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Tannic acid immune network and clenbuterol hydrochloride test strip detection method

A technology of clenbuterol hydrochloride and immune network, applied in the field of a method of terol detection, to achieve the effect of reduced antibody consumption, low consumption, and rapid analysis process

Pending Publication Date: 2022-03-01
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Since the test strip detection of clenbuterol hydrochloride is a competitive immunological assay, the detection sensitivity of the detection method itself is mainly determined by two factors: the signal output intensity and the concentration of the antibody used, and most studies at this stage only focus on There are few studies on the strength of signal output, and there are few studies on combining nanomaterials instead of colloidal gold to build an immune network to reduce the concentration of antibodies used and improve sensitivity

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preparation example Construction

[0027] Preparation of novel immunoprobes: see figure 1 , Add specific anti-clenbuterol hydrochloride monoclonal antibody (AbCLE) to the pre-prepared immune network in proportion, couple at room temperature for 40 minutes, centrifuge to remove excess BSA, resuspend and place at 4°C for use.

[0028] The monoclonal antibody against clenbuterol hydrochloride used in the present invention is obtained by the applicant's research group by using cell fusion technology to screen positive cells and induce a large amount of production in the peritoneal cavity of mice. Prepared with reference to the method described in the article "Production of ultrasensitivegeneric monoclonal antibodies against major aflatoxins using a modified two-step screening procedure" on pages 63-69 of volume 635 of the publication "Analytical Chimica Acta" by Zhang Daohong et al.: Clenbuterol hydrochloride artificial antigen, namely the commercially available clenbuterol hydrochloride CLE-bovine serum albumin co...

Embodiment 1

[0032] Preparation method of anti-clenbuterol monoclonal antibody

[0033] The preparation method of anti-clenbuterol hydrochloride monoclonal antibody comprises the following steps:

[0034] 1. Ascites collection:

[0035] Clenbuterol hydrochloride cells were cultured in advance, and when the cells grew to a good state, the cell line was injected into BALB / c mice that had been treated with liquid paraffin in advance, and the ascites of the mice was collected when the abdomen of the mice was obviously swollen.

[0036] 2. Antibody purification:

[0037] The antibody was purified by the octanoic acid-ammonium sulfate method. The specific operation was as follows: the ascites was filtered with double-layer filter paper, centrifuged at 12000 r / min for 15 min at 4 °C, and the supernatant was absorbed. The ascites supernatant obtained was mixed with 4 times the volume of acetate buffer, and n-octanoic acid 33 μL / mL ascites was slowly added under stirring, mixed at room temperatur...

Embodiment 2

[0043] 1. Polytannic acid nanospheres (PTAN) were prepared by formaldehyde-assisted cross-linking and hydrothermal reaction.

[0044] The specific method is: take 46 mL of distilled water, 8 mL of ethanol and 0.45 mL of ammonia water in a beaker, and after 60 min of magnetic stirring, slowly add 0.2 g of tannic acid powder, and observe that the color of the solution changes from colorless to light Yellow color, continue to add 0.38 mL formaldehyde under stirring and continue stirring for 24 h, then move to the reaction kettle at 100°C for 24 h. After being centrifuged and washed with water, the polytannic acid solution was obtained, and the product was stored in a refrigerator at 4 °C for later use. The particle size is about 131 nm.

[0045] 2. The preparation method of the immune network probe is as follows:

[0046] Take 6 mg / mL PTAN solution, add 20 μL 1 mg / mL goat anti-mouse immunoglobulin (GAMI) solution, incubate at room temperature for 40 min; add 10% bovine serum al...

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Abstract

The invention discloses a test strip for detecting clenbuterol hydrochloride through an immune network supported by tannic acid derived from biological resources and application of the test strip. A sensitive, effective and simple immunochromatography method is constructed by using a tannic acid nanosphere as a signal carrier for marking goat anti-mouse immunoglobulin, coupling a specific monoclonal antibody and using an immunochromatography test strip as a detection platform; meanwhile, the coupling of the signal carrier and the immune globulin is realized on the premise of not using any additional chemical reagent. Benefited from the effective protein enrichment capacity of the tannic acid, the constructed immune network realizes signal amplification of immunodetection, and further saves the consumption of specific monoclonal antibodies, so that the sensitivity (taking a test strip detection cutoff value as an example) is improved by 10 times, and the detection sensitivity is improved by 10 times. The application range of tannic acid derived from renewable biological resources is widened, and the constructed immune network provides a new path for development of a simple, high-repeatability and stable immunochromatography system.

Description

technical field [0001] The invention belongs to the field of biological detection, and specifically relates to a method for preparing a tannic acid immune network derived from biological resources and a method for detecting clenbuterol hydrochloride in meat products by using immune chromatography test strips as a detection platform, in particular to a A kind of tannin nanosphere constructs the immune network and prepares the probe and test strip for labeling specific monoclonal antibody, and its application for rapid detection of clenbuterol hydrochloride in meat products. . Background technique [0002] Clenbuterol (CLE), as a synthetic β2-adrenergic agonist, has been used not only to control bronchial asthma and lung diseases, but also as a growth-promoting drug in the past few decades. Considering the muscle tremors, acute poisoning, palpitations and other symptoms caused by excessive CLE intake, as well as the research status of CLE that can accumulate in animals for a ...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/558G01N33/545G01N33/543
CPCG01N33/94G01N33/577G01N33/54346G01N33/545G01N33/558Y02A50/30
Inventor 王建龙刘思杰张道宏舒蕊
Owner NORTHWEST A & F UNIV
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