CN102590519B - Kit or test strip for detecting aflatoxin - Google Patents

Kit or test strip for detecting aflatoxin Download PDF

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CN102590519B
CN102590519B CN201210045398.1A CN201210045398A CN102590519B CN 102590519 B CN102590519 B CN 102590519B CN 201210045398 A CN201210045398 A CN 201210045398A CN 102590519 B CN102590519 B CN 102590519B
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aflatoxins
kit
monoclonal antibody
sample
solution
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CN102590519A (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 kit or the test strips of aflatoxins
Technical field
The present invention relates to detect kit or the test strips of aflatoxins.
Background technology
Along with human living standard's improvement, food security becomes one of problem that on dining table, people pay close attention to the most day by day, and the milk that people every day all can be edible may contain the aflatoxins M harmful to health 1.Mammal is taken in by aflatoxins B 1after the feed or food polluting, can be generated aflatoxins M by hydroxylation in vivo 1.Research shows, aflatoxins M 1for strong carcinogen.It is 15 μ g/kg that the World Health Organization (WHO) formulates food aflatoxin maximum permissible concentration, and Federal Government relevant laws stipulate that the content in human consumption's milk can not exceed 0.5 μ g/kg, and the content in other animal feeds can not 300 μ g/kg.In " mycotoxin limitation in GB 2761-2011 food " national standard that China issues this year, stipulate aflatoxins M in food 1limitation in milk and milk products is 0.5 μ g/kg.And European Union member countries' regulation is stricter, M in former milk, thermal treatment milk and processing dairy products 1limitation is 0.05 μ g/kg, babym in food (comprising infant's milk) 1limitation 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 euzymelinked immunosorbent assay (ELISA) etc. both at home and abroad at present.The specificity of TLC method is poor, and sensitivity is also relatively poor, and required standard items concentration is higher, and potential contaminative is higher.The required instrument and equipment investment of HPLC method is large, and operative technique requirement is high, and decontaminating column consumes more, and testing cost is higher, and immune affinity column specificity is better, but need could quantitatively detect in conjunction with HPLC instrument and fluorophotometer, and detection technique requirement and cost are higher.Comparatively speaking, easy and simple to handle, quick, less pollution, sensitivity are also higher, are applicable to general inspection and the screening of batch sample for ELISA method, the kit of particularly succeeding in developing, bring great convenience to testing staff, also provide cost savings, meet at present the developing direction of detection field in the world simultaneously.Immune colloidal gold chromatography technology does not need any instrument and equipment and reagent, is particularly suitable for detection and large area generaI investigation that grass-roots unit is pressed for time in enormous quantities, and therefore enzyme immunoassay is widely used in Food Safety Analysis.
Summary of the invention
The object of this invention is to provide detect 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's common micro-organisms center on February 15th, 2012 and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number is CGMCC No.5779.
Described kit can be following 1) to 4) in any one:
1) comprise the kit of compound shown in formula (I) and the conjugate of carrier protein, described monoclonal antibody and enzyme labeling antiantibody; Wherein, described conjugate is as coating antigen;
2) comprise the kit of the enzyme labeling thing of compound shown in formula (I), described monoclonal antibody and antiantibody; Wherein, described antiantibody is as coating antigen;
3) comprise the enzyme labeling thing of compound shown in formula (I) and the kit of described monoclonal antibody; Wherein, described monoclonal antibody is as coating antigen;
4) comprise the kit of compound shown in formula (I) and the conjugate of carrier protein and the enzyme labeling thing of described monoclonal antibody; Wherein, described conjugate is as coating antigen.
Figure BDA0000138057080000021
Formula (I).
The conjugate of compound and carrier protein shown in formula (I) is specifically suc as formula shown in (II).
Figure BDA0000138057080000022
Formula (II).
Described carrier protein can be ovalbumin (OVA) or bovine serum albumin(BSA) (BSA).
In described conjugate, shown in formula (I), the coupling ratio of compound and carrier protein specifically can be (8-10): 1.Described coupling ratio refers to mol ratio.Compound and described carrier protein shown in formula (I) specifically can pass through active ester method coupling.
Described kit also comprises at least one in cleansing solution, sample concentration liquid, substrate nitrite ion and stop buffer.
