CN111693704A - Immunochromatographic test strip and preparation method thereof - Google Patents
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Abstract
The invention belongs to the technical field of immunodetection, and particularly relates to an immunochromatography test strip and a preparation method thereof. The invention prepares DON monoclonal antibody colloidal gold probe and FB1The monoclonal antibody colloidal gold probe, the fixed antigen and the fixed antibody are assembled on a PVC plastic rubber plate together with the sample pad and the water absorption pad, so that qualitative analysis and quantitative analysis combined with a mobile phone can be realized. The invention has high sensitivity, accuracy and stability, and is simple, convenient and quick to operate. Is suitable for Deoxynivalenol (DON) and fumonisin B in agricultural products such as grains1The field test of (1).
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to an immunochromatography test strip and a preparation method thereof.
Background
Deoxynivalenol (DON) belongs to trichothecene compounds and is a toxic secondary metabolite produced by fusarium. Fumonisins (FB)1) A mycotoxin is a water-soluble metabolite produced by Fusarium moniliforme. DON and FB1Mycotoxin widely existing in cereal grainsIn eating, the traditional Chinese medicine composition has the effects of teratogenicity, carcinogenesis, immunosuppressive toxicity, neurotoxicity and the like, not only causes great loss of crops such as yield reduction, but also poses great threat to the health of human and animals. The simultaneous detection and determination of these two mycotoxins is of great significance to food safety and health of the living body.
Currently, instrumental and immunoassay methods such as Thin Layer Chromatography (TLC), high performance liquid chromatography-mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA) are commonly used for detecting mycotoxins.
Compared with TLC and HPLC-MS, the immunoassay method has the characteristics of rapidness and low cost, and can realize high-throughput on-site screening of a large number of samples. However, ELISA has its own defects, such as complicated operation process, long detection time, and the need of a matched enzyme-labeling instrument, which cannot realize field detection. A simpler and faster immunoassay method is urgently needed. The colloidal gold immunochromatographic test strip is a novel immunolabeling technology which takes colloidal gold as a tracer marker to be applied to antigen and antibody, and has the advantages of simplicity, rapidness, accuracy and the like. However, most of the current commercial colloidal gold immunochromatographic test strip tests are single and qualitative tests. Even if quantitative analysis is performed, commercial instruments are used, which are not conducive to remote detection, and for simultaneous detection of DON and FB1The test paper strip has limited reports. It is reported that DON and FB can be detected1The sensitivity of the test strip is very low. Currently, colloidal gold immunochromatographic test strips are used to simultaneously detect DON and FB1The methods and test strips of (a) still remain to be improved.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. Therefore, the invention provides a colloidal gold immunochromatographic test strip and a method for preparing the same. The test strip prepared by the invention can be used for simultaneously, accurately and effectively detecting Deoxynivalenol (DON) and fumonisin B in a sample1（FB1) Content of (A), DON and FB1The qualitative and quantitative detection is fast and the sensitivity is high.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
according to a specific embodiment of the present invention, the present invention provides an immunochromatographic test strip, comprising a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), a water-absorbing pad, and a base plate; the combination pad is loaded with two colloidal gold probes which are respectively a DON monoclonal antibody colloidal gold probe and an FB1Monoclonal antibody colloidal gold probes; the nitrocellulose membrane is provided with two detection lines and a quality control line, one detection line is coated with DON-BSA, and the other detection line is coated with FB1-BSA。
The antibody in the DON monoclonal antibody colloidal gold probe is DON-BSA, and the FB is1The antibody in the monoclonal antibody colloidal gold probe is FB1-BSA。
The concentration of the DON-BSA is 0.1-0.2 mg/mL, and the FB is1-BSA concentration of 0.05-0.1 mg/mL.
GAM-IgG is coated on the quality control line; the concentration of GAM-IgG is 0.2 mg/mL.
