CN102590519A - Kit or test strip for detecting aflatoxin - Google Patents

Kit or test strip for detecting aflatoxin Download PDF

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CN102590519A
CN102590519A CN2012100453981A CN201210045398A CN102590519A CN 102590519 A CN102590519 A CN 102590519A CN 2012100453981 A CN2012100453981 A CN 2012100453981A CN 201210045398 A CN201210045398 A CN 201210045398A CN 102590519 A CN102590519 A CN 102590519A
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kit
aflatoxins
monoclonal antibody
sample
solution
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CN102590519B (en
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徐飞
吴小平
何丹婷
王照鹏
李娜
赵宁
李向梅
杨铮
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Beijing Ying Tai gray Testing Technology Co.,Ltd.
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BEIJING WDWK BIOTECHNOLOGY Co Ltd
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Abstract

The invention discloses a kit or a test strip for detecting aflatoxin. The enzyme linked immunosorbent assay kit for detecting aflatoxin comprises a monoclonal antibody secreted by anti-aflatoxin monoclonal antibody hybridoma cell 2A1 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.5779. The kit and the test strip have the advantages of high sensitivity, high accuracy, high precision, low cost, simpleness in operation, short test duration, simpleness in storage and long preservation time, can be used by various institutions, and can be used for quickly detecting massive samples, so that field high-flux quick detection can be realized. The kit and the test strip provided by the invention are applicable to detection of residual amount of aflatoxin in dairy products. The antibody, the kit, the test strip and the detection method play an important role in detecting aflatoxin.

Description

Detect the kit or the test strips of aflatoxins
Technical field
The present invention relates to detect the kit or the test strips of aflatoxins.
Background technology
Along with human living standard's improvement, food security becomes one of problem that people pay close attention to the most on the dining table day by day, and the milk that people can eat every day may contain the aflatoxins M harmful to health 1Mammal is taken in by aflatoxins B 1Behind the feed or food that pollutes, can be generated aflatoxins M by hydroxylation in vivo 1Research shows, aflatoxins M 1Be strong carcinogen.It is 15 μ g/kg that the World Health Organization (WHO) formulates food aflatoxin maximum permissible concentration, and the Federal Government relevant laws stipulate that the content in human consumption's the milk can not surpass 0.5 μ g/kg, and the content in other animal feeds can not 300 μ g/kg.Aflatoxins M in the regulation food in " mycotoxin is limited the quantity of in the GB 2761-2011 food " national standard of China's issue in this year 1Limiting the quantity of in milk and milk products is 0.5 μ g/kg.And European Union member countries' regulation is strict more, M in former milk, thermal treatment milk and the processing dairy products 1Limiting the quantity of is 0.05 μ g/kg, The babyM in the food (comprising that the infant suckles) 1Limiting the quantity of is 0.025 μ g/kg.Therefore, set up special, responsive aflatoxins M 1Detection method be the task of top priority.
Research mainly contains thin-layered chromatography (TLC), high performance liquid chromatography (HPLC), immune affinity column method and ELISA etc. both at home and abroad at present.The specificity of TLC method is relatively poor, and sensitivity is also relatively poor relatively, and required standard items concentration is higher, and potential contaminative is higher.The required instrument and equipment investment of HPLC method is big, and operative technique requires height, and decontaminating column consumption is more, and it is higher to detect cost, and the immune affinity column specificity is better, but needs to combine HPLC appearance and fluorophotometer ability detection by quantitative, and detection technique requirement and cost are higher.The ELISA method Comparatively speaking, and is easy and simple to handle, quick, pollution is less, sensitivity is also higher, is fit to the general inspection and the screening of sample in batches; The kit of particularly succeeding in developing; Bring great convenience to the testing staff, also provide cost savings simultaneously, meet at present the developing direction of detection range in the world.The immune colloidal gold chromatography technology does not need any instrument and equipment and reagent, be particularly suitable for detection and large tracts of land generaI investigation that grass-roots unit is pressed for time in enormous quantities, so enzyme immunoassay is widely used in Food Safety Analysis.
Summary of the invention
The purpose of this invention is to provide detect the aflatoxins kit or test strips.
The enzyme linked immunological kit of detection aflatoxins provided by the invention comprises the monoclonal antibody that aspergillus flavus resisting toxin monoclone antibody hybridoma 2A1 secretes.
Aspergillus flavus resisting toxin monoclone antibody hybridoma 2A1; Be called for short hybridoma 2A1; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 15th, 2012 and (be called for short CGMCC; The address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5779.
Said kit can be following 1) to 4) in any one:
1) comprises the kit of compound shown in the formula (I) and the conjugate of carrier protein, said monoclonal antibody and enzyme labeling antiantibody; Wherein, said conjugate is as coating antigen;
2) comprise the kit of the enzyme labeling thing of compound shown in the formula (I), said monoclonal antibody and antiantibody; Wherein, said antiantibody is as coating antigen;
3) comprise the enzyme labeling thing of compound shown in the formula (I) and the kit of said monoclonal antibody; Wherein, said monoclonal antibody is as coating antigen;
4) comprise the kit of enzyme labeling thing of conjugate and the said monoclonal antibody of compound shown in the formula (I) and carrier protein; Wherein, said conjugate is as coating antigen.
Figure BDA0000138057080000021
Formula (I).
The conjugate of compound and carrier protein shown in the formula (I) is specifically suc as formula shown in (II).
Figure BDA0000138057080000022
Formula (II).
Said carrier protein can be ovalbumin (OVA) or bovine serum albumin(BSA) (BSA).
In the said conjugate, the coupling ratio of compound and carrier protein specifically can be (8-10) shown in the formula (I): 1.Said coupling ratio refers to mol ratio.Compound and said carrier protein shown in the formula (I) specifically can pass through the active ester method coupling.
