阅读说明：本技术 一种检测组织中四环素的免疫胶体检测卡 (Immune colloidal detection card for detecting tetracycline in tissues ) 是由 杜霞 洪霞 袁超 于 2019-05-01 设计创作，主要内容包括：本发明四环素的免疫胶体金检测卡及其制备方法,涉及动物源食品兽药残留检测技术领域。本发明的检测卡外壳中的试纸条,由PVC胶板、样品垫、胶体金结合垫、包被膜和吸水垫组成；胶体金膜为含四环素单克隆抗体的玻璃纤维素膜,包被膜是硝酸纤维素膜,其上设有T线和C线,T线包被有四环素蛋白质偶联物,C线包被有羊抗鼠IgG抗体。本发明有效用于快速检测四环素,方便、快捷、结果准确。(The invention discloses an immune colloidal gold detection card for tetracycline and a preparation method thereof, and relates to the technical field of animal-derived food and veterinary drug residue detection. The test strip in the shell of the detection card consists of a PVC rubber plate, a sample pad, a colloidal gold combination pad, a coating film and a water absorption pad; the colloidal gold membrane is a glass cellulose membrane containing tetracycline monoclonal antibodies, the coating membrane is a nitrocellulose membrane, a T line and a C line are arranged on the nitrocellulose membrane, the T line is coated with tetracycline protein conjugates, and the C line is coated with goat anti-mouse IgG antibodies. The method is effectively used for quickly detecting the tetracycline, and is convenient, quick and accurate in result.)

1. The tetracycline colloidal gold detection card is characterized by being prepared according to the following steps:

(1) preparing a colloidal gold solution: 1L Erlenmeyer flask is taken, 495 mL of ultrapure water is added, and then 1% HAuCl chloroauric acid is added₄·3H₂O)5 mL, 500 mL of 0.01% chloroauric acid aqueous solution was prepared, heated to boiling, and 1% trisodium citrate Na was added under continuous stirring₃C₆H₅O₇·2H₂O solution 5-7 mL, stirring and heating, and maintaining 5 mi when the solution color is completely changed into transparent purple redStopping heating after n, supplementing water to the original volume, cooling to room temperature, and storing at 2-8 ℃ for later use;

(2) pretreatment of the antibody: centrifuging the tetracycline antibody to be labeled for 20 min at the temperature of 4 ℃ at the speed of 1000 r/min, taking the supernatant, and diluting the supernatant into 1 mg/mL by using 0.01 mol/L PBS; or diluting to 1 mg/ml with 0.01 mol/L PBS, and filtering with 0.22 mu m filter membrane;

(3) preparation of colloidal gold marker: taking 40 mL of the colloidal gold solution in the step (1), and adding 0.25 mol/L K₂CO₃Adjusting the pH value of the colloidal gold solution to 8.5, stirring the solution by a magnetic stirrer at 250 r/min, dropwise adding 4 mL of protein solution containing 0.3 mg of antibody protein, and reacting the solution for 10 min; dropwise adding 4 mL of 10% BSA, and continuously stirring for reaction for 10 min; centrifuging the gold-labeled antibody solution at the normal temperature of 1500 r/min for 10 min, and discarding the precipitate formed by the condensed gold particles; centrifuging the red supernatant solution for 20 min at 4 ℃ at 12000r/min, removing the supernatant, collecting the precipitate, and diluting the precipitate with a gold-labeled antibody diluent to a constant volume of 1 mL to prepare the tetracycline monoclonal antibody colloidal gold marker;

(4) preparing a colloidal gold film: uniformly spraying the colloidal gold marker of the tetracycline protein conjugate monoclonal antibody in the step (3) on a carrier glass cellulose membrane by using a membrane scratching instrument at the concentration of 5 muL/cm, and naturally airing at room temperature or drying for 3 hours at 37 ℃ to prepare a colloidal gold membrane containing the colloidal gold marker of the tetracycline protein conjugate monoclonal antibody;

(5) preparation of a coating film: diluting a goat anti-mouse IgG antibody and a tetracycline protein conjugate to 1 mg/mL, sequentially spraying the diluted goat anti-mouse IgG antibody and the tetracycline protein conjugate on a nitrocellulose membrane at the concentration of 1 muL/cm by using a membrane scratching instrument to prepare a coated membrane, and naturally airing at room temperature or drying at 37 ℃ after coating for 2 hours at 37 ℃;

