CN115356475A - Rapid detection test paper strip for beta receptor agonist and preparation method and application thereof - Google Patents

Abstract

The test strip comprises a sample pad, a combination pad, an NC membrane, absorbent paper and a rubber plate, wherein the combination pad comprises the combination pad and a conjugate of color microspheres and beta receptor agonist. Powerful technical support is provided for on-site rapid detection with huge sample size. Meanwhile, great convenience is brought to on-site investigation and detection for market supervision and management personnel.

Description

A quick test strip for beta-receptor agonist and its preparation method and application

technical field

The invention relates to the technical field of quantitative identification, in particular to a quick test strip for beta-receptor agonists and its preparation method and application.

Background technique

"Clenbuterol" is a β-receptor agonist, which is very harmful to humans. The main manifestations are: palpitations during acute poisoning, muscle tremors in the face, neck, and limbs, finger tremors, feeling of heaviness in the feet and even being unable to stand, headaches, Dizziness, nausea, vomiting, fatigue, facial flushing, skin allergic purple papules.

At present, the main methods for the detection of β-receptor agonists in food are: instrumental methods such as liquid chromatography-mass spectrometry, mass spectrometry, and gas chromatography-mass spectrometry. The size of the equipment is too large, the maintenance cost of the equipment is high, the detection time is long, and the detection operation steps are cumbersome. For the on-site rapid detection of large sample volume, these methods are difficult to realize.

Immunochromatography has become a hot spot in the field of rapid clinical diagnosis due to its advantages of simple operation and clear results. Existing clenbuterol test paper detection products are mainly a series of products such as colloidal gold immunochromatography test paper made on the basis of colloidal gold immunochromatography technology, such as patent document CN203786122U discloses a kind of clenbuterol detection with high measurement accuracy A test paper card, the gold standard binding pad of the test paper card is coated with a mixture of anti-clenbuterol hydrochloride conjugates, anti-ractopamine conjugates and anti-albuterol conjugate gold-labeled antibodies, prior art (Chen Yihong.β Technical research on gold-labeled test card for receptor agonists [D]. Fuzhou University, 2017.) disclosed that the preparation conditions of gold-labeled test card for β-receptor agonists and the pretreatment conditions of samples were optimized, and disclosed that β-receptor agonists The preparation of the antigen and antibody required by the gold standard test card for agonists, the prior art (Jiang Yanbin, Sun Guanru, Wang Hai, et al. Development of clenbuterol hydrochloride colloidal gold rapid detection test strips [C], 2011) discloses the use of colloidal gold Gold-labeled clenbuterol hydrochloride monoclonal antibody competitive membrane chromatography technology made rapid detection test strips for the detection of clenbuterol hydrochloride residues in pig urine.

However, the current colloidal gold immunochromatographic test strips mainly have the following defects: 1. The color development of colloidal gold is single, usually only showing a single red color; 2. The particle size of colloidal gold is not easy to control, and it is difficult to adjust the particle size according to the needs of the test paper 3. A strong reducing agent is needed in the labeling process of colloidal gold, which has a certain impact on human health; 4. There are no biologically active groups on the surface of colloidal gold, and the combination with antibodies is completely due to electrostatic interaction, which has poor stability; 5. 1. The particle size of colloidal gold is circular when the particle size is larger than h, and it is oval when the particle size is larger, and the shape is unstable.

In summary, there is an urgent need to develop an immunochromatographic detection method with low cost, simple operation, intuitive and accurate results, high sensitivity and good stability.

Contents of the invention

In order to overcome the problems mentioned in the above-mentioned prior art, the object of the present invention is to provide a quick test strip for beta receptor agonists and its detection method. The test strip provided by the present invention is low in cost, easy to operate, intuitive and accurate in results, High sensitivity and good stability.

In order to achieve the above object, the present invention provides the following technical solutions:

The first aspect of the present invention provides a kind of β-receptor agonist quick detection test strip, comprises sample pad, binding pad, NC film, absorbent paper and glue board, wherein, binding pad comprises binding pad and colored microsphere and β receptor Somatoagonist conjugates;

The binding pad is prepared by the following steps:

a. Soaking: Soak the bonding pad in the bonding pad soaking solution, take out the soaked bonding pad and dry;

b. Activation of colored microspheres: take colored microspheres, ultrasonically, take 200 μL of ultrasonically prepared colored microspheres, add MES solution and mix evenly, centrifuge, discard supernatant, repeat 2 times, then add MES solution, mix evenly, and sonicate;

Use the above MES solution to prepare NHS solution with a concentration of 5-20mol/L, take the NHS solution and add it to the colored microspheres that have been sonicated, and then use pure water to prepare EDC. The concentration of the EDC solution is 10-20mol/L. Add 5 μL to the above-mentioned colored microspheres, wrap them with tinfoil paper and react on a shaker in the dark for 5-30 minutes, and become the activated colored microspheres;

c. Preparation of colored microspheres and β-receptor agonist antibody conjugates: Take 1-5 μL of 1 μg/μL β-receptor agonist antibody and add it to the activated color microspheres, mix evenly, and react on a shaker in the dark for 0.5- 3h, add 5-15μL of 0.05-3mol/L ethanolamine solution and mix well, shake the reaction in the dark, centrifuge, discard the supernatant, mix with 300-1000μL microsphere diluent, ultrasonic for 5min, shake the reaction in the dark Centrifuge for 1 hour, discard the supernatant, mix evenly with 100-300 μL microsphere diluent, and sonicate to form a uniform β-receptor agonist antibody-colored microsphere solution, and store the solution at 2-8°C;

The β receptor agonist antibody can be 1 μL, 1.5 μL, 2 μL, 2.5 μL, 3 μL, 3.5 μL, 4 μL, 4.5 μL or 5 μL;

d, gold spraying and drying: take out the solution prepared in step c, add the gold spraying diluent to dilute, adjust the pH to 8-9.5 with potassium carbonate after dilution, carry out gold spraying on the binding pad prepared in step a, and spray gold Bake the bonding pad at 37-48°C for 1-1.5 hours;

Preferably, the bonding pad is baked at 37°C for 1.5h;

Preferably, the pH is adjusted to 9 with potassium carbonate in the step d;

The binding pad soaking solution is a 0.03-0.05mol/L Tris-HCL buffer solution with a pH of 7-7.5, which contains 3-5% trehalose, 0.3-0.5% BSA, and 0.05-0.1% Tween;

Preferably, the soaking solution of the binding pad is a 0.05 mol/L Tris-HCL buffer solution with pH=7.4, which contains 5% trehalose, 0.5% BSA, and 0.1% Tween;

Wherein, the β receptor agonist antibody is probuterol antibody, clopronaline antibody, cimaterol antibody, terbutaline antibody, tobuterol antibody, sibuterol antibody, salbutamol antibody, Zipaterol antibody, clenbuterol antibody, ractopamine antibody, fenoterol antibody, mapbuterol antibody, bromoclenbuterol antibody, mapenterol antibody, phenylethanolamine A antibody, bromobuterol One or more of Luo;

The microsphere diluent is: 0.01-0.02mol/LPBS and 0.5-1.2% BSA;

Preferably, the microsphere diluent is 0.01mol/LPBS and 1% BSA;

Preferably, the concentration of the β-receptor agonist antibody in the β-receptor agonist antibody-colored microsphere solution is 1-5 μL/mL;

The diluent used for the gold spraying consists of: 0.02% boric acid, 0.2% PEG20000, 0.02% preservative, 10% trehalose, 0.25% Tween, and 1% BSA.