Said kit also comprises at least a in cleansing solution, sample concentration liquid, substrate colour developing liquid and the stop buffer.
Per 1 liter of cleansing solution can obtain according to following method preparation: 10ml polysorbas20,5g sodium azide and 990ml phosphate buffer are mixed, obtain cleansing solution.Said phosphate buffer can be the phosphate buffer of pH7.2-7.6,0.005M-0.015M, and the concentration that specifically can be can be pH7.4,0.01M) sodium phosphate buffer.
Said sample concentration liquid can be the phosphate buffer that concentration is 0.03mol/L-0.05mol/L, is preferably the PBS damping fluid of pH7.4,0.04mol/L.
Said substrate colour developing liquid comprises colour developing liquid A and colour developing liquid B, can be the colour developing liquid A and colour developing liquid B of independent packaging, also can directly colour developing liquid A be mixed obtaining with colour developing liquid B equal-volume.Said colour developing liquid can be superoxol or urea peroxide solution; Said colour developing liquid B can be o-phenylenediamine (0PD) solution or tetramethyl benzidine (TMB) solution.Said colour developing liquid A specifically can be 2% (g/100ml) urea peroxide WS.Said colour developing liquid B specifically can be 1% (g/100ml) tetramethyl biphenyl amine aqueous solution.
Said stop buffer specifically can be the 0.2M aqueous sulfuric acid.
More than arbitrary said kit all can be used for detecting aflatoxins.
More than arbitrary said kit all can be used for detecting whether contain aflatoxins in the sample to be tested.
The present invention also protects a kind of colloidal gold strip that detects aflatoxins, is made up of absorption of sample pad, collaurum pad, reaction film and adsorptive pads; Along test strips axially; Said absorption of sample pad, said collaurum pad, said reaction film and said adsorptive pads are linked in sequence successively; The end of absorption of sample pad links to each other with the top of collaurum pad; The end of collaurum pad links to each other with the top of reaction film, and the end of reaction film links to each other with the top of adsorptive pads;
Be coated with the monoclonal antibody of colloid gold label on the said collaurum pad; Said monoclonal antibody is the monoclonal antibody of aspergillus flavus resisting toxin monoclone antibody hybridoma 2A1 secretion;
Detection zone and Quality Control district are arranged on the said reaction film, and detection zone (T line) is and the axial vertical ribbon of said test paper with Quality Control district (C line); Detection zone is positioned near the terminal side of collaurum pad; The Quality Control district is positioned at away from the terminal side of collaurum pad; Detection zone is coated with the conjugate (coating antigen) of compound shown in the formula (I) and carrier protein, and it is anti-that the Quality Control district encapsulates sheep anti mouse two.
Said absorption of sample pad is a cellulose filter membrane.Said collaurum pad is the glass fibre membrane that is coated with the said monoclonal antibody of colloid gold label.Said reaction film is nitrocellulose filter (a NC film).Said adsorptive pads is a thieving paper.
Said sample well is positioned on the sample absorbent away from the terminal end of collaurum pad.
Said colloidal gold strip can be used for detecting aflatoxins.
Said colloidal gold strip is used for detecting sample to be tested and whether contains aflatoxins.
More than arbitrary said aflatoxins can be aflatoxins M 1Or aflatoxins M 2
The present invention adopts the aflatoxins monoclonal antibody of high specific, and the method for inspection is convenient and easy, has specificity height, highly sensitive, characteristics such as degree of accuracy is high, accuracy height.Kit of the present invention, test strips and detection method are low to the pre-treatment requirement of sample, and sample pretreatment process is simple, simultaneously the fast detecting batch samples.That kit of the present invention and test strips (colloidal gold test paper card) have is highly sensitive, accuracy is high, precision is high, cost is low, simple to operate, detection time short, be fit to various units uses, stores advantage simple, long shelf-life; The fast detecting batch samples can realize on-the-spot high flux fast detecting simultaneously.Kit provided by the invention and test strips are applicable to the residual quantity of measuring aflatoxins in liquid milk, milk powder and the dairy produce.Antibody of the present invention, kit, test strips and detection method will be brought into play significant role in the detection of aflatoxins.
Description of drawings
Fig. 1 is aflatoxins M 1The ultraviolet spectrogram of artificial antigen.
Fig. 2 is for adopting aflatoxins M 1The canonical plotting of making.
Fig. 3 is the canonical plotting of kit.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is conventional method.Used test material among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.Used PBS damping fluid among the embodiment like no specified otherwise, is the PBS damping fluid of pH7.4,0.01M.Used carbonate buffer solution is the sodium carbonate buffer of pH9.6,0.05mol/L among the embodiment.Bovine serum albumin(BSA) is called for short BSA.Ovalbumin is called for short OVA.
Aflatoxins M 1Shown in (III), molecular weight is 328.27.
Figure BDA0000138057080000041
Formula (III)
Suc as formula shown in (IV), molecular weight is 152.15 to hydrazino-benzoic acid.
Figure BDA0000138057080000042
Formula (IV)
N, dinethylformamide (DMF) is suc as formula shown in (V).
Figure BDA0000138057080000043
Formula (V)
N-hydroxy-succinamide (NHS) is suc as formula shown in (VI).
Formula (VI)
1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is suc as formula shown in (VII).
Figure BDA0000138057080000052
Formula (VII)
Embodiment 1, preparation aflatoxins M 1Haptens
One, aflatoxins M 1Haptenic preparation
With 10mg aflatoxins M 1Be dissolved in the 5ml anhydrous pyridine, add to hydrazino-benzoic acid 10mg 70 ℃ of reflux stirring reaction 5h; Revolve to steam and remove pyridine, residue is used the 1mL dissolve with methanol, with methylene chloride: methyl alcohol (volume ratio 9: 1) is developping agent; Through thin-layer chromatography (TLC) separation and purification, collect Rf value (R fValue) is 0.35 sample, is aflatoxins M 1Haptens (obtaining 8mg).
