Colloidal gold test strip for detecting agricultural and veterinary drugs and illegal additives and preparation method thereof

Abstract

The present invention relates to the field of biological detection, and in particular to a colloidal gold test strip for detecting agricultural and veterinary drugs and illegal additives and a preparation method thereof. The test strip comprises a base plate and a sample pad, a colloidal gold binding pad, a buffer pad, a reaction membrane, a water-absorbing pad, and a self-adhesive sticker which are tightly connected in sequence on the base plate; the colloidal gold binding pad is coated with a labeled antibody that can bind to the agricultural and veterinary drugs and illegal additives; the buffer pad is coated with a buffer solution; an amino acid line, a detection line, and a quality control line are sequentially arranged along the chromatography direction on the reaction membrane; the amino acid line is coated with an amino acid solution; the detection line is coated with an artificially synthesized antigen, and the quality control line is coated with a sheep anti-mouse IgG secondary antibody that can bind to the labeled antibody. The test strip of the present invention can be quickly used for the detection of agricultural and veterinary drugs and illegal additives, with high sensitivity, good accuracy, high precision, and uniform color development, which effectively solves the problems of low sensitivity, uneven color development, and insufficient accuracy of existing products.

Description

Technical Field

[0001] The invention relates to the field of biological detection, and in particular to a colloidal gold test strip for detecting agricultural and veterinary drugs and illegal additives and a preparation method thereof. Background Art

[0002] Colloidal gold immunochromatography reagent plate has become one of the fastest, most sensitive and convenient immunological detection technologies today due to its single-sample detection, simple and fast operation, good specificity, high sensitivity, no need for auxiliary reagents and instruments, results can be observed with the naked eye, and long validity period. It is particularly suitable for on-site operations, time-sensitive inspections and large-scale surveys.

[0003] In the existing common immunocolloidal gold rapid detection reagent plate device, when the sample solution is dripped onto the sample pad, the analyte in the sample solution and the gold-labeled antibody on the gold-labeled antibody binding pad undergo a specific binding reaction between antigen and antibody, and soon with the capillary action of the nitrocellulose membrane, the analyte in the sample solution permeates to the other end of the membrane strip, and the gold-labeled antibody that has not been bound to the analyte in the sample solution will undergo a specific binding reaction between antigen and antibody with the specific binding antigen on the detection line, and the remaining gold-labeled antibody will bind to the secondary antibody (generally goat anti-mouse IgG) on the control line, so that the detection line and the control line show color changes.

[0004] During the reaction, if the analyte in the sample solution and the gold-labeled antibody on the gold-labeled antibody binding pad do not react sufficiently, the remaining unbound gold-labeled antibody will be relatively more, so that the gold-labeled antibody bound to the specific binding antigen on the detection line will be more, and the detection line will become darker. According to the detection result interpretation method of the immunocolloidal gold rapid detection reagent plate, the color depth difference between the detection line and the control line will be reduced, which may cause the detection line to be darker or the same as the control line, thereby changing the test result from positive to negative, reducing the product detection sensitivity. Therefore, whether the analyte in the sample solution and the gold-labeled antibody on the gold-labeled antibody binding pad react sufficiently determines the sensitivity of the immunocolloidal gold rapid detection reagent plate to a large extent. The factors that affect the adequacy of the reaction of the analyte in the sample solution and the gold-labeled antibody on the gold-labeled antibody binding pad are mainly nitrocellulose membrane (NC membrane), reaction time and stability of the reaction system in addition to the antigen and antibody self-affinity factors. The NC membrane has different pore sizes, hydrophobic and hydrophilic properties, etc., which will produce differences in the sensitivity of the same immunocolloidal gold rapid detection reagent plate. At present, the suppliers of nitrocellulose membranes with good stability and small batch-to-batch differences include Millipore (USA), Whatman and S&S, Sartorius (Germany), Yi Neng (China), and MDI (India). The more commonly used ones are Millipore, Whatman, and Sartorius. After many tests, these NC membrane manufacturers have screened out several membranes with the best universality to supply the market based on a large amount of experimental data. Therefore, there is little room for research on different NC membranes to improve the sensitivity of immunocolloidal gold rapid detection reagent plates. The more stable the reaction system of the analyte in the sample solution and the gold-labeled antibody on the gold-labeled antibody binding pad, and the more sufficient the reaction time, the more sufficient the reaction between the two. In ordinary immunocolloidal gold rapid detection reagent plate devices, the reaction time between the analyte in the sample solution and the gold-labeled antibody on the gold-labeled antibody binding pad is relatively cramped, which often causes the reaction between the two to be insufficient, thus affecting the sensitivity.

