CN100405063C - Citrinin immunochromatographic detection test paper and its production method and application - Google Patents

Abstract

The invention discloses a citrinin immunochromatographic detection test paper as well as a production method and application thereof. The detection test paper of the present invention comprises a liner, a sample pad, a tracer particle conjugate pad, a nitrocellulose membrane, a water-absorbent pad, a detection line, and a quality control line; on the liner, there are sample pads and tracer particle conjugates in sequence Pad, nitrocellulose membrane, water-absorbing pad; there is an antibody against citrinin labeled with tracer particles on the tracer particle conjugate pad, and a detection line and a quality control line are set on the nitrocellulose membrane, and there is citrinin on the detection line Antigens are detected with the same protein, and there are anti-antibodies on the quality control line, wherein the anti-antibodies are antibodies against citrinin. The advantage of the present invention is that: the present invention has the advantages of radioimmunoassay and enzyme-linked immunosorbent assay (ELISA). Simultaneously, the present invention has the advantages of simplicity, rapidity, storage at room temperature, single determination, citrinin can be detected in one test, and does not require any equipment except commercial reagents, and is especially suitable for on-site rapid detection of citrinin.

Description

Citrinin immunochromatographic detection test paper and its production method and application

technical field

The invention belongs to the field of biotechnology, and discloses a citrinin immunochromatographic detection test paper as well as a production method and application thereof.

Background technique

Citrinin is a mycotoxin produced by certain strains of Penicillium and Aspergillus, which was first isolated and purified in 1931. It can cause significant nephrotoxicity and is carcinogenic. The survey found that some citrinin-producing bacteria are widely distributed in nature and often cause mildew in crops such as corn and rice. In recent years, the pollution of citrinin has attracted more and more attention. In the Symposium on Mycotoxins, Endemic Nephropathy and Urinary Tract Tumors held in Lyon, France in 1991, the role of citrinin in the occurrence of Balcan endemic nephropathy was discussed, which aroused the great attention of the International Cancer Research Society. At that time, citrinin was listed as one of the toxins that must be detected by the European Branch of the Natural Toxin Detection Committee of the International Academy of Life Sciences. In 1995, French scholar Blanc confirmed that some Monascus strains can also produce citrinin. The discovery caused quite a stir in the food world. Prior to this, Monascus had been used as a strain for food production and processing. In our country, the use of Monascus to ferment and produce food and medicine has a history of thousands of years. Many traditional foods such as red yeast rice, red yeast rice wine and fermented bean curd are deeply loved by people. Modern research has also found that among the metabolites of Monascus, there are many valuable fermentation products in food, medicine and chemical industry. Such as natural monascus pigment with excellent quality, good coloring property and rich hue; Monacollin K and Lovastain which can significantly inhibit cholesterol synthesis and lower blood lipid levels; there are also very rich ergosterol, long-term Chain fatty acids and a variety of antibacterial active substances. In the early 1990s, Europe, America, Japan and other countries saw a sharp increase in the demand for my country's edible red yeast rice, medicinal red yeast rice and related products, which brought considerable economic benefits to my country's red yeast rice manufacturers. The problem of citrinin not only caused losses to the export of red yeast rice products in our country, but also seriously threatened people's health. Japan, Germany and other countries have formulated new standards for red yeast rice products exported from my country, stipulating that the content of citrinin must be lower than the specified value, otherwise it is strictly prohibited to import and sell. However, most of the red yeast rice products in our country do not meet these standards. Faced with this severe situation, a rapid detection method for citrinin should be established as soon as possible to protect people's health and save this ancient and young food production industry.

At present, the techniques for detecting citrinin can be divided into two categories, one is chromatographic analysis, including thin-layer chromatography, gas chromatography, liquid chromatography, etc., which has always been the most important chemical analysis method for mycotoxins. Issues such as sensitivity and separation efficiency have been adopted since 1985. The more common analytical method for mycotoxins is still liquid chromatography, including liquid chromatography-mass spectrometry. In terms of method applicability, separation efficiency and sensitivity, liquid chromatography and liquid chromatography-mass spectrometry are both It cannot be replaced by other methods. In recent years, capillary electrophoresis and capillary electrophoresis-mass spectrometry have also begun to be applied in the field of mycotoxin analysis due to their high separation efficiency and have attracted widespread attention. However, due to its expensive equipment, complicated operation and high requirements on the purity of samples, it is not suitable for testing large quantities of samples, so its use is limited. The other is the immunochemical method, which has high sensitivity and specificity due to low requirements on the purity of the sample. It is especially suitable for the detection of large batches of samples, and has been widely used in the detection of various mycotoxins in recent years. Such methods generally include radioimmunoassay RIA and enzyme-linked immunoassay ELISA. Although the RIA method has high sensitivity, it cannot be widely used because of the use of radioactive substances harmful to the human body. The ELISA method is widely used because of its simple operation and safe use.

Immunochromatography is a qualitative determination method developed on the basis of immunochemistry. It often uses strip-shaped fiber chromatography material as the solid phase, and makes the sample solution swim on the chromatography strip through capillary action, and at the same time makes the The analyte and the receptors on the chromatographic material for the analyte, such as antibodies or antigens, have a highly specific and high-affinity immune reaction, and the immune complex is enriched or trapped in a certain part of the chromatographic material during the chromatography process. Areas such as detection bands, through enzymatic reactions or direct application of visual markers, such as colloidal gold, to obtain intuitive experimental phenomena, such as color development. The free markers cross the detection zone to achieve the purpose of automatic separation from the bound markers. Common tracer particles in immunochromatography include colloidal gold, latex, colloidal selenium, gelatin, magnetic particles, etc. Among them, the most successful marker is colloidal gold.

If the commonly used chromogenic markers are used for the tracer particles, when the detection line and the quality control line develop normal color, they can be directly distinguished by the naked eye. If magnetic particles are used as the tracer particles, instrument interpretation is required.

Contents of the invention

The object of the present invention is to provide a citrinin immunochromatographic detection test paper and a manufacturing method thereof.

Another object of the present invention is to apply the detection test paper.

Technical scheme of the present invention is:

A citrinin immunochromatographic detection test paper, comprising liner, sample pad, tracer particle conjugate pad, nitrocellulose membrane, water-absorbing pad, detection line, quality control line; there are sample pads in order on the liner, Tracer particle conjugate pad, nitrocellulose membrane, water-absorbent pad; tracer particle-labeled antibody against citrinin is on the tracer particle conjugate pad, and a detection line and a quality control line are also set on the nitrocellulose membrane. There are citrinin detection antigens on the line, and anti-antibodies on the quality control line, where the anti-antibodies are antibodies against citrinin antibodies.

A method for making citrinin immunochromatographic detection test paper: the tracer particles are marked as any one of the following: colloidal gold particles; latex particles; colloidal selenium particles; gelatin particles; magnetic particles.

