CN112098641A - Application of alternan artificial antigen in enzyme linked immunosorbent assay kit - Google Patents
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Abstract
The invention provides an enzyme linked immunosorbent assay kit for detecting alternaria alternata, which comprises: the enzyme-linked immunosorbent assay kit comprises an ELISA plate coated with a coating antigen, an alternaria alternata standard solution, an alternaria alternata antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate color developing solution, a stop solution and a cleaning solution, wherein the coating antigen is an alternaria alternata coupling antigen, the enzyme conjugate is an enzyme-labeled alternaria alternata antibody, and the alternaria alternata antibody is obtained by immunizing animals with immunogen. The invention also discloses a method for detecting the alternaria alternata by applying the enzyme linked immunosorbent assay kit, which comprises the following steps: firstly, preprocessing a sample, then detecting by using a kit, and finally analyzing a detection result. The enzyme linked immunosorbent assay kit provided by the invention can be used for detecting the content of alternaria alternata in grain and feed samples, is simple and convenient to operate, low in cost and high in sensitivity, can be monitored on site, and is suitable for screening a large number of samples.
Description
Technical Field
The invention relates to an application technology of an alternariomycin artificial antigen in an enzyme linked immunosorbent assay kit, in particular to an alternariomycin artificial antigen molecular structure and an enzyme linked immunosorbent assay kit for detecting alternariomycin, which can qualitatively and quantitatively detect the residual quantity of alternariomycin drugs in grains and feeds.
Background
The Linzhou city of Henan province is an esophageal cancer high-incidence area, and researches prove that alternaria alternata and toxin thereof separated from grains in the city play a certain role in causing esophageal cancer. Although the content of the toxin in grains in the market is reduced, the toxin still exists, and the toxin exists in beverages such as fruit juice, and therefore, the alternaria alternata and the toxin thereof are closely related to human life.
In order to reduce the influence on human and livestock, it is important to detect alternaria alternata in grains and feed effectively and rapidly. Compared with an instrument method, the immunochemical detection method has the advantages of rapidness, specificity, sensitivity, accuracy, batch monitoring, simple sample treatment, automatic operation and the like when being used for detecting the mycotoxin. The precondition for immunochemical detection of alternaria alternata is that antibodies against alternaria alternata are required. The invention is a rapid detection method, has short time, simple operation and lower cost, and is suitable for detecting samples in large batch by a basic unit.
Disclosure of Invention
The invention aims to provide an enzyme linked immunosorbent assay kit capable of detecting the drug residue of alternarin in grains and feeds, and provides a qualitative and quantitative detection method which is efficient, accurate, simple and convenient and is suitable for screening large-batch samples.
The kit of the invention comprises: the enzyme-linked immunosorbent assay kit comprises an enzyme label plate coated with a coating antigen, an alternaria alternata standard solution, an alternaria alternata antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate color developing solution, a stop solution and a cleaning solution, wherein the coating antigen is an alternaria alternata coupling antigen, and the enzyme conjugate is an enzyme-labeled alternaria alternata antibody.
The specific antibody of alternaria alternata is prepared by taking an artificial antigen of alternaria alternata as immunogen, the specific antibody of alternaria alternata can be a monoclonal antibody of alternaria alternata or a polyclonal antibody of alternaria alternata, wherein the monoclonal antibody of alternaria alternata is preferred.
The labeled enzyme of the enzyme conjugate is horseradish peroxidase or alkaline phosphatase extracted from bacteria, wherein horseradish peroxidase is preferred; the enzyme conjugate is obtained by coupling enzyme and alternarin antibody.
In order to facilitate field monitoring and screening of a large number of samples, the kit further comprises an alternaria alternata standard solution, a substrate color development solution, a stop solution and a washing solution.
6 bottles of the alternaria alternata standard solution are respectively provided with the concentrations of 0 mug/L, 1 mug/L, 3 mug/L, 9 mug/L, 27 mug/L and 81 mug/L.
