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Goldmag particle-based acridinium ester chemiluminescence immunological detection method of HE4

A technology of acridinium ester chemistry and gold magnetic particles, which is applied in the field of detection of human epididymis secretory protein 4, can solve the problems affecting the separation effect, and achieve the effect of avoiding cross-reaction, good reaction specificity and good specificity

Active Publication Date: 2015-09-09
XIAN GOLDMAG NANOBIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The difficulty and complexity of establishing this method is that different detection objects have different requirements for detection methods, immune reaction conditions, and even the ratio of each component in the detection system, the composition of the luminescent liquid, etc., and the optimal result is determined through a large number of experiments In order to achieve the best detection range and sensitivity; in the process of magnetic separation, different magnetic field strength or magnetic separation time will directly affect the separation effect; in addition, in the process of antibody labeling, different antibodies or even different antibody dosages will directly affect the quality of the labeling system. (mainly reflected in labeling efficiency), labeling reaction conditions, purification and collection conditions, storage conditions, etc. need to design experiments repeatedly to determine

Method used

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  • Goldmag particle-based acridinium ester chemiluminescence immunological detection method of HE4
  • Goldmag particle-based acridinium ester chemiluminescence immunological detection method of HE4
  • Goldmag particle-based acridinium ester chemiluminescence immunological detection method of HE4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: A method for detecting human epididymis secretory protein 4 (HE4) by acridinium ester chemiluminescent immunology based on gold magnetic particles

[0072] (1) Coated

[0073] (1.1) Pretreatment: Take 100 μl of 10 mg / ml gold magnetic particles and wash them twice with 200 μl of pH 7.4, 0.02M Tris-HCl equilibrium buffer to balance the pH of the magnetic particles.

[0074] (1.2) Coupling: Dissolve 50 μg of HE4-coated antibody in 200 μl pH7.4, 0.02M Tris-HCl coupling buffer, mix well and add to the pretreated gold magnetic particles, at 37°C, 180rpm, react in a shaker for 30min. After the reaction was completed, it was taken out, magnetically separated, and the supernatant was discarded.

[0075] (1.3) Washing: add 300 μl pH7.4, 0.01M PBS washing buffer containing 0.05% Tween-20, magnetically separate, discard the supernatant, and wash 3 times.

[0076] (2) Blocking: Add 1ml of pH 7.4, 0.01M PBS buffer solution containing 5% skimmed milk powder and 2% fetal ...

Embodiment 2

[0086] Example 2: A method for detecting human epididymis secretory protein 4 (HE4) by acridinium ester chemiluminescent immunology based on gold magnetic particles

[0087] (1) Coated

[0088] (1.1) Pretreatment: Take 100 μl of 10 mg / ml gold magnetic particles and wash them twice with 200 μl of 0.01 M phosphate (PBS) equilibration buffer solution with pH 7.4 to balance the pH of the magnetic particles.

[0089](1.2) Coupling: Dissolve 50 μg of HE4-coated antibody in 200 μl of 0.01 M phosphate (PBS) coupling buffer at pH 7.4, mix well and add to pretreated gold magnetic particles, at 37 ℃, 180rpm, react in a shaker for 30min. After the reaction was completed, it was taken out, magnetically separated, and the supernatant was discarded.

[0090] (1.3) Washing: add 300 μl pH7.4, 0.01M PBS washing buffer containing 0.05% Tween-20, magnetically separate, discard the supernatant, and wash 3 times.

[0091] (2) Blocking: Add 1 ml of pH 7.4, 0.01M PBS buffer solution containing 5% ...

Embodiment 3

[0101] Example 3: A method for detecting human epididymis secretory protein 4 (HE4) by acridinium ester chemiluminescent immunology based on gold magnetic particles

[0102] (1) Coated

[0103] (1.1) Pretreatment: Take 100 μl of 10 mg / ml gold magnetic particles and wash them twice with 200 μl of pH 8.0, 0.1M Tris-HCl equilibrium buffer to balance the pH of the magnetic particles.

[0104] (1.2) Coupling: Dissolve 50 μg of HE4-coated antibody in 200 μl pH 8.0, 0.1M Tris-HCl coupling buffer, mix well and add to the pretreated gold magnetic particles, at 37°C, 180rpm, react in a shaker for 30min. After the reaction was completed, it was taken out, magnetically separated, and the supernatant was discarded.

[0105] (1.3) Washing: add 300 μl pH7.4, 0.1M Tris-HCl washing buffer containing 0.05% Tween-20, magnetically separate, discard the supernatant, and wash 3 times.

[0106] (2) Blocking: Add 1 ml of pH 8.0, 0.1M Tris-HCl buffer solution containing 5% skimmed milk powder and 2...

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Abstract

The invention provides a goldmag particle-based acridinium ester chemiluminescence immunological detection method of human epididymis secretory protein (HE4). The goldmag particle-based acridinium ester chemiluminescence immunological detection method mainly comprises following steps: (1) goldmag particle is taken as an immunoreaction and solid-phase separation carrier, and HE4 coated antibody is connected with the surface of the goldmag particle via coupling; (2) blank sites on the surface of the goldmag particle, which are not combined with the HE4 coated antibody, are blocked with a blocking solution; (3) acridinium ester (AE) is used for marking HE4 labelled antibody; (4) a sample to be detected, and the acridinium ester marked HE4 antibody are added into the blocked HE4 antibody coated goldmag particle for reaction so as to obtain a double-antibody sandwich compound, wherein the acridinium ester marked HE4 antibody is capable of realizing specific binding with HE4 antigen; (5) washing is carried out; and (6) chemiluminescence detection is carried out. The goldmag particle-based acridinium ester chemiluminescence immunological detection method is high in detection sensitivity, specificity, accuracy, and stability, and is simple and rapid; linearity range is wide; no radioactive contamination is caused; and operation is safe.

Description

technical field [0001] The invention relates to a method for detecting human epididymis secretion protein 4 (HE4). Background technique [0002] At present, the immunological detection methods for HE4 protein on the market mainly include enzyme-linked immunoassay, electrochemiluminescence and chemiluminescent enzyme immunoassay. Enzyme-linked immunoassay can be used for quantitative detection, but the operation steps are cumbersome, greatly affected by human factors, and the sensitivity, specificity and application range vary greatly; the accuracy of quantitative detection by electrochemiluminescence is high, and the level has quantitative and It has the advantage of stability, but it is not suitable for modern automatic immune instruments. It needs special supporting instruments to use, and the investment cost is high, so it is difficult to popularize and apply; The requirements for detection instruments and equipment are far lower than those of electrochemiluminescence, s...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N33/543G01N21/76
CPCG01N21/76G01N33/54326G01N33/577G01N33/68
Inventor 马乐郭博阳崔亚丽
Owner XIAN GOLDMAG NANOBIOTECH
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