[0005] In addition, when using traditional colloidal gold test strips, the test line (T line) may have uneven color development, such as dark color at the bottom of the line and light color at the top, or uneven color at the left and right ends of the line. This is mainly due to the influence of many factors such as raw materials, nitrocellulose membrane, and film dispenser. After the above situation occurs, it can be optimized by replacing raw materials (antigens, antibodies), screening and replacing nitrocellulose membranes, screening stabilizers and buffer systems, etc. Screening high-quality antigens and antibodies may improve the lines for a certain product, but it cannot improve the lines of all products and is not universal. The screening of stabilizers and buffer systems is relatively cumbersome and complicated, and different products are affected by stabilizers and buffer systems to different degrees, so the actual feasibility is not great.

[0006] Therefore, it is of great significance to develop a colloidal gold test strip that has uniform color development, high precision, high sensitivity, and is generally applicable to the detection of pesticides and veterinary drugs and illegal additives. Summary of the invention

[0007] The purpose of the present invention is to provide a colloidal gold test strip for detecting agricultural and veterinary drugs and illegal additives and a preparation method thereof. The colloidal gold test strip provided by the present invention has clear and uniform color development, high precision, good sensitivity, and is generally applicable to the detection of agricultural and veterinary drugs and illegal additives.

[0008] The present invention provides a colloidal gold test strip for detecting agricultural and veterinary drugs and illegal additives, comprising a bottom plate and a sample pad, a colloidal gold binding pad, a buffer pad, a reaction membrane, a water-absorbing pad, and a self-adhesive sticker which are tightly connected in sequence on the bottom plate;

[0009] The colloidal gold binding pad is coated with a labeled antibody that binds to the agricultural and veterinary drugs and illegal additives; the buffer pad is coated with a buffer solution; the reaction membrane is provided with an amino acid line, a detection line and a quality control line in sequence along the chromatography direction; the amino acid line is coated with an amino acid solution; the detection line is coated with an artificially synthesized antigen; and the quality control line is coated with a sheep anti-mouse IgG secondary antibody that can bind to the labeled antibody.

[0010] The colloidal gold test strip provided by the present invention is coated with an amino acid line at one end of the reaction membrane close to the colloidal gold binding pad. Experiments show that the colloidal gold test strip with the amino acid line has uniform color development, high precision and accuracy, and significantly improves the problem of uneven color development of the T line of the existing colloidal gold test strip and low accuracy of quantitative detection results caused by the uneven color development.

[0011] In some embodiments, the amino acid solution consists of leucine, phenylalanine, threonine, lysine, aspartic acid and a preservative in PBS buffer.

[0012] In some embodiments, the amino acid solution is a 0.01 M PBS buffer at pH 7.2-7.4 containing 0.05-0.2% leucine, 0.05-0.2% phenylalanine, 0.05-0.2% threonine, 0.05-0.2% lysine, 0.05-0.2% aspartic acid and 0.1-0.3% sodium azide.

[0013] In some specific embodiments, the amino acid solution is a 0.01 M PBS buffer at pH 7.4 containing 0.1% leucine, 0.1% phenylalanine, 0.1% threonine, 0.1% lysine, 0.1% aspartic acid and 0.2% sodium azide.

[0014] In some embodiments, the buffer solution for coating the buffer pad is a 0.1 mol / L phosphate buffer solution containing 1% polyethylene glycol octylphenyl ether and 1% bovine serum albumin.