(6) sample pad pretreatment: uniformly coating the sample pad treatment solution on a glass cellulose membrane, and naturally airing at room temperature under the condition that the air humidity is lower than 60%;

(7) assembling the detection card: the PVC rubber plate is sequentially adhered with a sample pad, a colloidal gold film, a coating film and absorbent cotton from top to bottom to be assembled into a test strip, the test strip is cut into a strip with a certain width, and then the test strip is arranged in a strip flat shell-shaped detection card shell.

2. The tetracycline colloidal gold assay card according to claim 1, wherein the detection line T coated in step (5) is provided below a quality control line C;

the sample pad treatment solution in step (6) was made up of 1 g of Bovine Serum Albumin (BSA) and 0.8 g of sodium chloride (NaCl) to 100 mL with 0.01 mol/L PBS containing 0.5% TRITON-100.

Technical Field

The invention relates to the technical field of veterinary drug residue detection in livestock, poultry and aquatic products, in particular to an immune colloidal gold detection card for tetracycline and a preparation method thereof.

Background

Tetracycline is the most basic compound in the tetracycline family antibiotics, and the tetracycline and the salts thereof are yellow or light yellow crystals, and are extremely stable in the F dry state, and except aureomycin, the aqueous solutions of other tetracycline families are quite stable. The tetracycline family is soluble in dilute acids, dilute bases, etc., and is slightly soluble in water and lower alcohols, but insoluble in ether and petroleum ether. The tetracycline antibiotics mainly comprise aureomycin, oxytetracycline and tetracycline. Tetracyclines antibiotics are broad-spectrum antibiotics with a phenanthrene mother nucleus discovered in 40 th of the 20 th century, and are widely applied to infections caused by gram-positive and gram-negative bacteria, intracellular mycoplasma, chlamydia and rickettsia. In addition, in some countries, including the united states, tetracycline is also used in large quantities as a growth promoter to be fed to animals, and thus tetracycline residues in animal foods pose a significant threat to human health.

At present, the determination method of tetracycline and metabolites thereof in animal derived foods mainly comprises a physicochemical detection method, for example, the chemical analysis method is used for determining the tetracycline residue in shrimp meat, the detection limit is 0.0125ug/kg, and the high performance liquid chromatography method is used for determining the tetracycline residue in fish meat, the detection limit is 0.0125 ug/kg. However, these methods have expensive instruments and equipment, complicated operation and low sensitivity, are not suitable for detecting large-scale samples, and cannot meet the urgent needs of the domestic food safety detection market.

The immunoassay technology shows wide application prospect in residue analysis of the agricultural and veterinary drugs by the advantages of high specificity of antigen-antibody reaction, simple and rapid determination method, high sensitivity, low cost, suitability for field large-batch sample screening and the like. With the continuous perfection and commercialization of the pesticide residue immunological detection technology, the immunological detection technology can become an effective and rapid detection means for food pesticide and veterinary drug residue and food safety and quality control. Based on the current research situation of veterinary drug residue diagnostic reagents, it is urgent to develop a rapid and sensitive detection reagent better than the current one. Although there are many methods for analyzing veterinary drug residues, the analysis by immunological techniques is the method with the best specificity and the shortest detection time.

Disclosure of Invention

In view of the above situation, the present invention aims to overcome the defects of the prior art and provide an immune colloidal gold detection card for tetracycline and a preparation method thereof, which can effectively solve the problem of rapid and simple tetracycline detection.

The tetracycline colloidal gold detection card comprises a colloidal gold combination pad coated with a monoclonal antibody colloidal gold marker, a nitrocellulose membrane coated with tetracycline-BSA and goat anti-mouse IgG, a sample pad, a water absorption pad, a PVC rubber plate and a plastic mould, wherein the sample pad and the combination pad are sequentially adhered to one end of the PVC rubber plate, the nitrocellulose membrane is adhered to the middle of the PVC rubber plate, and the water absorption pad is adhered to the other end of the PVC rubber plate.