According to a specific embodiment of the present invention, the present invention provides a method for preparing an immunochromatographic test strip, comprising the following steps:
(1) preparing a colloidal gold particle solution: adding 99 mL of ultrapure water and 1 mL of 1% chloroauric acid solution into the flask after the acid washing to prepare 100 mL of 0.01% chloroauric acid aqueous solution, heating to boil, adding 1 mL of trisodium citrate solution under the condition of continuous stirring, continuously reacting for 5min when the solution is completely changed into mauve, stopping heating, supplementing water to 100 mL, cooling to room temperature, and storing at 2-8 ℃ to obtain colloidal gold particle solution;
(2) combining DON monoclonal antibody and FB1The monoclonal antibody is marked on the colloidal gold particles in the step (1) to prepare a DON monoclonal antibody colloidal gold probe and FB1The monoclonal antibody colloidal gold probes are respectively sprayed on the bonding pads;
(3) two detection lines and one quality control line are arranged on the nitrocellulose membraneOne detection line is coated with DON-BSA, and the other detection line is coated with FB1-BSA, the quality control line coated with GAM-IgG;
(4) sticking the nitrocellulose membrane in the step (3) on a bottom plate, wherein one end of the nitrocellulose membrane is provided with a combination pad and a sample pad, and the other end of the nitrocellulose membrane is stuck with a water absorption pad; and overlapping the adjacent pads partially, and assembling to obtain the immunochromatographic test strip.
The diameter of the colloidal gold particles in the step (1) is 18-22 nm; the spraying amount of the monoclonal antibody colloidal gold probe in the step (2) is 47-49 mu L/cm2。
And (4) in the step (3), the detection line and the quality control line are parallel to each other and are separated by 5-8 mm.
In the step (3), the coincidence of the sample pad and the combination pad is 1-3 mm, and the overlapping of the water absorption pad and the upper part of the nitrocellulose membrane is 1-2 mm.
According to a specific embodiment of the present invention, the present invention further provides a method for qualitative and quantitative detection of deoxynivalenol and fumonisin B1The method comprises the following steps:
(1) and (3) qualitative detection: adding a detection sample on a sample pad of the immunochromatographic test strip of any one of claims 1 to 5, reacting for 3 to 5min, and observing the color development; when quality control line, DON detection line and FB1When the detection lines are all red, the detection lines indicate that the detected sample contains DON and FB1(ii) a When the quality control line shows red, DON detection line or FB1When only one detection line shows red color, the corresponding DON or FB in the detected sample is shown to be contained1One of (1); when only the quality control line shows red, the DON detection line and the FB1When the detection lines do not show red, the detected sample does not contain DON or FB1Or the content of the object to be detected is low, and the object cannot be detected and is regarded as absent; when the quality control line does not show color, the test strip is invalid;
(2) and (3) quantitative detection: taking a test strip color development result in a dark box by a mobile phone within ten minutes, analyzing by Alphaview software, and obtaining deoxynivalenol and/or fumonisin B in a detection sample according to a standard curve1The content of (a).
Compared with the prior art, the invention has the beneficial effects that:
the invention adopts low-cost colloidal gold as an antibody marker and simultaneously carries out the treatment on deoxynivalenol and fumonisin B1And the rapid detection is carried out, the nitrocellulose membrane is coated with two detection lines at the same time, and the rapid qualitative detection can be carried out on two kinds of mycotoxins simultaneously by comparing the color development depth with naked eyes, and meanwhile, the quantitative detection can be carried out by using specific software. The prepared chromatographic test strip has good sensitivity and stability, is simple and convenient to operate, is rapid and high in sensitivity, and can be used for simultaneously detecting DON and FB1The test strip has the lowest detection limits of 3.94 ng/mL and 2.65 ng/mL respectively by quantitative detection. Low cost and is especially suitable for on-site rapid diagnosis. Easy to popularize, and has wide market prospect and larger economic and social benefits.