Two, aflatoxins M 1Haptenic sign
Product to the step 1 preparation carries out ultimate analysis, and the result is following:
C：62.32；H：3.94；N：6.08；0：27.66。
The result shows that the product of step 1 preparation is a compound shown in the formula (I), is called M1-HBA again.
Figure BDA0000138057080000053
Formula (I).
Embodiment 2, aflatoxins M 1Preparation of artificial antigen and sign
One, aflatoxins M 1Immunogenic synthetic and sign
1, aflatoxins M 1Immunogenic synthetic
(1) compound is dissolved in 1mL N shown in the formula that 5mg embodiment 1 is prepared (I); In N '-dimethylformamide; Add 4mg N-hydroxy-succinamide and 4mg1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, the room temperature lower magnetic force stirs 2h, obtains solution I.
(2) the 30mg bovine serum albumin(BSA) is added in the 3mL PBS damping fluid, fully dissolving is solution II.
(3) solution I is added in the solution II, go into bag filter after slowly stirring 24h, 4 ℃ of dialysis 72h (water is changed 6 times in the centre) in physiological saline, under 4 ℃ of conditions, the centrifugal 30min of 8000rmp gets supernatant, i.e. aflatoxins M then 1Immunogen solution is sub-packed in the ampere bottle-20 ℃ of preservations, aflatoxins M 1Immunogene is called for short M1-BSA, aflatoxins M 1Immunogen solution is called for short M1-BSA solution.
(4) with M1-BSA solution with after the PBS damping fluid dilution, measure the spectrophotometric value of 280nm and 260nm, by formula calculate the protein concentration in the dilution, the protein concentration that records is on duty to be the M1-BSA concentration in the former M1-BSA solution behind its extension rate.Protein concentration (mg/ml)=1.45 * OD 280-0.74 * OD 260M1-BSA concentration in the M1-BSA solution is 6.1mg/ml.
2, aflatoxins M 1Immunogenic sign
M1-BSA solution is diluted (concentration that makes M1-BSA is 5mg/mL) with the PBS damping fluid, as the solution first; The PBS damping fluid that will contain 5mg/mL M1-HBA is as solution second; The PBS damping fluid that will contain 5mg/mL BSA is as solution third.Respectively solution first, solution second and solution third are carried out ultraviolet (200-380nm) spectral scan, the uv scan result sees Fig. 1.Significant change has taken place in the uv-spectrogram of comparing the solution first with solution third, and compound and BSA success coupling is described.
The maximum absorption wave long value of solution second is 262nm, and the maximum absorption wave long value of solution third is 280nm.Calculate the extinction coefficient (K) of each compound according to formula K=A/CL (A is the absorbance under the maximum absorption wave long value, and C is a solution concentration, and L is the thickness of liquid layer).
Adopt the maximum absorption wave long value of solution second and solution third that the solution first is carried out uv scan respectively; And according to the concentration of this compound of extinction coefficient backwards calculation in the solution first of this compound that has calculated; Obtain the volumetric molar concentration of this compound divided by molecular weight with concentration value; Calculate coupling ratio, the coupling ratio of compound and BSA is 8: 1 shown in the formula (I), and promptly compound shown in 8 formulas (I) combines 1 BSA.
Two, aflatoxins M 1The preparation of coating antigen and sign
1, aflatoxins M 1The preparation of coating antigen
Replace bovine serum albumin(BSA) with ovalbumin, other is with 1 of step 1.
Aflatoxins M 1Coating antigen is called for short M1-OVA, aflatoxins M 1Coating antigen solution is called for short M1-OVA solution.
M1-OVA concentration in the M1-OVA solution is 3.8mg/ml.
2, aflatoxins M 1The sign of coating antigen
Replace M1-BSA with M1-OVA, replace BSA with OVA, other is with 2 of step 1.
The coupling ratio of compound and OVA is 10: 1 shown in the formula (I), and promptly compound shown in 10 formulas (I) combines 1 OVA.
Embodiment 3, aflatoxins MONOCLONAL ANTIBODIES SPECIFIC FOR
Balb/c mouse: purchase in Beijing Vital River Experimental Animals Technology Co., Ltd.;
SP2/0 myeloma cell: available from Sigma-Aldrich company, catalog number is 08060101.
One, animal immune
With the M1-BSA solution immunity Balb/c mouse of embodiment 2 preparations, every mouse single immunization 100 μ gM1-BSA, immunity is 4 times altogether, and in each two weeks at interval, the immunization ways of first three time is the subcutaneous multi-point injection of nape portion, and back three times immunization ways is an intraperitoneal injection.
Two, Fusion of Cells and cloning
1, the 4th immunity be after 3 days, and extracting spleen cell merges in 5: 1 (quantitative proportion) ratios and SP2/0 myeloma cell, adopts indirect competitive ELISA to measure cell conditioned medium liquid, screens positive hole.
2, utilize limiting dilution assay that cloning is carried out in positive hole, obtain a strain and can secrete aflatoxins M 1The hybridoma of monoclonal antibody, called after aspergillus flavus resisting toxin monoclone antibody hybridoma 2A1 (being called for short hybridoma 2A1).Hybridoma 2A1 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 15th, 2012, and (be called for short CGMCC, the address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5779.
Three, cell cryopreservation and recovery
With cryopreserving liquid hybridoma 2A1 is processed 1 * 10 6The cell suspension of individual/ml is in the medium-term and long-term preservation of liquid nitrogen.During recovery, take out frozen pipe, put into 37 ℃ of water-bath middling speeds immediately and melt, move in the culture flask behind the centrifugal removal cryopreserving liquid and cultivate.