When the labeled enzyme is horseradish peroxidase, the substrate color development solution consists of a substrate solution A and a substrate solution B, wherein the A is hydrogen peroxide or carbamide peroxide, the B is o-phenylenediamine or tetramethylbenzidine, and the stop solution is 1-2 mol/L sulfuric acid solution or hydrochloric acid buffer solution; when the marker enzyme is bacteria extracted alkaline phosphatase, the substrate color developing solution is a para-nitrophosphate buffer solution, and the stop solution is 1-2 mol/L sodium hydroxide solution.
The washing liquid preferably has a pH value of 7.4, and contains 0.5-1.0% of Tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer solution, wherein the percentages are weight volume percentages.
The coating buffer solution used in the preparation process of the ELISA plate is carbonate buffer solution with the pH value of 9.6 and 0.05mol/L, and the confining solution is carbonate buffer solution with the pH value of 7.1-7.5, contains 1-3% of casein and 0.1-0.3 mol/L of phosphate buffer solution, and the percentage is weight volume percentage.
The preparation process of the ELISA plate comprises the following steps: diluting the coating source into 20 mu g/mL by using a coating buffer solution, adding 100 mu l into each hole, incubating for 2h in a dark place at 25 ℃ or overnight at 4 ℃, pouring off liquid in the holes, washing for 2 times by using a washing solution for 30s each time, patting to dry, then adding 150-200 mu l of a sealing solution into each hole, incubating for 1-2 h in a dark place at 25 ℃, pouring off liquid in the holes, patting to dry, drying, and performing vacuum sealing and storage by using an aluminum film.
The detection principle of the invention is as follows:
the kit adopts an indirect competition ELISA method, conjugate antigens are pre-coated on a micropore strip of an ELISA plate, the residual alternaria alternata in a sample and the conjugate antigens pre-coated on the micropore strip compete for antibodies against the alternaria alternata, after an enzyme-labeled secondary antibody is added, a TMB substrate is used for developing color, the absorbance value of the sample is in negative correlation with the content of the alternaria alternata contained in the sample, the absorbance value is compared with a standard curve, and the absorbance value is multiplied by the corresponding dilution multiple, so that the residual quantity of the alternaria alternata in the sample can be obtained.
The invention also provides a method for detecting the alternaria alternata by using the enzyme linked immunosorbent assay kit, which comprises the following steps:
(1) pretreating a sample;
(2) detecting by using the kit;
(3) and analyzing the detection result.
The enzyme linked immunosorbent assay kit for detecting the alternaria alternata mainly adopts an ELISA method to qualitatively or quantitatively detect the content of the alternaria alternata in a sample; the pretreatment requirement on the sample is low, the pretreatment process of the sample is simple, and a large amount of samples can be detected quickly; the main reagent is provided in the form of working solution, and the detection method is convenient and easy to implement and has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like. The enzyme linked immunosorbent assay kit has the advantages of simple structure, convenient use, low price, convenient carrying, high efficiency, accuracy, simplicity and convenience of the detection method, and is suitable for qualitative and quantitative screening of large-batch samples.
Drawings
FIG. 1: molecular structural formula of alternaria alternata hapten
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
EXAMPLE 1 preparation of kit Components
1. Preparation of an Interleaved Streptosporin hapten
Taking 30mg of alternaria alternata, adding 50ml of tetrahydrofuran for dissolving, adding 5ml of trifluoroacetic acid, fully stirring, adding 0.7g of glycolic acid, heating in an oil bath, carrying out reflux reaction for 12 hours, stopping the reaction, adding 100ml of water, adding 100ml of ethyl acetate, fully shaking, standing for layering, removing an aqueous phase, washing with 60ml of organic phase water, shaking for standing, separating the aqueous phase, evaporating the organic phase to dryness, washing with 1ml of dichloromethane/n-hexane (v/v,1/9), pulping, standing, separating a supernatant, drying the obtained precipitate, obtaining 26mg of glycolylated alternata hapten with the yield of 70%.