Drawings
FIG. 1 is a schematic structural diagram of a test strip;
FIG. 2 is a graph showing the color development results of the test strip;
FIG. 3 is a standard curve diagram of the prepared colloidal gold immunochromatographic test strip for detecting deoxynivalenol;
FIG. 4 is a standard curve diagram of the prepared colloidal gold immunochromatographic test strip for detecting fumonisin B1;
FIG. 5 is a graph showing the results of stability of the prepared colloidal gold test strip.
Detailed Description
The invention discloses a method for simultaneously detecting DON and FB1The immunochromatographic test strip and the preparation method thereof can be realized by appropriately improving the process parameters by taking the contents of the test strip as reference. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. The invention simultaneously detects DON and FB1While the immunochromatographic test strip has been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that the simultaneous detection of DON and FB described herein can be performed without departing from the scope, spirit and scope of the present invention1The immune layer ofThe analytical test strip is modified or properly changed and combined to realize and apply the technology of the invention.
The present invention provides a method for simultaneously detecting DON and FB1The immunochromatographic test strip is further described. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Materials and reagents: bovine serum albumin coupled vomitoxin (DON-BSA), bovine serum albumin coupled fumonisin B1（FB1BSA), DON monoclonal antibody and FB1Monoclonal antibodies were purchased from Power biotech co., Ltd (shenzhen, china); goat anti-mouse Secondary antibody (GAM-IgG) purchased from Life Sciences Research Products&Services. The negative sample is detected by liquid chromatography without containing DON and FB1The corn sample of (1).
Example 1: simultaneous detection of DON and FB1Preparation of immunochromatography test strip
(1) Preparing colloidal gold nanoparticles: adding 99 mL of ultrapure water and 1 mL of 1% chloroauric acid solution into the acid-washed flask to prepare 100 mL of 0.01% chloroauric acid aqueous solution, heating to boil, adding 1 mL of trisodium citrate solution under the condition of continuous stirring, continuously reacting for 5min when the solution is completely changed into mauve, stopping heating, supplementing water to 100 mL, cooling to room temperature, and storing at 2-8 ℃ to obtain the colloidal gold solution with the particle size of about 18-22 nm.
(2) DON and FB1Preparing a monoclonal antibody colloidal gold probe: taking two centrifuge tubes, respectively adding 1 mL of the colloidal gold solution prepared in the step (1) with 0.2 mol/L of K2CO3Adjusting the colloidal gold solution to pH 8.5; 1.5. mu.L of 1mg/mL DON monoclonal antibody and 2. mu.L of 0.5 mg/mL FB1The monoclonal antibody is added into each centrifugal tube and reacts for 30min at room temperature, so that the antibody is marked on the colloidal gold particles; then, 100. mu.L of 10% bovine serum albumin solution (0.01 mol/L BB buffer, pH 9.0) was added to each of the centrifuge tubesSealing for 30 min; after centrifugation at 8000 r/min for 30min at 4 ℃, the supernatant was removed. Redissolving the pellet in 600. mu.L Tris buffer (50 mmol/L containing 3% sucrose, 3% bovine serum albumin and 0.05% sulfadiazine as stabilizers) and preserving at 4 ℃; respectively obtaining stable DON monoclonal antibody colloidal gold probe and FB1Monoclonal antibody colloidal gold probes. 730 μ L each of the DON monoclonal antibody colloidal gold probe and FB1The monoclonal antibody colloidal gold probe is uniformly sprayed on the bonding pad, and the spraying amount is 47-49 mu L/cm2Naturally airing at room temperature or drying for 1h at 37 ℃, and performing vacuum drying and storage.
(3) Fixing the antigen: DON-BSA (initial concentration of 11.5 mg/mL) with concentration of 0.1-0.2 mg/mL and FB with concentration of 0.05-0.1 mg/mL are respectively added by using a colloidal gold spraying and film scratching instrument1BSA (initial concentration 3.6 mg/mL) was streaked on nitrocellulose membrane as detection line (T line); GAM-IgG (initial concentration 20 mg/mL) was diluted to 0.2mg/mL with 10 mmol/L PBS buffer, and 0.2mg/mL GAM-IgG was sprayed on the nitrocellulose membrane as a quality control line (line C) using a colloidal gold spraying striper. The scribing interval between the detection line and the quality control line is 5 mm, and the scribing speed is 1 mu L/cm; the scribed nitrocellulose membrane was dried at 65 ℃ for 5 h.