[0015] In the present invention, the colloidal gold binding pad is coated with a labeled antibody that binds to the antigen. In some embodiments, the labeled antibody is a monoclonal antibody labeled with colloidal gold.

[0016] In the present invention, the artificially synthesized antigen is a conjugate of agricultural veterinary drugs and illegal additives with a carrier protein.

[0017] In some specific embodiments, the pesticides and veterinary drugs and illegal additives are aflatoxin B1, chloramphenicol, clenbuterol hydrochloride or furazolidone metabolites.

[0018] In some embodiments, the carrier protein is one of bovine serum albumin, ovalbumin, and hemocyanin.

[0019] In some embodiments, the distance between the amino acid line and the detection line is 4 to 6 mm.

[0020] In some embodiments, the bottom plate is a PVC plate, the sample pad, the buffer pad and the colloidal gold binding pad are made of glass fiber, and the reaction membrane is an NC membrane.

[0021] In the colloidal gold test strip of the present invention, the sample pad, the colloidal gold binding pad, the nitrocellulose membrane and the water absorption pad have an overlap of 1 to 2 mm between adjacent parts. The purpose is, on the one hand, to ensure that the chromatography proceeds smoothly from the sample pad to the water absorption pad, and on the other hand, to allow the sample solution to have sufficient reaction time from the sample pad to the gold-labeled binding pad to the reaction membrane, so as to ensure that the effective components in the sample solution react smoothly with the gold-labeled antibodies in the gold-labeled binding pad.

[0022] The present invention also provides a method for preparing the colloidal gold test strip, comprising:

[0023] Prepare colloidal gold particles; label the antibody bound to the antigen with the colloidal gold particles to obtain a labeled antibody; coat the labeled antibody on a binding pad and dry it to obtain a colloidal gold binding pad; soak the glass fiber membrane in a buffer solution and then dry it to obtain a buffer pad;

[0024] The antigen and goat anti-mouse IgG secondary antibody were coated onto the reaction membrane in sequence along the chromatography direction, serving as the detection line and quality control line respectively;

[0025] An amino acid solution is prepared, and the amino acid solution is coated on one end of the reaction membrane close to the colloidal gold binding pad to obtain an amino acid line, wherein the distance between the amino acid line and the detection line is 4 to 6 mm;

[0026] The sample pad, the colloidal gold binding pad, the buffer pad, the reaction membrane and the water absorption pad are adhered to the bottom plate to obtain the colloidal gold test paper strip.

[0027] The present invention also provides a colloidal gold reagent plate, comprising a colloidal gold test strip of the present invention and a plastic shell 11 wrapping the colloidal gold test strip, wherein the plastic shell 11 is provided with a sample addition hole S and an observation window 12. The plastic shell 11 has the function of fixing the colloidal gold test strip and marking. When in use, the sample to be tested is dripped onto the sample pad 1 of the colloidal gold test strip through the sample addition hole S, and after standing for a few minutes, the color development of the C line and the T line is observed through the observation window 12, and the pesticides and veterinary drugs and illegal additives in the test sample are qualitatively determined according to the interpretation method, and the content of the pesticides and veterinary drugs and illegal additives can be quantitatively detected in combination with the colloidal gold reader. When a small molecule drug is a drug, the colloidal gold test strip can qualitatively and quantitatively detect the drug residue in the sample to be tested.

[0028] The colloidal gold test strip of the present invention is based on the principle of competition method. The pesticides and illegal additives in the sample and their reagents compete with the artificial antigen on the detection line for binding to the labeled antibody on the colloidal gold binding pad. The color development degree on the detection line is inversely proportional to the content of the pesticides and illegal additives in the sample to be tested.

[0029] Reading and interpretation method of qualitative test results: When the sample does not contain the detected pesticides and veterinary drugs and illegal additives or the content of the detected pesticides and veterinary drugs and illegal additives is lower than the detection limit, the T line of the test strip is stronger than or as strong as the C line, which is negative; the T line has no color or is weaker than the C line, which is positive; if the C line does not color, regardless of whether the T line is colored or not, the test strip is invalid.

