CN101393207A - Time-resolved fluorescence immunoassay kit of deoxynivalenol and detecting method thereof - Google Patents
Time-resolved fluorescence immunoassay kit of deoxynivalenol and detecting method thereof Download PDFInfo
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- CN101393207A CN101393207A CN 200810155490 CN200810155490A CN101393207A CN 101393207 A CN101393207 A CN 101393207A CN 200810155490 CN200810155490 CN 200810155490 CN 200810155490 A CN200810155490 A CN 200810155490A CN 101393207 A CN101393207 A CN 101393207A
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Abstract
The invention discloses a time-resolved fluoroimmunoassay reagent kit for deoxynivalenol (DON) and a detection method thereof, belonging to the technical field of time-resolved fluoroimmunoassay (TRFIA). The reagent kit adopts the TRFIA to detect the DON, and the basis of the measurement is a labeled immune reaction. A microporosity plate is enveloped with DON-BSA, a DON standard or sample is added, and then a DON antibody is added. The dissociative DON competes with the DON-BSA on the microporosity plate for the DON antibody, the unligated DON antibody is washed for removal, an EU<3+>-goat anti-rabbit antibody is added, and the unligated EU<3+>-goat anti-rabbit antibody is washed for removal after the labeled immune reaction. After an enhancement solution is added, a time-resolved fluorometer is used to determine the fluorescence intensity cps, the fluorescence intensity is inversely proportional to the DON concentration of the sample, and the content of the DON in the sample can be determined by contrasting with the standard curve. The reagent kit for detecting the DON has the advantages of simple structure, convenient use, low cost and high sensitivity, and can be used to detect the content of the DON in feedstuff, corn, grain and products thereof.
Description
Technical field
A kind of time resolved fluoro-immunoassay kit and detection method thereof of deoxynivalenol enol, belong to time resolved fluoro-immunoassay (TRFIA) technical field, be used for to feed the detection of deoxynivalenol enol in cereal and the goods thereof (being called for short DON) content.
Background technology
(deoxynivalenol DON) has another name called vomitoxin to deoxynivalenol, is a kind of trichothecene family toxin, is mainly produced by some sickle-like bacteria.DON extensively is present in the crops such as wheat, barley, corn.At present, DON pollution cereal occupies first of the various biotoxins in the world wide, is more serious situation.China 2006-2007 feed and raw material mycotoxin contamination survey report show: the vomitoxin recall rate is 99.1% in tested whole samples, exceeding standard rate 53.5%.Occupy first of six kinds of detected toxin.
Mycotoxin is poisoned and is often shown as tangible region and seasonality, and clinical manifestation is comparatively complicated, and acute poisoning, slow poisoning and teratogenesis and mutagenesis etc. are arranged.1998, in the appraisal report that international cancer research institution announces, DON was listed in three class carcinogenic substances.DON is under a cloud may to be got in touch with having closely of cancer of the esophagus disease.Therefore in order to ensure people's health, the health of DON detection research is necessary in the conducting food.
DON pollutes cereal and mainly causes health hazard and economic loss.Various countries formulate the limit standard of DON in cereal and the goods thereof one after another, and wherein area such as European Union country is comparatively strict to limiting the quantity of of DON.Limiting the quantity of of DON is 1000 μ g/kg in China's wheat, the corn.
The DON detection technique mainly is divided into two classes: physics and chemistry detection method and immunodetection.The physics and chemistry detection method comprises thin-layered chromatography (TLC), vapor-phase chromatography (GC), high performance liquid chromatography (HPLC), and GC or HPLC and mass spectrometer (MS), electron capture detector (EDC), UV-detector joint detection methods such as (UV).TLC is used for DON the earliest and detects.Minimum detectability is 20～300ng/g.Because therefore TLC detection method poor repeatability, the strict sensitivity of shortage generally only are used for qualitative or half-quantitative detection.GC method minimum detectability can reach 5ng/g, and DON contains three hydroxyls can form stronger hydrogen bond, needs sample is carried out derivatization before detection, makes the derivatization product have volatility, just can carry out the GC method then and detect; The GC method detection coefficient of variation is big in addition.Though HPLC has solved the derivatization problem of GC, it and GC method exist equally and need expensive instrument, and complex operation is treated sample product pre-service requirement height, uses multiple toxic organic solvent, detect shortcomings such as cost height.Immunodetection is mainly enzyme linked immunosorbent detection method (ELISA), and minimum detectability can reach 0.3～1ng/g.However, ELISA exists the marker enzyme molecule bigger, reduces sensitivity and the precision that detects thereby activity is subject to Effect of Environmental.
