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Magnetic particle-based hydrocortisone chemiluminescence immune assay method and kit

A chemiluminescence immunoassay, hydrocortisone technology, applied in chemical instruments and methods, chemiluminescence/bioluminescence, analysis by chemical reaction of materials, etc. Affect the accuracy and accuracy of experimental results, and achieve the effects of accurate detection results, stable substrates and high sensitivity

Inactive Publication Date: 2018-07-06
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional hydrocortisone immunoassay method mainly uses enzyme-linked immunosorbent assay, which is in an open space during the detection process, which is prone to cross-contamination, and the experiment is mainly performed manually, which makes the sample volume inaccurate and prone to operational errors Immediately affect the precision and accuracy of the experimental results

Method used

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  • Magnetic particle-based hydrocortisone chemiluminescence immune assay method and kit
  • Magnetic particle-based hydrocortisone chemiluminescence immune assay method and kit
  • Magnetic particle-based hydrocortisone chemiluminescence immune assay method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of magnetic particle chemiluminescence immunoassay method

[0055] 1. Preparation of rabbit-derived polyclonal antibody against hydrocortisone

[0056] (1) Antigen preparation: First, take 600mg hydrocortisone in 5mL N,N-dimethylformamide (DMF), add 44.2mg 4-dimethylaminopyridine (DMAP) and 163.8mg succinic anhydride and stir React for 24 hours, spin dry, dissolve the colloidal substance in chloroform, add 20mL distilled water and stir to obtain a large amount of light yellow crystals, carry out suction filtration, rinse the filter residue with excess distilled water and dry it, place the filter residue in 100mL saturated sodium bicarbonate solution, mix After uniformity, centrifuge to take the supernatant, acidify the supernatant with 1mol / L hydrochloric acid to pH 4, filter the filter residue obtained by suction, which is the hapten, and purify it through the column; take 10mg of the hapten and dissolve it in 500μL of anhydrous DMF, add 5.3mg N...

Embodiment 2

[0070] The establishment of embodiment 2 standard curve

[0071] 1. According to the optimal value determined by the optimization of the above conditions, the luminescence of a series of standard solutions with a solubility of 0.1, 5, 10, 50, 100, 500, 4000 and 40000 ng / mL prepared with 0.01 mol / L PBS solution respectively The value (RLU) is the ordinate, the logarithm of the standard concentration of hydrocortisone is the abscissa, and the standard curve is drawn using Origin9.0 software.

[0072] 2. The detection linear range (IC) of the standard curve based on magnetic particle chemiluminescence immunoassay 20 ~IC 80 ) is 0.42~72.27ng / mL, 50% competitive inhibition (IC 50 ) was 5.57ng / mL, the limit of detection (IC 10 ) is 0.12 ng / mL.

Embodiment 3

[0073] Example 3 Stability Evaluation Based on Magnetic Particle Chemiluminescence Immunoassay Method

[0074] 1. In this study, the stability of the method was evaluated by performing 5 measurements of hydrocortisone solutions with different concentrations of 0.1, 5, 10, 50, 100, 500, 4000 and 40000 ng / mL.

[0075] 2. The experimental results show that the coefficients of variation obtained by measuring 5 times of hydrocortisone solutions with concentrations of 0.1, 5, 10, 50, 100, 500, 4000 and 40000 ng / mL are 3.3%, 3.3%, and 2.1%, respectively. , 5.2%, 2.2%, 7.0%, 7.5% and 5.1%, indicating that the method has good reproducibility.

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Abstract

The invention discloses a fully-automatic magnetic particle-based hydrocortisone chemiluminescence immune assay method and application thereof. Immunomagnetic beads are coated by goat anti rabbit, antigen is detected by a chemiluminescent marker, a specific antibody of hydrocortisone is applied, a competitive response mode is established, a fully-automatic chemiluminescence immune assay serves asa detection tool, and the hydrocortisone is quantitatively detected according to the measured luminous value. According to the established method, the maximal detection range of the hydrocortisone reaches to 0.42 to 72.27 ng / mL, the detection limit is 0.12 ng / mL, the sensitivity can reach to 5.57 ng / mL and the recovery rate is 84.3 to 102.3 percent. According to the method, the detection process can realize full automation, the influence by professional testing personnel is not considered, the whole detection process is rapid and can be completed only by about 40 minutes, the detection limit is low, the sensitivity is high, the linear range is wide, the market requirement is met and good market application prospect is achieved.

Description

technical field [0001] The invention belongs to the technical field of food processing. More specifically, it relates to a magnetic particle hydrocortisone-based chemiluminescence immunoassay method and kit. Background technique [0002] Hydrocortisone, also known as cortisol, is a glucocorticoid produced by the adrenal cortex of the mammalian spine. It can regulate glucose metabolism in the living body and promote the conversion of protein into sugar. It has anti-inflammatory, anti-inflammatory, anti-inflammatory and anti-shock pharmacological properties. , anti-toxin and other effects. Individual farmers will illegally use glucocorticoids during the growth of animals, which will lead to their residues in animal-derived foods. Moreover, the effect of glucocorticoids is extremely strong. If people eat food containing glucocorticoids for a long time, even if the content is small, it will cause disorders of sugar, salt, protein and fat metabolism, insulin resistance and stag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07J7/00C07K14/765C07K14/77C07K16/44G01N21/76G01N33/543
CPCC07J7/008C07K14/765C07K14/77C07K16/44G01N21/76G01N33/54326
Inventor 徐振林傅慧君刘毅新袁利鹏沈玉栋孙远明雷红涛王弘杨金易
Owner SOUTH CHINA AGRI UNIV
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