2. Preparation of antigens
Immunogen preparation-the coupling of an alternaria alternata hapten and Bovine Serum Albumin (BSA) to obtain the immunogen.
Taking 14mg of glycolated alternaria alternata, adding 1ml of DMSO for dissolving, adding 9.7mg of NHS and 19mg of DCC, fully dissolving and uniformly mixing, and reacting for 3h to obtain a hapten activating solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.1M PB buffer solution for dissolving to obtain solution B, dropwise adding all solution A into solution B, reacting at room temperature for 4h, dialyzing and purifying with 0.02M PBS for 3 days, and changing the solution 3 times per day to obtain an alternaria alternata-BSA conjugate which is an immunogen, subpackaging at-20 ℃, and storing for later use.
Preparation of coating antigen-coupling of alternaria alternata hapten and Ovalbumin (OVA) to obtain immunogen.
Dissolving 9mg of glycolated alternaria alternata in 1ml of DMF (dimethyl formamide), adding 7.6mg of NHS (N-hydroxysuccinimide) and 9mg of EDC (diethyl formamide), fully dissolving and uniformly mixing, and reacting for 3 hours to obtain a hapten activating solution A; dissolving Ovalbumin (OVA)50mg in PB buffer solution 0.1M to obtain solution B, dripping all solution A into solution B, reacting at room temperature for 4h, dialyzing and purifying with PBS 0.02M for 3 days, and changing solution 3 times per day to obtain alternaria alternata-OVA conjugate, i.e. coating antigen, subpackaging at-20 deg.C, and storing for use.
3. Preparation of alternariosporin monoclonal antibody
Animal immunization: injecting the immunogen obtained in the above steps into Balb/c mice, wherein the immunization dose is 150 mug/mouse, so that antiserum is generated.
Cell fusion and cloning: after the serum determination result of the mouse is higher, spleen cells are taken and fused with SP2/0 myeloma cells according to the ratio of 8:1 (quantitative ratio), indirect competitive ELISA is adopted to determine cell supernatant, and positive holes are screened. Cloning the positive hole by limiting dilution method until obtaining hybridoma cell strain secreting alternaria alternate monoclonal antibody.
Freezing and recovering cells: preparation of monoclonal hybridoma cell line into 1 × 10 frozen stock solution6Cell suspension per mL, preserved for long period in liquid nitrogen. Taking out the frozen tube during recovery, immediately putting the tube into a water bath at 37 ℃ for fast melting, centrifuging to remove frozen liquid, and transferring the tube into a culture bottle for culture.
Production and purification of monoclonal antibodies: injecting sterilized paraffin oil 0.5 mL/mouse into the abdominal cavity of Balb/c mouse, and injecting stable monoclonal hybridoma cell strain 5X 10 into the abdominal cavity after 7 days5Ascites were collected 7 days later. Purifying ascites by octanoic acid-saturated ammonium sulfate method, and storing at-20 deg.C.
4. Preparation of enzyme conjugates
Taking a goat as an immune animal, and taking the alternaria alternata monoclonal antibody as an immunogen to immunize a goat without a pathogen to obtain the alternaria alternata antibody. And (3) coupling the alternaria alternata antibody with horseradish peroxidase (HRP) to obtain an enzyme conjugate.
5. Preparation of ELISA plates
Diluting the coating source to 20 mu g/mL by using a coating buffer solution, adding 100 mu l of the coating source into each hole, incubating for 2h at 25 ℃ in a dark place, pouring off liquid in the holes, washing for 2 times by using a washing solution for 30s each time, patting to dry, then adding 200 mu l of a sealing solution into each hole, incubating for 2h at 25 ℃ in a dark place, pouring off liquid in the holes, patting to dry, and performing vacuum sealing and storage by using an aluminum film after drying.