The amount of sprayed gold is 6-9.9 μL/cm, preferably 6 μL/cm.

The 6 μL/cm is 6 μL of the solution adjusted with potassium carbonate by adding the gold spray solution described in the gold spray step d per centimeter.

In step b, the pH of the MES solution is selected from 4-5, preferably 4.5-5;

In the present invention, the pH of the MES solution affects the combination of microspheres and antibodies, and when the pH is 4.5-5, the combination effect of microspheres and antibodies is the best.

Further, the amount of the β-receptor agonist antibody is 2-5 μL/mL;

For example, the amount of the β receptor agonist antibody can be 2, 3, 4, 5 μL/mL;

Further, the particle size of the colored microspheres is 100-300 nm,

For example, the particle size of the colored microspheres can be 100, 200, 300 nm, preferably 300 nm.

Further, the sample pad is made of glass fiber material and polyester fiber film;

Preferably a glass fiber membrane;

The thickness of the glass fiber membrane is 0.2-0.6 mm, preferably 0.3-0.45 mm;

The climbing speed of the glass fiber membrane is 5-70mm/60s; preferably, the climbing speed of the glass fiber membrane is 10-50mm/60s;

Preferably, the material of the bonding pad is glass fiber, and the water absorption is 450±50g/m ² ;

The NC film is selected from NC films with a width of 20-25mm and a climbing speed of 100-125s/4cm;

The absorbent paper is selected from absorbent paper with a climbing speed of 40-180 s/mm and a thickness of 0.87-1.13 mm;

More preferably, the model of the sample pad is selected from: DL42, RB45, RB65, CB06, SB06, RB37, KB50 or SB08, more preferably, the model of the sample pad is selected from: CB06, SB06, RB37, KB50 or SB08;

The model of the bonding pad is selected from VL98, VL78 or SAP-Z70, the model of the NC membrane is selected from 25mmJJ120 or 20mmJJ120, the model of the absorbent paper is selected from CH37, SH27, CH27 or SH37, the The type of rubber sheet is selected from PVC, SM31-40, SMNF31-40 or SMA31-40;

Particularly preferably, the model of the sample pad is SB06, the model of the bonding pad is SAP-Z70, the model of the NC membrane is 25mmJJ120, the model of the absorbent paper is CH37, the glue The model of the plate is PVC.

Further, the NC membrane contains T lines and C lines, the T lines contain β-receptor agonist antigens, the C line contains IgG, and the β-receptor agonist antigens are probutene, chlorprena Lin, cimaterol, terbutaline, tobuterol, sibuterol, albuterol, zilpaterol, clenbuterol, ractopamine, fenoterol, mabuterol, bromogram Renbuterol, mapenterol, phenylethanolamine A, bromobuterol, preferably, the β-receptor agonist antigen is ractopamine, albuterol or clenbuterol hydrochloride.

Another aspect of the present invention provides a kind of preparation method of above-mentioned test strip, comprises the following steps:

S1. Treatment of the sample pad: soak the sample pad in the sample pad soaking solution for 5-30 minutes, take it out and dry it in an oven at 36-42°C for 1.5-2.2 hours for later use;

Preferably, the soaking time in step S1 is 10min;

Preferably, the drying conditions in step S1 are a drying temperature of 37 degrees and a drying time of 2 hours;

S2. Treatment of the bonding pad: soak the bonding pad in the bonding pad soaking solution for 1.5-2.2 hours, take out the soaked bonding pad and place it in an oven and dry it at 37 for 2 hours for later use;

S3. Preparation of NC membrane: dilute the first solution with the first diluent, then dilute the second solution with the second diluent, use the diluted first solution as the T line, and the diluted second solution as the C line dashed;

S4. Lay the sample pad, conjugation pad, NC film, and absorbent paper on the PVC rubber sheet in sequence. The bonding sequence is: first paste the NC film, the conjugation pad is pressed against the NC film, the sample pad is pressed against the conjugation pad, and the absorbent paper is pressed. With the NC film, the overlapping length between each other is generally about 0.8-1.5mm. After the lamination is completed, use a cutter to cut it into test strips with a width of 3.8-4.2mm;

Preferably, the first solution is a solution comprising β receptor agonist antigen and BSA;

The composition of the first diluent is 0.01-0.03mol/LPBS+0.03-0.05% TritonX-100;

Preferably, the composition of the first diluent is 0.01mol/LPBS+0.03% TritonX-100;

The second solution is an IgG solution;

The composition of the second diluent is 0.01-0.03mol/LPBS+1-5% methanol+0.03-0.05% TritonX-100;

Preferably, the composition of the second diluent is 0.01mol/LPBS+3% methanol+0.03% TritonX-100;

Preferably, when the first solution is ractopamine and BSA solution, use the first diluent to dilute to a solution with a ractopamine concentration of 0.2-0.9 mol/L; use the second diluent to dilute the second solution to an IgG concentration of 0.4- 1.0mol/L solution;

For example, using the first diluent to dilute into a solution with a ractopamine concentration of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 mol/L, and using the second diluent to dilute to an IgG concentration of 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mol/L;

And/or, when the first solution is albuterol and BSA solution, use the first diluent to dilute to a solution with a salbutamol concentration of 0.02-0.08 mol/L; the second solution is diluted to a concentration of 0.4-0.8 mol with the second diluent /L IgG solution;

For example, use the first diluent to dilute to a solution with a salbutamol concentration of 0.02, 0.03, 0.04, 0.05, 0.06, 0.07 or 0.08mol/L, and use the second diluent to dilute to a concentration of 0.4, 0.5, 0.6, 0.7 or 0.8mol /L IgG solution;

And/or, when the first solution is clenbuterol hydrochloride and BSA solution, use the first diluent to dilute to a solution with a concentration of clenbuterol hydrochloride of 0.2-0.7mol/L; use the second diluent for the second solution Diluted to a solution with an IgG concentration of 0.4-0.6 mol/L;

For example, use the first diluent to dilute to a solution with a concentration of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 mol/L of clenbuterol hydrochloride, and use the second diluent to dilute to a concentration of 0.4, 0.5, 0.6, 0.7 or 0.8mol/L IgG solution;

More preferably, when the first solution is ractopamine and BSA solution, use the first diluent to dilute to a solution with a ractopamine concentration of 0.8 mol/L; L solution;

More preferably, when the first solution is albuterol and BSA solution, use the first diluent to dilute to a solution with a salbutamol concentration of 0.05mol/L; the second solution is diluted with a second diluent to an IgG concentration of 0.8mol/L solution;

More preferably, when the first solution is clenbuterol hydrochloride and BSA solution, use the first diluent to dilute to a solution with a clenbuterol hydrochloride concentration of 0.6mol/L; the second solution is diluted to A solution with an IgG concentration of 0.5mol/L;

Take the diluted first solution and the diluted second solution, draw a line on the NC membrane at a rate of 0.8-1.2 μL/cm, put the marked NC membrane into an oven at 36-42°C and dry for 12- 20min to prepare NC film;

Preferably, put the marked NC film into an oven at 37°C and dry it for 15 minutes to prepare the NC film.