Four, MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
1, increment cultivation
The preparation method of cell culture medium (7.4): in the RPMI-1640 nutrient culture media, add calf serum and soda mint, the final concentration of calf serum is 20% (quality percentage composition), and the final concentration of soda mint is 0.2% (quality percentage composition).
2A1 places cell culture medium with hybridoma, cultivates 2 days for 37 ℃, with sad-saturated ammonium sulfate method the nutrient solution that obtains is carried out purifying, obtains monoclonal anti liquid solution (20 ℃ of preservations).
Protein concentration in the monoclonal antibody (mg/ml)=1.45 * OD 280-0.74 * OD 260
Adopt above formula to calculate the protein concentration in the monoclonal antibody, be 18.9mg/ml.
2, ascites preparation
Balb/c mouse peritoneal injection sterilization paraffinum liquidum (0.4mL/ only).7 days pneumoretroperitoneum injection hybridoma cell strains (5 * 10 5Individual/only).Gather ascites after 7 days, carry out purifying, the ascites behind the purifying-20 ℃ preservation with sad-saturated ammonium sulfate method.
Five, the evaluation of monoclonal antibody
The monoclonal anti liquid solution that 1 of step 4 obtains is identified respectively as follows:
1, the mensuration of antibody titer
(1) adopt the M1-OVA solution (adopting carbonate buffer solution to regulate concentration) of embodiment 2 preparations to encapsulate 100 μ L/ holes; The concentration that encapsulates of M1-OVA is 1.0 μ g/mL.
Hatched 16 hours for (2) 4 ℃.
(3) seal and wash plate.
(4) every hole adds monoclonal anti liquid solution or its dilution (adopting the PBS damping fluid to carry out gradient dilution) that 1 of 100 μ L step 4 obtain.
(5) incubated at room 2h washes plate.
(6) every hole adds the sheep anti-mouse igg of 100 μ L horseradish peroxidase-labeled, incubated at room 2h.
(7) wash plate.
(8) add TMB colour developing liquid, lucifuge colour developing 15min.
(9) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD 450Value.
Be judged to be the positive when reaching 1.0 left and right sides with the OD value.Antibody titer is 1: 320000.
2, monoclonal antibody Sensitivity calculation
(1) to (3) with (1) of step 1 to (3).
(4) every hole adds 50 μ L aflatoxins M 1Standard solution is (by aflatoxins M 1Form with the PBS damping fluid; Aflatoxins M 1Concentration be respectively 0.03 μ g/L, 0.09 μ g/L, 0.18 μ g/L, 0.36 μ g/L, 0.72 μ g/L; With the hole that only adds the PBS damping fluid as control wells); Each concentration is provided with 3 multiple holes.
(5) every hole adds the monoclonal anti liquid solution that 1 of 50 μ L step 4 obtain.
(6) incubated at room 2h washes plate.
(7) every hole adds the sheep anti-mouse igg of 100 μ L horseradish peroxidase-labeled, incubated at room 2h.
(8) wash plate.
(9) add TMB colour developing liquid, lucifuge colour developing 15min.
(10) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD 450Value.
The light absorption value (mean values in three multiple holes) that the standard solution that adopts each concentration is obtained multiply by 100 as ordinate again divided by the light absorption value of control wells; Natural logarithm value with the aflatoxins M1 concentration (μ g/L) in each standard solution is horizontal ordinate curve plotting figure, sees Fig. 2.
Map 2 obtains the aflatoxins M1 concentration (μ g/L) that Y value equals 50% correspondence, is the IC50 value.The sensitivity (IC50 value) that monoclonal antibody detects aflatoxins M1 is 0.05 μ g/L.
3, affinity costant is measured
(1) with M1-OVA as the coating antigen coated elisa plate
Adopt the M1-OVA solution (adopting carbonate buffer solution to regulate concentration) of embodiment 2 preparations to encapsulate 100 μ L/ holes; Following OVA-SAL is set respectively encapsulates concentration: 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL.
Hatched 16 hours for (2) 4 ℃.
(3) seal and wash plate.
(4) every hole adds the dilution (diluting with the PBS damping fluid) of 100 μ L monoclonal anti liquid solutions; Protein concentration in the dilution is respectively 1.25,0.625,0.3125,1.5625 * 10 -1, 7.8 * 10 -2, 3.9 * 10 -2, 1.95 * 10 -2, 9.75 * 10 -3, 4.88 * 10 -3, 2.44 * 10 -3, 1.22 * 10 -3, 6.1 * 10 -4Mg/L.
(5) incubated at room 2h washes plate.
(6) every hole adds the sheep anti-mouse igg of 100 μ L horseradish peroxidase-labeled, incubated at room 2h.
(7) wash plate.
(8) add .TMB colour developing liquid, lucifuge colour developing 15min.
(9) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD 450Value.
Natural logarithm value with the protein concentration in the monoclonal antibody (mol/L) is a horizontal ordinate, is that ordinate is made curve with its corresponding absorbance.
Each antigen coated concentration obtains 1 S type curve, obtains 4 S type curves altogether.Find out the top of S curve, corresponding OD 450Value is set at ODMAX.Find out the corresponding AC of each bar curve 50%ODMAX respectively.Adopt 1 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 2.8 * 10 -12Mol/L.Adopt 0.5 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 27.7 * 10 -12Mol/L.Adopt 0.25 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 190.4 * 10 -12Mol/L.Adopt 0.125 μ g/mL to encapsulate concentration, the AC that 50%ODMAX is corresponding is 368.2 * 10 -12Mol/L.