The detection principle is as follows: add the sample dropwise on the sample pad, if there is citrinin in the sample, the citrinin will move to the tracer particle conjugate pad through chromatography, and combine with a limited amount of anti-citrinin antibody. When moving to the detection line, the anti-citrinin antibody labeled with the tracer particles has no redundant binding sites to combine with the detection antigen on the detection line, and the detection line does not develop color or magnetic enhancement. The anti-citrinin antibody labeled with the tracer particle continues to move forward to the quality control line, and reacts with the anti-antibody on the quality control line, and the quality control line develops color or magnetic enhancement. If there is no citrinin in the sample, the anti-citrinin antibody labeled on the tracer particle conjugate pad will bind to the detection antigen on the detection line, and the detection line will develop color or magnetic enhancement, and the quality control line will develop color. Or magnetic enhancement occurs.

A method for making citrinin immunochromatographic detection test paper: comprising the following steps, (A) preparation of citrinin antigen, including the preparation of monoclonal antibody immune antigen and citrinin detection antigen; (B) nitrocellulose Preparation of the test line and quality control line of the membrane; (C) preparation of the tracer particle conjugate pad; (D) assembly of the test strip.

A method for making citrinin immunochromatographic detection test paper: the preparation of citrinin antigen comprises the following steps: (A) citrinin is used to make its carboxyl group and a small molecular compound with carboxyl group and amino group at the same time through the active ester method The amino groups are connected by peptide bonds to form new compounds. (B) the compound is linked to the protein, polysaccharide, polynucleotide, polylysine, polystyrene or other artificially synthesized high-molecular-weight substances by the active ester method to form an artificial antigen; (C) One of the citrinin antigens was selected as the immune antigen. (D) One of the citrinin antigens was selected as the detection antigen.

A method for making citrinin immunochromatographic detection test paper: the preparation of the detection line and the quality control line of the nitrocellulose membrane comprises the following steps: (A) using citrinin to detect the antigen in a linear spot on the nitrocellulose membrane Prepare a test line; (B) Prepare a quality control line by linear spotting on a nitrocellulose membrane with an anti-antibody.

A method for making citrinin immunochromatographic detection test paper: the detection line and the quality control line are prepared in the following way: the nitrocellulose membrane is cut according to the size of 10-35mm wide; 0.01-5mg/mL citrinin detection antigen is spotted linearly on the membrane as a detection line, the detection line spotting position is 4-16mm away from the bottom edge of the membrane, and then the concentration is adjusted to 0.01-5mg/mL at the top of the membrane Anti-antibody spray quality control line, nitrocellulose membrane 4, dry at room temperature for 30 minutes, soak in 0.1-5% skimmed milk powder normal saline for 30 minutes, take out and blot dry, incubate at 37°C for 30 minutes, and place in a dry place at room temperature Store airtight.

A method for making citrinin immunochromatographic detection test paper: the preparation of the tracer particle conjugate pad comprises the following steps: (A) labeling the antibody against citrinin with the tracer particle, and the antibody includes IgG type antibody and IgM type antibody , IgA antibody; (B) Disperse the anti-citrinin antibody labeled with the tracer particles on the nitrocellulose membrane.

A method for making citrinin immunochromatographic detection test paper: the tracer particles for preparing the tracer particle conjugate pad are any one of the following: colloidal gold particles; latex particles; colloidal selenium particles; gelatin particles; magnetic particles.

A method for making citrinin immunochromatographic detection test paper: assembling the test strip, the steps are as follows: add a nitrocellulose membrane with a detection line and a quality control line on the liner 1; add a tracer on the liner 1 Particle conjugate pad; add sample pad to the pad; add absorbent pad to the pad; assemble into test strips.

Application of a citrinin immunochromatographic detection test paper in detection of citrinin.

The immunochromatography technique of the present invention detects citrinin, and the principle of determination is similar to the indirect competition ELISA method, and the antibody against citrinin marked with a certain concentration of tracer particles is coated on the glass fiber, and the antigen is adsorbed and detected on the nitrocellulose membrane respectively. (Since citrinin is not directly immobilized on the nitrocellulose membrane, it is necessary to prepare a conjugate of citrinin and carrier protein as the detection antigen) and anti-antibody as the detection line and quality control line. The detection test paper clip is composed of a plastic bottom plate, water-absorbing filter paper, glass fiber and nitrocellulose membrane. After the sample is added, the liquid permeates upward along the filter paper. If the sample contains the toxin to be detected, it will compete with the detection antigen on the nitrocellulose membrane for the labeled antibody on the glass fiber. When the toxin content is high, they will occupy the vast majority of the labeled antibody. The antigen-binding site on the detection line prevents the labeled antibody from combining with the detection antigen on the detection line, so no color develops on the detection line. On the contrary, if the sample does not contain the toxin to be detected, the labeled antibody will combine with the antigen on the detection line to develop color. The anti-antibody on the quality control line is developed by enriching the labeled antibody to prove the reliability of the result.

Citrinin is a small molecular compound, which has only reactogenicity but no immunogenicity, and cannot stimulate the body to produce an immune response. It must be coupled with a macromolecular carrier to make an artificial antigen to stimulate an effective immune response. Whether the monoclonal antibody with high affinity and specificity to citrinin can be successfully obtained, the preparation of human immune antigen is particularly important, and the coupling method needs to be carefully selected according to the molecular size, spatial conformation, and chemical properties of citrinin. . During the coupling process, the structural integrity of citrinin should be kept as far as possible, especially its molecular conformation should not be destroyed, because it is its special three-dimensional space structure, antigenic determinant, which matches the corresponding receptor on the surface of lymphocytes, and can Initiate an immune response against it. Slight changes in its conformation may lead to changes in its antigen. In addition, the immune response can be initiated only when the antigenic determinant comes into contact with the corresponding receptor on the surface of lymphocytes. When citrinin is a small molecular compound, when it is directly coupled to the carrier protein, it is not easy to access the receptor. Adding a connecting side chain between them will help the hapten to be exposed to the outside, forming an ideal space accessibility, and the effect may be better. Because there is a carboxyl group on the citrinin molecule, the present invention is designed to use the active ester method to connect it with a small molecular compound with both carboxyl and amino groups such as the amino group of ε-aminocaproic acid or δ-aminovaleric acid with a peptide bond, and then It is connected with the carrier protein by the active ester method to form a coupling bridge, and a relatively ideal effect has been achieved.

Advantages and scope of application of the present invention:

Compared with previous techniques for detecting citrinin in food, feed and red yeast rice products, the present invention has the following advantages: (1) easy and quick operation, only 5-10 minutes; (2) high sensitivity; (3) no special Instruments and equipment do not require any equipment except commercial reagents, and the cost is low; it is especially suitable for on-site rapid detection of citrinin. (4) There is no need to add additional substrate color indicator, and the result can be judged directly by color; (5) It can be stored at room temperature and measured in a single portion, and can be carried around.