Every 1 liter of cleansing solution can be prepared and obtain as follows: 10ml polysorbas20,5g sodium azide and 990ml phosphate buffer are mixed, obtain cleansing solution.Described phosphate buffer can be the phosphate buffer of pH7.2-7.6,0.005M-0.015M, and the concentration specifically can be can be pH7.4,0.01M) sodium phosphate buffer.
Described 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.
Described substrate nitrite ion comprises nitrite ion A and nitrite ion B, can be nitrite ion A and the nitrite ion B of independent packaging, also can directly nitrite ion A and nitrite ion B equal-volume be mixed to get.Described nitrite ion can be superoxol or urea peroxide solution; Described nitrite ion B can be o-phenylenediamine (0PD) solution or tetramethyl benzidine (TMB) solution.Described nitrite ion A specifically can be 2% (g/100ml) urea peroxide aqueous solution.Described nitrite ion B specifically can be 1% (g/100ml) tetramethyl biphenyl amine aqueous solution.
Described stop buffer specifically can be 0.2M aqueous sulfuric acid.
Arbitrary described kit all can be used for detecting aflatoxins above.
Arbitrary described kit all can be used for detecting in sample to be tested, whether to contain aflatoxins above.
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, described absorption of sample pad, described collaurum pad, described reaction film and described adsorptive pads are linked in sequence successively, the end of absorption of sample pad is connected with the top of collaurum pad, the end of collaurum pad is connected with the top of reaction film, and the end of reaction film is connected with the top of adsorptive pads;
On described collaurum pad, be coated with the monoclonal antibody of colloid gold label; Described monoclonal antibody is the monoclonal antibody of aspergillus flavus resisting toxin monoclone antibody hybridoma 2A1 secretion;
On described reaction film, have detection zone and Quality Control district, detection zone (T line) is and the axial vertical ribbon of described test paper with Quality Control district (C line); Detection zone is positioned at the side near collaurum pad end; Quality Control district is positioned at the side away from collaurum pad end; Detection zone is coated with the conjugate (coating antigen) of compound shown in formula (I) and carrier protein, and the coated sheep anti mouse two in Quality Control district is anti-.
Described absorption of sample pad is cellulose filter membrane.Described collaurum pad is the glass fibre membrane that is coated with the described monoclonal antibody of colloid gold label.Described reaction film is nitrocellulose filter (NC film).Described adsorptive pads is thieving paper.
Described sample well is positioned at one end away from collaurum pad end in sample absorbent.
Described colloidal gold strip can be used for detecting aflatoxins.
Described colloidal gold strip is for detection of whether containing aflatoxins in sample to be tested.
Arbitrary described aflatoxins can be aflatoxins M above 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 that specificity is high, highly sensitive, degree of accuracy is high, accuracy high.Kit of the present invention, test strips and detection method require low to the pre-treatment of sample, sample pretreatment process is simple, simultaneously fast detecting batch samples.That kit of the present invention and test strips (colloidal gold test paper card) have advantages of is highly sensitive, accuracy is high, precision is high, cost is low, simple to operate, detection time is short, be applicable to various units uses, stores simple, long shelf-life, fast detecting batch samples, can realize on-the-spot high flux fast detecting simultaneously.Kit provided by the invention and test strips, be applicable to measure the residual quantity of aflatoxins in liquid milk, milk powder and 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.
Accompanying drawing explanation
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 in following embodiment, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.PBS damping fluid used in embodiment, if no special instructions, is the PBS damping fluid of pH7.4,0.01M.In embodiment, carbonate buffer solution used is the sodium carbonate buffer of pH9.6,0.05mol/L.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)
To hydrazino-benzoic acid, suc as formula shown in (IV), molecular weight is 152.15.
Figure BDA0000138057080000042
Formula (IV)
DMF (DMF) is suc as formula shown in (V).
Figure BDA0000138057080000043
Formula (V)
N-hydroxy-succinamide (NHS) is suc as formula shown in (VI).
Figure BDA0000138057080000051
Formula (VI)
1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is suc as formula shown in (VII).
