.C201636
Antibody；Described 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.
2. immunochromatography time-resolved fluorescence kit according to claim 1, it is characterised in that：The list of the europium mark
Clonal antibody is to prepare in accordance with the following methods：
The aflatoxin B1 monoclonal antibody of europium mark：Europium after aflatoxin B1 monoclonal antibody and activation is marked into examination
Agent is mixed in borate buffer, oscillating reactions, and the Huang of target product europium mark is then obtained through centrifugation, redissolution, closing step
Aspertoxin B1 monoclonal antibodies；Europium labelled reagent after 1mL activation can be coupled aflatoxin B1 monoclonal antibody：30μg-
80μg。
The ochratoxin A monoclonal antibody of europium mark：By the europium labelled reagent after ochratoxin A monoclonal antibody and activation
Mixing, oscillating reactions in borate buffer, then obtain the reddish brown song of target product europium mark through centrifugation, redissolution, closing step
Mould toxin A monoclonal antibodies；Europium labelled reagent after 1mL activation can be coupled ochratoxin A monoclonal antibody：40μg-90μg.
The zearalenone monoclonal antibody of europium mark：Europium after zearalenone monoclonal antibody and activation is marked into examination
Agent is mixed in borate buffer, oscillating reactions, and the jade of target product europium mark is then obtained through centrifugation, redissolution, closing step
Zearlenone monoclonal antibody；Europium labelled reagent after 1mL activation can be coupled zearalenone monoclonal antibody：30μg-
90μg。
The fumonisin B1 monoclonal antibodies of europium mark：Europium labelled reagent after fumonisin B1 monoclonal antibodies and activation is existed
Mixed in borate buffer, oscillating reactions, the volt horse poison of target product europium mark is then obtained through centrifugation, redissolution, closing step
Plain B1 monoclonal antibodies；Europium labelled reagent after 1mL activation can be coupled fumonisin B1 monoclonal antibodies：30μg-80μg.
3. immunochromatography time-resolved fluorescence kit according to claim 1, it is characterised in that：Described activation method
Ultrasonic disperse in borate buffer to take the 0.2mol/L of europium labelled reagent pH 8.2, is then slowly added into carbodiimide molten
Supernatant is removed in liquid, shaken at room temperature activation, centrifugation, is redissolved with the 0.2mol/L of pH 8.2 borate buffer, standby, described work
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 adsorptive pads 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；Detection line and nitric acid in the fluorescent test paper strip on detecting pad close to nature controlling line is fine
The spacing on edge is 5-10mm on the plain film of dimension, 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 cillin bottle that described example reaction bottle is 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) conjugates
The package amount for joining thing is 50-250ng；It is coated with per cm required in the detection line of ochratoxin A-bovine serum albumin(BSA) conjugate
The package amount of the ochratoxin A wanted-bovine serum albumin(BSA) conjugate is 100-300ng；It is coated with zearalenone-cow's serum
The package amount of required zearalenone-bovine serum albumin(BSA) conjugate per cm is in the detection line of albumin conjugate
100-300ng；It is coated with the fumonisin B1- ox bloods required in the detection line of fumonisin B1- bovine serum albumin(BSA) conjugates
The package amount of pure protein conjugate is 50-150ng, and the package amount of the 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 mark is 0.1-0.3 μ g, the ochratoxin A list of europium mark
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 mark is
0.1-0.3 μ g, the content of the fumonisin B1 monoclonal antibody dried frozen aquatic productses of europium mark is 0.1-0.3 μ g.
7. immunochromatography time-resolved fluorescence kit according to claim 1, it is characterised in that：Described aspergillus flavus poison
Plain B1, ochratoxin A, zearalenone, fumonisin B1 immunochromatography time-resolved fluorescence quick testing reagent boxes also include
Sample diluting liquid and sample diluting liquid suction pipe, described sample diluting liquid are the Tween-20 that volume fraction is 0.01%-0.30%
The aqueous solution.