Further, the formula of the sample pad soaking solution includes: 25-35 mmol/LPBS, 0.3-0.5% SDS, 0.8-1.2% BSA and 0.3-0.8% PVA;

Preferably, the formula of the sample pad soaking solution comprises: 30mmol/LPBS, 0.5% SDS, 1% BSA and 0.5% PVA.

On the other hand, the present invention also provides a method for detecting β-receptor agonists, the specific steps are as follows:

(1) Prepare a series of β-receptor agonist standard solutions with concentration gradients, add 80-120 μL each to the test strips prepared by the above method, run on the board for 8-12 minutes, and observe and compare the weakening degree of the T-line brightness ;

(2) Take an appropriate amount of sample containing β-receptor agonist, soak it in the lysate, shake it 2-3 times and let it stand for 3-8 minutes, then take 80-120 μL and add it to the sample pad of the test strip, and wait for it to run After 8 to 15 minutes, observe the change of the T line of the test strip and judge whether it is negative or positive.

Preferably, the concentration gradient is: 1-100 ng/mL;

The degree of weakening of the brightness of the compared T line is: the standard substance concentration corresponding to the brightness of the T line in the recording step (1);

The change of observing the T-line of the test strip is as follows: comparing the brightness of the T-line in the step (2) with the brightness of the T-line of the standard product in the step (1), to obtain the content interval of the β-receptor agonist in the sample.

Another aspect of the present invention also provides an application of the aforementioned method in the detection of β-receptor agonists in animal-derived foods.

The food of animal origin includes: all edible animal tissues as well as eggs and milk;

Preferably, the animal-derived food includes artificially raised livestock and poultry, meat, organs and products of aquatic animals.

Beneficial effects of the present invention:

The test strip of the present invention has the advantages of small size, light weight, portability, fast analysis speed, simple operation steps, etc., breaks the limitations of traditional detection methods, and provides strong technical support for on-site rapid detection of large sample volumes. At the same time, it also brings great convenience to market supervision and management personnel for on-site investigation and testing.

Description of drawings

Figures 1 to 6 are screening diagrams of gold spray diluents;

Fig. 7～12 is the screening figure of potassium carbonate amount;

Figures 13 to 18 are screening diagrams for the amount of β-receptor agonist antibody

Figures 19 to 24 are screening diagrams of NC membranes;

Figures 25 to 30 are screening diagrams of antigen diluents;

Figures 31 to 36 are comparison diagrams of the drying effect of the bonding pad soaked and not soaked;

Figures 37 to 42 are comparison diagrams of the effects of soaking and non-immersing sample pads;

Figures 43 to 48 are the screening diagrams of gold spray concentration;

Figures 49 to 54 are T and C line concentration screening diagrams.

Detailed ways

Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains.

In the present invention, the term "%" refers to the mass-to-volume ratio, and the unit is g/mL. For example, 1% means that 1 g of solute is added to 100 mL of solution.

In the present invention, the term "beta receptor agonist" refers to the beta receptor agonist in veterinary medicine, which is a phenylethanolamine substance, and can be divided into aniline type (such as clenbuterol, clenbuterol) and phenol type (such as salbutamol, salbutamol; ractopamine, ractopamine), and phenol type β receptor agonists are divided into diphenol type (such as terbutaline, terbutaline). These compounds can change the metabolic pathways in animals, and have obvious effects of promoting growth, increasing lean meat percentage and reducing fat, generally including clenbuterol, albuterol, ractopamine, zilpaterol, clorprenaline, and special Butaline, cibuterol, sibuterol, mabuterol, bromobuterol, bambuterol, etc.

In the present invention, the term "running board" refers to the analysis process carried out after the sample to be tested is added dropwise on the test paper strip. The sample at one end slowly migrates to the other end, and through the combination of antigen and antibody, the T line develops color, so as to judge whether the sample contains the target object. After the passage, it can be qualitatively and quantitatively detected according to the result of color development.

In the present invention, the term "PBS" refers to PBS buffer, which is the most widely used buffer in biochemical research, and its main components are Na ₂ HPO ₄ , KH ₂ PO ₄ , NaCl and KCl, which are generally used as solvents, Effect of lysoprotective reagents. Due to the secondary dissociation of Na ₂ HPO ₄ and KH ₂ PO ₄ , the pH range of the buffer is very wide, while the main function of NaCl and KCl is to increase the concentration of salt ions. If necessary, 1mmol/L CaCl ₂ and 0.5mmol/L MgCl ₂ can be added to PBS to provide divalent cations.

In the present invention, the term "MES" refers to 2-morpholineethanesulfonic acid. When preparing the MES buffer solution, NaOH is used to adjust the pH to the target pH value (pH5.5-6.7). MES is easily soluble in water and is colorless and transparent.

In the present invention, the term "EDC" refers to the water system carbodiimide, which is mainly used to activate the carboxyl group, so that the carboxyl group, amino group, and hydroxyl group in the water system can undergo coupling reactions at room temperature or even low temperature, so it is often used in In the field of biochemistry, it is often used in amino acid reactions, such as the synthesis of peptides.

In the present invention, the Chinese alias of the term "Tris-HCL" is: Tris hydrochloride; Tris hydrochloride. Tris buffer is not only widely used as a solvent for nucleic acids and proteins, but also has many important uses. Tris was used for protein crystal growth under different pH conditions. The low ionic strength of Tris buffer can be used for the formation of intermediate fibers of C. elegans nuclear lamin (lamin). Tris is also one of the main components of the protein electrophoresis buffer, which forms a buffer system with glycine in the electrophoresis buffer to stabilize the pH during electrophoresis. In addition, Tris is also an intermediate for the preparation of surfactants, vulcanization accelerators and some drugs. Tris was also used as a titration standard. When used as a protein buffer, if mass spectrometry is required for follow-up work, Tris is not suitable, and it is better to replace it with a buffer that can be tolerated by other mass spectrometers.

In the present invention, the term "TritonX-100" refers to polyethylene glycol octyl phenyl ether with a structural formula of C ₁₄ H ₂₂ O(C ₂ H ₄ O) _n . It is colorless or almost colorless transparent viscous liquid. Soluble in water, toluene, xylene and ethanol, insoluble in petroleum ether. The refractive index is 1.4894 (25°C), and the viscosity is 24×10-3Pa·s. Can be used in biochemical research.

In the present invention, the term "BSA" refers to bovine serum albumin, which is a kind of albumin in bovine serum, contains 583 amino acid residues, has a molecular weight of 66.430kDa, and an isoelectric point of 4.7, and can be used as a blocking solution. Sample diluent, enzyme conjugate diluent.

In the present invention, the term "IgG" is the abbreviation of Immunoglobulin G (Immunoglobulin G, IgG), which is the main antibody component of serum, accounting for about 75% of serum Ig.

The following will clearly and completely describe the technical solution in the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

Examples 1-24

1.1 Activation of colored microspheres

The particle size of colored microspheres is 300nm.