Time resolved fluoro-immunoassay method (TRFIA) is the new immunoassay that last century, early eighties grew up.Its principle is to utilize the sequestrant with bifunctional group structure, one end and lanthanide series combination, and the free amino group on the other end and the antibody molecule connects, and makes EU
3+Antigen in the labelled antibody, it and testing sample is combined into immune complex.Ideally, the fluorescence intensity of measuring lanthanide series in the compound just can be determined the amount of antigen in the sample, but the fluorescence intensity of in fact this compound quite a little less than, have only and add a kind of enhancing solution (Enhancement solution) again, lanthanide series is disintegrated down from compound, and with strengthen β-naphthoyltrifluoroacetone contained in the liquid (β-NTA) form microcapsules again, the very strong fluorescence of emission under the exciting of light such as ultraviolet, up to a million times of enhancing effects.Differentiate luminoscope with the time and measure its fluorescence intensity cps, can determine the amount of antigen in the sample.
Summary of the invention
The object of the present invention is to provide kit and the detection method thereof of a kind of DON of detection, be used for detection feed, cereal and goods DON content thereof.
Technical scheme of the present invention: a kind of time resolved fluoro-immunoassay kit of deoxynivalenol enol, the deoxynivalenol enol is called for short DON, it is by wrapping by plate (1), damping fluid (2), DON standard (3), the antibody dried frozen aquatic products (4) of DON, the goat anti-rabbit antibody of europium mark (5), cleansing solution (6) and enhancing liquid (7) are formed.
Described kit wherein wraps by plate (1) bag by solid phase antigen, with the Na of 50mmol/L pH9.6
2CO
3-NaHCO
3Damping fluid is diluted to 0.5mg/L as coating buffer with DON-BSA, and the every hole of microwell plate adds 100 μ L, and 4 ℃ of placements are spent the night; Discard coating buffer, with cleansing solution (6) washed twice; Every then hole adds the sealing of 200 μ L gelatin, places 2 hours for 37 ℃; Discard confining liquid, vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing.
Described kit, DON standard (3) is wherein diluted from the pure product of DON and is obtained, and dilution is the PBS of 0.01mmol/L, pH 7.4, totally 6 bottles of DON standards, every bottle DON concentration is respectively: 0ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, 500ng/mL.
Described kit, wherein the goat anti-rabbit antibody of europium mark (5) with the goat anti-rabbit antibody of buying, to pH 9.0, is collected protein peak through the conversion buffered condition of PD-10 post, gets switched goat anti-rabbit antibody and adds Eu
3+-N
2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl, reaction is spent the night, and reactant liquor is through column chromatography, collects protein peak, and dilution, packing are standby.
Described kit, wherein damping fluid (2): 8mmol/L NaCl, mass concentration 0.1% BSA, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s, 0.1mL/L Tween-80 and mass concentration 0.1% NaN
3The Tris-HCl of 50mmol/L pH7.8; Cleansing solution (6): 14.5mmol/L NaCl, 0.2mL/LTween-80 and mass concentration 0.2% NaN
3The Tris-HCl of 50mmol/L pH7.8; Strengthen liquid (7): every liter of pH 3.2 Potassium Hydrogen Phthalate damping fluids that contain 15 μ mol β-naphthoyltrifluoroacetones, 50 μ mol trioctyl-phosphine oxide and 1mL triton x-100.
Carry out the detection method of deoxynivalenol enol with described kit, it is operating as: get be coated with DON-BSA the micropore bag by plate, add the DON standard of 50 μ L or the sample handled well in micropore separately; Add the DON antibody of 50 μ L with damping fluid (2) dilutions in 1: 20,37 ℃ vibrated 1 hour, and it is inferior to give a baby a bath on the third day after its birth with cleansing solution (6); The goat anti-rabbit antibody of 100 μ L europium marks of damping fluid (2) dilutions in 1: 20 in addition, 37 ℃ of vibrations 0.5 hour are washed six times with cleansing solution (6); Add 200 μ L and strengthen liquid, vibrate and measure fluorescence intensity cps after 5 minutes, to add the detection data drawing standard curve of deoxynivalenol enol standard, to add the deoxynivalenol enol content of detection data from the typical curve calculation sample of sample.
The detection method of described deoxynivalenol enol, its sample preparation: the sample preparation of feed, cereal and goods thereof: sample is crushed to 20 orders, getting 5 gram samples is placed in the test tube, add 25mL distilled water, jump a queue and vibrated 3 minutes, filter, filter paper adopts No. 1 paper of Xinhua, get 1mL filtrate and dilute redundant detection with 1mL distilled water or deionized water; Beer sample is handled: direct sample detects or dilutes the standby detection in back in proportion with distilled water.