[0030] The present invention provides a colloidal gold test strip for detecting agricultural and veterinary drugs and illegal additives and a preparation method thereof. The colloidal gold test strip comprises a bottom plate and a sample pad, a colloidal gold binding pad, a buffer pad, a reaction membrane, and a water-absorbing pad which are tightly connected in sequence on the bottom plate; an amino acid line, a detection line, and a quality control line are sequentially arranged on the reaction membrane along the chromatography direction; the detection line is coated with an antigen, and the colloidal gold binding pad is coated with a labeled antibody that binds to the antigen; and the amino acid line is coated with an amino acid solution. Compared with the existing colloidal gold test strip, the test strip of the present invention has the advantages of high sensitivity, uniform color development, high precision, etc., and is easy to use and operate, and does not require professional personnel to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0032] Figure 1 It is a structural schematic diagram and a top view of the colloidal gold test strip of the present invention;

[0033] Among them, 1 indicates a sample pad, 2 indicates a colloidal gold binding pad, 3 indicates a buffer pad, 4 indicates a reaction membrane, 5 indicates an amino acid line, 6 indicates a detection line, 7 indicates a quality control line, 8 indicates a water-absorbing pad, 9 indicates a sticker, and 10 indicates a PVC base plate;

[0034] Figure 2 The operation schematic diagram of the colloidal gold reagent plate of the present invention; wherein S indicates a sample addition hole, 6 indicates a detection line, 7 indicates a quality control line, 11 indicates a plastic housing, and 12 indicates an observation window;

[0035] Figure 3 The figure is a schematic diagram of the result judgment of the colloidal gold test strip and the reagent plate of the present invention, wherein T is the test line and C is the quality control line; if the color of the T line is darker than that of the C line or the color intensity is the same, it is negative; if the T line does not color or the color is lighter than that of the C line, it is positive; if the C line does not color, it is invalid;

[0036] Figure 4 The test results of chloramphenicol using the conventional colloidal gold test strip and the colloidal gold test strip of the present invention are compared, wherein 4-a to 4-b are the test results when the concentration of the five amino acids is 0.05%, 4-c to 4-d are the test results when the concentration of the five amino acids is 0.1%, and 4-e to 4-f are the test results when the concentration of the five amino acids is 0.2%;

[0037] Figure 5 It is a schematic diagram of the structure of a traditional colloidal gold test strip; wherein 1 indicates a sample pad, 2 indicates a colloidal gold binding pad, 3 indicates a reaction membrane, 4 indicates a detection line, 5 indicates a quality control line, 6 indicates a water absorption pad, and 7 indicates a PVC bottom plate;

[0038] Figure 6 6-a is the effect of different concentrations of amino acid lines on the color development of aflatoxin B1 colloidal gold test strips, 6-b is the test result of the colloidal gold test strips of the present invention;

[0039] Figure 7 The test results of clenbuterol hydrochloride using the traditional colloidal gold test strip and the colloidal gold test strip of the present invention are compared. 7-a to 7-b are the test results of the traditional colloidal gold test strip, and 7-c to 7-d are the test results of the colloidal gold test strip of the present invention.

[0040] Figure 8 The test results of chloramphenicol using the conventional colloidal gold test strip and the colloidal gold test strip of the present invention are compared. 8-a to 8-b are the test results of the conventional colloidal gold test strip, and 8-c to 8-d are the test results of the colloidal gold test strip of the present invention.

[0041] Fig. 9The present invention compares the detection results of furazolidone metabolites using the conventional colloidal gold test strip and the colloidal gold test strip of the present invention. 9-a to 9-b are the detection results of the conventional colloidal gold test strip, and 9-c to 9-d are the detection results of the colloidal gold test strip of the present invention. DETAILED DESCRIPTION

[0042] The present invention discloses a colloidal gold test strip for detecting agricultural and veterinary drugs and illegal additives and a preparation method thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0043] The disclosed embodiments are described to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

[0044] In the colloidal gold test strip for detecting agricultural and veterinary drugs and illegal additives and the preparation method thereof provided by the present invention, the reagents, materials and instruments used are all common commercial products and can be purchased in the market.

