CN103575885A - Enzyme linked immunoassay kit for detecting T-2 toxin, and application thereof - Google Patents
Enzyme linked immunoassay kit for detecting T-2 toxin, and application thereof Download PDFInfo
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- G—PHYSICS
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Abstract
The present invention provides an enzyme linked immunoassay kit for detecting T-2 toxin. The kit contains a T-2 toxin conjugated antigen coated enzyme label plate, a T-2 toxin standard substance solution, a concentrated enzyme conjugate, an enzyme conjugate working solution, a substrate coloration solution and a termination solution. The invention further discloses a method for detecting T-2 toxin in a sample by using the enzyme linked immunoassay kit. The method comprises: carrying out a sample pretreatment, detecting by using the kit, and analyzing the detection result. The enzyme linked immunoassay kit can be used for detecting T-2 toxin residue in feed (raw materials, matching materials and concentrated materials) samples, has characteristics of simple operation, low cost, high sensitivity and on-site monitoring, and is suitable for screening of mass samples.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, be specifically related to a kind of enzyme linked immunological kit for detection of T-2 toxin, it is applicable to T-2 toxin determination in feed (raw material, batch, concentrate feed).
Technical background
T-2 toxin (T-2 toxin) is the strongest a kind of of the Trichothecenes toxin poisoning that produces of sickle-like bacteria, can cause animal to produce the cometabolism material of toxic reaction, it is distributed widely in nature, in be everlasting contaminated field crops and stock's cereal, finds.No matter in feed the content of T-2 toxin be high level or low-levelly all can cause that animal feed intake declines, weightening finish slowly, immune system damage etc.Research shows, T-2 toxin shows the multiple toxicity such as genotoxicity, immunotoxicity, hematotoxicity to humans and animals, and can cause apoptosis of many kinds.In the joint conference that FAO (Food and Agriculture Organization of the United Nation) (FAO) in 1973 and the World Health Organization (WHO) (WHO) hold in Geneva, using T-2 toxin with the same the most dangerous food pollution source as naturally existing of aflatoxin.
The assay method of T-2 toxin mainly contains thin-layered chromatography (TCL), vapor-phase chromatography, high performance liquid chromatography, liquid chromatography-mass spectrography, radioimmunoassay detection method, euzymelinked immunosorbent assay (ELISA) etc., TLC method is one of the most frequently used the earliest detection method, have simple to operate, expense is cheap, good separating effect, the advantages such as velocity of separation is fast, it is a kind of common detection method, but it is loaded down with trivial details also to exist sample to purify, quantitatively accurate not, the shortcomings such as cost is higher, and while TLC method relates to the problem of a visual color quantification, or it is convenient to be not so good as gas chromatography and liquid chromatography, accurately.Gas chromatography and liquid phase chromatography analytical method need to carry out derivatization treatment to T-2 toxin; Liquid chromatograph mass spectrography sensitivity is higher, do not need to carry out derivatization treatment, thereby liquid chromatograph mass spectrography is to detect the more satisfactory method of T-2 toxin.The many methods of report are that one pole mass spectrum is selected ion technology (SIM) at present.Yet for complex matrices sample (as cereal), the interference that adopts plural serial stage mass-spectrometric technique can more effectively remove matrix, guarantees the accuracy that trace materials is analyzed.It is too complicated that TCL method, chromatography etc. have, some shortage sensitivity, and what have is expensive, is unsuitable for conventional toxin analysis.Immunology detection is sensitive, quick, simple because of it, and is used to the mensuration of T-2 toxin.The immunological method that is applied to T-2 toxin determination mainly contains radioimmunology, enzyme linked immunosorbent assay and immunofluorescence technique etc.Because radioactive isotope stores, uses inconvenience, nonradioactive labeling applies more in recent years.Enzyme linked immunosorbent assay (ELISA) was highly sensitive with it, specificity good, easy and simple to handle, low cost and other advantages Yi Bei China was decided to be examination criteria in 1992, ELISA method, as a kind of accurate, reliable, quick, special detection method, is suitable for the rapid screening of a large amount of samples.
Summary of the invention
The object of the present invention is to provide a kind of enzyme linked immunological kit of measuring for T-2 content of toxins, it is simple to operate, is applicable to the screening of on-the-spot batch samples.
