Time resolution fluorescence immunoassay method based on magnetic separation

A technology of time-resolved fluorescence and analysis methods, applied in the field of time-resolved fluorescence immunoassay analysis based on magnetic separation, can solve the problems of limited application development in the field of immunodiagnosis, easy to be interfered by environmental substances, short luminescence time, etc., to overcome the luminous intensity Weak, no radioactive contamination, wide standard curve effect

Inactive Publication Date: 2019-02-01
江苏美克医学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some shortcomings of CLIA limit its application and development in the field of immunodiagnosis, such as: short luminescence time, a single sample can only be detected once, some items have a high background and are easily interfered by environmental substances, etc.

Method used

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  • Time resolution fluorescence immunoassay method based on magnetic separation
  • Time resolution fluorescence immunoassay method based on magnetic separation
  • Time resolution fluorescence immunoassay method based on magnetic separation

Examples

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Effect test

Embodiment 1

[0033] The specific operation steps of a time-resolved fluorescence immunoassay method based on magnetic separation in the present invention:

[0034] Step 1: After activation, the magnetic beads are coupled with monoclonal antibodies, blocked, and washed to obtain immunomagnetic beads, which are stored for later use; the specific steps are as follows:

[0035] Pretreatment: Take 25 μL of magnetic beads with 1% solid content and 1 μm surface carboxyl group modification in a 2 mL imported centrifuge tube, add 500 μL of 50 mmol / L MES solution with pH 6.0, vortex mix, centrifuge at 15000 rpm, 10 min, 4 °C, remove Add 500μL of 50mmol / L pH6.0 MES solution to the supernatant, and ultrasonically reconstitute;

[0036] Activation: Add 2 μL of 25mg / mL NHS (50mmol / L pH6.0 MES configuration), vortex mixing, then add 10mg / mL EDC (50mmol / L pH6.0 MES configuration) 2μL, vortex mixing, shaker at 250r / min, 15min at 37°C;

[0037] Coupling: 15000rpm, 10min, centrifuge at 4°C, remove superna...

Embodiment 2

[0059] The specific operation steps of a time-resolved fluorescence immunoassay analysis method based on magnetic separation of the present invention:

[0060] Step 1: After activation, the magnetic beads are coupled with monoclonal antibodies, blocked, and washed to obtain immunomagnetic beads, which are stored for later use; the specific steps are as follows:

[0061] Pretreatment: Take 25 μL of magnetic beads with 1% solid content and 2 μm surface carboxyl modification in a 2 mL imported centrifuge tube, add 500 μL of 20 mmol / L MES solution with pH6.0, vortex mix, centrifuge at 15000 rpm, 10 min, 4 °C, remove Add 500μL of 20mmol / L pH6.0 MES solution to the supernatant, and reconstitute by ultrasonication;

[0062] Activation: Add 2μL of 15mg / mL NHS (20mmol / L pH6.0 MES configuration), vortex to mix, then add 10mg / mL EDC (20mmol / L pH6.0 MES configuration) 2μL, vortex to mix, shake at 250r / min, 15min at 37°C;

[0063] Coupling: centrifuge at 15000rpm, 10min, 4°C, remove the...

Embodiment 3

[0085] The specific operation steps of a time-resolved fluorescence immunoassay analysis method based on magnetic separation of the present invention:

[0086] Step 1: After activation, the magnetic beads are coupled with monoclonal antibodies, blocked, and washed to obtain immunomagnetic beads, which are stored for later use; the specific steps are as follows:

[0087] Pretreatment: Take 25 μL of magnetic beads with 1% solid content and particle size of 500 nm surface hydroxyl modification in a 2 mL imported centrifuge tube, add 500 μL of 50 mmol / L MES solution with pH6.0, vortex mix, centrifuge at 15000 rpm, 10 min, 4 °C, remove Add 500μL of 50mmol / L pH6.0 MES solution to the supernatant, and ultrasonically reconstitute;

[0088] Activation: Add 2μL of 25mg / mL NHS (50mmol / L pH6.0 MES configuration), vortex mixing, then add 10mg / mL EDC (50mmol / L pH6.0 MES configuration) 2μL, vortex mixing, shaker 100r / min, 30°C for 30min;

[0089] Coupling: 15000rpm, 10min, centrifuge at 4...

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Abstract

The invention provides a time resolution fluorescence immunoassay method based on magnetic separation; the method mainly comprises the following steps of firstly, coupling magnetic beads with an antibody to form immunomagnetic beads; meanwhile, enabling time resolution fluorescent microsphere to be coupled with antibody to form immunofluorescent microspheres; then enabling the immunomagnetic beadsand the immunofluorescent microspheres and the antigen in a sample to be oscillated and incubated in a reaction tube to form an immunomagnetic bead-antigen-immunofluorescent microsphere compound; andfinally, testing the fluorescence intensity, emitted by excitation of the compound at 360 nm excitation, by a time resolution instrument, wherein a standard curve is used as reference for determiningthe amount of the antigen in the sample. According to the analysis method, the reaction time is greatly shortened, and the detection efficiency and sensitivity are improved.

Description

technical field [0001] The invention belongs to the fields of bioanalytical chemistry and nano-biotechnology, and in particular relates to a magnetic separation-based time-resolved fluorescent immunoassay analysis method. Background technique [0002] Clinical detection of new protein indicators generally uses enzyme-linked immunosorbent assay (ELISA). The general sensitivity and detection range of ELISA are limited, and there are defects such as requiring professionals to operate, cumbersome steps, and long time-consuming. Especially for the detection of new indicators, the comprehensiveness of data is lacking, and accurate quantitative data cannot be provided for clinicians, which lacks the guiding significance for clinical treatment. [0003] Chemiluminescent Immunoassay (CLIA) is a combination of highly sensitive chemiluminescent assay technology and highly specific immune response, used for various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/58G01N33/533G01N33/536G01N21/64
CPCG01N21/6428G01N33/533G01N33/536G01N33/5434G01N33/582G01N33/585
Inventor 黄宝福孙康俊潘为民王学锋
Owner 江苏美克医学技术有限公司
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