8. immunochromatography time-resolved fluorescence kit according to claim 1, it is characterised in that：Described time resolution
The preparation method of fluorescent test paper strip is as follows：
(1) blotting paper is cut out into obtain adsorptive pads；
(2) preparation of detecting pad：
By aflatoxin B1-bovine serum albumin(BSA) conjugate, ochratoxin A-bovine serum albumin(BSA) conjugate, Gibberella zeae
Ketenes-bovine serum albumin(BSA) conjugate, fumonisin B1- bovine serum albumin(BSA) conjugates are configured to concentration for 0.10-0.50mg/
ML coating buffer, mode is sprayed with line by it in carrying out being respectively separated coating on nitrocellulose filter, 4 detection lines are obtained, then
Dried 60-120 minutes 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, its transverse direction is coated in line spray mode
On nitrocellulose filter, nature controlling line is obtained, is 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 soaked, is taken out, is dried 4-6 hours under the conditions of 37-40 DEG C, obtains sample pad, so
Room temperature preservation in rearmounted drier；
(4) assembling of immunochromatography time-resolved fluorescence test strips：
Adsorptive pads, detecting pad, sample pad are pasted successively from top to bottom in the one side of cardboard, and adjacent each pad is in the overlapping company in junction
Connect, produce fluorescent test paper strip.
9. the immunochromatography time-resolved fluorescence quick testing reagent box described in claim 1 is in aflatoxin B1, ochratoxin
Application in A, zearalenone, fumonisin B1 content detections, it is characterised in that：Testing sample is treated through pre-treatment
After survey sample solution, add in example reaction bottle, mix, insert time-resolved fluorescence test strips, after 37 DEG C are reacted 10 minutes, use
Time-resolved fluorescence tester is detected, obtains detection line (T) fluorescence intensity and nature controlling line (C) fluorescence on fluorescent test paper strip strong
The ratio of degree；Ratio based on the fluorescent test paper strip detection line time-resolved fluorescence intensity being obtained ahead of time with nature controlling line fluorescence intensity
(T/C) respectively with aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 concentration relation curve, obtain
Aflatoxin B1, ochratoxin A, zearalenone, fumonisin B1 content in testing sample solution are obtained, is most passed through afterwards
Conversion produces aflatoxin B1 in testing sample, ochratoxin A, zearalenone, fumonisin B1 content.
10. application according to claim 9, it is characterised in that：By such scheme, described immunochromatography time resolution is glimmering
The ratio (T/C) of light ELISA test strip line fluorescence intensity and nature controlling line fluorescence intensity is malicious with aflatoxin B1, Aspergillus ochraceus respectively
Plain A, zearalenone, the relation curve of fumonisin B1 concentration are obtained using following methods：
(1) aflatoxin B1, fumonisin B1, ochratoxin A and the Gibberella zeae alkene for obtaining graded series concentration are prepared
Ketone standard liquid；
(2) by the aflatoxin B1 of appropriate above-mentioned each gradient, ochratoxin A, zearalenone, fumonisin B1 standards
Product solution is added separately in example reaction bottle, is mixed, and inserts fluorescent test paper strip, and 37 DEG C are reacted 6-10 minutes, use time resolution
Fluorescence immunity analyzer detects the fluorescence intensity level for obtaining detection line (T) and nature controlling line (C) on each fluorescent test paper strip, is derived from
The ratio (T/C) of each fluorescent test paper strip detection line time-resolved fluorescence intensity and nature controlling line fluorescence intensity；
(3) ratio of immunochromatography time-resolved fluorescence ELISA test strip line fluorescence intensity and nature controlling line fluorescence intensity is obtained through fitting
It is worth (T/C) and aflatoxin B1, fumonisin B1, ochratoxin A, the relation curve of zearalenone.
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| CN113552359A (en) * | 2021-07-16 | 2021-10-26 | 江南大学 | Duplex fluoroimmunoassay quantitative test strip for simultaneously detecting AFB1 and Cd in grains |
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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 |
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| CN114660294A (en) * | 2020-12-23 | 2022-06-24 | 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 |
| CN113552359A (en) * | 2021-07-16 | 2021-10-26 | 江南大学 | Duplex fluoroimmunoassay quantitative test strip for simultaneously detecting AFB1 and Cd in grains |
| CN113588962A (en) * | 2021-07-28 | 2021-11-02 | 江南大学 | Method for detecting fumonisin B1 based on fluorescent immune quantitative test strip |
| CN113588962B (en) * | 2021-07-28 | 2024-04-30 | 江南大学 | Method for detecting fumonisin B1 based on fluorescent immunity quantitative test strip |
| WO2023207166A1 (en) * | 2022-04-28 | 2023-11-02 | 江南大学 | Five-linked fluorescence immunoassay quantitative test strip for simultaneously testing various mycotoxins and heavy metal cd in cereals |
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