Figure BDA0000138057080000052
Formula (VII)
Embodiment 1, prepare aflatoxins M 1haptens
One, aflatoxins M 1haptenic preparation
By 10mg aflatoxins M 1be dissolved in 5ml anhydrous pyridine, add hydrazino-benzoic acid 10mg, 70 ℃ of heating return stirring reaction 5h, revolve to steam and remove pyridine, residue is dissolved with 1mL methyl alcohol, take methylene chloride: methyl alcohol (volume ratio 9: 1), as developping agent, by thin-layer chromatography (TLC) separation and purification, is collected Rf value (R fvalue) be 0.35 sample, be aflatoxins M 1haptens (obtaining 8mg).
Two, aflatoxins M 1haptenic sign
Product prepared by step 1 carries out ultimate analysis, and result is as follows:
C：62.32；H：3.94；N：6.08；0：27.66。
Result shows, product prepared by step 1 is compound shown in formula (I), is called again M1-HBA.
Figure BDA0000138057080000053
Formula (I).
Embodiment 2, aflatoxins M 1the Preparation and characterization of artificial antigen
One, aflatoxins M 1immunogenic synthetic and sign
1, aflatoxins M 1immunogenic synthetic
(1) shown in the formula (I) 5mg embodiment 1 being prepared, compound is dissolved in 1mL N, in N '-dimethylformamide, add 4mg N-hydroxy-succinamide and 4mg1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, room temperature lower magnetic force stirs 2h, obtains solution I.
(2) 30mg bovine serum albumin(BSA) is added in 3mL PBS damping fluid, fully dissolve, be solution II.
(3) solution I is added in solution II, enter bag filter after slowly stirring 24h, 4 ℃ of dialysis 72h (water is changed 6 times in centre) in physiological saline, then, under 4 ℃ of conditions, the centrifugal 30min of 8000rmp, gets supernatant, i.e. aflatoxins M 1immunogen solution, is sub-packed in 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) after M1-BSA solution is diluted with PBS damping fluid, measure the spectrophotometric value of 280nm and 260nm, press formula and calculate the protein concentration in dilution, be the M1-BSA concentration in former M1-BSA solution after the protein concentration value recording is multiplied by its extension rate.Protein concentration (mg/ml)=1.45 × OD 280-0.74 × OD 260.M1-BSA concentration in M1-BSA solution is 6.1mg/ml.
2, aflatoxins M 1immunogenic sign
By PBS damping fluid dilution (concentration that makes M1-BSA is 5mg/mL) for M1-BSA solution, as solution first; Using the PBS damping fluid containing 5mg/mL M1-HBA as solution second; Using the PBS damping fluid containing 5mg/mL BSA as solution third.Respectively solution first, solution second and solution third are carried out to ultraviolet (200-380nm) spectral scan, uv scan the results are shown in Figure 1.There is significant change in the uv-spectrogram of solution first compared with solution third, and compound and BSA success coupling are 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 maximum absorption wave long value, and C is solution concentration, the thickness that L is liquid layer).
Adopt respectively the maximum absorption wave long value of solution second and solution third to carry out uv scan to solution first, and according to this compound of extinction coefficient backwards calculation of this compound having calculated the concentration in solution first, obtain the volumetric molar concentration of this compound divided by molecular weight with concentration value, calculate coupling ratio, shown in formula (I), the coupling ratio of compound and BSA is 8: 1, and compound shown in 8 formulas (I) is in conjunction with 1 BSA.
Two, aflatoxins M 1the Preparation and characterization of coating antigen
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 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.
Shown in formula (I), the coupling ratio of compound and OVA is 10: 1, and compound shown in 10 formulas (I) is in conjunction with 1 OVA.
The preparation of embodiment 3, aflatoxins monoclonal antibody
Balb/c mouse: be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.;
SP2/0 myeloma cell: purchased from Sigma-Aldrich company, catalog number is 08060101.
One, animal immune
M1-BSA solution immunity Balb/c mouse prepared by embodiment 2, every mouse single immunization 100 μ gM1-BSA, immunity 4 times altogether, every minor tick two weeks, the immunization ways of first three time is the subcutaneous multi-point injection of nape portion, the immunization ways of latter three times is 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 to carry out cloning to 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's common micro-organisms center (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on February 15th, 2012, and preserving number is CGMCC No.5779.
Three, cell cryopreservation and recovery
Hybridoma 2A1 is made to 1 × 10 with cryopreserving liquid 6the cell suspension of individual/ml is preserved for a long time in liquid nitrogen.When recovery, take out cryopreservation tube, put into immediately 37 ℃ of water-bath middling speeds and melt, after centrifugal removal cryopreserving liquid, move in culture flask and cultivate.