(4) Assembling the colloidal gold test strip: assembling the colloidal gold immunochromatographic test strip as shown in fig. 1, wherein a nitrocellulose membrane is adhered to a PVC base plate, a combination pad and a sample pad are arranged at one end of the nitrocellulose membrane, and a water absorption pad is adhered at the other end of the nitrocellulose membrane; the assembly sequence is sample pad, combination pad, nitrocellulose membrane, and absorbent pad, and the connection parts of the pads are partially overlapped; the coincidence of the sample pad and the combination pad is 1-3 mm, the overlap of the water absorption pad and the upper part of the nitrocellulose membrane is 1-2 mm, and DON and FB are respectively sprayed on the nitrocellulose membrane1Spraying GAM-IgG as a quality control line as a detection line, wherein the detection line and the quality control line are parallel to each other, are 5-8 mm apart from each other, and are vertical to the long phase of the immune colloidal gold test strip, the detection line is close to one end of the binding pad, and the quality control line is close to one end of the water absorption pad; cut into test strips with the width of 4 mm by a numerical control high-speed cutting machine and stored in a dark and dry environment for later use.
Example 2: DON and FB1Qualitative detection of
This example determines the limit of detection of the immunochromatographic test strip prepared in example 1. Respectively with DON and FB1The DON standard substance solution with concentration of 0, 1.5, 6, 12, 35, 45, 50mg/L and FB solution with concentration of 0, 1.5, 3, 6, 10, 20, 25 mg/L are prepared for detection1A standard solution; respectively adding 1g of negative sample into a centrifuge tube, and then adding DON and FB with different concentrations1Standard solution to form mixed solution containing 50/25, 45/20, 35/10, 12/6, 6/3, 1.5/1.5 and 0/0mg/L standard solution, mixing uniformly and standing overnight; adding 10 mL of distilled water, fully and uniformly mixing for 5min, and centrifuging at 4000 rpm for 10 min; extracting the supernatant as a sample solution to be detected (the serial numbers are 1-7 respectively), respectively dripping the sample solution to be detected onto the immunochromatographic test strip prepared in the embodiment 1 for detection, completing the immunochromatographic process after 3-5min, and observing the color development result as shown in fig. 2. The test paper strip has DON and FB1The lowest detection limits (sensitivity) were 50. mu.g/kg and 25. mu.g/kg, respectively.
When quality control line, DON detection line and FB1When the detection lines are all red, the detection lines indicate that the detected sample contains DON and FB1(ii) a When the quality control line shows red, DON detection line or FB1When the detection line shows red, the detection line indicates that the detected sample contains DON or FB1(ii) a Only the quality control line shows red, DON detection line and FB1When the detection lines do not show red, the detected sample does not contain DON or FB1Or the content of the object to be detected is low, and the object cannot be detected and is regarded as absent; when the quality control line shows no color, the test strip is invalid.
Example 3: DON and FB1Quantitative analysis of
The test strips obtained in example 2 were photographed in a dark box, analyzed with Alphaview software, and a standard curve was established to determine DON and FB in the test samples1The specific content of (a). FIG. 3 is a standard curve diagram of the prepared colloidal gold immunochromatographic test strip for detecting deoxynivalenol; FIG. 4 is a standard curve diagram of the prepared colloidal gold immunochromatographic test strip for detecting fumonisin B1; from fig. 3 and 4, it can be determinedDON and FB1The lowest detection limit of (2) is 3.94 ng/mL and 2.65 ng/mL respectively; the linear ranges are respectively 5.54-42.7 ng/mL and 3.44-16.44 ng/mL. The prepared colloidal gold immunochromatographic test strip has high sensitivity, and can be applied to quantitative analysis of deoxynivalenol and fumonisin B1 in a sample; this result is more accurate than qualitative analysis.