The invention belongs to the technical field of immunobiology and is mainly used for citrinin detection. The units using the test strip of the present invention are mainly food and feed processing and production departments, food hygiene inspection departments, product quality supervision departments, and the like.

Description of drawings

Fig. 1 is a visual structure schematic diagram of the citrinin immunochromatographic detection test paper of the present invention.

Fig. 2 is a partially enlarged view of the tracer particle conjugate pad of the citrinin immunochromatographic detection test paper of the present invention.

Fig. 3 is a partially enlarged view of the detection line of the citrinin immunochromatographic detection test paper of the present invention.

Fig. 4 is a partially enlarged view of the quality control line of the citrinin immunochromatographic detection test paper of the present invention.

Fig. 5 is a schematic diagram of the working principle of the citrinin immunochromatographic detection test paper of the present invention.

Detailed ways

Example 1

A citrinin immunochromatographic detection test paper, comprising a liner 1, a sample pad 2, a tracer particle conjugate pad 3, a nitrocellulose membrane 4, an absorbent pad 5, a detection line 6, a quality control line 7, and the liner On 1, there are sample pad 2, tracer particle conjugate pad 3, nitrocellulose membrane 4, and water-absorbing pad 5 in sequence; tracer particle conjugate pad 3 has anti-ciritrinin antibody 10 labeled with tracer particle 9, The nitrocellulose membrane 4 is also provided with a test line 6 and a quality control line 7, the test line 6 has a citrinin detection antigen 8, and the quality control line 7 has an anti-antibody 1

1, wherein the anti-antibody 11 is an antibody against antibody 10 of citrinin.

The detection principle is as follows: add sample 12 dropwise on the sample pad 2, if there is citrinin in the sample 12, the citrinin will move to the tracer particle conjugate pad 3 through chromatography, and the limited amount of citrinin-resistant When moving to the detection line 6, the anti-citrinin antibody 10 labeled with the tracer particle 9 has no redundant binding sites to bind to the detection antigen 8 on the detection line 6, and the detection line 6 does not develop color or no magnetic enhancement occurs. The anti-citrinin antibody 10 marked by the tracer particle 9 continues to move forward to the quality control line 7, and reacts with the anti-antibody 11 on the quality control line 7, and the quality control line 7 develops color or magnetic enhancement. If there is no citrinin 8 in the sample 12, the antibody 10 against citrinin marked by the tracer particle 9 on the tracer particle conjugate pad 3 is combined with the detection antigen 8 on the detection line 6, and the detection line 6 develops color or Magnetic enhancement occurs, and quality control line 7 develops color or magnetic enhancement occurs.

The preparation method of citrinin immunochromatography detection test paper comprises the following steps, (A) the preparation of citrinin antigen, including the preparation of the immune antigen of monoclonal antibody and citrinin detection antigen 8; (B) nitrocellulose membrane Preparation of detection line 6 and quality control line 7 of 4; (C) preparation of tracer particle conjugate pad 3; (D) assembly of test strips.

Example 2

The tracer particles 9 are colloidal gold particles. All the other are with embodiment 1.

Example 3

The tracer particles 9 are latex particles. All the other are with embodiment 1.

Example 4

The tracer particles 9 are colloidal selenium particles. All the other are with embodiment 1.

Example 5

The tracer particles 9 are selected from gelatin particles. All the other are with embodiment 1.

Example 6

The tracer particles 9 are latex particles. All the other are with embodiment 1.

Example 7

The tracer particles 9 are magnetic particles. All the other are with embodiment 1.

In Examples 2-7:

If the commonly used chromogenic markers are used for the tracer particles, when the detection line and the quality control line develop normal color, they can be directly distinguished by the naked eye. If magnetic particles are used as the tracer particles, instrument interpretation is required. The sensitivity of the instrument to distinguish magnetic particles is higher.

Example 8

A method for making citrinin immunochromatographic detection test paper, the preparation of citrinin antigen comprises the following steps: (A) citrinin makes its carboxyl group and a small molecular compound with both carboxyl group and amino group through the active ester method The amino groups are connected by peptide bonds to form new compounds. (B) the compound is linked to the protein, polysaccharide, polynucleotide, polylysine, polystyrene or other artificially synthesized high-molecular-weight substances by the active ester method to form an artificial antigen; (C) One of the citrinin antigens was selected as the immune antigen. (D) One of the citrinin antigens was selected as the detection antigen. All the other are with embodiment 1.

Example 9

The preparation of the detection line 6 and the quality control line 7 of the nitrocellulose membrane 4 comprises the following steps: (A) using citrinin to detect the antigen 8 on the nitrocellulose membrane 4 to prepare a detection line 6 in a linear spot; (B) Use anti-antibody 11 to perform line spotting on nitrocellulose membrane 4 to prepare a quality control line 7 . All the other are with embodiment 1.

Example 10

A method for making citrinin immunochromatographic detection test paper, the detection line 6 and the quality control line 7 are prepared in the following way: the nitrocellulose membrane 4 is cut according to the size of 10-35mm wide; citrinin detection antigen 8 with a concentration adjusted to 0.01-5mg/mL was spotted linearly on the membrane as the detection line 6, and the spotting position of the detection line 6 was 4-16mm away from the bottom edge of the membrane, and then the concentration was adjusted at the top of the membrane to Spray quality control line 7 with 0.01-5mg/mL anti-antibody 11, dry nitrocellulose membrane 4 at room temperature for 30 minutes, soak in 0.1-5% skimmed milk powder normal saline for 30 minutes, remove and blot dry, and incubate at 37°C 30 minutes, and store in a dry place at room temperature. All the other are with embodiment 1.

Example 11

A method for making a citrinin immunochromatographic detection test paper, the preparation of the tracer particle conjugate pad 3 comprises the following steps: (A) labeling the antibody 10 against citrinin with the tracer particle, the antibody 10 includes an IgG type antibody, IgM antibody, IgA antibody; (B) Disperse the anti-citrinin antibody 10 labeled with the tracer particles 9 on the nitrocellulose membrane 4 . All the other are with embodiment 1.

Example 12

A method for making citrinin immunochromatographic detection test paper, assembling the test strip, the steps are as follows: add a nitrocellulose membrane 4 with a detection line 6 and a quality control line 7 on the liner 1; Add the tracer particle conjugate pad 3; add the sample pad 2 on the pad 1; add the water-absorbing pad 5 on the pad 1; assemble it into a detection test strip. All the other are with embodiment 1.

Example 13

A test paper for detecting citrinin by immunochromatography is prepared, and it is specifically applied to specific detection.

