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Method of detecting small molecule substances based on chemiluminescence immunology of golden-magnetic particles

A technology of chemiluminescent immunity and small molecule substances, which is applied in the direction of measuring devices, scientific instruments, and analytical materials, can solve the problems of antigen or antibody inactivation, complex coupling process, and restrictions on popularization and application, and achieve high precision and detection The effect of high sensitivity and safe operation

Inactive Publication Date: 2012-10-17
XIAN GOLDMAG NANOBIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most traditional magnetic particles are modified by surface functionalization with active groups such as carboxyl (-COOH), hydroxyl (-OH), amino (-NH2), aldehyde (-CHO) and other active groups that interact with antigens or antibodies. The covalent effect of these antigens and antibodies on the surface of magnetic beads is coupled, the coupling process is complicated, and it is easy to cause the inactivation of the antigen or antibody during the coupling, not only the coupling efficiency is low, but also the cost is wasted. For these reasons, based on The popularization and application of chemiluminescence of magnetic particles is limited

Method used

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  • Method of detecting small molecule substances based on chemiluminescence immunology of golden-magnetic particles
  • Method of detecting small molecule substances based on chemiluminescence immunology of golden-magnetic particles
  • Method of detecting small molecule substances based on chemiluminescence immunology of golden-magnetic particles

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Effect test

Embodiment 1

[0061] Embodiment 1: Method for Quantitative Detection of Morphine in Human Blood or Urine by Chemiluminescent Immunology Based on Gold Magnetic Particles

[0062] (1) Coated

[0063] (1.1) Pretreatment: Take 200uL of 5mg / mL gold magnetic particles, wash them twice with 200uL of Tris-HCl coupling buffer solution with pH=7.4 and a concentration of 0.02M to balance the pH of the magnetic particles.

[0064] (1.2) Coupling: dissolve the morphine synthetic antigen in 200uL pH=7.4, concentration 0.02M Tris-HCl coupling buffer, mix well and add to the pretreated gold magnetic particles, at 37°C, 180rpm, shake React in bed for 20min. After the reaction was completed, it was taken out, magnetically separated, and the supernatant was discarded.

[0065] (1.3) Washing: add 400 uL of 0.02M Tris-HCl washing buffer, perform magnetic separation, and discard the supernatant.

[0066] (2) Blocking: add 1 mL of Tris-HCl buffer solution containing 5% skimmed milk powder to the gold magnetic ...

Embodiment 2

[0070] Embodiment 2 Method for Quantitative Detection of Testosterone in Human Serum by Chemiluminescence Immunology Based on Gold Magnetic Particles

[0071] In addition to using testosterone synthetic antigen coated gold magnetic particles, anti-testosterone polyclonal antibody detection, alkaline phosphatase-labeled goat anti-rabbit antibody, the chemiluminescent system uses 1,2-dioxane derivatives such as 3-(2 -spiroadamantane)-4-methoxy-4-(3-phosphooxy)-phenyl-1,2-dioxetane (AMPPD), and the testosterone standard concentration used in the establishment of the standard curve Except, other concrete steps are with embodiment 1.

[0072] in conclusion: Figure 4 It is based on the dose-response curve of the chemiluminescent detection of morphine with gold magnetic particles. It has a good linearity from 5ng / ml to 1000ng / ml, IC50 is 59.8ng / ml, and the lowest detection line reaches 0.46ng / ml.

[0073] Figure 5 The dose-response curve of testosterone detected by chemiluminesc...

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Abstract

The invention provides a method of detecting small molecule substances based on chemiluminescence immunology of golden-magnetic particles. The method mainly comprises the following steps: (1) coating: a step of using a golden-magnetic particle as carriers for immunoreaction and solid separation, using a small molecule substance corresponding to a small molecule substance to be detected to synthesize an antigen and coupling the antigen onto the surface of the golden-magnetic particle; (2) enclosing: a step of enclosing blank sites on the surface of the golden-magnetic particle which have not bound to the antigen by using blocking buffer , carrying out magnetic separation and discarding supernatant; (3) reaction with the substance to be detected: a step of adding a sample to be detected, an antibody against the small molecule substance to be detected and enzyme labeled secondary antibody capable of specifically binding to the antibody into the golden-magnetic particle which has bonded with the antigen synthesized from the small molecule substance so as to form a specific antigen-antibody-enzyme labeled secondary antibody compound; (4) cleaning and (5) detection. The detection method provided in the invention has the advantages of high sensitivity, good specificity, a wide linear range, high precision, good stability, no radioactive pollution, safe operation, simpleness and rapidity.

Description

technical field [0001] The invention relates to a method for chemiluminescent immunological detection of small molecular substances. Background technique [0002] At present, the general immunological detection methods for the detection of small molecular substances such as certain hormones, drugs, and drugs on the market mainly include colloidal gold immunochromatographic test strip detection (Immunochromatographic Assay), enzyme-linked immunosorbent assay (Enzyme-linked Immunosorbent Assay), radioimmunoassay (Radioimmunoassay, RIA) and so on. Radioimmunoassay is the earliest established classical immunoassay method with high detection accuracy and the minimum detection limit can reach 10 -3 ~10 -6 ug / ml, the amount of sample used is small, and no pretreatment is required. However, due to the high investment cost of radioimmunoassay, low cost of consumables, and relatively simple operation, it is difficult to popularize and apply because it is harmful to operators and po...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/551
Inventor 张秦鲁杜海平崔亚丽马乐
Owner XIAN GOLDMAG NANOBIOTECH
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