1. Activation steps of colored microspheres 1: Sonicate the colored microspheres for 5 minutes, take 200 μL of the sonicated colored microspheres and add 1ml 0.05mol/L MES (pH=5.0) solution, mix evenly, centrifuge at 13000r/min for 20min, discard Add 1ml of 0.05mol/L MES (pH=4.5) to the supernatant, mix evenly and sonicate for 5min, centrifuge at 13000r/min for 20min, discard the supernatant, add 200μL of 1ml 0.05mol/L MES(pH=4.5) to mix evenly, sonicate For 5 minutes, use 0.05mol/LMES solution to prepare NHS10mol/L, take 15μL and add it to the colored microspheres that have been sonicated, then prepare EDC15mol/L with pure water, take 2μL and add it to the above colored microspheres, wrap the shaker with tinfoil paper to avoid light React for 15 minutes;

2. Activation steps of colored microspheres 2: Sonicate the colored microspheres for 5 minutes, take 200 μL of the sonicated colored microspheres and add 1ml 0.05mol/LMES (pH=5.0) solution to mix evenly, centrifuge at 13000r/min for 20min, discard the supernatant solution, add 1ml of 0.05mol/LMES (pH=5.0) boric acid solution, mix evenly and sonicate for 5min, centrifuge at 13000r/min for 20min, discard the supernatant, add 200μL of 1ml 0.05mol/LMES(pH=5.0) boric acid solution, mix evenly, and sonicate for 5min. Use 0.05mol/LMES solution to prepare NHS10mol/L, take 15μL and add it to the colored microspheres that have been sonicated, then prepare EDC15mol/L with pure water, take 2μL and add it to the above colored microspheres, wrap it in tin foil paper and react on a shaker in the dark for 15min ;

3. Activation steps of colored microspheres 3: Sonicate the colored microspheres for 5 minutes, take 200 μL of the sonicated colored microspheres and add 1ml 0.05mol/LMES (pH=5.0) solution to mix evenly, centrifuge at 13000r/min for 20min, discard the supernatant Add 1ml of 0.05mol/LMES solution, mix evenly and sonicate for 5min, centrifuge at 13000r/min for 20min, discard the supernatant, add 200μL 0.01mol/LpH=8.99 boric acid solution, mix evenly, sonicate for 5min, prepare NHS10mol with 0.05mol/LMES solution /L, take 15 μL and add it to the colored microspheres that have been sonicated, then prepare EDC15mol/L with pure water, take 2 μL and add it to the above colored microspheres, wrap it in tinfoil paper and react on a shaker in the dark for 15 minutes.

1.2 Preparation of colored microspheres and β receptor agonist antibody conjugates:

(1) Preparation of colored microsphere 1 and ractopamine antibody conjugate: Take 5 μL of ractopamine antibody at 1 μg/μL and add it to the activated colored microspheres, mix evenly, react on a shaker in the dark for 2 hours, and add 10 μL of 0.1mol/L The ethanolamine solution was mixed evenly, reacted on a shaker in the dark for 30min, centrifuged at 13000r/min for 10min, discarded the supernatant, mixed evenly with 500μL microsphere diluent (0.01mol/LPBS+1%BSA), ultrasonicated for 5min, and shaken in the dark React for 1h, centrifuge at 13000r/min for 10min, discard the supernatant, mix evenly with 200μL microsphere diluent (the amount of ractopamine antibody is 4μL/mL), and sonicate for 5min. Store at 2-8 degrees; the colored microspheres 1 is purple or black, and the manufacturer of ractopamine antibody is Shenzhen Kejie Industrial Development Co., Ltd.

(2) Preparation of colored microsphere 2 and albuterol conjugate: Take 5 μL of 1 μg/μL albuterol antibody and add it to the activated colored microspheres, mix evenly, react on a shaker in the dark for 2 hours, add 10 μL of 0.1mol/L ethanolamine solution Mix evenly, react on a shaker in the dark for 30 minutes, centrifuge at 13000r/min for 10min, discard the supernatant, mix evenly with 500μL microsphere diluent (0.01mol/LPBS+1%BSA), ultrasonicate for 5min, and react on a shaker in the dark for 1h. Centrifuge at 13000r/min for 10min, discard the supernatant, mix evenly with 200μL of microsphere diluent (the amount of albuterol antibody is 4μL/mL), and sonicate for 5min. Store at 2-8 degrees; the color microsphere 2 is purple or Black, the manufacturer of albuterol antibody is Antibiotics.

(3) Preparation of color microsphere 3 and clenbuterol hydrochloride conjugate: Take 5 μL of 1 μg/μL clenbuterol hydrochloride antibody and add it to the activated color microspheres, mix evenly, react on a shaker in the dark for 2 hours, add Mix 10 μL of 0.1mol/L ethanolamine solution evenly, react on a shaker in the dark for 30min, centrifuge at 13000r/min for 10min, discard the supernatant, mix well with 500μL microsphere diluent (0.01mol/LPBS+1%BSA), and ultrasonicate for 5min , reacted on a shaker in the dark for 1h, centrifuged at 13000r/min for 10min, discarded the supernatant, mixed evenly with 200μL microsphere diluent (the amount of clenbuterol hydrochloride antibody was 4μL/mL), and ultrasonicated for 5min. The color microsphere 3 is purple or black, and the clenbuterol hydrochloride antibody manufacturer is Xi'an Qiyue Biology.

1.3 Handling of sample pads

Soak the sample pad in the sample pad soaking solution for 10 minutes, take it out and dry it in an oven at 37 degrees for 2 hours for later use. The formula of the sample pad soaking solution is: 30 mmol/LPBS, 0.5% SDS, 1% BSA and 0.5 %PVA.

1.4 Treatment of the bonding pad: Soak the bonding pad in the bonding pad soaking solution for 2 hours, take out the soaked bonding pad and dry it in an oven at 37 for 2 hours for later use. The bonding pad soaking solution is 0.05mol of pH=7.4 /L Tris-HCL buffer solution, which contains 5% trehalose, 0.5% BSA, 0.1% Tween;

1.5 Gold spraying steps for bonding pads:

Take out the ractopamine antibody conjugate, sonicate for 5 minutes, take 1 μL of the sonicated microsphere antibody conjugate and add 100 μL of gold spray diluent (the components are: 0.02% boric acid, 0.2% PEG20000, 0.02% preservative, 10% seaweed sugar, 0.25% Tween and 1% BSA), adjust the pH with potassium carbonate after dilution, and prepare a gold-spraying mixed solution with pH=9. In the mixed solution, the concentration of potassium carbonate is 2 μL/mL, and the gold-sprayed mixed solution is again Ultrasound for 5 minutes to fully mix the microsphere antibody conjugates. The antibody volume of the gold spray mixture is 4 μL/mL, and to prevent the aggregation of the microsphere antibody conjugates, take out the ultrasonic gold spray mixture, and use XYZ three-dimensional gold spray The scribing instrument adopts XYZ three-dimensional gold-spraying scribing instrument, and sprays gold on the treated bonding pad at a speed of 9.9 μL/cm. Put the gold-sprayed bonding pad in an oven and dry it at 37°C for 1 hour for later use;

Take the albuterol antibody conjugate, the method is the same as the aforementioned ractopamine antibody conjugate;

Take the clenbuterol hydrochloride antibody conjugate, the method is the same as the aforementioned ractopamine antibody conjugate;

The formula of the gold spray diluent: 0.2% PEG20000, 0.02% preservative, 10% trehalose, 0.25% Tween, 1% BSA.