[0045] The present invention will be further described below in conjunction with embodiments:

[0046] Example 1 Preparation of colloidal gold test strips of the present invention

[0047] 1) Preparation of sample pad: The glass fiber membrane is soaked in a buffer solution and then dried. The specific preparation method is: soak in sample pad treatment solution for more than 15 minutes, place in a drying oven or drying room at 37°C, and dry for more than 16 hours. Preparation method of sample pad treatment solution: weigh 38.137g Na2B4O7·10H2O, 10g PVP, 1g BSA, 10mL Triton X-100, add 1L H2O to dissolve, and adjust pH to 9.3.

[0048] 2) Preparation of colloidal gold binding pad: It is prepared by soaking the glass fiber membrane in a buffer solution and then drying it. The specific preparation method is: soak it in the sample pad treatment solution for more than 30 minutes, place it in a drying oven or drying room at 37°C, and bake it for more than 16 hours. Preparation method of gold-labeled antibody binding pad treatment solution: weigh 5g PVA, 7.1g Na2HPO4, 5g BSA, 1mL Triton X-100, add 1L H2O to dissolve, and adjust the pH to 7.4. Prepare colloidal gold particles, use colloidal gold particles to label the antibody that binds to the detection line antigen, and use a gold dot machine to coat the gold-labeled antibody onto the binding pad according to the gold-labeled antibody spray volume determined by repeated debugging.

[0049] 3) Preparation of the cushion pad: The glass fiber membrane was soaked in a mixed solution of 0.1 mol / L phosphate buffer solution (PBS) + 1% triton X-100 + 1% bovine serum albumin (BSA) and then dried.

[0050] 4) Coating of C-line and T-line: According to the working concentration and spray volume of C-line goat anti-mouse IgG and the working concentration and spray volume of T-line specific binding antigen determined through repeated debugging, use a film dispenser to coat the goat anti-mouse IgG and specific binding antigen onto the nitrocellulose membrane as C-line and T-line.

[0051] 5) Coating of amino acid line: prepare an amino acid solution (0.1% leucine, 0.1% phenylalanine, 0.1% threonine, 0.1% lysine, 0.1% aspartic acid, 0.2% sodium azide and the balance PBS (0.01M pH 7.4), coat the amino acid solution on one end of the reaction membrane close to the colloidal gold binding pad to obtain an amino acid line, the distance between the amino acid line and the detection line is 4 to 6 mm;

[0052] 6) Assembly of the reagent strip: stick the sample pad, colloidal gold binding pad, buffer pad, nitrocellulose membrane, and absorbent pad on the PVC backing in sequence to form a reagent card, and use a cutting machine to cut the card into test strips.

[0053] The structural diagram is as follows Figure 1 shown.

[0054] See also Figure 1The colloidal gold test strip of the utility model comprises a bottom plate 10 and a sample pad 1, a colloidal gold binding pad 2, a buffer pad 3, a reaction membrane 4, a water-absorbing pad 8, and a self-adhesive label 9 which are tightly connected in sequence on the bottom plate 10; the colloidal gold binding pad 1 is coated with a labeled antibody that binds to the agricultural veterinary drugs and illegal additives; the buffer pad 3 is coated with a buffer solution; an amino acid line 5, a detection line 6, and a quality control line 7 are sequentially arranged on the reaction membrane 4 along the chromatography direction; the amino acid line 5 is coated with an amino acid solution; the detection line 6 is coated with an artificially synthesized antigen; the quality control line 7 is coated with a sheep anti-mouse IgG secondary antibody that can bind to the labeled antibody.

[0055] The operation process of the colloidal gold reagent plate of the present invention is shown in Figure 2 , wherein S indicates a sample addition hole, 6 indicates a detection line, 7 indicates a quality control line, 11 indicates a plastic housing, and 12 indicates an observation window.