Kit of the present invention, it comprises:
(1) be coated with the ELISA Plate of the toxin conjugated antigen of T-2;
(2) T-2 toxin standard solution;
(3) concentrated enzyme conjugates;
(4) enzyme combination diluent;
(5) substrate nitrite ion;
(6) stop buffer.
The toxin conjugated antigen of described T-2 is the conjugate of T-2 toxin haptens and carrier protein, and described carrier protein can be ovalbumin, bovine serum albumin(BSA), hemocyanin, thyroprotein, mouse haemocyanin, human albumin.
The haptenic structure of described T-2 toxin is as follows:
Described T-2 toxin haptens preparation process: 24mg T-2 toxin, 30mg phthalic anhydride and the mixed liquor of 0.1ml pyridine in 2ml DMSO, at 80 ℃, stirring reaction is 15 hours, and steaming desolventizes, and column chromatography purification obtains phthalic acid list T-2 toxin ester.
The T-2 toxin monoclone antibody that described concentrated enzyme conjugates is enzyme labeling, described marker enzyme is horseradish peroxidase; Enzyme conjugates adopts sodium periodate method that marker enzyme and antibody are carried out to coupling and obtains.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises T-2 toxin standard solution, enzyme combination diluent, substrate nitrite ion, stop buffer.
Described stop buffer is sulfuric acid or the hydrochloric acid solution of 1 ~ 2mol/L, and described substrate nitrite ion is comprised of substrate solution A liquid and substrate solution B liquid, and substrate solution A liquid is urea peroxide solution, and substrate solution B liquid is tetramethyl biphenyl amine aqueous solution.
Described enzyme combination diluent is pH7.2 ~ 7.4, and containing 0.05% ~ 0.1% bovine serum albumin(BSA), the phosphate buffer of 0.1mol/L, described number percent is weight percentage.
Wherein ELISA Plate coated damping fluid used in preparation process is pH9.0 ~ 9.6,0.2mol/L carbonate buffer solution, and confining liquid used is pH7.2 ~ 7.4,0.1mol/L phosphate buffer, described number percent is weight percentage.
In the present invention, the preparation process of ELISA Plate is: with coated damping fluid, coating antigen is diluted to 0.1 ~ 0.2 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h or 4 ℃ spend the night, and the coating buffer that inclines, with cleansing solution washing 2 times, each 30s, pat dry, then in every hole, add 150 ~ 200 μ l confining liquids, 37 ℃ of incubation 1 ~ 2h, the liquid in hole that inclines pats dry, and preserves after dry with the vacuum seal of aluminium film.
Detection principle of the present invention is:
When the toxin conjugated antigen of pre-coated T-2 on capillary strip, add after sample solution or standard solution, the enzyme conjugates of the pre-coated toxin conjugated antigenic competition T-2 of T-2 toxin in residual T-2 toxin and ELISA Plate in sample, with substrate nitrite ion, develop the color, the content of sample light absorption value and contained T-2 toxin is negative correlation, relatively can draw the content of T-2 toxin in sample with typical curve.Simultaneously according to the depth of color in ELISA Plate, with the comparison of the T-2 toxin standard solution color of the series concentration concentration range of T-2 toxin in judgement sample roughly.
The present invention also provides a kind of method that above-mentioned enzyme linked immunological kit detects T-2 toxin of applying, and it comprises step:
(1) sample pre-treatments;
(2) with kit, detect;
(3) analyzing and testing result.
The enzyme linked immunological kit that the present invention detects T-2 toxin mainly adopts competitive ELISA method to detect the content of T-2 toxin in sample; The pre-treatment of sample is required low, sample pretreatment process is simple, fast detecting batch samples simultaneously, and main agents provides with the form of working fluid, and the method for inspection is convenient and easy, has that specificity is high, highly sensitive, accuracy is high, degree of accuracy high.Enzyme linked immunological kit of the present invention, carrying convenience, easy to use, simple in structure, detection method is quick, easy, be suitable for batch samples screening.
Accompanying drawing explanation
Fig. 1: T-2 toxin haptens composite diagram
Fig. 2: T-2 toxin haptens hydrogen nuclear magnetic resonance spectrogram
Fig. 3: the canonical plotting of kit
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only for the present invention is described, and be not used for limiting the scope of the invention.
The preparation of embodiment 1 kit components
1, T-2 toxin haptens preparation
24mg T-2 toxin, 30mg phthalic anhydride and the 0.1ml pyridine mixed liquor in 2ml dimethyl sulfoxide (DMSO) (DMSO), at 80 ℃, stirring reaction is 15 hours, steaming desolventizes, and column chromatography purification obtains phthalic acid list T-2 toxin ester, and synthetic route chart is as Fig. 1.