Example 2 construction of enzyme-linked immunosorbent assay kit for detecting alternaria alternata
An enzyme linked immunosorbent assay kit for detecting the alternaria alternata is constructed, and comprises the following components:
(1) an ELISA plate coated with alternariomycin coupling antigen;
(2) 6 bottles of alternariosporin standard solution, wherein the concentrations are respectively 0 mug/L, 1 mug/L, 3 mug/L, 9 mug/L, 27 mug/L and 81 mug/L;
(3) using horseradish peroxidase labeled alternariosporin antibody;
(4) the substrate color development liquid consists of a liquid A and a liquid B, wherein the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(5) the stop solution is 2mol/L sulfuric acid;
(6) the washing liquid has a pH value of 7.4, and contains 0.5-1.0% of tween-20, 0.01-0.03% of sodium azide preservative and 0.1-0.3 mol/L of phosphate buffer solution, wherein the percentages are weight volume percentages;
example 3 detection of alternan in cereals and feeds
1. Detection with a kit
And numbering the corresponding micropores of the samples and the standard products in sequence, making 2 holes in parallel for each sample and standard product, and recording the positions of the standard holes and the sample holes. Adding 20-60 mul of standard substance/sample into corresponding micropore, adding 20-60 mul of enzyme-labeled secondary antibody into each micropore, adding 50-100 mul of antibody working solution into each pore, and reacting for 30min at 25 ℃ in a dark environment. And (5) spin-drying the liquid in the holes, washing for 4-5 times, and patting dry. Adding 50 mul/hole of the substrate solution A, adding 50 mul/hole of the substrate solution B, mixing uniformly, and reacting for 15min at 25 ℃ in a dark environment. Adding 50 mul/well of stop solution, mixing, setting an enzyme-labeling instrument at 450nm, and measuring the OD value of each well.
2. Analysis of detection results
The percent absorbance of the standard or sample is equal to the average of the absorbance values of the standard or sample (double well) divided by the average of the absorbance values of the first standard (0 standard) and multiplied by 100% to obtain the percent absorbance value of the standard or sample. And drawing a standard curve graph by taking the percent absorbance of the standard substance as an ordinate and taking the logarithm of the concentration (mu g/L) of the alternaria alternate standard substance as an abscissa. And substituting the percent absorbance of the sample into the standard curve, reading out the concentration corresponding to the sample from the standard curve, and multiplying the concentration by the corresponding dilution multiple to obtain the actual concentration of the alternaria alternata in the sample.
3. Pretreatment method
The pretreatment method of the grain sample (soybean meal, rice meal, corn meal, flour and the like) comprises the following steps:
homogenizing the grain sample with a homogenizer; weighing the homogenized grain sample into a 50ml polystyrene centrifuge tube; adding organic solvent and deionized water, respectively, shaking with oscillator for 5min, and centrifuging for 5 min; transferring supernatant into a 50ml polystyrene centrifuge tube, adding organic solvent, shaking for 5min with an oscillator, and centrifuging for 5 min; removing the upper liquid layer, taking the lower organic phase layer into a clean and dry glass tube, and drying by blowing under water bath nitrogen flow; adding organic solvent, whirling with vortex instrument, adding redissolution working solution, and centrifuging for 5 min; the upper organic solvent phase was removed and 50. mu.l of the lower aqueous phase was taken for analysis.
A feed (raw material, batch and concentrate) sample pretreatment method comprises the following steps:
weighing feed samples into a 50ml polystyrene centrifuge tube, adding an organic solvent, oscillating for 5min by using an oscillator, and centrifuging for 5 min; transferring supernatant, adding redissolution working solution, shaking with oscillator for 1min, and mixing; 50 μ l was taken for analysis.