1.6 Treatment of T-line and C-line on NC film

Take clenbuterol hydrochloride+BSA solution and IgG solution respectively, dilute the ractopamine+BSA solution with 0.01mol/LPBS+0.03%TritonX-100, and dilute the IgG solution with 0.01mol/LPBS+3%methanol+0.03%TritonX- 100 of the antigen diluent was diluted, and the concentrations of the diluted solutions were 0.8mol/L and 1.0mol/L respectively, and then the XYZ three-dimensional spray gold scriber was used to draw a line on the NC membrane at a speed of 1 μL/cm, and the drawn Put the finished NC film in an oven at 37°C for 15 minutes for later use;

The T line and C line on the NC membrane are salbutamol + BSA solution and IgG solution respectively, the salbutamol + BSA solution is diluted with 0.01mol/LPBS + 0.03% TritonX-100, and the IgG solution is diluted with 0.01mol/LPBS + 3% methanol + Dilute with 0.03% TritonX-100 antigen diluent, the concentration of the diluted solution is 0.05mol/L and 0.8mol/L, and then use the XYZ three-dimensional spray gold scriber to draw on the NC membrane at a speed of 1μL/cm Line, put the marked NC film into the oven at 37 degrees to dry for 15 minutes for later use;

The T line and C line on the NC membrane are clenbuterol hydrochloride + BSA solution and IgG solution respectively. Dilute clenbuterol hydrochloride + BSA with 0.01mol/LPBS + 0.03% TritonX-100, and dilute the IgG solution with 0.01mol/ LPBS + 3% methanol + 0.03% TritonX-100 antigen diluent to dilute, the concentration of the diluted solution is 0.6mol/L and 0.5mol/L respectively, and then use XYZ three-dimensional gold-sprayed streaking instrument to spray 1μL on the NC membrane Scribe at a speed of 1/cm, put the NC film that has been marked in an oven at 37 degrees and dry for 15 minutes for later use.

1.7 Assembly of test strips

Lay the processed sample pad, conjugation pad, NC film, and absorbent paper on the PVC rubber sheet in sequence. The lamination sequence is: first paste the NC film, the conjugation pad presses the NC film, the sample pad presses the conjugation pad, and the absorbent paper Press the NC film. The overlapping length between each other is generally about 1mm. After lamination, cut it into test strips with a width of 4 mm with a cutter. (In the process of bonding, it is customary to place the T line on the NC film close to the bonding pad, and the C line close to the absorbent paper).

Among them, the NC film is 25mmJJ120.

1.8 How to use test strips

Taking the detection of β-receptor agonists in hair as an example, use the test strip prepared by the present invention to detect β-receptor agonists (including ractopamine, salbutamol or clenbuterol hydrochloride) in hair, the specific steps as follows

(1) Prepare a series of β-receptor agonist standard solutions with concentration gradients, add 100 μL each to the test strip, run the board for 10 minutes, and observe the brightness of the T and C lines. The concentration gradient is β receptor agonist concentration 1, 2, 5, 10, 20, 50, 100 ng/ml.

(2) Take an appropriate amount of 1g of the sample containing β-receptor agonist, crush it and add 1ml of lysate (volume ratio of ethanol/water: 1:9), shake it for 2-3 times and let it stand for 5min. After 5min, take 100μL and add Put it on the sample pad of the test strip, and observe the brightness of the T line of the test strip after it runs for 10 minutes, so as to determine the negative or positive judgment of the content of the β-receptor agonist.

Embodiment 2～24 spray gold diluent formulation optimization

The preparation method of the β-receptor agonist quick test strip is the same as in Example 1, the only difference is that in step 1.5, the formula of the gold spray diluent is as follows:

Composition of No. 1 gold spray diluent: 0.02% boric acid, 0.2% PEG20000, 0.02% preservative;

Composition of No. 2 gold spray diluent: 0.02% boric acid, 0.2% PEG20000, 0.02% preservative and 10% trehalose;

Composition of No. 3 gold spray diluent: 0.02% boric acid, 0.2% PEG20000, 0.02% preservative, 10% trehalose and 0.25% Tween;

The composition of No. 4 spray gold dilution solution: 0.02% boric acid, 0.2% PEG20000, 0.02% preservative, 10% trehalose, 0.25% Tween, 1% BSA.

The preservatives are those well known in the art.

The test results are shown in Figures 1 to 6. From top to bottom in the figure are No. 1 to No. 4 gold spray diluents. The results of the ractopamine group are shown in Figures 1 and 4, and the results of the salbutamol group are shown in Figures 2 and 5. The results of the clenbuterol hydrochloride group are shown in Figure 3 and Figure 6. From top to bottom in the figure are No. 1 to No. 4 gold-sprayed diluents. The gold spray diluent of Luo group was the best when the gold spray solution was No. 4.

The optimization of embodiment 25～61 potassium carbonate content

The preparation method of the β-receptor agonist quick test strip is the same as in Example 1, the only difference is that in step 1.5, the amount of potassium carbonate added is different in the gold spraying of the binding pad: the figures from top to bottom are Figures 7 to 12 , adjust the pH with potassium carbonate, and prepare a gold-spraying mixed solution. In the mixed solution, the concentration of potassium carbonate is: (1) 1.5 μL/mL, (2) 2 μL/mL, (3) 2.5 μL/mL, (4) ) 3 μL/mL, (5) 3.5 μL/mL, (6) 4 μL/mL.

In the ractopamine group, as shown in Figure 7 and Figure 10, the concentrations of potassium carbonate from left to right were 1.5 μL/mL, 2 μL/mL, 2.5 μL/mL, 3 μL/mL, 3.5 μL/mL, 4 μL/mL. The effect of 2μL/mL is the best, other collocations have insufficient brightness combined with T line or C line.

As shown in Figure 8 and Figure 11, the concentrations of potassium carbonate in the albuterol group from left to right were 1.5 μL/mL, 2 μL/mL, 2.5 μL/mL, 3 μL/mL, 3.5 μL/mL, 4 μL/mL, and the comparison shows that 4 μL /mL has the best effect, and other collocations may have T-line or C-line combined with insufficient brightness.

The concentrations of potassium carbonate from left to right in the clenbuterol hydrochloride group are 1.5 μL/mL, 2 μL/mL, 2.5 μL/mL, 3 μL/mL, 3.5 μL/mL, 4 μL/mL as shown in Figure 9 and Figure 12 , Comparison shows that the effect of 2μL/mL is the best, other collocations appear T-line or C-line combined with insufficient brightness.

Example 61-85 Optimization of Antibody Quantity

The preparation method of the β-receptor agonist quick test strip is the same as in Example 1, the only difference is that in step 1.2, the amounts of β-receptor agonist antibodies are: (1) 2 μL/mL, (2) 3 μL/mL , (3) 4 μL/mL, (4) 5 μL/mL.

As shown in Figure 13 and Figure 16 for the ractopamine group, the amounts of β-receptor agonist antibodies added from top to bottom were 2 μL/mL, 3 μL/mL, 4 μL/mL, and 5 μL/mL. The effect is the best, and other collocations have poor running board effects.

