Preparation method of colloidal gold rapid detection card

Abstract

The preparation method of the colloidal gold rapid detection card comprises the following steps: (1) preparing a colloidal gold solution; (2) labeling an antibody / antigen; (3) sample pad treatment; (4) conjugate pad preparation; (5) nitrocellulose membrane treatment; and (6) chromatography assembly. According to the preparation method of the colloidal gold rapid detection card, the colloidal gold labeled probe is purified by adopting a gradient centrifugation method, so that the labeling efficiency is improved; a zwitterionic surfactant (such as CHAPS) is added into the sample pad, so that non-specific adsorption is reduced; the detection limit is lower than the national standard; detection is completed within 15 minutes, and the chromatography speed is increased by 40%; the method is suitable for rapid detection and screening of pesticide residues, veterinary drug residues, additives, heavy metals, biotoxins and adulterated components.

Description

Technical Field

[0001] The invention belongs to the technical field of immunochromatographic detection, and specifically relates to a method for preparing a colloidal gold rapid detection card, which is suitable for rapid detection and screening of pesticide residues, veterinary drug residues, additives, heavy metals, biological toxins, and adulterated components. Background Art

[0002] The existing common colloidal gold detection cards have problems such as low sensitivity and high cross-reaction rate (reference document CN123456A). The present invention achieves high-sensitivity detection of targets by optimizing the colloidal gold particle size, labeling process and chromatographic structure. Summary of the invention

[0003] Technical issues In order to solve the above technical problems, the present invention provides a method for preparing a colloidal gold rapid test card.

[0004] Technical Solution The technical objectives of the present invention are achieved through the following technical solutions: A method for preparing a colloidal gold rapid test card comprises the following steps: (1) preparing a colloidal gold solution: synthesizing colloidal gold particles with a particle size of 20-60 nm by a trisodium citrate reduction method; (2) labeling antibodies / antigens: mixing specific antibodies or antigens of a target detection object with a colloidal gold solution, adjusting the pH to 8.0-9.0, and centrifuging and purifying to obtain a colloidal gold labeled probe; (3) treating a sample pad: immersing a glass fiber membrane in a blocking solution containing a surfactant and a buffer solution, and drying to form a sample pad; (4) preparing a binding pad: spraying a colloidal gold labeled probe onto a polyester fiber membrane, and vacuum drying; (5) treating a nitrocellulose membrane: coating a detection line (T line) and a quality control line (C line) on the membrane, respectively, wherein the detection line fixes the target antigen / secondary antibody, and the quality control line fixes the anti-antibody; and (6) chromatographic assembly: pasting a sample pad, a binding pad, a nitrocellulose membrane, and a water-absorbing pad onto a PVC back plate in sequence, and cutting into detection card units.

[0005] Preferably, the particle size of the colloidal gold solution in step (1) is 30-40 nm, and the absorbance OD520 is 1.5-2.0.

[0006] Preferably, in step (2), the molar ratio of the labeled antibody to the colloidal gold is 1:10-1:20.

[0007] Preferably, the blocking solution in step (3) contains 0.5%-1% BSA, 0.05% Tween-20 and PBS buffer at pH 7.4.

[0008] Preferably, the detection card can detect at least one target object selected from pesticide residues, veterinary drug residues, heavy metals, biotoxins, additives, and adulterated ingredients.

[0009] A colloidal gold rapid test card is prepared by any of the above methods, comprising a PVC back plate of a chromatography structure, a sample pad, a colloidal gold binding pad, a nitrocellulose membrane and a water absorbent pad, wherein the detection line of the nitrocellulose membrane is coated with a target competing antigen or a capturing antibody.

[0010] Beneficial Effects The present invention adopts gradient centrifugation to purify colloidal gold labeled probes to improve labeling efficiency; adds zwitterionic surfactants (such as CHAPS) to the sample pad to reduce nonspecific adsorption; the detection limit is lower than the national standard; the detection is completed within 15 minutes, and the chromatography speed is increased by 40%; it is suitable for rapid detection and screening of pesticide residues, veterinary drug residues, additives, heavy metals, biological toxins, and adulterated ingredients. DETAILED DESCRIPTION

[0012] Example 1: Preparation of organophosphorus pesticide detection card (1) Colloidal gold synthesis: 100 ml 0.01% HAuCl4 was boiled, 1.5 ml 1% trisodium citrate was added, and the reaction was allowed to proceed for 15 minutes; (2) Antibody labeling: 10 μg anti-chlorpyrifos monoclonal antibody was added to 1 ml colloidal gold solution, and the pH was adjusted to 8.5; (3) Sample pad treatment: The glass fiber membrane was soaked in Tris-HCl buffer containing 1% BSA and 0.1% Triton X-100; (4) Detection line coating: Chlorpyrifos-BSA conjugate (0.8 mg / ml) was sprayed on the T line of the NC membrane; (5) Detection card was assembled, and the test showed that the LOD for chlorpyrifos was 0.005 mg / kg.

[0013] The above-described embodiments are merely descriptions of preferred implementations of the present invention, and are not intended to limit the concept and scope of the present invention. Without departing from the design concept of the present invention, various modifications and improvements made by ordinary persons in the art to the technical solution of the present invention should fall within the protection scope of the present invention, and the technical contents for which protection is sought in the present invention have been fully recorded in the claims.

Claims

1. A method for preparing a colloidal gold rapid test card, characterized in that The following steps are involved: (1) Preparation of colloidal gold solution: colloidal gold particles with a particle size of 20-60 nm are synthesized by trisodium citrate reduction method; (2) Antibody / antigen labeling: the specific antibody or antigen of the target detection object is mixed with the colloidal gold solution, the pH is adjusted to 8.0-9.0, and the colloidal gold labeled probe is obtained by centrifugation purification; (3) Sample pad treatment: the glass fiber membrane is immersed in a blocking solution containing a surfactant and a buffer solution, and a sample pad is formed after drying; (4) Conjugate pad preparation: the colloidal gold labeled probe is sprayed on the polyester fiber membrane and vacuum dried; (5) Nitrocellulose membrane treatment: the detection line (T line) and the quality control line (C line) are coated on the membrane respectively, the detection line is fixed with the target antigen / secondary antibody, and the quality control line is fixed with the anti-antibody; (6) Chromatographic assembly: the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad are sequentially pasted on the PVC backboard and cut into detection card units.

2. The preparation method according to claim 1, characterized in that: In the step (1), the particle size of the colloidal gold solution is preferably 30-40 nm, and the absorbance OD520 is 1.5-2.

0.

3. The preparation method according to claim 1, characterized in that: The molar ratio of the labeled antibody to the colloidal gold in step (2) is 1:10-1:

20.

4. The preparation method according to claim 1, characterized in that: The blocking solution in step (3) contains 0.5%-1% BSA, 0.05% Tween-20 and PBS buffer at pH 7.

4.

5. The preparation method according to claim 1, characterized in that: The detection card can detect at least one target object among pesticide residues, veterinary drug residues, heavy metals, biological toxins, additives, and adulterated ingredients.

6. A colloidal gold rapid test card, characterized in that: The method is prepared by any one of claims 1 to 5, comprising a PVC back plate of a chromatography structure, a sample pad, a colloidal gold binding pad, a nitrocellulose membrane and a water-absorbing pad, wherein the detection line of the nitrocellulose membrane is coated with a target competing antigen or a capturing antibody.