[0056] The method for reading the colloidal gold test strip of the present invention is shown in Figure 3 , where T is the test line and C is the quality control line; when the sample does not contain the tested pesticides and veterinary drugs and illegal additives or the content of pesticides and veterinary drugs and illegal additives is lower than the detection limit, the T line of the test strip is stronger than or as strong as the C line, which is negative; the T line has no color or is weaker than the C line, which is positive; the C line does not color, regardless of whether the T line is colored or not, the test strip is invalid.

[0057] Example 2 Optimization of amino acid lines coated with amino acid solutions of different concentrations

[0058] Taking chloramphenicol as the pesticide and veterinary drug to be tested and illegal additives, the concentration of amino acid solution was optimized. The effects of five amino acid solutions in the amino acid solution at concentrations of 0.05%, 0.1% and 0.2% on the color development of chloramphenicol test strips were investigated. The rest of the test strips were prepared according to the method of Example 1. The results are shown in Figure 4 .

[0059] The experimental results show that the colloidal gold test strip prepared by coating the amino acid line with 0.1% amino acid solution concentration has a clear T line color and more uniform color.

[0060] Example 3 Precision Detection Experiment

[0061] Aflatoxin B1 was used as the agricultural and veterinary drug to be tested and the illegal additives, and a colloidal gold test strip for detecting aflatoxin B1 was prepared according to the method of Example 1. The aflatoxin B1 solution with a concentration of 0 ppb, 0.5 ppb, 1 ppb, 2 ppb, 4 ppb and 8 ppb was tested, and the test data was measured with a colloidal gold reader. The coefficient of variation (CV) was calculated based on the 4 measurement results.

[0062] At the same time, using the traditional colloidal gold test strips (structure see Figure 5 ) were used to test the samples, and the results are shown in Tables 1-2.

[0063] Table 1 Test results of traditional colloidal gold test strips

[0064]

[0065] Table 2 Colloidal gold test strip test results of the present invention

[0066]

[0067] From the test results in Tables 1 and 2, it can be seen that the coefficient of variation of the colloidal gold test strip of the present invention is between 1.5% and 7.2%, which is significantly smaller than the coefficient of variation of the traditional colloidal gold test strip, indicating that the test strip of the present invention has higher precision.

[0068] Embodiment 4 Colloidal gold test strip of the present invention

[0069] Aflatoxin B1, clenbuterol hydrochloride, chloramphenicol, and furazolidone metabolites were respectively used as the pesticides and veterinary drugs to be tested and the illegal additives. The method of Example 1 was used to prepare the method for detecting aflatoxin B1, clenbuterol hydrochloride, chloramphenicol, and furazolidone of the present invention. The schematic diagram of the structure is shown in FIG. Figure 1 .

[0070] Example 5 Comparative experiment on the color development of small molecule drugs by conventional colloidal gold test strips and the colloidal gold test strips of the present invention

[0071] Four conventional colloidal gold test strips were prepared for the detection of aflatoxin B1, clenbuterol hydrochloride, chloramphenicol, and furazolidone metabolites. The schematic diagrams are shown in Figure 5 .

[0072] Aflatoxin B1, clenbuterol hydrochloride, chloramphenicol, and furazolidone metabolites were tested using conventional colloidal gold test strips and colloidal gold test strips of Example 4, respectively. The results are shown in Figures 6 to 9 .

[0073] Depend on Figure 6-9 It can be seen that compared with the traditional colloidal gold test strip, the colloidal gold test strip of the present invention has clearer and more uniform color after adding the amino acid line, and is suitable for the detection of aflatoxin B1, clenbuterol hydrochloride, chloramphenicol, and furazolidone metabolites.

[0074] Example 6 Comparative Experiment on Sensitivity of Conventional Colloidal Gold Test Strip and Colloidal Gold Test Strip of the Present Invention

[0075] Taking the chloramphenicol immunocolloidal gold reagent plate as an example, compared with the traditional one without a buffer pad (structure see Figure 5 ) and the reagent plate using a buffer pad in Example 1 of the present application (see Figure 1), chloramphenicol standard solution was added to the negative solution to a final concentration of 0.05, 0.1, 0.2 and 0.4 μg / kg, and the reagent plate with a buffer pad and the reagent plate without a buffer pad were titrated respectively. The test data and test results were determined using a colloidal gold reader. The experimental results are shown in Table 3.