Including the following steps:

(1). Preparation of citrinin artificial antigen, including immunization antigen for immunizing animals to prepare monoclonal antibody and detection antigen for preparation of detection line.

(2). Preparation of nitrocellulose membrane test line 6 and quality control line 7.

①Use the conjugate of citrinin and carrier protein as the detection antigen linear spotting as the detection line 6.

②Use the anti-citrinin antibody 10 antibody line as the quality control line 7.

(3). Preparation of tracer particle conjugate pad 3:

The tracer particle 9-labeled antibody 10 against citrinin is dispersed on the glass fiber to make the tracer particle conjugate pad 3 .

(4). Assembly of test strips:

① Add a nitrocellulose membrane 4 with a detection line 6 and a quality control line 7 on the liner 1;

② Add tracer particle conjugate pad 3 on pad 1;

③ Add sample pad 2 on pad 1;

④Add water-absorbing pad 5 on pad 1; assemble it into a test strip.

(5). Processing of samples to be tested.

The program can be subdivided into five parts:

(1). Preparation of citrinin artificial antigen, including immunization antigen for immunizing animals to prepare monoclonal antibody and detection antigen for preparation of detection line; (2). Nitrocellulose membrane detection line 6 and quality control line The preparation of 7; (3). The preparation of the tracer particle conjugate pad 3; (4). The assembly of the test strip; (5). The processing of the sample to be tested.

in

1 Preparation of citrinin artificial antigen:

① Dissolve 1.25 mg (5 μmol) of citrinin in 0.3 mL tetrahydrofuran (THF), 0.9 mg (7.5 μmol) of nitrogen hydroxysuccinic acid (NHS) and 2 mg (10 μmol) of dicyclohexylcarbodiimide (DCC) in 0.18 In mL THF, the two were mixed evenly, and shaken and reacted at room temperature for 24 hours in the dark.

② After the reaction is complete, centrifuge at 10,000rpm for 10 minutes to remove the precipitate. Vacuum-dry the supernatant, add 600 μL nitrogen, nitrogen-dimethylformamide (DMF) to dissolve, slowly drop into 10 μmol ε-aminocaproic acid or δ-aminovaleric acid solution, dissolve in 400 μL 1M Na ₂ CO ₃ , The reaction was shaken at room temperature for 2 hours (protected from light).

③ After the reaction, extract three times with 2 mL of chloroform. The organic phase was evaporated to dryness under reduced pressure. Redissolve in 0.1-3mL THF, repeat the above steps, and further activate the citrinin that has been linked with aminocaproic acid.

④ After centrifuging at 10,000rpm for 10 minutes to remove the precipitate, the organic phase was vacuum-dried, dissolved in 0.3-3mL dimethyl sulfoxide, and then 0.9mL of carrier substances (keyhole limpet hemocyanin, bovine serum albumin, thyroglobulin, egg Albumin, skimmed milk powder, polylysine or other artificially synthesized macromolecular substances with amino groups) solution (0.5mg dissolved in 0.13M NaHCO ₃ ), shaking reaction at room temperature for 2 hours (protected from light) . After the reaction, 0.01M PBS (pH7.2) was dialyzed at 4° C. for 72 hours, purified with Sephadex G-25, and analyzed by ultraviolet scanning to determine the molar ratio of the coupling product, and then vacuum freeze-dried for use.

⑤ Select one of the artificial antigens formed by coupling with different carrier substances as the immune antigen, and the other as the detection antigen.

2. The preparation of nitrocellulose membrane detection line 6 and quality control line 7 comprises the following steps:

The nitrocellulose membrane 4 was cut to a size of 10 mm wide. Fully dialyzed with normal saline buffer solution, the detection antigen whose concentration is adjusted to 0.01mg/mL is spotted linearly on the membrane as detection line 6. Spray quality control line 7 with anti-antibody 11 corresponding to antibody 10 against citrinin at 0.01 mg/mL. The nitrocellulose membrane 4 was dried at room temperature for 30 minutes, soaked in 0.1% skim milk powdered saline for 30 minutes, removed and blotted dry, incubated at 37°C for 30 minutes, and sealed in a dry place at room temperature for storage.

3. Preparation of Tracer Particle Conjugate Mat 3

(1) Preparation and labeling of tracer particles 9

①Colloidal gold sol preparation mark:

a) Colloidal gold sol preparation: take 200mL of 0.01% gold tetrachloride aqueous solution, heat to boiling, add 1-20mL 1% sodium citrate aqueous solution, boil for 5 minutes, orange-red appears, colloidal gold particle diameter is measured as 10- by electron microscope 80nm;

b) Colloidal gold-labeled antibody: take 50 mL of colloidal gold, adjust the pH (8.0) with 0.1 mol/L potassium carbonate, mix the colloidal gold sol and antibody with stirring, and then add polyethylene glycol aqueous solution to make the final concentration 0.05%. Centrifuge the crude product at 6000 rpm for 45 minutes, suspend the precipitate to 1.5 mL with saline, and store it at 4°C.

②Colloidal selenium sol preparation and antibody labeling:

a) Preparation of colloidal selenium sol: 550 mL of deionized water and 10 g of polyacrylic acid were added to a four-neck round-bottomed flask with a capacity of 1 liter, stirred at room temperature with nitrogen, and 69.5 mL of hydrazine hydrate was added, and the stirring was continued for 20 minutes. 9.63 g of selenous acid was dissolved in 280 mL of water, stirred at room temperature and dropped into the reaction mixture to obtain a 120 nm red selenium sol.

b) Colloidal selenium antibody labeling: Anti-citrinin antibody 10 was prepared with 20mmol/L pH7.3 phosphate buffer solution to a concentration of 4.6mg/mL, added 25μL to 25mL selenium sol with pH7.3, and stirred at room temperature for 10 minutes Then add 1% PEG80001mL, mix well, and centrifuge at 5000rpm at 4°C for 5 minutes to obtain a soft red precipitate, which is made into 1mL suspension with phosphate buffer containing 0.05% NaN ₃ .

③ Preparation of latex and gelatin and antibody labeling:

Colored latex particles and gelatin particles are blue in color. Dilute the latex particles or gelatin particles to 1% concentration with pH 7.1 phosphate buffer solution, take 50mL respectively, mix the latex or gelatin solution and the antibody 10 against citrinin under stirring, stir at room temperature for 30 minutes, and then store at 37°C Incubate in a water bath for 60 minutes and place at 4°C for 4-5 hours. Centrifuge at 15,000 rpm for 30 minutes, and resuspend with 2 mL of phosphate buffer.

④Magnetic particle antibody labeling:

Magnetic particles were purchased from Bangs Laboratories. Dilute the magnetic particles to 1% concentration with pH 7.1 phosphate buffer solution, take 50mL, mix it with anti-citrinin antibody 10 under stirring, stir at room temperature for 30 minutes, incubate in 37°C water bath for 60 minutes, and place at 4°C 4 hours. Centrifuge at 15,000 rpm for 30 minutes, and resuspend with 2 mL of phosphate buffer.

