Direct competitive enzyme immunoassay method based on nano-gold plasma excimer

A plasmon and analysis method technology, which is applied in the field of direct competitive enzyme-linked immunosorbent assay and the detection of small molecule compounds, can solve the problems of high cost, increased detection cost, hindered application, etc., and achieves easy operation and low cost. , the effect of highly sensitive detection

Active Publication Date: 2014-09-17
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Many studies have adopted new detection modes, such as chemiluminescence and electrochemiluminescence, which have more sensitive detection capabilities compared with traditional enzyme-linked immunoassay methods, but these

Method used

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  • Direct competitive enzyme immunoassay method based on nano-gold plasma excimer
  • Direct competitive enzyme immunoassay method based on nano-gold plasma excimer
  • Direct competitive enzyme immunoassay method based on nano-gold plasma excimer

Examples

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Embodiment 1

[0063] Example 1 The present embodiment relates to the detection method of methyltestosterone, comprises the steps:

[0064] (1) Preparation of methyltestosterone derivatives – CAT enzyme-labeled antigen

[0065] Oximate methyltestosterone by carboxymethylhydroxylamine hemihydrochloride to obtain derivative 3-carboxymethyloxime-methyltestosterone (3-CMO-MT), and then use carbodiimide method to convert 3-CMO-MT Conjugated with CAT enzyme. details as follows:

[0066] Take 2.5 mg of 3-CMO-MT, 1.5 mg of NHS, and 2.5 mg of EDC·HCl, add it to 1 mL of DMF, and shake at room temperature for 2 h in the dark. Another 27 mg of catalase was dissolved in 1.5 mL of PBS containing 20% ​​DMF, and pre-cooled at 4°C. The 3-CMO-MT activation solution was added dropwise to the CAT solution, stirred continuously, and reacted at 4°C for 2 h; after the reaction, the reaction solution was first dialyzed with 20% DMF in PBS, and finally dialyzed with 0.01M PBS for 2 days. Freeze at -20°C.

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Embodiment 2

[0076] Example 2 The present embodiment relates to the detection method of clenbuterol, comprising the following steps:

[0077] (1) Preparation of clenbuterol-CAT enzyme-labeled antigen

[0078] Conjugate clenbuterol hydrochloride (CL) to CAT enzyme using the diazonium method as follows:

[0079] Dissolve 2.5 mg of clenbuterol hydrochloride in 0.1 mol / L HCl solution, place in a refrigerator at 4°C and stir. After 20 min, add 0.2 mol / L NaNO three times continuously into the beaker 2 Solution, 20uL each time, with an interval of 25s. Stir at low speed for about 1 hour at 4°C until the solution is colorless. Whether sodium nitrite is excessive can be tested with starch potassium iodide test paper. Weigh 39.4mg of CAT and dissolve in 20mM borate buffer. Under the condition of ice bath, add the activated CL dropwise to the CAT solution. During the dropwise addition, keep stirring and adjust the pH value with 1mol / L NaOH to maintain it between 8.0 and 8.5. After the dropwis...

Embodiment 3

[0089] Example 3 The present embodiment relates to the detection method of gentamicin, comprises the steps:

[0090] (1) Preparation of gentamicin-CAT enzyme-labeled antigen

[0091] Gentamicin was coupled to CAT enzyme using the glutaraldehyde method. details as follows:

[0092] Dissolve 3 mg of gentamicin in 0.5 mL of 0.01M PBS, add 0.1 mL of 2.0% glutaraldehyde to the above solution, and activate the reaction for 30 minutes. After 38.7 mg of CAT was dissolved in 1.2 mL of 0.01M PBS, the activated solution was added dropwise to the CAT solution, and the reaction was stirred overnight. Finally dialyze against 0.01M PBS for 2 days. After the dialysis is completed, centrifuge at 4°C and 5000rpm for 10 minutes to remove the precipitate, and store the supernatant at -20°C for later use.

[0093] (2) Preparation of coated plates

[0094] Use 0.05M carbonate buffer solution with pH 9.6 as the coating diluent to dilute the gentamicin antibody, the antibody concentration is a...

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Abstract

The invention discloses a direct competitive enzyme immunoassay method based on a nano-gold plasma excimer, and immunoassay visible detection on small-molecule compounds can be realized. The direct competitive enzyme immunoassay method comprises the steps of competitively combining a test sample possibly containing the small-molecule compounds and haptens labeled with catalase with small-molecule compound antibodies covering a solid phase, decomposing hydrogen peroxide through the combined enzyme-labeled antigens, reducing chloroauric acid into nano-gold through the hydrogen peroxide under a certain buffering condition, and detecting the small-molecule compounds in the sample by observing the color or the light absorption intensity of a developing liquid. The method disclosed by the invention has the advantages of high sensitivity, simplicity in operation, low cost, visible result distinguishing and the like, and the shortcomings of the conventional small-molecule compound enzyme-linked immunoassay detection technology are overcome.

Description

technical field [0001] The invention relates to a detection method of small molecular compounds, in particular to a direct competition enzyme-linked immunoassay method based on nano-gold plasmons, belonging to the field of immunological detection and analysis. Background technique [0002] Enzyme-linked immunoassay technology is a very mature solid-phase immunoassay mode, which has been applied in clinical diagnosis and analysis for many years. Wide range of applications. In the detection of small molecule compounds, ELISA mainly adopts two modes, indirect competition method and direct competition method. In the two detection modes, enzyme-labeled secondary antibodies and enzyme-labeled haptens are required respectively, which serve as signal amplification for immunosorbent assays. In environmental monitoring and food hazard detection, the most commonly used enzyme molecule in enzyme-linked immunoassay is horseradish peroxidase (HRP) enzyme, and its most commonly used subs...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N21/31
CPCG01N33/54346G01N33/54393
Inventor 彭池方段小慧泮秋立刘春丽
Owner JIANGNAN UNIV
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