[0076] Table 3 Sensitivity test results

[0077]

[0078] It can be seen from the experimental data in Table 3 that the detection limit of the reagent plate with a buffer pad used in the present invention is significantly lower than the detection limit of the traditional reagent plate without a buffer pad.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A colloidal gold test strip for detecting agricultural and veterinary drugs and illegal additives, characterized in that: It includes a bottom plate and a sample pad, a colloidal gold binding pad, a buffer pad, a reaction membrane, a water-absorbing pad, and a self-adhesive sticker which are tightly connected in sequence on the bottom plate; The colloidal gold binding pad is coated with a labeled antibody that binds to the agricultural veterinary drugs and illegal additives; the buffer pad is coated with a buffer solution; the reaction membrane is provided with an amino acid line, a detection line and a quality control line in sequence along the chromatography direction; the amino acid line is coated with an amino acid solution; the detection line is coated with an artificially synthesized antigen; the quality control line is coated with a goat anti-mouse IgG secondary antibody that can bind to the labeled antibody; The amino acid solution is composed of leucine, phenylalanine, threonine, lysine, aspartic acid and a PBS buffer solution of a preservative; The buffer solution for coating the cushion pad is a 0.1 mol / L phosphate buffer solution containing 1% polyethylene glycol octylphenyl ether and 1% bovine serum albumin; The labeled antibody is a monoclonal antibody labeled with colloidal gold; The artificially synthesized antigen is a conjugate of agricultural veterinary drugs and illegal additives with carrier proteins.

2. The colloidal gold test strip according to claim 1, characterized in that: The amino acid solution is a 0.01 M PBS buffer solution at pH 7.2-7.4 containing 0.05-0.2% leucine, 0.05-0.2% phenylalanine, 0.05-0.2% threonine, 0.05-0.2% lysine, 0.05-0.2% aspartic acid and 0.1-0.3% sodium azide.

3. The colloidal gold test strip according to claim 1, characterized in that: The amino acid solution is a 0.01 M PBS buffer solution at pH 7.4 containing 0.1% leucine, 0.1% phenylalanine, 0.1% threonine, 0.1% lysine, 0.1% aspartic acid and 0.2% sodium azide.

4. The colloidal gold test strip according to claim 1, characterized in that: The carrier protein is one of bovine serum albumin, ovalbumin and hemocyanin.

5. The colloidal gold test strip according to claim 1, characterized in that: The distance between the amino acid line and the detection line is 4 to 6 mm.

6. The colloidal gold test strip according to claim 1, characterized in that: The bottom plate is a PVC plate, the sample pad, the buffer pad and the colloidal gold binding pad are made of glass fiber, and the reaction membrane is an NC membrane.

7. The method for preparing the colloidal gold test strip according to claim 1, characterized in that: include: preparing colloidal gold particles; Using colloidal gold particles to label the labeled antibody bound to the pesticide and veterinary drug and the illegal additive to obtain a labeled antibody; coating the labeled antibody onto a conjugate pad and drying the conjugate pad to obtain a colloidal gold conjugate pad; The glass fiber membrane is soaked in a buffer solution and then dried to obtain a buffer pad; The synthetic antigen and goat anti-mouse IgG secondary antibody were coated onto the reaction membrane in sequence along the chromatography direction, serving as the detection line and quality control line respectively; An amino acid solution is prepared, and the amino acid solution is coated on one end of the reaction membrane close to the colloidal gold binding pad to obtain an amino acid line, wherein the distance between the amino acid line and the detection line is 4 to 6 mm; The sample pad, the colloidal gold binding pad, the buffer pad, the reaction membrane and the water absorption pad are adhered to the bottom plate to obtain the colloidal gold test paper strip.

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