The preparation method of the tetracycline colloidal gold detection card is realized by the following steps:

(1) preparing a colloidal gold solution: 1L Erlenmeyer flask is taken, 495 mL of ultrapure water is added, and then 1% chloroauric acid (HAuCl) is added₄·3H₂O)5 mL, 500 mL of 0.01% aqueous chloroauric acid solution was prepared, heated to boiling, and 1% trisodium citrate (Na) was added with constant stirring₃C₆H₅O₇·2H₂O) 5-7 mL of solution, continuously stirring and heating, maintaining for 5 min when the color of the solution is completely changed into transparent mauve, stopping heating, replenishing water to the original volume, cooling to room temperature, and storing at 2-8 ℃ for later use;

(2) pretreatment of the antibody: centrifuging the tetracycline antibody to be labeled for 20 min at the temperature of 4 ℃ at the speed of 1000 r/min, taking the supernatant, and diluting the supernatant into 1 mg/mL by using 0.01 mol/L PBS; or diluting to 1 mg/ml with 0.01 mol/L PBS, and filtering with 0.22 mu m filter membrane;

(3) preparation of colloidal gold marker: taking 40 mL of the colloidal gold solution obtained in the step (1), adjusting the pH value of the colloidal gold solution to 8.5 by using 0.25 mol/L K2CO3, stirring by using a magnetic stirrer at 250 r/min, dropwise adding 4 mL of protein solution containing 0.3 mg of antibody protein, and reacting for 10 min; dropwise adding 4 mL of 10% BSA, and continuously stirring for reaction for 10 min; centrifuging the gold-labeled antibody solution at normal temperature and low speed (1500 r/min) for 10 min, and discarding the precipitate formed by the condensed gold particles; centrifuging the red supernatant solution for 20 min at 4 ℃ at 12000r/min, removing the supernatant, collecting the precipitate, and diluting the precipitate with a gold-labeled antibody diluent to a constant volume of 1 mL to prepare the tetracycline monoclonal antibody colloidal gold marker;

(4) preparing a colloidal gold film: uniformly spraying the colloidal gold marker of the tetracycline protein conjugate monoclonal antibody in the step (3) on a carrier glass cellulose membrane by using a membrane scratching instrument at the concentration of 5 muL/cm, and naturally airing at room temperature or drying for 3 hours at 37 ℃ to prepare a colloidal gold membrane containing the colloidal gold marker of the tetracycline protein conjugate monoclonal antibody;

(5) preparation of a coating film: diluting a goat anti-mouse IgG antibody and a tetracycline protein conjugate to 1 mg/mL, sequentially spraying the diluted goat anti-mouse IgG antibody and the tetracycline protein conjugate on a nitrocellulose membrane at the concentration of 1 muL/cm by using a membrane scratching instrument to prepare a coated membrane, and naturally airing at room temperature or drying at 37 ℃ after coating for 2 hours at 37 ℃;

(6) sample pad pretreatment: uniformly coating the sample pad treatment solution on a glass cellulose membrane, and naturally airing at room temperature under the condition that the air humidity is lower than 60%;

(7) assembling the detection card: the PVC rubber plate is sequentially adhered with a sample pad, a colloidal gold film, a coating film and absorbent cotton from top to bottom to be assembled into a test strip, the test strip is cut into a strip with a certain width, and then the test strip is arranged in a strip flat shell-shaped detection card shell.

The detection line T coated in the step (5) is arranged below the quality control line C;

the sample pad treatment solution in the step (6) is prepared by adding 1 g of calf serum albumin (BSA) and 0.8 g of sodium chloride (NaCl) into 0.01 mol/L PBS containing 0.5% TRITON-100 to make a volume of 100 mL;

the method can be effectively used for measuring the tetracycline, and is simple, convenient, quick and accurate in result.

Drawings

FIG. 1 is a structural diagram of an immune colloidal gold test card for tetracycline of the present invention: in the figure, 1, a sample adding hole 2, a detection line 3, a quality control line 4, a detection hole 5, a test strip 6 and a detection card shell.

FIG. 2 is a sectional view of the test strip in the immuno-colloidal gold test card for tetracycline of the present invention, in which 7 is a sample pad 8, a colloidal gold conjugate pad 9, a PVC plate 10, a nitrocellulose membrane 11, and a water-absorbing pad.

Detailed Description