(2) Preparation of tracer particle conjugate pad

The anti-citinillin antibody 10 labeled with the tracer particles 9 was mixed and dispersed in proportion on glass fiber paper with a thickness of 1 mm, and then freeze-dried and stored in a sealed and dry place.

4. Assembly of note:

① Add a nitrocellulose membrane 4 with a detection line 6 and a quality control line 7 on the liner 1;

② Add tracer particle conjugate pad 3 on pad 1;

③ Add sample pad 2 on pad 1;

④Add water-absorbing pad 5 on pad 1; assemble it into a test strip.

5. Processing of samples to be tested

① Solid sample processing method: In a 50mL Erlenmeyer flask, weigh 1-5g of red yeast rice, and extract three times with 15mL methanol-chloroform solution (1:1). Each time of extraction, continue for 1h under magnetic stirring, and combine the extractions liquid. Evaporate to dryness at 40°C with a rotary evaporator, and then dissolve with PBS (pH 7.6) containing 10% methanol.

②Processing method for samples containing fat to be tested: Weigh 1-5g of the sample, soak it in 10-20mL of methanol for 12h, then degrease it twice with _isooctane ; then add distilled water, adjust the pH to 4.5 with _H2SO4 , and extract with chloroform Afterwards, evaporate to dryness in a water bath, dilute to 1 mL with PBS (pH 7.6) containing 10% methanol, and set aside.

3. the processing method of feed sample: take sample 3.0-5.0g, add acetonitrile (or methyl alcohol): sulfuric acid (20%)=99: 1 mixed solution 20-30mL, after ultrasonic treatment 15 minutes, centrifuge and get supernatant ( 3000rpm/min, 20 minutes), repeated 3 times, the extracts were combined, concentrated in a 60°C water bath to dryness, dissolved in PBS (pH7.6) containing 10% methanol, and used directly for determination.

④ Liquid sample processing method: Take a certain volume of sample, filter it with filter paper, add an equal volume of PBS (pH 7.6) containing 10% methanol, and set aside.

Example 14:

The colloidal gold immunochromatography method for detecting citrinin and the preparation of test paper were used to detect red yeast rice samples.

The present embodiment is divided into 5 parts: (1) processing of sample to be tested; (2) preparation of artificial antigen; (3) preparation of nitrocellulose membrane 4; (4) preparation of colloidal gold conjugate pad; (5) Assembly of test strips; (6) detection and result judgment

(1) Handling of samples to be tested

In a 50mL Erlenmeyer flask, weigh 5g of red yeast rice, extract three times with 10mL methanol-chloroform solution (1:1), each time of extraction, continue for 1h under magnetic stirring, and combine the extracts. Evaporate to dryness at 40°C with a rotary evaporator, and then dissolve in PBS (pH7.6) containing 10% methanol.

(2) Preparation of artificial antigen

① Dissolve 1.25 mg (5 μmol) of citrinin in 0.3 mL of tetrahydrofuran (THF). Nitrohydroxysuccinic acid (NHS) 0.9 mg (7.5 μmol) and dicyclohexylcarbodiimide (DCC) 2 mg (10 μmol) were dissolved in 0.2 mL THF. The two were mixed, placed at room temperature in the dark and shaken for 24 hours.

② After the reaction is complete, centrifuge at 10,000rpm for 10 minutes to remove the precipitate. The supernatant was vacuum-dried, dissolved in 200 μL nitrogen, nitrogen-dimethylformamide (DMF), slowly dropped into 10 μmol ε-aminocaproic acid solution (dissolved in 400 μL 1M Na ₂ CO ₃ ), and shaken at room temperature for 2 hours ( away from light).

③After the reaction, extract three times with 0.2mL chloroform. The organic phase was evaporated to dryness under reduced pressure. Redissolve in 0.4mL THF, repeat the above steps, and further activate the citrinin that has been linked with aminocaproic acid.

④ Centrifuge at 10,000 rpm for 10 minutes to remove the precipitate, vacuum-dry the organic phase, dissolve in 0.2 mL dimethyl sulfoxide, add 400 μL keyhole limpet hemocyanin solution (0.5 mg dissolved in 0.13 M NaHCO ₃ ), shake at room temperature React for 2 minutes (protect from light).

⑤After the reaction, dialyze with 0.01M PBS (pH7.2) at 4C for 72 hours, purify with Sephadex G-25, measure the molar ratio of the coupling product by UV scanning analysis, and freeze-dry in vacuum for use.

(3) Preparation of nitrocellulose membrane 4

The nitrocellulose membrane 4 was cut to a size of 35 mm wide. Fully dialyzed with normal saline buffer solution, the concentration of the detection antigen adjusted to 5mg/mL was spotted linearly on the membrane as the detection line. Spray quality control line 7 with anti-antibody 11 corresponding to antibody 10 against citrinin. The nitrocellulose membrane 4 was dried at room temperature for 30 minutes, soaked in 5% skimmed milk powdered physiological saline for 30 minutes, removed and blotted dry, incubated at 37°C for 30 minutes, and sealed in a dry place at room temperature for storage.

(4) Preparation of colloidal gold conjugate pad

① Colloidal gold sol preparation mark: take 200mL of 0.01% gold tetrachloride aqueous solution, heat to boiling, add 10mL of 1% sodium citrate aqueous solution, boil for 5 minutes, orange-red appears, and the diameter of colloidal gold particles is 10-80nm as measured by electron microscope; Take 50 mL of colloidal gold, adjust the pH (8.0) with 0.1 mol/L potassium carbonate, mix the colloidal gold sol and anti-citrinin antibody 10 under stirring, and then add polyethylene glycol aqueous solution to make the final concentration 0.05%. Centrifuge the crude product at 6000 g for 45 minutes, suspend the precipitate to 1.5 mL with physiological saline, and store it at 4°C.

② Mix and disperse the colloidal gold-labeled anti-citrinin antibody 10 in proportion on glass fiber paper with a thickness of 1m, freeze-dry and store in a sealed and dry place.

(5) Assembly of note:

① Add a nitrocellulose membrane 4 with a detection line 6 and a quality control line 7 on the liner 1;

② Add tracer particle conjugate pad 3 on pad 1;

③ Add sample pad 2 on pad 1;

④Add water-absorbing pad 2 to pad 1; assemble it into a test strip.

(6) Detection and result judgment:

Take 0.5 mL of the processed sample, centrifuge for a while, insert the test strip vertically, the insertion depth does not exceed the sample pad 2, stay in the sample for about 5 seconds, take out the test paper vertically, and judge the result after 5 minutes: at the detection line and quality control line If there is red on the test line, it is negative; if there is no red on the test line, it is positive when the quality control line is red; if there is no red on the quality control line, it is invalid.