Four, the preparation and purification of monoclonal antibody
1, increment cultivation
The preparation method of cell culture medium (7.4): add calf serum and sodium bicarbonate in RPMI-1640 nutrient culture media, the final concentration of calf serum is 20% (quality percentage composition), and the final concentration of sodium bicarbonate is 0.2% (quality percentage composition).
Hybridoma 2A1 is placed in to cell culture medium, cultivates 2 days for 37 ℃, by sad-saturated ammonium sulfate method, the nutrient solution obtaining is carried out to purifying, obtain monoclonal antibody solution (20 ℃ of preservations).
Protein concentration (mg/ml)=1.45 × OD in monoclonal antibody 280-0.74 × OD 260.
Adopting above formula to calculate the protein concentration in monoclonal antibody, is 18.9mg/ml.
2, ascites preparation
Balb/c mouse peritoneal injection sterilizing paraffin oil (0.4mL/ only).7 days pneumoretroperitoneum injection hybridoma cell strains (5 × 10 5individual/only).After 7 days, gather ascites, carry out purifying, ℃ preservation of ascites-20 after purifying by sad-saturated ammonium sulfate method.
Five, the evaluation of monoclonal antibody
1 of the step 4 monoclonal antibody solution obtaining is identified respectively as follows:
1, the mensuration of antibody titer
(1) adopt M1-OVA solution (adopting carbonate buffer solution to regulate concentration) prepared by embodiment 2 to be coated with 100 μ L/ holes; The coated concentration of M1-OVA is 1.0 μ g/mL.
Hatch 16 hours for (2) 4 ℃.
(3) seal and wash plate.
(4) every hole adds 1 monoclonal antibody solution obtaining or its dilution (adopting PBS damping fluid to carry out gradient dilution) of 100 μ L step 4.
(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 nitrite ion, lucifuge colour developing 15min.
(9) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD 450value.
While reaching 1.0 left and right with OD value, be judged to be the positive.Antibody titer is 1: 320000.
2, the calculating of monoclonal antibody sensitivity
(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 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; The hole in contrast, hole of PBS damping fluid will only be added); Each concentration arranges 3 multiple holes.
(5) every hole adds 1 monoclonal antibody solution obtaining of 50 μ L step 4.
(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 nitrite ion, lucifuge colour developing 15min.
(10) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD 450value.
The light absorption value that adopts the standard solution of each concentration to obtain (mean values in three multiple holes) is multiplied by 100 as ordinate again divided by the light absorption value of control wells, take the natural logarithm value of the aflatoxins M1 concentration (μ g/L) in each standard solution as horizontal ordinate curve plotting figure, see Fig. 2.
Contrast Fig. 2, obtains Y value and equals the aflatoxins M1 concentration (μ g/L) of 50% correspondence, is IC50 value.The sensitivity (IC50 value) that monoclonal antibody detects aflatoxins M1 is 0.05 μ g/L.
3, affinity costant is measured
(1) use M1-OVA as coating antigen coated elisa plate
Adopt M1-OVA solution (adopting carbonate buffer solution to regulate concentration) prepared by embodiment 2 to be coated with 100 μ L/ holes; The coated concentration of following OVA-SAL is set respectively: 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL, 0.125 μ g/mL.
Hatch 16 hours for (2) 4 ℃.
(3) seal and wash plate.
(4) every hole adds the dilution (diluting with PBS damping fluid) of 100 μ L monoclonal antibody solutions; Protein concentration in 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 nitrite ion, lucifuge colour developing 15min.
(9) every hole adds 100 μ L 2mol/L sulfuric acid stopped reactions; Read OD 450value.
Take the natural logarithm value of the protein concentration in monoclonal antibody (mol/L) as horizontal ordinate, make curve take its corresponding absorbance as ordinate.
Each antigen coated concentration obtains 1 S type curve, obtains altogether 4 S type curves.Find out the top of S curve, corresponding OD 450value is set as ODMAX.Find out respectively each the antibody concentration that curve 50%ODMAX is corresponding.Adopt 1 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 2.8 × 10 -12mol/L.Adopt 0.5 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 27.7 × 10 -12mol/L.Adopt 0.25 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 190.4 × 10 -12mol/L.Adopt 0.125 μ g/mL to be coated with concentration, the antibody concentration that 50%ODMAX is corresponding is 368.2 × 10 -12mol/L.