Beneficial effect of the present invention: detection DON kit provided by the invention is simple in structure, easy to use, cheap, highly sensitive, is used for the detection to feed, corn, cereal and goods DON content thereof.
Description of drawings
Fig. 1: the TRFIA kit synoptic diagram that detects DON.1, bag is by plate, and 2, damping fluid, 3, the DON standard, 4, the antibody dried frozen aquatic products of DON, 5, the goat anti-rabbit antibody of europium mark, 6, cleansing solution, 7, strengthen liquid.
Fig. 2: DON-TRFIA canonical plotting.
Specific embodiments
EU
3+The preparation of-goat anti-rabbit antibody:
Get the PBS 5g/L that is dissolved in 50mmol/L pH7.0, goat anti-rabbit antibody 1-2mL, to pH9.0, eluent is the Na that contains the 50mmol/L pH9.0 of 0.155mol/L NaCl through the conversion buffered condition of PD-10 post
2CO
3-NaHCO
3Damping fluid.Collect protein peak, through the quantitative (1.46A of uv absorption analysis
280-0.74A
260), dilute goat anti-rabbit antibody to 2g/L with above-mentioned eluent.The goat anti-rabbit antibody of getting after 500-1000 μ L dilutes adds the Eu that contains 0.2-0.4mg
3+-N
2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl (Eu
3+In-DTTA) the bottle, 30 ℃ of magnetic agitation reactions 20 hours.Sepharose CL-6B post (1 * 40cm) chromatography, the A of the Tris-HCl damping fluid balance of reactant liquor through using 80mmol/LpH7.8
280Protein peak is collected in monitoring, and the dilution packing is standby.
Bag is prepared by the plate solid phase antigen:
With the Na of DON-BSA with 50mmol/L pH 9.6
2CO
3-NaHCO
3Damping fluid is diluted to the coating buffer of 0.5mg/L, and 96 each hole of hole microwell plate add 100 μ L, and 4 ℃ of placements are spent the night; Discard coating buffer, with cleansing solution flushing twice, every hole adds the sealing of 200 μ L gelatin, places 2 hours for 37 ℃; Discard confining liquid, vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing.
The preparation of reagent:
(1) DON standard: (0ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL 500ng/mL), dilutes from the pure product of DON and obtains, and dilution is PBS (0.01mmol/L, pH7.4).
(2) damping fluid: contain 8mmol/L NaCl, 0.1%BSA, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s (DTPA), 0.1ml/L Tween-80 and 0.1%NaN
3The Tris-HCl of 50mmol/L pH7.8.
(3) cleansing solution: contain 14.5mmol/L NaCl, 0.2ml/L Tween-80 and 0.2%NaN
3The Tris-HCl of 50mmol/L pH 7.8.
(4) strengthen the preparation of liquid: every L contains 15 μ mol β-naphthoyltrifluoroacetones (β-NTA), 50 μ mol trioctyl-phosphine oxide (TOPO), the Potassium Hydrogen Phthalate damping fluid of the pH 3.2 of 1mL triton x-100 (Triton X-100).
The composition of kit:
(1), 1 * 96 orifice plate (8 * 12 hole can be split as single hole) is coated with DON-BSA.
(2), 6 * DON titer, the 1.0mL/ bottle, concentration of standard solution is: 0,0.1,1,10,100,500ng/mL.
(3), 1 * DON antibody dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
(4), 1 * EU
3+-goat anti-rabbit antibody dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
(5), 1 * enhancing liquid: 15mL.
(6), 1 * cleansing solution: 30mL, the time spent is with distilled water 1: 25 dilution.
(7), 1 * damping fluid: 30mL.
Points for attention before measuring
1, uses before all reagent to be gone up to room temperature (18-30 ℃).
2, immediately all reagent are put back to 2-8 ℃ after the use.
If the hyperchannel pipettor is used in the big suggestion of 3 sample sizes.
4, hatch at all constant temperature and avoid irradiate light in the process, use the cap covers micropore.
5, taking-up needs microwell plate and the framework with quantity, no microwell plate is put in the former Fresco Bag and with the drying agent that provides reseal, and is stored in 2-8 ℃.
Concrete detection step is as follows:
Sample preparation: corn sample is crushed to 20 orders, gets 5 gram samples and be placed in the test tube, add 25mL distilled water.Jump a queue and vibrated 3 minutes, filter, filter paper adopts No. 1 paper of Xinhua.Get 1mL filtrate and dilute with 1mL distilled water or deionized water, standby.