Example 15

The method for detecting citrinin by immunochromatography of latex particles or gelatin particles and the preparation of test paper are used to detect red yeast bean curd samples.

The present embodiment is divided into 5 parts: (1) processing of sample to be tested; (2) preparation of artificial antigen; (3) preparation of nitrocellulose membrane; (4) preparation of latex particle or gelatin particle conjugate pad; ( 5) Assembly of test strips; (6) Detection and result judgment

(1) Red yeast bean curd sample processing method

Weigh 2 g of the sample, soak it in 10 mL of methanol for 1 h, then degrease it twice with isooctane; then add distilled water, adjust the pH to 4.5 with H ₂ SO ₄ , extract with chloroform, evaporate to dryness in a water bath, and wash with 10% methanol in PBS ( pH7.6) dissolved and set aside.

(2 preparation of artificial antigen

① Dissolve 1.25 mg (5 μmol) of citrinin in 0.3 mL of tetrahydrofuran (THF). Nitrohydroxysuccinic acid (NHS) 0.9 mg (7.5 μmol) and dicyclohexylcarbodiimide (DCC) 2 mg (10 μmol) were dissolved in 0.2 mL THF. The two were mixed, placed at room temperature in the dark and shaken for 24 hours.

② After the reaction is complete, centrifuge at 10,000rpm for 10 minutes to remove the precipitate. The supernatant was vacuum-dried, dissolved in 200 μL of nitrogen, nitrogen-dimethylformamide (DMF), slowly dropped into 10 μmol of ε-aminocaproic acid solution (dissolved in 400 μL of 1M Na ₂ CO ₃ ), and shaken at room temperature for reaction 2 hours (protect from light).

③After the reaction, extract three times with 0.2mL chloroform. The organic phase was evaporated to dryness under reduced pressure. Redissolve in 0.4mL THF, repeat the above steps, and further activate the citrinin that has been linked with aminocaproic acid.

④ After centrifuging at 10000rpm for 10 minutes to remove the precipitate, the organic phase was vacuum-dried, dissolved in 0.2mL dimethyl sulfoxide, and then added to 400μL skimmed milk powder solution (0.5mg dissolved in 0.13M NaHCO ₃ ), shaken and reacted at room temperature for 2 hours ( away from light).

⑤After the reaction, dialyze with 0.01M PBS (pH7.2) at 4C for 72 hours, purify with SephadexG-25, measure the molar ratio of the coupling product by UV scanning analysis, and freeze-dry in vacuum for use.

(3) Preparation of nitrocellulose membrane 4

The nitrocellulose membrane 4 was cut to a size of 15 mm wide. Fully dialyzed with normal saline buffer solution, the concentration of the detection antigen adjusted to 3mg/mL was spotted linearly on the membrane as the detection line 6, and the spot position of the detection line 6 was 8mm away from the bottom edge of the membrane, and then adjusted to the top of the membrane with a concentration of The anti-antibody 11 corresponding to the anti-citrinin antibody 10 at 3 mg/mL was sprayed on the quality control line 7 . The nitrocellulose membrane 4 was dried at room temperature for 30 minutes, soaked in 1% skimmed milk powdered saline for 30 minutes, removed and blotted dry, incubated at 37°C for 30 minutes, and sealed in a dry place at room temperature for storage.

(4) Preparation of latex and gelatin combination pad:

Colored latex particles and gelatin particles are blue in color. Dilute latex particles or gelatin particles to 1% concentration with pH 7.1 phosphate buffer solution, take 50mL respectively, mix the solution with anti-citrinin antibody 10 under stirring, stir at room temperature for 30 minutes, and incubate in a water bath at 37°C for 60 Minutes, 4-5 hours at 4°C. Centrifuge at 15,000 rpm for 30 minutes, and resuspend with 2 mL of phosphate buffer. Mix and disperse the antibodies labeled latex particles or gelatin particles on glass fiber paper with a thickness of 1 mm, freeze-dry and store in a sealed and dry place.

(5) Assembly of note:

① Add a nitrocellulose membrane 4 with a detection line 6 and a quality control line 7 on the liner 1;

② Add tracer particle conjugate pad 3 on pad 1;

③ Add sample pad 2 on pad 1;

④Add water-absorbing pad 5 on pad 1; assemble it into a test strip.

(6) Detection and result judgment:

Take 0.5 mL of the processed sample, centrifuge for a while, insert the test strip vertically, the insertion depth does not exceed the sample pad 2, stay in the sample for about 5 seconds, take out the test paper vertically, and judge the result after 5 minutes: test line 6 and quality control line If there is blue color at all 7 points, it is negative; if there is no blue color on the test line 6, it is positive if the blue color appears on the quality control line 7; if there is no blue color on the quality control line 7, it is invalid.

The detection principle is as follows: the sample is added dropwise on the sample pad 2, if there is foreign citrinin in the sample, the citrinin in the sample will move to the tracer particle conjugate pad 3 through chromatography, and the limited amount of citrinin will The antibody 10 of citrinin is combined. When moving to the detection line 6, the antibody 10 against citrinin marked by the tracer particle 9 has no redundant binding site and the detection antigen 8 on the detection line 6. The detection antigen 8 is For the combination of citrinin and the coupling product of the carrier, the detection line 6 does not develop color or magnetic enhancement. The anti-citrinin antibody 10 marked by the tracer particle 9 continues to move forward to the quality control line 7, and reacts with the antibody 11 of the anti-citrinin antibody 10 on the quality control line 7, and the quality control line 7 develops color or appears magnetic enhanced. If there is no citrinin 8 in the sample, the antibody 10 against citrinin marked by the tracer particle 9 on the tracer particle conjugate pad 3 and the detection antigen (the coupling product of citrinin and the carrier) on the detection line 6 )8 combination, the detection line 6 develops color or exhibits magnetic enhancement, and the quality control line 7 develops color or exhibits magnetic enhancement as above.

Example 16

Colloidal selenium immunochromatography detection method for citrinin and preparation of test paper for detection of feed samples

The present embodiment is divided into 5 parts: (1) processing of sample to be tested; (2) preparation of artificial antigen; (3) preparation of nitrocellulose membrane 4; (4) preparation of colloidal selenium conjugate pad; (5) Assembly of test strips; (6) detection and result judgment

(1) Handling of samples to be tested

In a 50mL centrifuge tube, weigh 3.0g of the feed sample, add acetonitrile (or methyl alcohol): sulfuric acid (20%)=99: 1 mixed solution 20mL, after ultrasonic treatment for 15 minutes, centrifuge to get the supernatant (3000rpm/min, 20 minutes), repeating a total of 3 times, the extracts were combined, concentrated to dryness in a 60°C water bath, dissolved in PBS (pH 7.6) containing 10% methanol, and used directly for determination.