By one group between two 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 formula, n be every group in the multiple of two coated concentration, [Ab] t 1, [Ab] t 2be respectively two antibody concentration (mol/L) that 50%ODMAX is corresponding in every group.For example: 1 μ g/mL is coated with concentration 50%OD 450corresponding antibody concentration is 2.8 × 10 -12mol/L, the coated concentration 50%OD of coated 0.5 μ g/mL 450corresponding antibody concentration is 27.7 × 10 -12mol/L, Ka=(2-1)/2 (2 × 27.7 × 10 -12-2.8 × 10 -12)=9.5 × 10 9m -1.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
By 30 generations of external hybridoma 2A1 continuous biography, get culture supernatant every 5 generations and carry out ELISA mensuration (method is with 1 of step 5).Result shows tiring without significant difference of continuous 30 generations, and hybridoma 2A1 can stablize and goes down to posterity.
The preparation of embodiment 4, aflatoxins polyclonal antibody
New zealand white rabbit: be purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
Aflatoxins M by new zealand white rabbit with preparation in 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 BSA amount), immunity in every three weeks once, when immunity for the first time by aflatoxins M 1immunogene and Freund's complete adjuvant are mixed and made into emulsifying agent and carry out immunity, and six immunity of for the second time to the are by aflatoxins M 1immunogene and incomplete Freund's adjuvant are mixed and made into emulsifying agent and carry out immunity, and the 7th immunity is only by aflatoxins M 1immunogene is carried out immunity, and the 7th immunity be 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: every 1 liter of cleansing solution is prepared and obtained as follows: 10ml polysorbas20,5g sodium azide and 990ml phosphate buffer (pH7.4,0.01M) are mixed, obtain cleansing solution.
2, the ELISA Plate of coated M1-OVA
The M1-OVA solution dilution (concentration that makes M1-OVA is 0.5 μ g/mL) of with carbonate buffer solution being prepared by embodiment 2, is coating buffer; Coating buffer is added to 96 hole polystyrene ELISA Plate (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, pats dry; Then in every hole, add 200 μ L confining liquids, 37 ℃ of incubation 2h, liquid in the hole of inclining, dry rear with aluminium film vacuum seal preservation.
Every 1 liter of described confining liquid is prepared as follows: by 5ml horse serum, 1g sodium azide and 30g for casein phosphate buffer (pH7.2,0.02M) dissolve 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: 1 of the step 4 of embodiment 3 monoclonal antibody solution obtaining is diluted 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 purchased from Sigma-Aldrich company of the U.S., 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.In 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.
Sample diluting liquid is as aflatoxins M 1concentration is the standard solution (0 standard) of 0 μ g/L.
7, substrate nitrite ion
Mixed by A liquid and B liquid equal-volume, A liquid is 2% (g/100ml) urea peroxide aqueous solution, and B liquid is 1% (g/100ml) tetramethyl biphenyl amine aqueous solution.
8, stop buffer: 0.2M aqueous sulfuric acid.
Two, kit test method
The kit that adopts step 1 to prepare detects as follows:
(1) sample pre-treatments
When detection sample is liquid milk or recombined milk: get sample to be checked in centrifuge tube, fully balance, to room temperature, adds sample diluting liquid, fully, after whirling motion, get 70 μ L as sample to be tested solution.
When detection sample is milk powder: take powdered milk sample in centrifuge tube, add deionized water, violent whirling motion to milk powder dissolves completely, adds sample diluting liquid, fully, after whirling motion, gets 70 μ L as sample to be tested solution.
Detection sample is Yoghourt: take Yoghourt 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 of coated M1-OVA, add standard solution (70 μ L/ holes; Each standard solution arranges three multiple holes), then add antibody working fluid (30 μ L/ hole), with cover plate film shrouding, in 37 ℃ of constant temperature ovens, react 30min; Pour out liquid in hole, wash 5 times (step of each washing is: every hole adds 250 μ L cleansing solutions, pours out liquid in hole after 30s), pat dry with thieving paper; Add ELIAS secondary antibody working fluid (100 μ L/ hole), in 37 ℃ of constant temperature ovens, react 30min, pour out liquid in hole, wash 5 times (step is the same); Every hole adds substrate nitrite ion 100 μ L, and vibration mixes gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min; Every hole adds stop buffer 50 μ L, and vibration mixes gently, measures every hole absorbance (OD630 and OD450 dual wavelength, the 450th, predominant wavelength, the 630th, reference wavelength) by microplate reader.