Get the DON-BSA lath, the sample that adds the DON standard of 50 μ L or handle well is in micropore separately, each standard and sample must use new suction nozzle, DON antibody 50 μ L with damping fluid dilution in 1: 20, the pipettor tip must not touch the liquid of putting in the hole, 37 ℃ vibrated 1 hour, and cleansing solution is given a baby a bath on the third day after its birth inferior, with the EU of damping fluid dilution in 1: 20
3+-goat anti-rabbit antibody 100 μ L, 37 ℃ vibrated 0.5 hour, and cleansing solution is washed six times, adds 200 μ L and strengthens liquid vibration measurement after 5 minutes.DON content from the typical curve calculation sample.The results are shown in Table 1, trying to achieve the contained DON concentration of this example sample according to typical curve is 69.2ng/mL.
Table 1
Embodiment 2 preparation kits: with embodiment 1.
Bag is prepared by the plate solid phase antigen: with embodiment 1.
The preparation of reagent: with embodiment 1.
The reagent that kit provides
Reagent in each box enough carries out 48 measurements, and the material in the box is as follows:
(1), 1 * 48 orifice plate (4 * 12 hole can be split as single hole) is coated with DON-BSA.
(2), 6 * DON titer, the 1.0mL/ bottle, concentration of standard solution is: 0,0.1,1,10,100,1000ng/mL.
(3), 1 * DON antibody dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
(4), 1 * EU
3+-goat anti-rabbit antibody dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
(5), 1 * enhancing liquid: 15mL.
(6), 1 * cleansing solution: 30mL, the time spent is with distilled water 1: 25 dilution.
(7), 1 * damping fluid: 30mL.
The reagent that the laboratory should be provided for oneself is identical with embodiment 1.
Points for attention are with embodiment 1 before measuring.
The concrete step that detects is with embodiment 1.
Embodiment 3 wheat samples are measured
The reagent that kit provides is identical with embodiment 1, is used to detect wheat samples.
Concrete detection step is as follows:
Wheat samples is handled: wheat samples is crushed to 20 orders, gets 5 gram samples and be placed in the test tube, add 25mL distilled water.Jump a queue and vibrated 3 minutes, filter, filter paper adopts No. 1 paper of Xinhua.Get 1mL filtrate and dilute with 1mL distilled water or deionized water, standby.
Get the DON-BSA lath, the sample that adds the DON standard of 50 μ L or handle well is in micropore separately, each standard and sample must use new suction nozzle, DON antibody 50 μ L with damping fluid dilution in 1: 20, the pipettor tip must not touch the liquid of putting in the hole, 37 ℃ vibrated 1 hour, and cleansing solution is given a baby a bath on the third day after its birth inferior, with the EU of damping fluid dilution in 1: 20
3+-goat anti-rabbit antibody 100 μ L, 37 ℃ vibrated 0.5 hour, and cleansing solution is washed six times, adds 200 μ L and strengthens liquid vibration measurement after 5 minutes.DON content from the typical curve calculation sample.The results are shown in Table 2, trying to achieve the contained DON concentration of this example sample according to typical curve is 7.11ng/mL.
Table 2
Embodiment 4 beer samples are measured
The reagent that kit provides is identical with embodiment 1, is used to detect beer sample.
Concrete detection step is as follows:
Beer sample is handled: directly extract beer sample or with 5 times of dilutions of distilled water.
Get the DON-BSA lath, the sample that adds the DON standard of 50 μ L or handle well is in micropore separately, each standard and sample must use new suction nozzle, DON antibody 50 μ L with damping fluid dilution in 1: 20, the pipettor tip must not touch the liquid of putting in the hole, 37 ℃ vibrated 1 hour, and cleansing solution is given a baby a bath on the third day after its birth inferior, with the EU of damping fluid dilution in 1: 20
3+-goat anti-rabbit antibody 100 μ L, 37 ℃ vibrated 0.5 hour, and cleansing solution is washed six times, adds 200 μ L and strengthens liquid vibration measurement after 5 minutes.DON content from the typical curve calculation sample.The results are shown in Table 3, trying to achieve the contained DON concentration of this example sample according to typical curve is 2.67ng/mL.
Table 3
Claims (7)
1, a kind of time resolved fluoro-immunoassay kit of deoxynivalenol enol, the deoxynivalenol enol is called for short DON, it is characterized in that by bag by plate (1), damping fluid (2), DON standard (3), the antibody dried frozen aquatic products (4) of DON, the goat anti-rabbit antibody of europium mark (5), cleansing solution (6) and enhancing liquid (7) are formed.