With one group in twos of 4 concentration, calculate the affinity costant of monoclonal antibody according to formula
Ka＝(n-1)/2(n[Ab]t 1-[Ab]t 2)
In the formula, two multiples that encapsulate concentration during n is every group, [Ab] t 1, [Ab] t 2Be respectively two ACs (mol/L) that 50%ODMAX is corresponding in every group.For example: 1 μ g/mL encapsulates concentration 50%OD 450Corresponding AC is 2.8 * 10 -12Mol/L encapsulates 0.5 μ g/mL and encapsulates concentration 50%OD 450Corresponding AC is 27.7 * 10 -12Mol/L, Ka=(2-1)/2 (2 * 27.7 * 10 -12-2.8 * 10 -12)=9.5 * 10 9M -1And the like, obtain all the other 5 Ka values, be respectively 1.41 * 10 9M -1, 0.91 * 10 9M -1, 1.97 * 10 9M -1, 1.03 * 10 9M -1, 1.18 * 10 9M -1, the affinity costant that calculates monoclonal antibody of averaging is 2.67 * 10 9M -1
Six, the stability of hybridoma cell strain
With 30 generations of the external continuous biography of hybridoma 2A1, whenever get culture supernatant at a distance from 5 generations and carry out ELISA mensuration (method is with 1 of step 5).The result shows the no significant difference of tiring of continuous 30 generations, and hybridoma 2A1 can stablize and goes down to posterity.
Embodiment 4, aflatoxins Polyclonal Antibody Preparation
New zealand white rabbit: purchase in Beijing Vital River Experimental Animals Technology Co., Ltd..
With the aflatoxins M of new zealand white rabbit with preparation among the embodiment 2 1Immunogene (M1-BSA) is carried out immunity (immunization ways is the subcutaneous multi-point injection of nape portion).Every each immune 1.5mg of rabbit (in the BSA amount), per three all immunity once, during immunity for the first time with aflatoxins M 1Immunogene and Freund's complete adjuvant are mixed and made into emulsifying agent and carry out immunity, and the second time to the 6th immunity is with aflatoxins M 1Immunogene and incomplete Freund are mixed and made into emulsifying agent and carry out immunity, and the 7th immunity is only with aflatoxins M 1Immunogene is carried out immunity, and the 7th immunity be the heart blood sampling after 10 days, obtains the polyclonal antibody of purifying with ammonium sulfate precipitation.
The preparation of the enzyme linked immunological kit of embodiment 5, detection aflatoxins
One, the composition of enzyme linked immunological kit
Enzyme linked immunological kit is made up of following substances:
1, cleansing solution: per 1 liter of cleansing solution obtains according to following method preparation: 10ml polysorbas20,5g sodium azide and 990ml phosphate buffer (pH7.4,0.01M) are mixed, obtain cleansing solution.
2, encapsulate the ELISA Plate of M1-OVA
With the M1-OVA solution dilution (concentration that makes M1-OVA be 0.5 μ g/mL) of carbonate buffer solution, be coating buffer with embodiment 2 preparations; Coating buffer is added 96 hole polystyrene ELISA Plates (48 holes also can), every hole 100 μ L, 37 ℃ of incubation 2h; The coating buffer that inclines, with cleansing solution washing 3 times, each 30s claps and does; In every hole, add 200 μ L confining liquids then, 37 ℃ of incubation 2h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
Per 1 liter of said confining liquid is prepared according to following method: 5ml horse serum, 1g sodium azide and 30g casein are dissolved with phosphate buffer (pH7.2,0.02M) and be settled to 1000ml, obtain confining liquid.
3, sample concentration liquid: PBS damping fluid (pH7.4,0.04M).
Sample concentration liquid is diluted with water to 20 times of volumes, is sample diluting liquid.
4, antibody working fluid: the monoclonal anti liquid solution that 1 of the step 4 of embodiment 3 is obtained dilutes with sample diluting liquid, and making protein concentration is 5.5ng/mL.
5, ELIAS secondary antibody working fluid
The sheep anti-mouse antibody of horseradish peroxidase (HRP) mark is available from U.S. Sigma-Aldrich company, and catalog number is A7058, by specification preparation ELIAS secondary antibody working fluid.
6, standard solution
Standard items are aflatoxins M 1(FERMENTEK product; Catalog number is FER005).
With sample diluting liquid dilution dissolving aflatoxins M 1, obtain each standard solution.Be respectively 0.03 μ g/L, 0.09 μ g/L, 0.18 μ g/L, 0.36 μ g/L, 0.72 μ g/L in each standard solution.
Sample diluting liquid is as aflatoxins M 1Concentration is the standard solution (0 standard) of 0 μ g/L.
7, substrate colour developing liquid
Mixed by A liquid and B liquid equal-volume, A liquid is 2% (g/100ml) urea peroxide WS, and B liquid is 1% (g/100ml) tetramethyl biphenyl amine aqueous solution.
8, stop buffer: 0.2M aqueous sulfuric acid.
Two, kit test method
Adopt the kit of step 1 preparation to detect as follows:
(1) sample pre-treatments
When the detection sample was liquid milk or recombined milk: get sample to be checked in centrifuge tube, fully balance added sample diluting liquid to room temperature, fully after the whirling motion, gets 70 μ L as sample to be tested solution.
When the detection sample was milk powder: take by weighing powdered milk sample in centrifuge tube, add deionized water, violent whirling motion to milk powder dissolved fully, adds sample diluting liquid, fully after the whirling motion, gets 70 μ L as sample to be tested solution.
The detection sample is a sour milk: take by weighing the sour milk sample in centrifuge tube, add sample diluting liquid, fully whirling motion 30s gets 70 μ L as sample to be tested solution.
