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Bovine cell factor electrochemical label-free impedimetric immunodetection method

An immunodetection method and cytokine technology, applied in the field of electrochemical immunoassay and immunodetection of bovine cytokines, can solve the problems of unsatisfactory sensitivity, sensitivity to cytokines, and easy interference, so as to reduce non-specific adsorption, The effect of maintaining biological activity and broadening the linear range

Inactive Publication Date: 2013-12-25
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, biological analysis is the commonly used detection method for bovine IFN-γ and IL-4. These biological analysis methods are low in sensitivity, laborious, time-consuming, and difficult to standardize.
In addition, they are sensitive to buffers and other cytokines in the test sample and are susceptible to interference
Although enzyme-linked immunosorbent assays have also been established to detect bovine IFN-γ and IL-4, these methods are also cumbersome, laborious, time-consuming, and the sensitivity is not satisfactory
Due to the small molecular weight of bovine IEN-γ and IL-4 and the extremely small amount in body fluids, the sensitivity of the current method for determining bovine IEN-γ and IL-4 is still far from clinical practice.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1. Make a linear relationship diagram between the logarithmic value of bovine IFN-γ concentration and the relative change value before and after impedance:

[0030] (1) Prepare reduced graphene nanomaterials by chemical dispersion method: mix 0.1 g of graphite oxide with 100 mL of distilled water, and oscillate ultrasonically for 30 min. Afterwards, the graphite oxide dispersed by shaking was added into the three-necked flask, and 1 g of sodium borohydride was added, and heated to reflux at 100° C. for 8 hours. Static filtration, natural drying and collection of the generated is the reduced graphene.

[0031] (2) Prepare the mixed solution of reduced graphene / chitosan / bovine IFN-γ antibody: ultrasonically disperse 2 mg of reduced graphene in 1 mL of chitosan-acetic acid solution with a mass percentage of 1% chitosan to form reduced graphite ene / chitosan complexes.

[0032] Then add bovine IFN-γ antibody 400 μg / mL to the above complex, stir and mix evenly to form a mix...

Embodiment 2

[0044] 1. Make a linear relationship diagram between the logarithmic value of bovine IL-4 concentration and the relative change value before and after impedance:

[0045] (1) Synthesis of rod-shaped titanium dioxide: In an ice-water bath, slowly add 1.8 mL of titanium tetrachloride into a 50 mL beaker containing 19 mL of distilled water, and stir vigorously for 10 min to obtain a white suspension. Add 15 mL of chloroform solvent to the suspension, stir it magnetically for 10 min, then transfer it to a polytetrafluoroethylene reactor, place it in an oven at 160 °C, and conduct a hydrothermal reaction for 12 h. The obtained product was centrifuged and washed with absolute ethanol until neutral, dried in a vacuum oven at 60°C for 24 h, and ground at room temperature to obtain the product.

[0046] (2) Preparation of titanium dioxide / chitosan / bovine IL-4 antibody mixture: 2 mg titanium dioxide nanorods were ultrasonically dispersed in 1 mL of 1 % chitosan acetic acid solution, the...

Embodiment 3

[0058] 1. Make a linear relationship diagram between the logarithmic value of bovine IL-4 concentration and the relative change value before and after impedance:

[0059] (1) Synthesis of mesoporous silica nanoparticles: Weigh 10 g of cetyltrimethylammonium bromide and dissolve in 480 mL of distilled water, and add sodium hydroxide solution (2.00 M, 3.50 mL) to keep the solution temperature at 80°C. 5 mL of triethoxysilane was added dropwise to the solution and stirred at 80 °C for 2 h, and the resulting white precipitate was separated by filtration. The separated silica particles were calcined in a 550 °C horse-boiling furnace for 5 h to obtain the final product.

[0060] (2) Preparation of silica / chitosan / bovine IFN-γ antibody mixture: ultrasonically disperse 2 mg of mesoporous silica in 1 mL of 1 % chitosan-acetic acid solution, and then add bovine IFN-γ Antibody, stir and mix evenly to form a mixture of silica / chitosan / bovine IFN-γ antibody.

[0061] (3) Clean the glass...

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Abstract

The invention relates to a bovine cell factor electrochemical label-free impedimetric immunodetection method belonging to the technical field of electrochemical immunoassay. The method comprises the following steps: ultrasonically dispersing a nano material in a chitosan solution, thus preparing a nano material / chitosan compound; modifying a bovine cell factor antibody fixed on a glassy carbon electrode with the nano compound, thus obtaining a novel bovine cell factor immunosensor; and then, using the immunosensor for bovine cell factor electrochemical label-free impedimetric immunodetection. The detection method does not need labels, is simple, quick, low in cost, high in sensitivity and favorable in reproducibility and stability, and can be used for early diagnosis of bovine tuberculosis and research on bovine cell immune mechanism.

Description

technical field [0001] The invention belongs to the technical field of electrochemical immunoanalysis, in particular to the technical field of immunological detection of bovine cytokines. Background technique [0002] Tuberculosis is one of the most serious infectious diseases affecting the health and milk quality of dairy cows. γ-interferon (IFN-γ) is a type of glycoprotein secreted by T cells under the stimulation of a specific inducer, which has biological activities such as anti-virus, anti-tumor and immune regulation functions. It is the earliest discovery and the most concerned cytokines. Interleukin-4 (IL-4), discovered in the 1980s, is a pleiotropic T cell activating factor secreted by Th2 cells and an important regulator of the humoral immune response of the organism. In the early stage of tuberculosis infection, the secretion levels of IFN-γ and IL-4 in the bovine body were significantly increased. Bovine immune memory cells during latent infection also prod...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N27/26G01N27/327
Inventor 杨占军赵洁陈祥
Owner YANGZHOU UNIV
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