As shown in Figure 14 and Figure 17 for the albuterol group, the amount of β-agonist antibody added from top to bottom was 2 μL/mL, 3 μL/mL, 4 μL/mL, and 5 μL/mL. The effect comparison shows that the effect of 4 μL/mL Best, other collocations have poor running board effect.

As shown in Figure 15 and Figure 18 for the clenbuterol hydrochloride group, the amounts of β-receptor agonist antibodies added from top to bottom were 2 μL/mL, 3 μL/mL, 4 μL/mL, and 5 μL/mL, and the effect comparison shows that 4 μL /mL has the best effect, and other collocations have poor running board effect.

Example 85-96 Matching optimization of NC membranes and bonding pads

The preparation method of the β-receptor agonist quick test strip is the same as in Example 1, the only difference is that in step 1.7, the NC membranes matched with the binding pads are: 20mmJJ120 or 25mmJJ120.

The results of the ractopamine group are shown in Figure 19 and Figure 22. From top to bottom in the figure, they are 25mmJJ120 and 20mmJJ120. From the effect comparison, it can be seen that the effect of NC film 25mmJJ120 is the best. Other collocations have incomplete release and poor running board effect.

The results of the albuterol group are shown in Figure 20 and Figure 23, in which the numbers from top to bottom are 25mmJJ120 and 20mmJJ120. From the effect comparison, it can be seen that the effect of NC film 25mmJJ120 is the best. Other collocations have incomplete release and poor running board effect.

The results of the clenbuterol hydrochloride group are shown in Figure 21 and Figure 24. From top to bottom in the figure, they are 25mmJJ120 and 20mmJJ120. From the effect comparison, it can be seen that the effect of NC film 25mmJJ120 is the best. Other collocations have incomplete release and poor running board effect.

The optimization of embodiment 97-114 antigen diluent

The preparation method of the β-receptor agonist quick test strip is the same as in Example 1, the only difference being that the antigen diluent for diluting the IgG solution in step 1.6: (1) PBS+15% sucrose (2) PB+15% sucrose ( 3) PBS+3% methanol+0.03% TritonX-100, the specific results are shown in Figures 25-30.

In the ractopamine group, the antigen diluents for diluting the IgG solution from left to right are PBS+15% sucrose, PB+15% sucrose, PBS+3% methanol+0.03% TritonX-100 running board effect, different antigen diluents lead to The combination effects are different, as can be seen from Figure 25 and Figure 28, PBS+3% methanol+0.03% TritonX-100 running board has the best effect.

The antigen diluents of the IgG solution diluted from left to right in the salbutamol group are PBS+15% sucrose, PB+15% sucrose, PBS+3% methanol+0.03% TritonX-100 running board effect, different antigen diluents lead to binding The effects are different, as can be seen from Figure 26 and Figure 29, the PBS+15% sucrose running board has the best effect.

The antigen diluents for diluting the IgG solution from left to right in the clenbuterol hydrochloride group are PBS+15% sucrose, PB+15% sucrose, PBS+3% methanol+0.03% TritonX-100 running board effect, different antigens Diluents lead to different binding effects. From Figure 27 and Figure 30, it can be seen that PBS+15% sucrose has the best running effect.

Example 115-126 Optimization of the processing method of the binding pad

The preparation method of the β-receptor agonist quick test strip is the same as that in Example 1, the only difference is that in step 1.4, the binding pad is treated in the following ways: (1) soaking the binding pad; (2) not soaking.

The soaking solution is 0.05 mol/L Tris-HCL buffer solution with pH=7.4, which contains 5% trehalose, 0.5% BSA, and 0.1% Tween.

The results of the ractopamine group are shown in Figure 31 and Figure 34. From left to right in the figure are the board running effects of unsoaked and soaked mats. The sensitivity of the assay is greatly reduced as the number of antibodies that can bind to T and C lines decreases.

The results of the albuterol group are shown in Figure 32 and Figure 35. From left to right in the figure are the running board effects of unsoaked and soaked bonding pads. The sensitivity of the assay is greatly reduced as the number of antibodies combined with T and C lines decreases.

The results of the clenbuterol hydrochloride group are shown in Figure 33 and Figure 36. From left to right in the figure are the running board effects of the bonding pad without soaking and soaking. After the bonding pad is not soaked and dried, the bonding pad spray gold solution is basically not released. , resulting in fewer antibodies that can bind to T and C lines, and the sensitivity of the assay is greatly reduced.

The processing mode of embodiment 127～138 sample pad is optimized

The preparation method of the β-receptor agonist quick test strip is the same as that in Example 1, the only difference is that in step 1.3, the sample pad is treated in the following ways: (1) soak the sample pad; (2) not soak.

The results of the ractopamine group are shown in Figure 37 and Figure 40. From top to bottom in the figure, they show the running effect of the binding pad soaked and unsoaked. After the sample pad is not soaked and dried, there will be less antibodies bound to the T and C lines. The sensitivity of the assay Greatly reduced.

The results of the salbutamol group are shown in Figure 38 and Figure 41. From top to bottom in the figure are the running board effects of soaked and unsoaked binding pads. After the sample pad is not soaked and dried, there will be fewer antibodies bound by T and C lines. The sensitivity of the measurement is greatly improved. reduce.

The results of the clenbuterol hydrochloride group are shown in Figure 39 and Figure 42. From top to bottom in the figure, they show the running effect of the binding pad soaked and unsoaked. After the sample pad is not soaked and dried, there will be fewer antibodies bound to the T and C lines. The sensitivity of the assay is greatly reduced.

Example 139-150 Optimization of concentration of different gold spraying amounts

The preparation method of the β-receptor agonist quick test strip is the same as in Example 1, the only difference is that the amount of gold sprayed in step 1.5 is different:

The results of the ractopamine group are shown in Figure 43 and Figure 46. After adding 20ng/ml of the standard substance in the figure, 6μL/cm and 9.9μL/cm were added in order from left to right. The comparison shows that the effect of 6μL/cm is better, and the concentration is too high. A high amount of gold spraying will lead to problems such as insensitivity to the content determination of β-receptor agonists.

The results of the albuterol group are shown in Figure 44 and Figure 47. After adding 20ng/ml of the standard substance in the figure, 6μL/cm and 9.9μL/cm are added in order from left to right. Through the comparison effect, it can be seen that the effect of 6μL/cm is better, and the gold spray with too high concentration A large amount will lead to problems such as insensitivity in the determination of β-receptor agonist content.

The results of the clenbuterol hydrochloride group are shown in Figure 45 and Figure 48. After adding 20ng/ml of the standard substance in the figure, 6μL/cm and 9.9μL/cm are added from left to right in the figure. The comparison results show that 6μL/cm is better, and the concentration is too high. A high amount of gold spraying will lead to problems such as insensitivity to the content determination of β-receptor agonists.

Example 151 ~ 180T, C line concentration optimization

The preparation method of the ractopamine group is basically the same as that of Example 1, the difference is that: from left to right, the concentrations of T and C lines are: T line: 0.2mol/L, C line: 0.4mol/L;

T line: 0.4mol/L, C line: 0.6mol/L;

T line: 0.5mol/L, C line: 0.8mol/L;

T line: 0.8mol/L, C line: 1.0mol/L;

T line: 0.2mol/L, C line: 0.5mol/L.

The results are shown in Figure 49 and Figure 52.