(2) kit test method
1, the making of typical curve
In the ELISA Plate that encapsulates M1-OVA, add standard solution (70 μ L/ holes; Each standard solution is provided with three multiple holes), add antibody working fluid (30 μ L/ hole) again, with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens; Pour out liquid in the hole, wash 5 times (step of each washing is: every hole adds 250 μ L cleansing solutions, pours out liquid in the hole behind the 30s), clap with thieving paper and do; Add ELIAS secondary antibody working fluid (100 μ L/ hole), react 30min in 37 ℃ of constant temperature ovens, pour out liquid in the hole, wash 5 times (step is the same); Every hole adds substrate colour developing liquid 100 μ L, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min; Every hole adds stop buffer 50 μ L, and the mixing that vibrates is gently measured every hole absorbance (OD630 and OD450 dual wavelength, the 450th, predominant wavelength, the 630th, reference wavelength) with ELIASA.
The absorbance mean value (B) of the standard solution of each concentration of usefulness multiply by 100% again divided by the absorbance (B0) of first standard solution (0 standard), obtains the percentage absorbance.Utilization Originpro 7.0 softwares are analyzed the data result, are the X axle with the natural logarithm value of standard items concentration (μ g/L), and the percentage absorbance simulates typical curve for the Y axle.Typical curve is seen Fig. 3.
2, the mensuration of aflatoxins concentration in the sample
In the ELISA Plate that encapsulates M1-OVA, add sample to be tested solution or its dilution (70 μ L/ holes; Three multiple holes are set), add antibody working fluid (30 μ L/ hole) again, with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens; Pour out liquid in the hole, wash 5 times (step is the same), clap with thieving paper and do; Add ELIAS secondary antibody working fluid (100 μ L/ hole), react 30min in 37 ℃ of constant temperature ovens, wash 5 times (step is the same); Every hole adds substrate colour developing liquid 100 μ L, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min; Every hole adds salt stop buffer 50 μ L, and the mixing that vibrates is gently measured every hole absorbance (OD630 and OD450 dual wavelength, the 450th, predominant wavelength, the 630th, reference wavelength) with ELIASA.
The result judges: use each sample to be tested solution absorbency mean value (B) to multiply by 100% again divided by the absorbance (B0) of first standard solution (0 standard), obtain the percentage absorbance.The percentage absorbance of corresponding each sample to be tested solution then can be read aflatoxins M from typical curve 1Concentration value, multiply by the extension rate of respective sample again, converse aflatoxins M in the sample to be tested solution 1Content.
Three, kit detects effect assessment
(1) accuracy and precision test
In the milk sample that does not contain aflatoxins, add aflatoxins M 1(standard items) make aflatoxins M 1Final concentration in sample is respectively 0.1 μ g/kg, 0.3 μ g/kg, 0.5 μ g/kg; With the sample after adding respectively according to step 2
(1) method described in is carried out pre-treatment, obtains sample to be tested solution.
From the kit of three different batches, respectively extract 3 kits and detect, (two) of detection method such as step 22 described in, each experiment repetition 5 times is according to aflatoxins M in the sample to be tested solution 1Cubage detect aflatoxins M in the sample 1Content, the result sees table 1.
Table 1 is used each kit and is detected aflatoxins M in the milk sample that draws 1Content (μ g/kg)
Kit 1 Kit 2 Kit 3
Aflatoxins M 1Final concentration in sample is 0.1 μ g/kg 0.084 0.087 0.091
Aflatoxins M 1Final concentration in sample is 0.3 μ g/kg 0.28 0.26 0.24
Aflatoxins M 1Final concentration in sample is 0.5 μ g/kg 0.44 0.42 0.41
The difference calculate recovery rate and the coefficient of variation, the result sees table 2.
The aflatoxins M that the recovery=application kit detection computations goes out 1Content ÷ milk in the actual aflatoxins M that adds 1Content * 100%.
The coefficient of variation (CV)=mensuration result's the standard deviation and the number percent of its mean value.
The computing method of plate within variance coefficient: certain sample (being generally medium level) replication number of times gained result's the coefficient of variation in plate within variance coefficient=same same block of plate of once measuring.
The computing method of variation within batch coefficient: the variation within batch coefficient=coefficient of variation of each parallel samples in once measuring together.
The computing method of interassay coefficient of variation: interassay coefficient of variation=same sample is got its mean value in different batches mensuration result's the coefficient of variation.
Table 2 recovery and coefficient of variation result
Figure BDA0000138057080000131
The result shows: the recovery of all samples is 79.3%～94.5%, and the variation within batch coefficient is 4.3%～8.6%, and interassay coefficient of variation is 12.8%～15.1%.
(2) kit storage life
The kit preservation condition is 2-8 ℃, through 12 months mensuration, and the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, aflatoxins M 1Add the practical measurement value all within normal range.Consider in transportation and the use, have improper preservation condition and occur that kit the condition held of 37 ℃ of preservations 8 days, is carried out accelerated deterioration and tests, and the result shows that each item index of this kit meets the requirements fully.Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 8 days, measure the result and show that also kit each item index is normal fully.Can draw kit from above result can preserve more than 12 months at 2-8 ℃ at least.
(3) cross reacting rate test
(the analogue standard solution is made up of analogue and PBS damping fluid, and the concentration of analogue is respectively 0.03 μ g/L, 0.09 μ g/L, 0.18 μ g/L, 0.36 μ g/L, 0.72 μ g/L in the ELISA Plate that encapsulates M1-OVA, to add the analogue standard solution; With the hole that only adds the PBS damping fluid as control wells; 50 μ L/ holes; Each concentration is provided with 3 multiple holes), add antibody working fluid (50 μ L/ hole) again, with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens; Pour out liquid in the hole, wash 5 times (step is the same), clap with thieving paper and do; Add ELIAS secondary antibody working fluid (100 μ L/ hole), react 30min in 37 ℃ of constant temperature ovens, wash 5 times (step is the same); Every hole adds substrate colour developing liquid 100 μ L, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min; Every hole adds stop buffer 50 μ L, and the mixing that vibrates is gently measured every hole absorbance (OD630 and OD450 dual wavelength, the 450th, predominant wavelength, the 630th, reference wavelength) with ELIASA.