Get above-mentioned product through nuclear magnetic resonance hydrogen spectruming determining, as shown in Figure 2, nuclear magnetic spectrum explanation: the carboxyl signal peak explanation haptens that two groups of aromatic ring signal peaks that 8.0ppm left and right increases and 13.3ppm left and right increase synthesizes successfully.
2, the preparation of antigen
Immunogene preparation---T-2 toxin haptens and bovine serum albumin(BSA) (BSA) coupling obtains immunogene.
Get 0.5ml dimethyl formamide for haptens 4.5mg (DMF) dissolving and obtain I liquid; Get BSA 30mg and obtain II liquid with the PBS dissolving of 2.0ml 0.1mol/L pH 7.0; Get carbodiimides (EDC) 10mg and obtain III liquid with the PBS dissolving of 0.5ml 0.1mol/L pH 7.0; I liquid is mixed with II liquid, under agitation slowly drop to III liquid, room temperature reaction 2 hours, dialyses three days, changes liquid every day three times for 4 ℃.
Coating antigen preparation---T-2 toxin haptens and ovalbumin (OVA) coupling obtains coating antigen.
Get haptens 4.5mg and dissolve with 0.5ml DMF, be cooled to 10 ℃, add 2 μ l isobutyl chlorocarbonates, 10 ℃ of stirring reaction 30min obtain IV liquid; Get OVA 18mg and obtain V liquid with the sodium carbonate dissolving of 2.5ml 0.05mol/L; IV liquid is slowly added in V liquid, and 4 ℃ are reacted 18 hours; Dialyse three days, change liquid every day three times for 4 ℃.
3, the preparation of T-2 toxin monoclone antibody
A. animal immune
The immunizing antigen that above-mentioned steps is obtained is injected in Balb/c Mice Body, and immunizing dose is 150 μ g/, makes it produce antiserum.
B. Fusion of Cells and cloning
Get immune Balb/c mouse boosting cell, in 9:1(quantitative proportion) ratio and SP2/0 myeloma cell's fusion, screening obtains the T-2 toxin monoclone antibody hybridoma cell strain of stably excreting T-2 toxin monoclone antibody.
C. cell cryopreservation and recovery
Hybridoma is made to 1 * 10 with cryopreserving liquid
9the cell suspension of individual/ml is preserved for a long time in liquid nitrogen.During 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.
D. the preparation and purification of monoclonal antibody
Increment cultivation: hybridoma is placed in to cell culture medium, cultivates under 37 ℃ of conditions, by sad-saturated ammonium sulfate method, the nutrient solution obtaining is carried out to purifying, obtain monoclonal antibody ,-20 ℃ of preservations.
4, the preparation of ELISA Plate
With coated damping fluid, coating antigen is diluted to 0.1 ~ 0.2 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h or 4 ℃ spend the night, and the coating buffer that inclines, with cleansing solution washing 2 times, each 30s, pat dry, then in every hole, add 150 ~ 200 μ l confining liquids, 37 ℃ of incubation 1 ~ 2h, the liquid in hole that inclines pats dry, and preserves after dry with the vacuum seal of aluminium film.
5, the preparation of enzymic-labelled antibody
Adopt sodium periodate method to carry out coupling antibody and horseradish peroxidase (HRP) and prepare enzymic-labelled antibody, the molar concentration rate of enzyme and antibody is 2:1.
Embodiment 2 detects the establishment of the enzyme linked immunological kit of T-2 toxin
Set up the enzyme linked immunological kit that detects T-2 toxin, make it comprise following component:
(1) ELISA Plate of the coated toxin conjugated antigen of T-2;
(2) T-2 toxin standard solution is 6 bottles, and concentration is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L, 81 μ g/L;
(3) concentrated enzyme conjugates;
(4) enzyme combination diluent;
(5) substrate nitrite ion is comprised of substrate solution A liquid and substrate solution B liquid, and substrate solution A liquid is urea peroxide, and substrate solution B liquid is tetramethyl benzidine;
(6) stop buffer is 2mol/L sulfuric acid.