The preparation method of the albuterol group is basically the same as that of Example 1, the difference is that the concentrations of the T and C lines from left to right are respectively:

T line: 0.04mol/L, C line: 0.4mol/L;

T line: 0.02mol/L, C line: 0.6mol/L;

T line: 0.08mol/L, C line: 0.7mol/L;

T line: 0.04mol/L, C line: 0.7mol/L;

T line: 0.05mol/L, C line: 0.8mol/L.

The results are shown in Figure 50 and Figure 53.

The preparation method of clenbuterol hydrochloride group is basically the same as that of Example 1, the difference is that: from left to right, the concentrations of T and C lines are: T line: 0.4mol/L, C line: 0.4mol/L; T line: 0.5mol/L, C line: 0.4mol/L; T line: 0.6mol/L, C line: 0.4mol/L; T line: 0.6mol/L, C line: 0.5mol/L; T line: 0.2mol /L, C line: 0.4mol/L. The results are shown in Figure 51 and Figure 54.

The test strip of the present invention has obvious change in T/C when dripping the β receptor agonist (that is, the test strip has certain sensitivity to the concentration of the β receptor agonist), and the brightness of the T line when the blank running board Brighter than the C line, when the β-receptor agonist is determined, the β-receptor agonist antigen in the test sample combines with the antibody on the binding pad, so that the amount of antibody that can bind to the T-line antigen is greatly reduced, and the brightness of the T-line Significantly weakened, thereby realizing that the test strip has higher sensitivity to the concentration of the β-receptor agonist in the sample to be tested. By comparing the measured values and considering from the perspective of economic benefits, the T and C line concentrations of ractopamine are 0.8mol/L and 1.0mol/L respectively, and the T and C line concentrations of salbutamol are 0.05mol/L and 0.8mol/L respectively. L, clenbuterol hydrochloride T and C line concentrations of 0.6 mol/L and 0.5 mol/L respectively are better experimental schemes. The setting of this concentration can not only meet the sensitivity of the determination but also achieve certain economic benefits.

Experimental example 1 test strip precision test

Using the test strip prepared in Example 1, using ractopamine with a concentration of 1 ng/ml, take 100 μL and add it to the test strip. After running for 10 minutes, observe the T-line brightness with the naked eye after running for 10 minutes. Repeat 5 times. The results are shown in Table 1.

Table 1 Test results of test strip precision

It can be seen from Table 1 that the test strips for ractopamine have high precision.

Using the test strips prepared in Example 1, using albuterol with a concentration of 1 ng/ml and clenbuterol hydrochloride with a concentration of 1 ng/ml, take 100 μL and add it to the test strip. After running the board for 10 minutes, observe the running board for 10 minutes with the naked eye The final T-line brightness was repeated 5 times, and the specific results are shown in Table 2 and Table 3.

Table 2 Precision test results of test strips

Salbutamol concentration (1ng/ml) T-line brightness (purple) T line brightness (black) 1 disappear disappear 1 disappear disappear 1 disappear disappear 1 disappear disappear 1 disappear disappear

It can be seen from Table 2 that the test strip of albuterol has high precision.

Table 3 Precision test results of test strips

It can be seen from Table 3 that the test strips of clenbuterol hydrochloride have high precision.

Experimental example 2 (determination of test strip specificity)

Adopt the prepared test strip of embodiment 1, adopt the sulfa powder of 1ng/ml (Sichuan Ruibiao Quality Inspection Technology Service Co., Ltd., specification is 100ug/ml), the aflatoxin B1 of 1ng/ml and the aflatoxin B1 of 1ng/ml For cocaine, use purified water as a blank, add 100 μL each to the test strip, and after running the board for 10 minutes, observe the degree of decrease in brightness of the T line with the naked eye, and repeat 5 times. The specific results are shown in Table 4 and Table 5.

The remaining drugs are aflatoxin B1 and cocaine of common specifications on the market.

Table 4 Test strip specificity test results (purple microspheres)

As can be seen from Table 4, the purple test strip of the present invention has good specificity and can quickly distinguish different medicines.

Table 5 Test strip specificity test results (black microspheres)

As can be seen from Table 5, the black test strip of the present invention has good specificity and can quickly distinguish different medicines.

Experimental example 3 test strip stability test

Using the test strip prepared in Example 1, using a β-receptor agonist with a concentration of 1 ng/ml, take 100 μL and add it to the test strip. After using different running times, observe the degree of brightness reduction of the T line with the naked eye, and repeat 5 times, see Table 6 and Table 7 for specific results.

The beta receptor agonist contains ractopamine, albuterol, and clenbuterol hydrochloride.

Table 6 (purple) test strip stability test results

As can be seen from Table 6, the stability of the purple microsphere test paper strip of the present invention is very good.

Table 7 (black) test strip stability test results

As can be seen from Table 7, the stability of the black microsphere test paper strip of the present invention is very good.

Experimental Example 4 Actual Sample Detection

Take an appropriate amount of positive sample, break it up and add 1ml of lysate, shake it 2-3 times and let it stand for 5 minutes. After 5 minutes, take 100 μL and add it to the sample pad of the test strip. After running for 10 minutes, observe the content of β-receptor agonist with naked eyes. For the judgment of positive and negative, see Table 8 and Table 9 for specific results.

Table 8 Determination results of test strips (purple) on the content of β-receptor agonists in sample meat hair

Table 9 Determination results of test strips (black) on the content of β-receptor agonists in sample meat hair

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (10)

1. The beta receptor agonist rapid detection test paper strip is characterized by comprising a sample pad, a combination pad, an NC (numerical control) membrane, absorbent paper and a rubber plate, wherein the combination pad comprises a combination pad matrix, colored microspheres and beta receptor agonist conjugates;

the bonding pad substrate is prepared by the following steps:

a. soaking: soaking the bonding pad in a bonding pad soaking solution, taking out the soaked bonding pad, and drying;

b. activation of the colored microspheres: taking color microspheres, performing ultrasonic treatment, adding MES solution into 200 μ L of the ultrasonic treated color microspheres, mixing, centrifuging, discarding supernatant, repeating for 2 times, adding MES solution, mixing, and performing ultrasonic treatment;

preparing an NHS solution with the concentration of 5-20 mol/L by using the MES solution, adding the NHS solution into the color microspheres which are well subjected to ultrasonic treatment, preparing EDC by using pure water, wherein the concentration of the EDC solution is 10-20 mol/L, adding 1-5 mu L of the EDC solution into the color microspheres, wrapping the color microspheres by using tinfoil paper, and reacting for 5-30 min by using a light-proof shaking table to obtain activated color microspheres;

c. preparation of the colored microsphere and beta receptor agonist antibody conjugate: adding 1-5 mu L of beta receptor agonist antibody of 1 mu g/mu L into the activated colored microspheres, uniformly mixing, carrying out a shaking table reaction in a dark place for 0.5-3 h, adding 5-15 mu L of 0.05-3 mol/L ethanolamine solution, uniformly mixing, carrying out a shaking table reaction in a dark place, centrifuging, discarding the supernatant, uniformly mixing with 300-1000 mu L of microsphere diluent, carrying out ultrasonic treatment for 5min, carrying out a shaking table reaction in a dark place for 1h, centrifuging, discarding the supernatant, uniformly mixing with 100-300 mu L of microsphere diluent, and carrying out ultrasonic treatment to form uniform beta receptor agonist antibody-colored microsphere solution, and storing the solution at 2-8 ℃;