With the cross reacting rate of computes kit to other analogue.
Figure BDA0000138057080000141
The result sees table 3.
The specificity of table 3 kit
The analogue title Available from Business Name and products thereof catalog number (Cat.No.) Cross reacting rate
Aflatoxins M 1 FERMENTEK company; Catalog number is FER005 100％
Aflatoxins M 2 FERMENTEK company; Catalog number is FER006 About 94%
Aflatoxins B 1 FERMENTEK company; Catalog number is FER001 About 18.7%
Aflatoxins B 2 FERMENTEK company; Catalog number is FER002 About 5.4%
Aflatoxins G 1 FERMENTEK company; Catalog number is FER003 ＜1％
Aflatoxins G 2 FERMENTEK company; Catalog number is FER004 ＜1％
Bovine serum albumin(BSA) Fluorochem Limi ted company; Catalog number is S02375 ＜1％
Deoxynivalenol FERMENTEK company; Catalog number is FER008 ＜1％
Experiment shows, with aflatoxins M 2Cross reacting rate be 94%, with aflatoxins B 1, aflatoxins B 218.7% and 5.4% cross reaction is arranged, with other architecture analog no cross reactions respectively.
Embodiment 6, the test strips that detects aflatoxins and preparation and application
One, the structure of test strips
Said test strips is made up of absorption of sample pad, collaurum pad, reaction film and adsorptive pads;
Along test strips axially; Absorption of sample pad, collaurum pad, reaction film and adsorptive pads are linked in sequence successively; The end of absorption of sample pad links to each other with the top of collaurum pad, and the end of collaurum pad links to each other with the top of reaction film, and the end of reaction film links to each other with the top of adsorptive pads;
Be coated with 1 monoclonal antibody that obtains of step 4 of the embodiment 3 of colloid gold label on the said collaurum pad;
Detection zone and Quality Control district are arranged on the said reaction film, and detection zone (T line) is and the axial vertical ribbon of test strips with Quality Control district (C line); Detection zone is positioned near the terminal side of collaurum pad; The Quality Control district is positioned at away from the terminal side of collaurum pad; Detection zone encapsulates the M1-OVA of embodiment 2 preparations, and it is anti-that the Quality Control district encapsulates sheep anti mouse two.
Sample well is positioned on the sample absorbent away from the terminal end of collaurum pad.
Two, the preparation of test strips
(1) colloid gold label antibody
1. the preparation of colloidal gold solution
Get 0.01% aqueous solution of chloraurate 100mL and be heated to boiling with the constant temperature magnetic stirrer, continue to add 1% trisodium citrate aqueous solution 2.5mL under the condition of stirring, continue agitating heating 20min, solution is bright redness.The room temperature cooling returns to original volume with deionized water, 2-8 ℃ of preservation.
The preparation of 2. golden labeling antibody solution
Use 0.1mol/L K 2CO 3The WS is regulated the pH to 8.2 of colloidal gold solution; Getting 10mL then adds in the 50mL beaker; Magnetic stirrer 250r/min stirs; Dropwise add the 1 monoclonal anti liquid solution (containing 0.35mg protein) that obtains of the step 4 of embodiment 3, dropwise add the 3mL 5g/100mL BSA WS, continue to stir 10min.
3. with the centrifugal 20min of golden labeling antibody solution normal temperature low speed (1500r/min), discard the deposition that forms by the gold grain that condenses, get red supernatant solution.
4. with 4 ℃ of step solution 3., the centrifugal 40min of 11000r/min; Solution is divided into three layers (fine and close gold grain layers of black on transparent supernatant, the flowable kermesinus deposition in the pipe end and the pipe diapire); Flowable kermesinus deposition is transferred in the another one centrifuge tube, used the 0.01mol/L phosphate buffer that contains 1g/100mL BSA to be suspended to the volume of former golden labeling antibody solution, spend the night; 4 ℃, the centrifugal 40min of 11000r/min, collecting precipitation.
5. with the 0.01mol/L phosphate buffer that contains 1g/100mL BSA and 0.02g/100mL NaN3 step deposition 4. is suspended to former golden labeling antibody solution volume 1/40,2-8 ℃ of preservation.
(2) metal spraying: the suspension that step (1) is obtained is sprayed onto on the glass fibre membrane, processes the collaurum pad.
(3) spray film: the T line position on reaction film is sprayed M1-OVA solution, the C line position of embodiment 2 preparations and is sprayed sheep anti-mouse antibody.
(4) assembling: absorption of sample pad (cellulose filter membrane), collaurum pad, nitrocellulose filter (NC film), adsorptive pads (thieving paper) are assembled by conventional method, and slitting is then packed test strips in the plastics fabrication into, forms test card.
Three, detect with test card
(1) sample pre-treatments and detection
The detection sample is a liquid milk; The liquid milk of getting the 3mL mixing adds 3mL ethyl acetate in centrifuge tube, leave standstill 2-3min behind the mixing; The centrifugal 10min of 4000g gets the 1.5mL supernatant liquid, and nitrogen dries up; Get the fully whirling motion in sample hose of 150 μ L sample diluting liquids, leave standstill 5min, be sample to be tested solution.
Take out test card, lie against desktop behind the Kaifeng, draw sample to be tested solution and dropwise add 4 in sample well; The 5-10min judged result, the judged result behind the 15min is invalid.