Be multiplied by again 100% divided by the absorbance (B0) of first standard solution (0 standard) with the absorbance mean value (B) of the standard solution of each concentration, obtain percentage absorbance.Use Originpro 7.0 softwares to analyze data result, take the natural logarithm value of standard items concentration (μ g/L) as X-axis, percentage absorbance is that Y-axis simulates typical curve.Typical curve is shown in Fig. 3.
2, the mensuration of aflatoxins concentration in sample
In the ELISA Plate of coated M1-OVA, add sample to be tested solution or its dilution (70 μ L/ holes; Three multiple holes are set), then add antibody working fluid (30 μ L/ hole), with cover plate film shrouding, in 37 ℃ of constant temperature ovens, react 30min; Pour out liquid in hole, wash 5 times (step is the same), pat dry with thieving paper; Add ELIAS secondary antibody working fluid (100 μ L/ hole), in 37 ℃ of constant temperature ovens, react 30min, wash 5 times (step is the same); Every hole adds substrate nitrite ion 100 μ L, and vibration mixes gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min; Every hole adds salt stop buffer 50 μ L, and vibration mixes gently, measures every hole absorbance (OD630 and OD450 dual wavelength, the 450th, predominant wavelength, the 630th, reference wavelength) by microplate reader.
Result judgement: be multiplied by again 100% divided by the absorbance (B0) of first standard solution (0 standard) with the absorbance mean value (B) of each sample to be tested solution, obtain percentage absorbance.The percentage absorbance of corresponding each sample to be tested solution, can read aflatoxins M from typical curve 1concentration value, then be multiplied by the extension rate of respective sample, converse aflatoxins M in sample to be tested solution 1content.
Three, kit detects effect assessment
(1) accuracy and precision test
To not adding aflatoxins M containing in the milk sample of aflatoxins 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; By 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, 3 kits of each extraction detect, and detection method is described in (twos') of step 22, and each experiment repeats 5 times, according to aflatoxins M in sample to be tested solution 1cubage detect aflatoxins M in sample 1content, the results are shown in Table 1.
Each kit of table 1 application detects aflatoxins M in the milk sample drawing 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
Calculate recovery rate and the coefficient of variation, the results are shown in Table 2 respectively.
The aflatoxins M that the recovery=application kit detection computations goes out 1content ÷ milk in the actual aflatoxins M adding 1content × 100%.
The coefficient of variation (the CV)=standard deviation of measurement result and the number percent of its mean value.
The computing method of plate within variance coefficient: the coefficient of variation of certain sample (being generally medium level) replication number of times acquired results in plate within variance coefficient=same same plate of once measuring.
The computing method of variation within batch coefficient: the coefficient of variation of each parallel samples in variation within batch coefficient=same once mensuration.
The computing method of interassay coefficient of variation: interassay coefficient of variation=same sample, in the coefficient of variation of different batches measurement result, is got its mean value.
Table 2 recovery and coefficient of variation result
Result shows: the recovery of all samples is 79.3%～94.5%, and variation within batch coefficient is 4.3%～8.6%, and interassay coefficient of variation is 12.8%～15.1%.
(2) kit storage life
Kit preservation condition is 2-8 ℃, through the mensuration of 12 months, and the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, aflatoxins M 1add practical measurement value all within normal range.Consider in transportation and use procedure, have improper preservation condition and occur, kit is placed 8 days under the condition of 37 ℃ of preservations, carry out accelerated deterioration experiment, result shows that the indices of this kit meets the requirements completely.Consider that the freezing situation of kit occurs, kit is put into-20 ℃ of refrigerator freezings 8 days, measurement result also shows that kit indices is completely normal.Can show that from above result kit can at least can preserve more than 12 months at 2-8 ℃.