The detection of T-2 toxin in embodiment 3 samples
1. sample pre-treatments
With homogenizer homogeneous feed sample; Take feed sample after 5.0g ± 0.05g homogeneous to 50ml polystyrene centrifuge tube, add 25ml 50% methyl alcohol, with oscillator thermal agitation 5min, more than 3000g, the centrifugal 5min of room temperature (20-25 ℃/68-77 ℉); Get 500 μ l supernatants to 2ml polystyrene centrifuge tube, add the oscillator 1min that vibrates for 500 μ l 10% sodium-chloride water solutions, mix; Get 20 μ l for analyzing.
2. with kit, detect
To being coated with in the ELISA Plate micropore of the toxin conjugated antigen of T-2, add T-2 toxin standard solution/sample 20 μ l, add again enzyme conjugates working fluid 100 μ l(that concentrated enzyme conjugates is diluted according to the volume of 1:10 with enzyme combination diluent), with cover plate mould shrouding, 25 ℃ of lucifuge reaction 10min, pour out liquid in hole, every hole adds 250 μ l deionized waters fully to wash 4-5 time, every minor tick 10s, with thieving paper, pat dry, every hole adds substrate solution A liquid urea peroxide 50 μ l, substrate solution B liquid tetramethyl benzidine (TMB) 50 μ l, vibration mixes gently, 25 ℃ of constant temperature oven lucifuge colour developing 5min, every hole adds 2mol/L stop buffer sulfuric acid 50 μ l, vibration mixes gently, by microplate reader wavelength set at 450nm place, measure every hole absorbance (OD value).
3. Analysis of test results
Absorbance (B with the absorbance mean value (B) of the standard solution of each obtained concentration divided by first standard solution (0 standard)
0) be multiplied by again 100%, obtain percentage absorbance.The logarithm value of T-2 toxin standard items concentration (μ g/L) of take is X-axis, and percentage absorbance is Y-axis, drawing standard curve map.By the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample, can read from typical curve the content of T-2 toxin.
Embodiment 4 T-2 toxin enzyme linked immunological kit sensitivity, preci-sion and accuracy, storage life experiment
1. kit sensitivity and detectability
According to conventional method, measure kit sensitivity test, kit sensitivity is 1 μ g/L, respectively 20 parts of blank feeds (raw material, batch, concentrate feed) sample is detected, from typical curve, find the concentration corresponding to each percentage absorptance, mean value with 20 this T-2 of increment toxin concentrations adds that 3 times of standard deviations represent detectability, and result obtains the method feed (raw material, batch, concentrate feed) pattern detection is limited to 10 μ g/kg.
2. kit accuracy and precision
The testing result coefficient of variation (CV%) of a certain concentration sample of the replication of usining is as precision evaluation index.Using the recovery as accuracy estimating index.Coefficient of variation CV% computing formula is: CV%=SD/ X * 100%; Wherein SD is standard deviation, the mean value that X is determination data.Recovery computing formula is: the recovery (%)=practical measurement value/theoretical value * 100%.The interpolation concentration that wherein theoretical value is analog sample.
By 10 μ g/kg, 20 μ g/kg, tri-concentration T-2 toxin of 40 μ g/kg, raw material, batch, concentrate feed sample are added to reclaim and measure, each sample do 4 parallel, with three batches of different reagent, measure, average recovery rate and the precision of calculation sample the results are shown in following table.
Table 1 precision and accuracy test
T-2 toxin with tri-concentration of 10,20,40 μ g/kg adds feed (raw material, batch, concentrate feed) sample, and average recovery rate is between 78.6% ~ 101.6%; The coefficient of variation is all less than 20%.The preci-sion and accuracy of testing result all meets relevant criterion requirement.
3. kit storage life test
Kit preservation condition is 2 ~ 8 ℃, and through the mensuration of 12 months, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, T-2 toxin added practical measurement value all within normal range.Consideration, in transportation and use procedure, has improper preservation condition and occurs, kit is placed 7 days under 37 ℃ of preservation conditions, carries out accelerated deterioration experiment, and result shows that this kit indices meets the requirements completely.Consider that the freezing situation of kit occurs, kit is put into-20 ℃ of refrigerator freezings 7 days, measurement result also shows that kit indices is completely normal.From above result, can show that kit can preserve 12 months at 2 ~ 8 ℃.