Criterion as a result:
Negative: the colour developing of C line, T line naked eyes are visible, and no matter shade all is judged to feminine gender;
Positive: the colour developing of C line, the T line does not develop the color, and is judged to the positive;
Invalid: the C line does not develop the color, and no matter whether the T line develops the color, and it is invalid that this test card all is judged to.
Four, the effect of test card
(1) false positive rate and false negative rate
The negative milk of the conclusive evidence of learning from else's experience (contains aflatoxins M 1＜0.5 μ g/L) 50 parts, the positive milk of the conclusive evidence of learning from else's experience (contains aflatoxins M 1>=0.5 μ g/L) 50 part.Sample is detected with test card respectively, calculate false positive rate and false positive rate.
The result: in 50 parts of negative milk samples, test card detects totally 1 part of positive, and false positive rate is 2%.In 50 parts of positive milk samples were measured, test card detected 0 part of negative sample, and false negative rate is 0%.
(2) test card storage life
Stability test is the result show, this test card can be preserved 1 year at shady and cool dry place under 2-8 ℃ or room temperature condition.

Claims (8)

1. an enzyme linked immunological kit that detects aflatoxins comprises the monoclonal antibody that aspergillus flavus resisting toxin monoclone antibody hybridoma 2A1 secretes; The deposit number of said aspergillus flavus resisting toxin monoclone antibody hybridoma 2A1 is CGMCC No.5779.
2. kit as claimed in claim 1 is characterized in that: said kit is following 1) to 4) in any one:
1) comprises the kit of compound shown in the formula (I) and the conjugate of carrier protein, said monoclonal antibody and enzyme labeling antiantibody; Wherein, said conjugate is as coating antigen;
2) comprise the kit of the enzyme labeling thing of compound shown in the formula (I), said monoclonal antibody and antiantibody; Wherein, said antiantibody is as coating antigen;
3) comprise the enzyme labeling thing of compound shown in the formula (I) and the kit of said monoclonal antibody; Wherein, said monoclonal antibody is as coating antigen;
4) comprise the kit of enzyme labeling thing of conjugate and the said monoclonal antibody of compound shown in the formula (I) and carrier protein; Wherein, said conjugate is as coating antigen;
Figure FDA0000138057070000011
Formula (I).
3. kit as claimed in claim 2 is characterized in that: said carrier protein is ovalbumin or bovine serum albumin(BSA).
4. like arbitrary described kit in the claim 1 to 3, it is characterized in that: said kit also comprises at least a in cleansing solution, sample concentration liquid, substrate colour developing liquid and the stop buffer.
5. a colloidal gold strip that detects aflatoxins is made up of absorption of sample pad, collaurum pad, reaction film and adsorptive pads;
Along test strips axially; Said absorption of sample pad, said collaurum pad, said reaction film and said adsorptive pads are linked in sequence successively; The end of absorption of sample pad links to each other with the top of collaurum pad; The end of collaurum pad links to each other with the top of reaction film, and the end of reaction film links to each other with the top of adsorptive pads;
Be coated with the monoclonal antibody of colloid gold label on the said collaurum pad; Said monoclonal antibody is the monoclonal antibody of aspergillus flavus resisting toxin monoclone antibody hybridoma 2A1 secretion; The deposit number of said aspergillus flavus resisting toxin monoclone antibody hybridoma 2A1 is CGMCC No.5779;
Detection zone and Quality Control district are arranged on the said reaction film, and detection zone is and the axial vertical ribbon of said test paper with the Quality Control district; Detection zone is positioned near the terminal side of collaurum pad; The Quality Control district is positioned at away from the terminal side of collaurum pad; Detection zone is coated with the conjugate of compound shown in the formula (I) and carrier protein, and it is anti-that the Quality Control district encapsulates sheep anti mouse two.
6. arbitrary said kit in the claim 1 to 4, or, the said colloidal gold strip of claim 5, the application in the inspection aflatoxins.
7. arbitrary said kit in the claim 1 to 4, or whether the said colloidal gold strip of claim 5 contains the application in the aflatoxins detecting sample to be tested.
8. like arbitrary described kit in the claim 1 to 4, or the described colloidal gold strip of claim 5, or like claim 6 or 7 described application, it is characterized in that: said aflatoxins is aflatoxins M 1Or aflatoxins M 2
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CN108828205A (en) * 2018-04-09 2018-11-16 国家食品安全风险评估中心 Ochratoxin A haptens, artificial antigen and preparation method thereof, kit and ochratoxin A detection method

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Publication number Priority date Publication date Assignee Title
CN107083368A (en) * 2017-04-10 2017-08-22 北京勤邦生物技术有限公司 A kind of hybridoma cell strain for secreting anti-total aflatoxina monoclonal antibody and its application
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CN107561273A (en) * 2017-08-29 2018-01-09 联合益康（北京）生物科技有限公司 A kind of time-resolved fluorescence test strips for detecting Aflatoxins M1 and its application
CN107589265A (en) * 2017-08-29 2018-01-16 联合益康（北京）生物科技有限公司 A kind of time-resolved fluorescence test strips for detecting aflatoxin B1 and its application
CN107677810A (en) * 2017-09-11 2018-02-09 扬州市伊绿鲜生态农业科技有限公司 A kind of aflatoxin chemoluminescence method quantitative determination reagent kit
CN108828205A (en) * 2018-04-09 2018-11-16 国家食品安全风险评估中心 Ochratoxin A haptens, artificial antigen and preparation method thereof, kit and ochratoxin A detection method
CN108828205B (en) * 2018-04-09 2021-11-02 国家食品安全风险评估中心 Ochratoxin A hapten, artificial antigen, preparation method and kit of artificial antigen, and ochratoxin A detection method

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