Example 4 determination of technical parameters of Alternalisin
1. Sensitivity and detection limit of kit
The sensitivity of the kit is determined according to a conventional method, the range of a standard curve is 1-81 mu g/L, and IC50(50% inhibitory concentration) the floating range is 4.3-7.5 mug/L; the 20 samples were tested and the concentrations corresponding to the percent absorbance values were determined from the standard curve, and the detection limit was expressed as the average of the 20 sample concentrations plus 3 standard deviations, showing that the method has a limit of 10. mu.g/kg for alternaria alternata in cereals and feeds.
2. Sample precision and accuracy testing
The recovery rate is used as an accuracy evaluation index, and the relative standard deviation (RSD%) of the detection result of a sample with a certain concentration is repeatedly measured and used as a precision evaluation index. The calculation formula is as follows: recovery (%) — actual measurement/theoretical value × 100%, where theoretical value is the added concentration of the sample; relative standard deviation RSD% ═ SD/X × 100%, where SD is the standard deviation and X is the average of the measured data.
The grain samples are subjected to addition recovery measurement according to the alternaria alternata with two concentrations of 10 mu g/kg and 20 mu g/kg, the feed samples are subjected to addition recovery measurement according to the alternaria alternata with two concentrations of 10 mu g/kg and 20 mu g/kg, 4 samples are subjected to parallel measurement by using three different reagents, and the average recovery rate and precision result of the samples are calculated and shown in the following table.
TABLE 1 grain and feed sample precision and accuracy tests
The grain and feed samples are subjected to addition recovery measurement according to the alternaria alternata with two concentrations of 10 mug/kg and 20 mug/kg, and the average recovery rates are respectively 70.6% -83.5% and 79.4% -87.1%; the relative standard deviation in and between batches was less than 15%.
3. Stability test of kit
The storage condition of the kit is 2-8 ℃, and the maximum absorbance value (zero standard), 50% inhibition concentration and the actual measurement value of adding the alternaria alternata are all within the normal range after 12 months of measurement. Considering that abnormal storage conditions occur in the transportation and use processes, the kit is placed for 7 days under the storage condition of 37 ℃ for accelerated aging experiments, and the results show that all indexes of the kit completely meet the requirements. And in consideration of the occurrence of the freezing condition of the kit, the kit is frozen for 7 days in a refrigerator at the temperature of-20 ℃, and the determination result also shows that all indexes of the kit are completely normal. From the above results, it can be obtained that the kit can be stored at 2 to 8 ℃ for at least 12 months.
Claims (4)
1. An enzyme linked immunosorbent assay kit for detecting alternaria alternata, which is characterized by comprising: the enzyme-linked immunosorbent assay kit comprises an enzyme label plate coated with a coating antigen, an alternaria alternate standard solution, an alternaria alternate antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate color developing solution, a stop solution and a cleaning solution, wherein the coating antigen is an alternaria alternate coupling antigen, the enzyme conjugate is an enzyme-labeled alternaria alternate antibody, the alternaria alternate antibody is obtained by immunizing an animal with immunogen, the alternaria alternate coupling antigen is obtained by coupling an alternaria alternate hapten and a carrier protein, and the alternaria alternate hapten is obtained by carrying out a series of chemical reactions on the alternaria alternate and glycolic acid and other substances.
2. The kit of claim 1, wherein said alternan hapten has the formula:
3. the kit according to claim 1, characterized in that the immunogen is prepared as follows:
taking 30mg of alternaria alternata, adding 50ml of tetrahydrofuran for dissolving, adding 5ml of trifluoroacetic acid, fully stirring, adding 0.7g of glycolic acid, heating in an oil bath, carrying out reflux reaction for 12 hours, stopping the reaction, adding 100ml of water, adding 100ml of ethyl acetate, fully shaking, standing for layering, removing an aqueous phase, washing with 60ml of organic phase water, shaking for standing, separating the aqueous phase, evaporating the organic phase to dryness, washing with 1ml of dichloromethane/n-hexane (v/v,1/9), pulping, standing, separating a supernatant, drying the obtained precipitate, obtaining 26mg of glycolylated alternata hapten with the yield of 70%.