2, kit according to claim 1 is characterized in that bag is wrapped by solid phase antigen by plate (1), with the Na of 50mmol/L pH 9.6
2CO
3-NaHCO
3Damping fluid is diluted to 0.5mg/L as coating buffer with DON-BSA, and the every hole of microwell plate adds 100 μ L, and 4 ℃ of placements are spent the night; Discard coating buffer, with cleansing solution (6) washed twice; Every then hole adds the sealing of 200 μ L gelatin, places 2 hours for 37 ℃; Discard confining liquid, vacuum is drained, rearmounted-20 ℃ of freezing preservations of lath sealing.
3, kit according to claim 1, it is characterized in that DON standard (3), dilute from the pure product of DON and obtain, dilution is the PBS of 0.01mmol/L, pH 7.4, totally 6 bottles of DON standards, every bottle DON concentration is respectively: 0ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, 500ng/mL.
4, kit according to claim 1 is characterized in that the goat anti-rabbit antibody (5) of europium mark, with the goat anti-rabbit antibody of buying, to pH 9.0, collects protein peak through the conversion buffered condition of PD-10 post, gets switched goat anti-rabbit antibody and adds Eu
3+-N
2-[p-isocyanic acid-benzyl]-diethylenetriamine tetraacethyl, reaction is spent the night, and reactant liquor is through column chromatography, collects protein peak, and dilution, packing are standby.
5, kit according to claim 1 is characterized in that damping fluid (2): 8mmol/L NaCl, mass concentration 0.1% BSA, 50 μ mol/L diethylene triamine pentacetic acid (DTPA)s, 0.1mL/L Tween-80 and mass concentration 0.1% NaN
3The Tris-HCl of 50mmol/L pH 7.8; Cleansing solution (6): 14.5mmol/LNaCl, 0.2mL/L Tween-80 and mass concentration 0.2% NaN
3The Tris-HCl of 50mmol/L pH 7.8; Strengthen liquid (7): every liter of pH 3.2 Potassium Hydrogen Phthalate damping fluids that contain 15 μ mol β-naphthoyltrifluoroacetones, 50 μ mol trioctyl-phosphine oxide and 1mL triton x-100.
6, carry out the detection method of deoxynivalenol enol with the described kit of claim 1, it is characterized in that being operating as: get be coated with DON-BSA the micropore bag by plate, add the DON standard of 50 μ L or the sample handled well in micropore separately; Add the DON antibody of 50 μ L with damping fluid (2) dilutions in 1: 20,37 ℃ vibrated 1 hour, and it is inferior to give a baby a bath on the third day after its birth with cleansing solution (6); The goat anti-rabbit antibody of 100 μ L europium marks of damping fluid (2) dilutions in 1: 20 in addition, 37 ℃ of vibrations 0.5 hour are washed six times with cleansing solution (6); Add 200 μ L and strengthen liquid, vibrate and measure fluorescence intensity cps after 5 minutes, to add the detection data drawing standard curve of deoxynivalenol enol standard, to add the deoxynivalenol enol content of detection data from the typical curve calculation sample of sample.
7, the detection method of deoxynivalenol enol according to claim 6, it is characterized in that sample preparation: the sample preparation of feed, cereal and goods thereof: sample is crushed to 20 orders, getting 5 gram samples is placed in the test tube, add 25mL distilled water, jump a queue and vibrated 3 minutes, filter, filter paper adopts No. 1 paper of Xinhua, get 1mL filtrate and dilute redundant detection with 1mL distilled water or deionized water; Beer sample is handled: direct sample detects or dilutes the standby detection in back in proportion with distilled water.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102830232A (en) * | 2012-09-20 | 2012-12-19 | 重庆市科学技术研究院 | Time resolution fluoroimmunoassay kit for detecting tylosin and tilmicosin and detection method of kit |
| CN105445463A (en) * | 2014-09-25 | 2016-03-30 | 苏州新波生物技术有限公司 | Hepatitis E virus IgG antibody detection kit and preparation method and application thereof |
-
2008
- 2008-10-07 CN CN 200810155490 patent/CN101393207A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102830232A (en) * | 2012-09-20 | 2012-12-19 | 重庆市科学技术研究院 | Time resolution fluoroimmunoassay kit for detecting tylosin and tilmicosin and detection method of kit |
| CN105445463A (en) * | 2014-09-25 | 2016-03-30 | 苏州新波生物技术有限公司 | Hepatitis E virus IgG antibody detection kit and preparation method and application thereof |
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Application publication date: 20090325 |