(3) cross reacting rate test
To adding analogue standard solution in the ELISA Plate of coated M1-OVA, (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; The hole in contrast, hole of PBS damping fluid will only be added; 50 μ L/ holes; Each concentration arranges 3 multiple holes), then add antibody working fluid (50 μ L/ hole), with cover plate film shrouding, in 37 ℃ of constant temperature ovens, react 30min; Pour out liquid in hole, wash 5 times (step is the same), pat dry with thieving paper; Add ELIAS secondary antibody working fluid (100 μ L/ hole), in 37 ℃ of constant temperature ovens, react 30min, wash 5 times (step is the same); Every hole adds substrate nitrite ion 100 μ L, and vibration mixes gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min; Every hole adds stop buffer 50 μ L, and vibration mixes gently, measures every hole absorbance (OD630 and OD450 dual wavelength, the 450th, predominant wavelength, the 630th, reference wavelength) by microplate reader.
Cross reacting rate with following formula calculating kit to other analogue.
Figure BDA0000138057080000141
The results are shown in Table 3.
The specificity of table 3 kit
Analogue title Purchased 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 Approximately 94%
Aflatoxins B 1 FERMENTEK company; Catalog number is FER001 Approximately 18.7%
Aflatoxins B 2 FERMENTEK company; Catalog number is FER002 Approximately 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 2there is respectively 18.7% and 5.4% cross reaction, with other structure analogue no cross reactions.
Test strips and the preparation and application thereof of embodiment 6, detection aflatoxins
One, the structure of test strips
Described 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 is connected with the top of collaurum pad, and the end of collaurum pad is connected with the top of reaction film, and the end of reaction film is connected with the top of adsorptive pads;
On described collaurum pad, be coated with 1 monoclonal antibody obtaining of the step 4 of the embodiment 3 of colloid gold label;
On described reaction film, have detection zone and Quality Control district, detection zone (T line) is and the axial vertical ribbon of test strips with Quality Control district (C line); Detection zone is positioned at the side near collaurum pad end; Quality Control district is positioned at the side away from collaurum pad end; M1-OVA prepared by the coated embodiment 2 in detection zone, the coated sheep anti mouse two in Quality Control district is anti-.
Sample well is positioned at one end away from collaurum pad end in sample absorbent.
Two, the preparation of test strips
(1) colloidal gold labeled monoclonal antibody
1. the preparation of colloidal gold solution
Get 0.01% aqueous solution of chloraurate 100mL thermostatic electromagnetic stirrer and be heated to boiling, in the situation that continues to stir, add 1% trisodium citrate aqueous solution 2.5mL, continue agitating heating 20min, solution is bright redness.Room temperature is cooling, returns to original volume, 2-8 ℃ of preservation with deionized water.
2. the preparation of golden labeling antibody solution
Use 0.1mol/L K 2cO 3aqueous solution regulates the pH to 8.2 of colloidal gold solution, then getting 10mL adds in 50mL beaker, magnetic stirrer 250r/min stirs, dropwise add 1 monoclonal antibody solution obtaining (containing 0.35mg protein) of the step 4 of embodiment 3, dropwise add 3mL 5g/100mL BSA aqueous solution, continue to stir 10min.
3. by the centrifugal 20min of golden labeling antibody solution normal temperature low speed (1500r/min), discard the precipitation being formed by the gold grain condensing, get red supernatant solution.
4. by 4 ℃ of step solution 3., the centrifugal 40min of 11000r/min, solution is divided into three layers (gold grain layers of black densification on transparent supernatant, the flowable kermesinus precipitation in the pipe end and pipe diapire), flowable kermesinus precipitation is transferred in another one centrifuge tube, with the volume that is suspended to former golden labeling antibody solution containing the 0.01mol/L phosphate buffer of 1g/100mL BSA, spend the night, 4 ℃, the centrifugal 40min of 11000r/min, collecting precipitation.
5. with containing the 0.01mol/L phosphate buffer of 1g/100mL BSA and 0.02g/100mL NaN3, step precipitation is 4. 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 glass fibre membrane, makes collaurum pad.
(3) spray film: the T line position on reaction film is sprayed M1-OVA solution, C line position prepared by embodiment 2 and 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 according to a conventional method, then slitting, test strips is packed in plastics fabrication, form test card.