4. A method for detecting the content of alternaria alternata in a sample, comprising the following steps:
(1) pretreating a sample;
(2) detecting with the kit according to any one of claims 1 to 3;
(3) and analyzing the detection result.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103848913A (en) * | 2014-03-26 | 2014-06-11 | 中国农业科学院油料作物研究所 | Artificial antigen of aflatoxin biosynthesis precursor ST and preparation method for artificial antigen |
| CN105712970A (en) * | 2016-01-27 | 2016-06-29 | 华南农业大学 | Hapten, artificial antigen and antibody directly targeted to alternariol and preparation method and application thereof |
| CN106093381A (en) * | 2016-07-08 | 2016-11-09 | 北京农业质量标准与检测技术研究中心 | Rod method toxin rod method phenol monomethyl ether colloidal gold immuno-chromatography test paper strip |
| CN106565737A (en) * | 2016-10-08 | 2017-04-19 | 华南农业大学 | Aflatoxin B1 hapten, aflatoxin B1 artificial antigen and aflatoxin B1 egg yolk antibody preparation method |
| CN108912090A (en) * | 2018-02-12 | 2018-11-30 | 北京农业质量标准与检测技术研究中心 | A kind of test strips of quick detection alternariol and alternariol monomethyl ether total amount |
-
2020
- 2020-08-17 CN CN202010823404.6A patent/CN112098641B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103848913A (en) * | 2014-03-26 | 2014-06-11 | 中国农业科学院油料作物研究所 | Artificial antigen of aflatoxin biosynthesis precursor ST and preparation method for artificial antigen |
| CN105712970A (en) * | 2016-01-27 | 2016-06-29 | 华南农业大学 | Hapten, artificial antigen and antibody directly targeted to alternariol and preparation method and application thereof |
| CN106093381A (en) * | 2016-07-08 | 2016-11-09 | 北京农业质量标准与检测技术研究中心 | Rod method toxin rod method phenol monomethyl ether colloidal gold immuno-chromatography test paper strip |
| CN106565737A (en) * | 2016-10-08 | 2017-04-19 | 华南农业大学 | Aflatoxin B1 hapten, aflatoxin B1 artificial antigen and aflatoxin B1 egg yolk antibody preparation method |
| CN108912090A (en) * | 2018-02-12 | 2018-11-30 | 北京农业质量标准与检测技术研究中心 | A kind of test strips of quick detection alternariol and alternariol monomethyl ether total amount |
Non-Patent Citations (7)
| Title |
|---|
| CHAN-YUAN YAO等: "High affinity antibody based on a rationally designed hapten and development of a chemiluminescence enzyme immunoassay for quantification of Alternariol in fruit Juice, maize and flour.", FOOD CHEMISTRY, vol. 283 * |
| CHRISTIAN CERVINO等: "Novel Aflatoxin Derivatives and Protein Conjugates", MOLECULES, vol. 12, no. 3 * |
| GURMIT SINGH等: "Development of an Indirect Competitive ELISA for Analysis of Alternariol in Bread and Bran Samples", FOOD ANALYTICAL METHODS, vol. 11 * |
| JIANYI WANG 等: "A novel hapten and monoclonal antibody-based indirect competitive ELISA for simultaneous analysis of alternariol and alternariol monomethyl ether in wheat", FOOD CONTROL, vol. 94 * |
| 安玉会 等: "林县互隔交链孢霉素B的分离纯化研究", 河南医科大学学报, vol. 22, no. 3 * |
| 王瑾瑾 等: "不同浓度的互隔交链孢霉素对 NIH/ 3T3 细胞 cAMP水平的影响", 肿瘤基础与临床, vol. 22, no. 1 * |
| 郑其岚 等: "交链孢酚单甲醚(AME)特异性抗体研制(Ⅰ)", 肿瘤基础与临床, vol. 4, no. 3 * |
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