(2) Preparation of artificial antigen

① Dissolve 1.25 mg (5 μmol) of citrinin in 0.3 mL of tetrahydrofuran (THF). Nitrohydroxysuccinic acid (NHS) 0.9 mg (7.5 μmol) and dicyclohexylcarbodiimide (DCC) 2 mg (10 μmol) were dissolved in 0.2 mL THF. The two were mixed, placed at room temperature in the dark and shaken for 24 hours.

② After the reaction is complete, centrifuge at 10,000rpm for 10 minutes to remove the precipitate. The supernatant was vacuum-dried, dissolved in 200 μL nitrogen, nitrogen-dimethylformamide (DMF), slowly dropped into 10 μmol ε-aminocaproic acid solution (dissolved in 400 μL 1M Na ₂ CO ₃ ), and shaken at room temperature for 2 hours ( away from light).

③After the reaction, extract three times with 0.2mL chloroform. The organic phase was evaporated to dryness under reduced pressure. Redissolve in 0.4mL THF, repeat the above steps, and further activate the citrinin that has been linked with aminocaproic acid.

④ Centrifuge at 10,000rpm for 10 minutes to remove the precipitate, vacuum-dry the organic phase, dissolve in 0.2mL dimethyl sulfoxide, add 400μL ovalbumin solution (0.5mg dissolved in 0.13M NaHCO ₃ ), shake and react at room temperature for 2 hours (protect from light).

⑤After the reaction, dialyze with 0.01M PBS (pH7.2) at 4C for 72 hours, purify with SephadexG-25, measure the molar ratio of the coupling product by UV scanning analysis, and freeze-dry in vacuum for use.

(3) Preparation of nitrocellulose membrane 4

The nitrocellulose membrane 4 was cut to a size of 25 mm wide. Fully dialyzed with saline buffer solution, the concentration of the detection antigen adjusted to 0.8mg/mL was spotted linearly on the membrane as the detection line 6, the position of the detection line 6 was 6mm away from the bottom edge of the membrane, and then the concentration was adjusted on the top of the membrane. Control line 7 was sprayed with anti-antibody 11 corresponding to antibody 10 against citrinin at 0.5 mg/mL. The nitrocellulose membrane 4 was dried at room temperature for 30 minutes, soaked in 1% skimmed milk powdered saline for 30 minutes, removed and blotted dry, incubated at 37°C for 30 minutes, and sealed in a dry place at room temperature.

(4) Preparation of colloidal selenium conjugate pad:

Colloidal selenium antibody labeling: Anti-citrinin antibody was made into 4.6mg/mL concentration solution with 20mmol/L pH7.3 phosphate buffer solution, 25μL was added to 25mL selenium sol, stirred at room temperature for 10 minutes, and then 1% PEG80001mL was added. Mix well, and centrifuge at 5000 rpm for 5 minutes at 4°C to obtain a soft red precipitate, which is made into 1 mL suspension with phosphate buffer containing 0.05% NaN ₃ . The antibody 10 against citrinin labeled with colloidal selenium particles was mixed and dispersed on glass fiber paper with a thickness of 1 mm, and then freeze-dried and stored in a sealed and dry place.

(5) Assembly of note:

① Add a nitrocellulose membrane 4 with a detection line 6 and a quality control line 7 on the liner 1;

② Add tracer particle conjugate pad 3 on pad 1;

③ Add sample pad 2 on pad 1;

④Add water-absorbing pad 5 on pad 1; assemble it into a test strip.

(6) Detection and result judgment:

Take 0.5mL of the processed sample, centrifuge for a while, insert the test strip vertically, the insertion depth does not exceed the sample pad, stay in the sample for about 5 seconds, take out the test paper vertically, and judge the result after 5 minutes: test line 6 and quality control line 7 If there is a red line at the center, it is negative; if there is no red line on the test line 6, it is positive if the red line appears on the quality control line 7; if there is no red line on the quality control line 7, it is invalid.

Example 17

Method for detecting citrinin by immunochromatography with magnetic particles as tracer particles and preparation of test paper for detection of red koji wine samples

This embodiment is divided into 5 parts: (1) processing of the sample to be tested; (2) preparation of artificial antigen; (3) preparation of nitrocellulose membrane 4; (4) preparation of magnetic particle conjugate pad; (5) Assembly of test strips; (6) detection and result judgment

(1) Handling of samples to be tested

Take a certain volume of sample, filter it with filter paper, add an equal volume of PBS (pH7.6) containing 10% methanol, and set aside.

(2) Preparation of artificial antigen

① Dissolve 1.25 mg (5 μmol) of citrinin in 0.3 mL of tetrahydrofuran (THF). Nitrohydroxysuccinic acid (NHS) 0.9 mg (7.5 μmol) and dicyclohexylcarbodiimide (DCC) 2 mg (10 μmol) were dissolved in 0.2 mL THF. The two were mixed, placed at room temperature in the dark and shaken for 24 hours.

② After the reaction is complete, centrifuge at 10,000rpm for 10 minutes to remove the precipitate. The supernatant was vacuum-dried, dissolved in 200 μL nitrogen, nitrogen-dimethylformamide (DMF), slowly dropped into 10 μmol ε-aminocaproic acid solution (dissolved in 400 μL 1M Na ₂ CO ₃ ), and shaken at room temperature for 2 hours ( away from light).

③After the reaction, extract three times with 0.2mL chloroform. The organic phase was evaporated to dryness under reduced pressure. Redissolve in 0.4mL THF, repeat the above steps, and further activate the citrinin that has been linked with aminocaproic acid.

④ After centrifuging at 10000rpm for 10 minutes to remove the precipitate, the organic phase was vacuum-dried, dissolved in 0.2mL dimethyl sulfoxide, and then added to 400μL thyroglobulin solution (0.5mg dissolved in 0.13M NaHCO ₃ ), shaken at room temperature for reaction 2 hours (protect from light).

⑤After the reaction, 0.01M PBS (pH7.2) was dialyzed at 4°C for 72 hours, purified with Sephadex G-25, and the molar ratio of the coupling product was determined by UV scanning analysis, and vacuum freeze-dried for use.

(3) Preparation of nitrocellulose membrane 4

The nitrocellulose membrane 4 is cut to a width of 10-35 mm. Fully dialyzed with normal saline buffer solution, the concentration of the detection antigen adjusted to 0.01-5mg/mL was spotted linearly on the membrane as the detection line 6, and the spotting position of the detection line 6 was 4-16mm away from the bottom edge of the membrane, and then on the top of the membrane. Spray the quality control line with anti-antibody 11 corresponding to antibody 10 against citrinin, adjusted to a concentration of 0.01-5 mg/mL. Nitrocellulose membrane 4 was dried at room temperature for 30 minutes, soaked in 0.1-5% skimmed milk powdered physiological saline for 30 minutes, removed and blotted dry, incubated at 37°C for 30 minutes, and sealed in a dry place at room temperature for storage.