d. spraying gold and drying: c, taking out the solution prepared in the step c, adding a gold spraying diluent for dilution, adjusting the pH value to 8-9.5 by using potassium carbonate after dilution, spraying gold on the bonding pad prepared in the step a, and baking the bonding pad after gold spraying for 1-1.5 h at the temperature of 37-48 ℃;

the bonding pad soaking solution is a Tris-HCl buffer solution with the pH = 7-7.5 and the concentration of 0.03-0.05 mol/L, wherein the Tris-HCl buffer solution comprises trehalose, BSA and Tween, and the mass volume ratio of the trehalose to the BSA is 3-5%, and the Tween is 0.05-0.1%;

wherein the beta receptor agonist antibody is selected from the group consisting of: one or more of an isoprochane antibody, a chlorpropaline antibody, a cimaterol antibody, a terbutaline antibody, a tulobuterol antibody, a sibutrol antibody, a salbutamol antibody, a zilpaterol antibody, a clenbuterol antibody, a ractopamine antibody, a fenoterol antibody, a mabuterol antibody, a bromoclenbuterol antibody, a mabuterol antibody, a phenylethanolamine a antibody, a bromobutaro;

the microsphere diluent is as follows: 0.01 to 0.02mol/LPBS and 0.5 to 1.2% BSA;

in the step c, the addition amount of the microsphere diluent is 300-1000 mu L;

the diluent used for spraying the gold comprises the following components: 0.02% boric acid, 0.2% PEG20000,0.02% preservative, 10% trehalose, 0.25% Tween and 1% BSA;

the amount of the gold spraying is 6-9.9 mu L/cm, and preferably 6 mu L/cm.

2. The test strip of claim 1, wherein the beta receptor agonist antibody is selected from the group consisting of a ractopamine antibody, a salbutamol antibody, and a clenbuterol hydrochloride antibody.

3. The test strip of claim 1, wherein the amount of the beta receptor agonist antibody is 2 to 5 μ L/mL.

4. The test strip of claim 1, wherein the particle size of the colored microspheres is 100-300 nm, preferably 300nm.

5. The test strip of claim 1, wherein the sample pad is a glass fiber material, a polyester fiber film;

preferably a glass fibre membrane;

the thickness of the glass fiber membrane is 0.2-0.6 mm, preferably 0.3-0.45 mm;

the climbing speed of the glass fiber membrane is 5-70 mm/60s;

preferably, the climbing speed of the glass fiber membrane is 5-50 mm/60s;

preferably, the bonding pad is made of glass fiber and has the water absorption capacity of 450 +/-50 g/m ² ；

The NC film is selected from NC films with the width of 20-25 mm and the climbing speed of 100-125 s/4 cm;

the absorbent paper is selected from absorbent paper with the climbing speed of 40-180 s/mm and the thickness of 0.87-1.13 mm.

6. The test strip of claim 1, wherein the NC membrane comprises a T line comprising a beta receptor agonist antigen and a C line comprising IgG;

the beta receptor stimulant antigen is ractopamine, salbutamol or clenbuterol hydrochloride.

7. A method for preparing the test strip of any one of claims 1 to 6, which comprises the following steps:

s1, processing a sample pad: placing the sample pad in the sample pad soak solution to soak for 5-30 min, taking out and placing in a drying oven to dry for 1.5-2.2 h at 36-42 ℃ for later use;

preferably, the soaking time in the step S1 is 10min;

preferably, the drying condition in the step S1 is that the drying temperature is 37 ℃ and the drying time is 2h;

s2, treatment of the bonding pad: soaking the bonding pad in a bonding pad soaking solution for 1.5-2.2 h, taking out the soaked bonding pad, and drying the bonding pad in a drying oven at 36-48 ℃ for 1.5-3 h for later use;

preferably, the bonding pad is soaked for 2 hours in the step S2, the soaked bonding pad is taken out and placed in a drying oven to be dried for 2 hours for standby after being dried for 37 hours at the temperature of 36-48 ℃;

s3, preparing an NC film: diluting the first solution by using a first diluent, then diluting the second solution by using a second diluent, and scribing a T line by using the diluted first solution and a C line by using the diluted second solution;

s4, laminating sample pad, combination pad, NC membrane, absorbent paper on the PVC offset plate in proper order, the laminating order is: firstly pasting an NC film, pressing the NC film by a combination pad, pressing the combination pad by a sample pad, pressing the NC film by absorbent paper, overlapping the NC film and the sample pad with each other, wherein the overlapping length is generally about 0.8-1.5 mm, and cutting the NC film into test strips with the width of 3.8-4.2 mm by a cutter after the pasting is finished;

preferably, the first solution is a solution comprising a beta receptor agonist antigen and BSA;

the composition of the first diluent is 0.01-0.03 mol/LPBS + 0.03-0.05% Triton X-100;

the second solution is an IgG solution;

the composition of the second diluent is 0.01-0.03 mol/LPBS + 1-5% methanol + 0.03-0.05% Triton X-100;

preferably, when the first solution is ractopamine and BSA solution, the first diluent is adopted to dilute the first solution into a solution with the ractopamine concentration of 0.2-0.9 mol/L; diluting the second solution into a solution with the IgG concentration of 0.4-1.0 mol/L by using a second diluent;

and/or when the first solution is the salbutamol and BSA solution, diluting the solution into a solution with the salbutamol concentration of 0.02-0.08 mol/L by using a first diluent; diluting the second solution into a solution with the IgG concentration of 0.4-0.8 mol/L by using a second diluent;

and/or when the first solution is a clenbuterol hydrochloride solution and a BSA (bovine serum albumin) solution, diluting the first solution into a solution with the clenbuterol hydrochloride concentration of 0.2-0.7 mol/L by adopting a first diluent; diluting the second solution into a solution with the IgG concentration of 0.4-0.6 mol/L by using a second diluent;

and (3) taking the diluted first solution and the diluted second solution, scribing on the NC film at the speed of 0.8-1.2 mu L/cm, and drying the scribed NC film in an oven at the temperature of 36-42 ℃ for 12-20 min to obtain the NC film.

8. The method for preparing the test strip of claim 7, wherein: the formula of the sample pad soaking solution comprises: 25-35 mmol/LPBS, 0.3-0.5% SDS, 0.8-1.2% BSA and 0.3-0.8% PVA.

9. A method for detecting a beta receptor agonist, which is characterized by using the test strip prepared in claim 7 to detect, and comprises the following specific steps:

(1) Preparing a series of beta receptor agonist standard solutions with concentration gradients, adding 80-120 mu L of each beta receptor agonist standard solution to a test strip, running the test strip for 8-12 min, and observing and comparing the brightness reduction degree of a T line;

(2) Taking a proper amount of a sample containing the beta receptor stimulant, soaking the sample into the lysate, shaking for 2-3 times, standing for 3-8 min, then adding 80-120 mu L of the lysate to a sample pad of a test strip, and observing the change of a test strip T line and the judgment of negative and positive after the sample pad runs for 8-15 min.

10. Use of the method of claim 9 for detecting a beta receptor agonist in a food product of animal origin.

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