Claims (10)
1. detect an enzyme-linked immunologic detecting kit for T-2 toxin, it is characterized in that including:
(1) be coated with the ELISA Plate of the toxin conjugated antigen of T-2;
(2) T-2 toxin standard solution;
(3) concentrated enzyme conjugates;
(4) enzyme combination diluent;
(5) substrate nitrite ion;
(6) stop buffer.
2. kit as claimed in claim 1, is characterized in that the toxin conjugated antigen of described T-2 is to be obtained by T-2 toxin haptens and carrier protein couplet, and described T-2 toxin haptens structure is as follows:
3. kit as claimed in claim 2, is characterized in that the haptenic preparation method of described T-2 toxin mainly comprises the steps:
24mg T-2 toxin, 30mg phthalic anhydride and the mixed liquor of 0.1ml pyridine in 2ml DMSO, at 80 ℃, stirring reaction is 15 hours, and steaming desolventizes, and column chromatography purification obtains phthalic acid list T-2 toxin ester.
4. kit as claimed in claim 1, is characterized in that the T-2 toxin monoclone antibody that described concentrated enzyme conjugates is enzyme labeling.
5. kit as claimed in claim 1, the marker enzyme that it is characterized in that described concentrated enzyme conjugates is horseradish peroxidase; Enzyme conjugates is to adopt sodium periodate method that marker enzyme and antibody coupling are obtained.
6. kit as claimed in claim 1, it is characterized in that: the sulfuric acid that described stop buffer is 1 ~ 2mol/L or hydrochloric acid solution, described substrate nitrite ion is comprised of substrate solution A liquid and substrate solution B liquid, and substrate solution A liquid is urea peroxide solution, and substrate solution B liquid is tetramethyl biphenyl amine aqueous solution.
7. kit as claimed in claim 1, is characterized in that coated damping fluid used is pH9.0 ~ 9.6,0.2mol/L carbonate buffer solution, and confining liquid used is pH7.2 ~ 7.4,0.1mol/L phosphate buffer, described number percent is weight percentage.
8. kit as claimed in claim 1, is characterized in that described enzyme combination diluent is pH7.2 ~ 7.4, and containing 0.05% ~ 0.1% bovine serum albumin(BSA), the phosphate buffer of 0.1mol/L, described number percent is weight percentage.
9. kit as claimed in claim 1, is characterized in that described T-2 toxin standard solution concentration is respectively 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L, 81 μ g/L.
10. a method that detects T-2 content of toxins in sample, key step comprises:
1) testing sample is carried out to pre-treatment;
2) with the enzyme linked immunological kit described in claim 1-9 any one, detect;
3) analyzing and testing result.
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| CN104788468A (en) * | 2015-04-02 | 2015-07-22 | 东北农业大学 | Method for extracting T-2 toxin from potatoes |
| CN105021593A (en) * | 2015-06-12 | 2015-11-04 | 青岛科技大学 | Method for determining T-2 toxin based on foot point domain and hybridization chain reaction |
| CN105301253A (en) * | 2015-09-07 | 2016-02-03 | 北京勤邦生物技术有限公司 | T-2 toxin-enriched immunomagnetic bead as well as preparation method and application thereof |
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| CN111273018A (en) * | 2020-04-13 | 2020-06-12 | 北京维德维康生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting bromadiolone and preparation and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104788468A (en) * | 2015-04-02 | 2015-07-22 | 东北农业大学 | Method for extracting T-2 toxin from potatoes |
| CN105021593A (en) * | 2015-06-12 | 2015-11-04 | 青岛科技大学 | Method for determining T-2 toxin based on foot point domain and hybridization chain reaction |
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| CN105301253A (en) * | 2015-09-07 | 2016-02-03 | 北京勤邦生物技术有限公司 | T-2 toxin-enriched immunomagnetic bead as well as preparation method and application thereof |
| CN105606574A (en) * | 2016-01-21 | 2016-05-25 | 湖南科技大学 | Method and kit for detecting T-2 toxins |
| CN105606574B (en) * | 2016-01-21 | 2018-07-24 | 湖南科技大学 | The detection method and detection kit of T-2 toxin |
| CN111273018A (en) * | 2020-04-13 | 2020-06-12 | 北京维德维康生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting bromadiolone and preparation and application thereof |
| CN111273018B (en) * | 2020-04-13 | 2024-02-20 | 北京维德维康生物技术有限公司 | ELISA kit for detecting bromadiolone and preparation and application thereof |
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| Publication number | Publication date |
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| CN103575885B (en) | 2016-06-01 |
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