(4) Preparation of magnetic particle conjugate pad:

Magnetic particles were purchased from Bangs Laboratories. Dilute the magnetic particles to 1% concentration with pH 7.1 phosphate buffer solution, take 50mL, mix it with anti-citrinin antibody 10 under stirring, stir at room temperature for 30 minutes, then incubate in 37°C water bath for 60 minutes, 4°C Leave for 4-5 hours. Centrifuge at 15,000 rpm for 30 minutes, and resuspend with 2 mL of phosphate buffer. The antibodies labeled with magnetic particles were mixed and dispersed on glass fiber paper with a thickness of 1 mm, and then freeze-dried and stored in a sealed and dry place.

(5) Assembly of note:

① Add a nitrocellulose membrane 4 with a detection line 6 and a quality control line 7 on the liner 1;

② Add tracer particle conjugate pad 3 on pad 1;

③ Add sample pad 2 on pad 1;

④Add water-absorbing pad 5 on pad 1; assemble it into a test strip.

(6) Detection and result judgment:

Take 0.5 mL of the processed sample, centrifuge for a while, insert the test strip vertically, the insertion depth does not exceed the sample pad 2, stay in the sample for about 5 seconds, take out the test paper vertically, and judge the result with a magnetic reader after 5 minutes: test line If there is magnetic enhancement at the quality control line, it is negative; if there is no magnetic enhancement at the detection line, it is positive if there is magnetic enhancement at the quality control line; if there is no magnetic enhancement at the quality control line, it is invalid.

Example 18

1% bovine serum albumin was used instead of 0.1-5% skimmed milk powder, and the rest were tested according to the method in Example 15.

After comparison, it was found that compared with 1% bovine serum albumin, the cost of using 1% skim milk powder was lower, and the background adsorption could be reduced by 20%. A 20% reduction in the background meant that the detection sensitivity was increased by 20%.

Example 19

Detect according to the method for embodiment 15, detect to a plurality of samples, the content of citrinin in the red yeast rice is 3.2mg/kg-1500mg/kg, the content of citrinin in the red yeast rice pigment is 193mg/kg-17452mg/ kg, the citrinin content in the feed is 5mg/kg-632mg/kg.

The off-line sensitivity of the sample detection in the above embodiment is high.

Claims (9)

1. citrinin immunity chromatography detection test paper, comprise liner (1), sample pad (2), trace particle bond pad (3), nitrocellulose filter (4), adsorptive pads (5), detection line (6), nature controlling line (7), sample pad (2) is arranged on liner (1) in order, trace particle bond pad (3), nitrocellulose filter (4), adsorptive pads (5); The antibody (10) that the anti-citrinin of trace particle (9) mark is arranged on the trace particle bond pad (3), also be provided with detection line (6) and nature controlling line (7) on the nitrocellulose filter (4), there is citrinin to detect antigen (8) on the detection line (6), antiantibody (11) is arranged on the nature controlling line (7), and wherein antiantibody (11) is the antibody of the antibody (10) of anti-citrinin.

2. as a kind of method for making of citrinin immunity chromatography detection test paper, it is characterized in that: comprise the steps that (A) preparation of citrinin antigen comprises the immunizing antigen of monoclonal antibody and the preparation that citrinin detects antigen (8); (B) preparation of the detection line (6) of nitrocellulose filter (4) and nature controlling line (7); (C) preparation of trace particle bond pad (3); (D) assembling test strips.

3. the method for making of a kind of citrinin immunity chromatography detection test paper as claimed in claim 2 is characterized in that: trace particle (9) be labeled as following any one: colloid gold particle; Latex particle; The electroselenium particle; Gelatin particle; Magnetic-particle.

4. the method for making of a kind of citrinin immunity chromatography detection test paper as claimed in claim 2, it is characterized in that: the preparation of citrinin antigen, comprise the steps: that (A) citrinin links to each other with peptide bond by the amino that active ester method makes its carboxyl and have the carboxyl and the micromolecular compound of amino simultaneously, forms new compound; (B) this compound links to each other with the high molecular weight material of protein, polysaccharide, polynucleotide, poly-D-lysine, lustrex or other synthetic of high molecular carrier mass by active ester method once more and forms artificial antigen; (C) select a kind of of citrinin antigen as immunizing antigen; (D) select a kind of of citrinin antigen as detecting antigen.

5. the method for making of a kind of citrinin immunity chromatography detection test paper as claimed in claim 2, it is characterized in that: the detection line (6) of nitrocellulose filter (4) and the preparation of nature controlling line (7) comprise the steps: that (A) detects antigen (8) with citrinin and go up a linear spotting preparation detection line (6) at nitrocellulose filter (4); (B) prepare a nature controlling line (7) with antiantibody (11) at the enterprising line shape of nitrocellulose filter (4) point sample.

6. the method for making of a kind of citrinin immunity chromatography detection test paper as claimed in claim 5 is characterized in that: detection line (6) and nature controlling line (7) are to prepare like this: nitrocellulose filter (4) is cut out by the wide size of 10-35mm; To fully dialyse through normal saline buffer solution, the citrinin that concentration is adjusted into 0.01-5mg/mL detect antigen (8) on film linear spotting as detection line (6), detection line (6) point sample position is from film base 4-16mm, be adjusted into antiantibody (11) the spraying nature controlling line (6) of 0.01-5mg/mL then with concentration on the top of film, nitrocellulose filter (4) drying at room temperature 30 minutes, place the physiological saline of 0.1-5% skimmed milk power to soak 30 minutes, take out suck dry moisture, hatched 30 minutes in 37 ℃, put dry place hermetically storing under the room temperature.

7. the method for making of a kind of citrinin immunity chromatography detection test paper as claimed in claim 2, it is characterized in that: the preparation of trace particle bond pad (3) comprises the steps: (A) antibody (10) with the anti-citrinin of trace particle mark, and antibody (10) comprises IgG type antibody, IgM type antibody, IgA antibody; (B) antibody (10) of the good anti-citrinin of trace particle (9) mark is dispersed on the nitrocellulose filter (4).

8. the method for making of a kind of citrinin immunity chromatography detection test paper as claimed in claim 2 is characterized in that: the assembling test strips, and step is as follows: the nitrocellulose filter (4) that adds detection line (6) and nature controlling line (7) on liner (1); On liner (1), add trace particle bond pad (3); On liner (1), add sample pad (2); On liner (1), add adsorptive pads (5); Be assembled into test strip.

9. the application of citrinin immunity chromatography detection test paper in detecting citrinin of adopting the described method for making of claim 2 to make.

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