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One-step method enzyme-linked immunosorbent assay method and kit for detection of beta-excitants

An enzyme-linked immunoassay and stimulant technology, which is applied in the field of immunoassay, can solve problems such as troublesome use, and achieve the effect of reducing operating procedures, high sensitivity, and reducing operating steps

Active Publication Date: 2014-02-19
浙江迪恩生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Solution A is generally composed of PBS buffer, citric acid, ED-TA ethylenediaminetetraacetic acid, ProcLin-300, hydrogen peroxide, and solution B is generally composed of PBS buffer, citric acid, ED-TA ethylenediaminetetraacetic acid, ProcLin -300, composed of sodium thiosulfate; the two liquids need to be mixed in proportion before use, and both liquids need to be kept away from light, which is troublesome to use

Method used

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  • One-step method enzyme-linked immunosorbent assay method and kit for detection of beta-excitants
  • One-step method enzyme-linked immunosorbent assay method and kit for detection of beta-excitants
  • One-step method enzyme-linked immunosorbent assay method and kit for detection of beta-excitants

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The preparation of embodiment 1β-agonist specific antibody

[0030] Clenbuterol, salbutamol, terbutaline, mabuterol, bromobuterol, tobuterol and sibuterol each take 0.1mol, dissolved in 50ml of PBS (0.01mol / L) solution, fully After dissolving, 250 μL of 25% glutaraldehyde was added dropwise and reacted for 30 minutes. Separately weigh polylysine (or bovine serum albumin, or chicken gamma globulin, or ovalbumin), dissolve it in 13mL PBS (0.01mol / L), add the above reaction solution dropwise, and stir at room temperature for 3h .

[0031] Take a 50cm dialysis bag, boil it in boiling water for 5 minutes, rinse it with deionized water at 60°C for 3 minutes, and store it in deionized water at 4°C for later use.

[0032] Transfer the artificial antigen mixture into a dialysis bag, and dialyze with 2x2L of 0.01M pH7.4 PBS buffer and 2x2L of deionized water for 3 days. Finally, the liquid in the dialysis bag was made into powder by freeze-drying method to obtain the specific ...

Embodiment 2

[0036] The determination of embodiment 2β-agonist antiserum potency

[0037] Measured by direct ELISA method. The enzyme-labeled reaction plate was coated with β-agonist specific antibody, washed, blocked, and the negative serum of the same dilution was used as a control, and the doubling dilution of β-agonist peroxidase conjugate was sequentially added to wells 1 to 12 in the first row , both 100uL / well, incubate at 22-23°C for 10 minutes; add horseradish peroxidase (ARP) substrate 100uL / well, incubate for 10 minutes, and measure OD after terminating the reaction 450 value. Antibody OD 450 The value is the same as the negative serum OD of the same dilution 450 The titer of the beta-agonist-specific antibody was defined as the maximum dilution for which the ratio of values ​​was greater than 2. The titer of the β-agonist-specific antibody was determined to be 1000.

Embodiment 3

[0038] Example 3 Checkerboard Titration Determines Antibody Coating Concentration and Enzyme-labeled Antigen Working Concentration

[0039] IC by checkerboard titration 50 Value, maximum absorbance value and sample addition recovery rate comparison, determine the coating concentration of the antibody and the working concentration of the enzyme-labeled antigen.

[0040] Table 1 Maximum absorbance value (OD value) comparison

[0041]

[0042] Note: The ones with "-" indicate that the OD value exceeds the reading range of the instrument (>4.0).

[0043] Table 2IC 50 (ng / mL) vs.

[0044]

[0045] Table 30.5ng / mL sample addition recovery (%) comparison

[0046]

[0047] It can be seen from Table 1-3 that as the concentration of the coated β-agonist-specific antibody decreases, the maximum absorbance value decreases, and the IC 50 The value also decreases within a certain range. The coating antibody was determined to be 4.0×10 3 Dilution times, its IC 50Concentratio...

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Abstract

The invention discloses a one-step method enzyme-linked immunosorbent assay method and kit for detection of beta-excitants and belongs to the technical field of immunodetection. The kit comprises beta-excitant specific antibodies, an ELISA plate coated with beta-excitant specific antibodies, beta-excitant horse radish peroxidase markers, clenbuterol standard substances, an enzyme-labeled substance diluent, a developer, a cleaning solution, a stopping solution and a concentrated sample diluents. The kit has advantages of high sensitivity, strong specificity, high precision, high accuracy, low requirements to instruments and devices, long reagent storage life, high automaticity, no radioactive isotope pollution and the like, and can play an important role in detection of animal-origin food and feed-products.

Description

technical field [0001] The invention belongs to the technical field of immunoassay, and in particular relates to a one-step enzyme-linked immunoassay method for detecting beta-stimulants and a kit thereof. Background technique [0002] Beta stimulants are a type of human and veterinary drug used as a treatment for asthma. Overdosage in livestock can convert adipose tissue into muscle tissue, and beta stimulants are often abused in livestock production because they can significantly improve fat percentage and production efficiency. In recent years, cases of poisoning by albuterol, tobuterol, bromobuterol, clenbuterol, sibuterol, mabuterol, and terbutaline have been reported. [0003] Among the β-agonists, albuterol, tobuterol, bromobuterol, clenbuterol, sibuterol, mabuterol, and terbutaline are more toxic, and the detection of these substances is generally synthesized by albuterol and a carrier Antigen or hapten, immunize animals, and obtain polyclonal antibodies, which can...

Claims

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Application Information

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IPC IPC(8): G01N33/544G01N21/31
CPCG01N33/535G01N33/544G01N33/94
Inventor 徐华莉李亮张明洲王旻子魏建良程晔
Owner 浙江迪恩生物科技股份有限公司
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