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Hormone mass spectrometric detection method based on antibody coupling magnetic bead enrichment technology

A technology of mass spectrometry detection and antibody coupling, which is applied in mass spectrometry analysis, measuring devices, instruments, etc., can solve the problems of narrow linear range, limited application of liquid mass spectrometry and poor specificity, etc.

Active Publication Date: 2021-06-22
HANGZHOU BAICHEN MEDICAL LAB CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the clinical detection of endogenous hormones usually uses an antibody-based chemiluminescence method, which uses specific antibodies to capture the required detection substances, separates the required detection substances through antibody magnetic beads, and then detects them with an enzyme-labeled method. However, this method has poor specificity, The linear range is narrow, and the concentration of target compounds such as hormones cannot be accurately detected; liquid mass spectrometry (LC-MS / MS) detection is a new type of detection technology, due to its high specificity, wide linear range, and Multiple indicators can be detected at the same time, which can specifically overcome the defects of the chemiluminescence method in the detection of hormones
[0003] However, the current sample pretreatment method for the detection of hormones by liquid chromatography-mass spectrometry is a non-specific enrichment method, which contains more unnecessary matrix substances, resulting in a higher matrix effect during the detection process, and can be separated by Substances with similar structures, so liquid mass spectrometry is not widely used in hormone detection
[0004] In addition, the current method is difficult to apply to the detection of endogenous small molecular compounds of hormones expressed by antibodies. When antibodies are expressed in serum, endogenous small molecular compounds of hormones will be produced, and antibodies will tightly bind to endogenous small molecular compounds of hormones. , the use of simple antibody purification steps cannot effectively separate the endogenous small molecule compounds of hormones, resulting in the inability to perform subsequent accurate detection of such hormones

Method used

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  • Hormone mass spectrometric detection method based on antibody coupling magnetic bead enrichment technology
  • Hormone mass spectrometric detection method based on antibody coupling magnetic bead enrichment technology
  • Hormone mass spectrometric detection method based on antibody coupling magnetic bead enrichment technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: The antibody contains small molecular compounds of endogenous hormones.

[0029] Reagent preparation:

[0030] Four antibodies of cortisol, testosterone, progesterone and aldosterone were selected, and the antibodies were purchased from Thermo; methanol was purchased from Merck, and methanol was mixed with water to prepare an 80% aqueous methanol solution;

[0031] Experimental equipment preparation:

[0032] 1.5ml centrifuge tube self-made magnetic stand; 1.5ml centrifuge tube; Vortex shaker (Scientific Industries * Vortex-Genie2 company, Vortex-Genie 2); Waters TQS mass spectrometer (Waters company);

[0033] experiment procedure:

[0034] Take 5μl antibody (aldosterone, progesterone, testosterone or cortisol) into 1.5ml centrifuge tube, add 100μl 80% methanol aqueous solution, vortex in vortex shaker for 3min, centrifuge at 12000g 4℃ for 10min, take supernatant Put 50 μl into a 96-well plate, then add 50 μl of water, mix well and detect on Waters TQS. ...

Embodiment 2

[0051] Example 2: Both 0.1M glycine pH2.8 eluent and 20% methanol solution cannot effectively remove endogenous hormones on antibody-coupled magnetic beads

[0052] Reagent preparation:

[0053] Glycine was purchased from Sigma, prepared as 0.1M glycine pH2.8; ​​phosphate buffer saline (PBS) was purchased from Sigma, deionized water was purchased from Merck; From sigma, add water to prepare 50mM Tris-HCl, pH7.4, and carboxyl magnetic beads were purchased from Yingruicheng Biochemical Technology Co., Ltd. Other reagents are with embodiment one.

[0054] Experimental instrument preparation: same as implementation case 1

[0055] experiment procedure:

[0056] 1. Antibody magnetic bead coupling: mix the magnetic beads evenly, take 10mg magnetic beads and add them to a 1.5mL EP tube, remove the supernatant by magnetic separation on the magnetic stand, add deionized water and mix evenly, remove the supernatant by magnetic separation , and repeat 2 more times. Add 100 μL PBS to...

Embodiment 3

[0066] Example 3: 80% methanol can effectively remove endogenous hormones on antibody-coupled magnetic beads without affecting antibody activity. 10% methanol is used as an enrichment system and mixed with 4 kinds of antibodies to achieve better enrichment effect

[0067] Reagent preparation:

[0068] All hormone compounds and internal standards were purchased from Sigma, and other reagents were the same as those in Example 1 or 2, and 10% methanol was used to prepare internal standards (Table 4) and calibrator (Table 5) respectively.

[0069] Table 4 internal standard concentration of each compound

[0070]

[0071] Table 5 Concentration of each compound in calibrator

[0072]

[0073]

[0074] Other reagents and experimental equipment are prepared with embodiment one or two.

[0075] experiment procedure:

[0076] The method of preparing antibody-coupled magnetic beads is the same as that described in Example 2.

[0077] 1. Removal of endogenous hormones on anti...

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Abstract

The invention provides a hormone mass spectrometric detection method based on an antibody coupling enrichment technology, the method comprises the following steps: coupling an antibody and a magnetic bead, adding a high-concentration methanol solution, vortex to remove an endogenous hormone small molecule compound carried on the antibody, and recovering the activity of the antibody to obtain an antibody magnetic bead capable of enriching the hormone small molecule compound; adding detection serum or a standard curve sample to obtain a first solution; adding an internal standard substance into the first solution, performing vortex incubation at room temperature, and adding low-concentration methanol for washing to obtain antibody magnetic beads enriched with hormone small molecule compounds; placing the antibody magnetic beads enriched with the hormone small molecule compounds in 80% methanol aqueous solution for resuspension vortex to obtain eluent containing the hormone small molecule compounds; detecting a hormone small molecule compound by mass spectrometry, immobilizing an antibody by using magnetic beads, eluting the hormone small molecule compound by using 80% methanol aqueous solution, and then detecting the hormone small molecule compound by mass spectrometry, so that the detection specificity is improved.

Description

technical field [0001] The invention relates to the field of hormone detection, in particular to a hormone mass spectrometry detection method based on antibody-coupled magnetic bead enrichment technology. Background technique [0002] At present, the clinical detection of endogenous hormones usually uses an antibody-based chemiluminescence method, which uses specific antibodies to capture the required detection substances, separates the required detection substances through antibody magnetic beads, and then detects them with an enzyme-labeled method. However, this method has poor specificity, The linear range is narrow, and the concentration of target compounds such as hormones cannot be accurately detected; liquid mass spectrometry (LC-MS / MS) detection is a new type of detection technology, due to its high specificity, wide linear range, and Multiple indicators can be detected at the same time, and the defects existing in the detection of hormones by chemiluminescent method...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/08G01N30/14G01N30/72
CPCG01N30/02G01N30/06G01N30/08G01N30/14G01N30/72G01N2030/045G01N33/54326G01N33/743G01N2560/00G01N2030/8813G01N2030/027
Inventor 薛建有杨方星吴超超高强
Owner HANGZHOU BAICHEN MEDICAL LAB CO LTD
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