Example 4: colloidal gold test strip accuracy detection
Respectively weighing 1g of DON/FB1The corn flour and the wheat are used as test samples and are added with DON/FB in a centrifuge tube1The standard substance is prepared into three recovery samples (3 samples are parallel) with different concentrations (1.2 ng/mL, 2.0 ng/mL and 2.7 ng/mL) respectively, the samples are uniformly mixed and placed overnight, 10 mL of distilled water is added into each sample to be used as an extracting solution, the mixture is fully and uniformly mixed for 5min, the mixture is centrifuged at 4000 rpm for 10 min, supernatant is taken, and the sample recovery rate is calculated according to the formula that the recovery rate is the detected recovery concentration divided by the adding concentration × 100%.
Table 2 shows Deoxynivalenol (DON) and Fumonisin (FB)1) Additive recovery in samples
As can be seen from Table 1, the detection reagent strip provided by the invention is suitable for the residue determination of deoxynivalenol and fumonisin in corn flour and wheat. Average recovery rate is calculated from the measured/added values, FB in corn flour and wheat1The average recovery rate of the DON is 81.6-97.3%, the average recovery rate of the DON is 80.5-108.6%, and the accuracy and the feasibility of sample detection are proved.
Example 5: colloidal gold test strip stability detection
The prepared colloidal gold test strips are respectively sealed and stored in an environment with the temperature of 4 ℃ and the temperature of 37 ℃, 1g of negative samples are respectively detected every month according to the method described in the embodiment 2, and the color change condition of the test strips is observed. The results are shown in fig. 5, the prepared colloidal gold test strip has good detection effects after being stored for 1, 2, 3 and 4 months at 4 ℃ and 37 ℃, and the detection results have no obvious difference, which indicates that the prepared colloidal gold immune test strip can be stored for at least 4 months at 4-37 ℃.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The immunochromatographic test strip comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, and is characterized in that the combination pad is loaded with two colloidal gold probes which are respectively a DON monoclonal antibody colloidal gold probe, a FB1Monoclonal antibody colloidal gold probes; the nitrocellulose membrane is provided with two detection lines and a quality control line, one detection line is coated with DON-BSA, and the other detection line is coated with FB1-BSA。
2. The immunochromatographic test strip according to claim 1, wherein the DON monoclonal antibody colloidal gold probe is coated with a colloidal gold-labeled DON monoclonal antibody, and the FB is coated with the colloidal gold-labeled DON monoclonal antibody1Monoclonal antibody colloidal gold probe coated with colloidal gold labeled FB1A monoclonal antibody.
3. The immunochromatographic test strip according to claim 1, wherein the concentration of DON-BSA in the detection line is 0.1-0.2 mg/mL, and the FB concentration in the detection line is 0.1-0.2 mg/mL1-BSA concentration of 0.05-0.1 mg/mL.
4. The immunochromatographic test strip according to claim 1, wherein GAM-IgG is coated on a quality control line; the concentration of GAM-IgG is 0.2 mg/mL.
5. A preparation method of an immunochromatographic test strip is characterized by comprising the following steps:
(1) preparing a colloidal gold particle solution;
(2) respectively mixing DON monoclonal antibody and FB1The monoclonal antibody is marked on the colloidal gold particles in the step (1) to prepare a DON monoclonal antibody colloidal gold probe and FB1The monoclonal antibody colloidal gold probes are respectively sprayed on the bonding pads;
(3) spraying two detection lines and a quality control line on the nitrocellulose membrane, wherein one detection line is coated with DON-BSA, and the other detection line is coated with FB1-BSA, the quality control line coated with GAM-IgG;
(4) sticking the nitrocellulose membrane in the step (3) on a bottom plate, wherein one end of the nitrocellulose membrane is provided with a combination pad and a sample pad, and the other end of the nitrocellulose membrane is stuck with a water absorption pad; and overlapping the adjacent pads partially, and assembling to obtain the immunochromatographic test strip.