Three, detect with test card
(1) sample pre-treatments and detection
Detection sample is liquid milk; Get liquid milk that 3mL mixes in centrifuge tube, add 3mL ethyl acetate, mix rear standing 2-3min; The centrifugal 10min of 4000g, gets 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, behind Kaifeng, lie against desktop, draw sample to be tested solution and dropwise add 4 in sample well; 5-10min judged result, the judged result after 15min is invalid.
Result criterion:
Negative: the colour developing of C line, T line naked eyes are visible, and no matter shade is all judged to feminine gender;
Positive: the colour developing of C line, T line does not develop the color, and is judged to the positive;
Invalid: C line does not develop the color, no matter whether T line develops the color, and it is invalid that this test card is all judged to.
Four, the effect of test card
(1) false positive rate and false negative rate
The negative milk of the confirmation of learning from else's experience is (containing aflatoxins M 1< 0.5 μ g/L) 50 parts, the positive milk of the confirmation of learning from else's experience is (containing aflatoxins M 1>=0.5 μ g/L) 50 parts.Sample is detected with test card respectively, calculate false positive rate and false positive rate.
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 are measured, test card detects 0 part of negative sample, and false negative rate is 0%.
(2) test card storage life
Stability test result shows, this test card can be preserved 1 year at shady and cool dry place under 2-8 ℃ or room temperature condition.

Claims (6)

1. detect an enzyme linked immunological kit for aflatoxins, comprise the monoclonal antibody of aspergillus flavus resisting toxin monoclone antibody hybridoma 2A1 secretion; The deposit number of described aspergillus flavus resisting toxin monoclone antibody hybridoma 2A1 is CGMCC No.5779; Described aflatoxins is aflatoxins M 1or aflatoxins M 2;
Described kit is following 1) to 4) in any one:
1) comprise the kit of compound shown in formula (I) and the conjugate of carrier protein, described monoclonal antibody and enzyme labeling antiantibody; Wherein, described conjugate is as coating antigen;
2) comprise the kit of the enzyme labeling thing of compound shown in formula (I), described monoclonal antibody and antiantibody; Wherein, described antiantibody is as coating antigen;
3) comprise the enzyme labeling thing of compound shown in formula (I) and the kit of described monoclonal antibody; Wherein, described monoclonal antibody is as coating antigen;
4) comprise the kit of compound shown in formula (I) and the conjugate of carrier protein and the enzyme labeling thing of described monoclonal antibody; Wherein, described conjugate is as coating antigen;
Figure FDA0000486117990000011
Formula (I).
2. kit as claimed in claim 1, is characterized in that: described carrier protein is ovalbumin or bovine serum albumin(BSA).
3. kit as claimed in claim 1 or 2, is characterized in that: described kit also comprises at least one in cleansing solution, sample concentration liquid, substrate nitrite ion and stop buffer.
4. detect a colloidal gold strip for aflatoxins, formed by absorption of sample pad, collaurum pad, reaction film and adsorptive pads;
Along test strips axially, described absorption of sample pad, described collaurum pad, described reaction film and described adsorptive pads are linked in sequence successively, the end of absorption of sample pad is connected with the top of collaurum pad, the end of collaurum pad is connected with the top of reaction film, and the end of reaction film is connected with the top of adsorptive pads;
On described collaurum pad, be coated with the monoclonal antibody of colloid gold label; Described monoclonal antibody is the monoclonal antibody of aspergillus flavus resisting toxin monoclone antibody hybridoma 2A1 secretion; The deposit number of described aspergillus flavus resisting toxin monoclone antibody hybridoma 2A1 is CGMCC No.5779;
On described reaction film, have detection zone and Quality Control district, detection zone is and the axial vertical ribbon of described test paper with Quality Control district; Detection zone is positioned at the side near collaurum pad end; Quality Control district is positioned at the side away from collaurum pad end; Detection zone is coated with the conjugate of compound shown in formula (I) and carrier protein, and the coated sheep anti mouse two in Quality Control district is anti-;
Described aflatoxins is aflatoxins M 1or aflatoxins M 2.
5. arbitrary described kit in claims 1 to 3, or, colloidal gold strip described in claim 4, the application in inspection aflatoxins; Described aflatoxins is aflatoxins M 1or aflatoxins M 2.
6. arbitrary described kit in claims 1 to 3, or whether colloidal gold strip described in claim 4, contain the application in aflatoxins detecting sample to be tested; Described aflatoxins is aflatoxins M 1or aflatoxins M 2.
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