6. The preparation method of the immunochromatographic test strip according to claim 5, wherein the diameter of the colloidal gold particles in the step (1) is 18 to 22 nm.
7. The preparation method of the immunochromatographic test strip according to claim 5, wherein the spraying amount of the monoclonal antibody colloidal gold probe in the step (2) is 47-49 μ L/cm2。
8. The preparation method of the immunochromatographic test strip according to claim 5, wherein in the step (3), the detection line and the quality control line are parallel to each other and are separated by 5-8 mm.
9. The preparation method of the immunochromatographic test strip according to claim 5, wherein in the step (3), the sample pad and the binding pad are overlapped for 1-3 mm, and the water absorption pad and the upper part of the nitrocellulose membrane are overlapped for 1-2 mm.
10. Qualitative and quantitative detection of deoxynivalenol and fumonisin B1OfThe method is characterized by comprising the following steps:
(1) and (3) qualitative detection: adding a detection sample on a sample pad of the immunochromatographic test strip of any one of claims 1 to 4, reacting for 3 to 5min, and observing the color development; when quality control line, DON detection line and FB1When the detection lines are all red, the detection lines indicate that the detected sample contains DON and FB1(ii) a When the quality control line shows red, DON detection line or FB1When only one detection line shows red color, the corresponding DON or FB in the detected sample is shown to be contained1One of (1); when only the quality control line shows red, the DON detection line and the FB1When the detection lines do not show red, the detected sample does not contain DON or FB1Or the content of the object to be detected is low, and the object cannot be detected and is regarded as absent; when the quality control line does not show color, the test strip is invalid;
(2) and (3) quantitative detection: taking a test strip color development result in a dark box by a mobile phone within ten minutes, analyzing by Alphaview software, and obtaining deoxynivalenol or fumonisin B in a detection sample according to a standard curve1The content of (a).
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101281195A (en) * | 2008-05-19 | 2008-10-08 | 中国计量学院 | Colloidal gold immune chromatography test paper for detecting biotoxin and detecting method thereof |
| CN104569405A (en) * | 2014-12-19 | 2015-04-29 | 南昌大学 | Preparation method of golden flower nanoparticle immune chromatography test strip capable of simultaneously detecting deoxynivalenol and fumonisins B1 in grains |
| CN204314301U (en) * | 2014-12-26 | 2015-05-06 | 北京康源泰博生物科技有限公司 | The immuno-chromatographic test paper strip of a kind of synchronous detection vomitoxin and fumonisin |
| CN111141903A (en) * | 2020-01-16 | 2020-05-12 | 上海交通大学 | Colloidal gold immunochromatographic test strip for triple simultaneous detection of fusarium toxin, and preparation method and application thereof |
-
2020
- 2020-05-28 CN CN202010468250.3A patent/CN111693704A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101281195A (en) * | 2008-05-19 | 2008-10-08 | 中国计量学院 | Colloidal gold immune chromatography test paper for detecting biotoxin and detecting method thereof |
| CN104569405A (en) * | 2014-12-19 | 2015-04-29 | 南昌大学 | Preparation method of golden flower nanoparticle immune chromatography test strip capable of simultaneously detecting deoxynivalenol and fumonisins B1 in grains |
| CN204314301U (en) * | 2014-12-26 | 2015-05-06 | 北京康源泰博生物科技有限公司 | The immuno-chromatographic test paper strip of a kind of synchronous detection vomitoxin and fumonisin |
| CN111141903A (en) * | 2020-01-16 | 2020-05-12 | 上海交通大学 | Colloidal gold immunochromatographic test strip for triple simultaneous detection of fusarium toxin, and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 黄馨雨: "同时检测谷物及中药材中伏马毒素B1和脱氧雪腐镰刀菌烯醇免疫层析方法的研究DON的", 《万方数据知识服务平台》 * |
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Application publication date: 20200922 |