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Immunomagnetic bead-based time-resolved fluorescence immunoassay kit of cardiac myosin binding protein C (cMyBP-C)

A time-resolved fluorescence, immunomagnetic bead technology, applied in the fields of bioanalytical chemistry and nano-biology, 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 weak luminescence intensity, The effect of no radioactive contamination and a wide range of standard curves

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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  • Immunomagnetic bead-based time-resolved fluorescence immunoassay kit of cardiac myosin binding protein C (cMyBP-C)
  • Immunomagnetic bead-based time-resolved fluorescence immunoassay kit of cardiac myosin binding protein C (cMyBP-C)
  • Immunomagnetic bead-based time-resolved fluorescence immunoassay kit of cardiac myosin binding protein C (cMyBP-C)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The specific preparation process of a time-resolved fluorescence immunoassay kit for cardiac myosin binding protein C (cMyBP-C) based on immunomagnetic beads of the present invention:

[0036] Step 1: Preparation of immunomagnetic beads

[0037] Pretreatment: Take 25μL of 1% solid content and particle size of 1μm surface carboxyl modified magnetic beads in a 2mL imported centrifuge tube, add 500μL of 50mmol / L pH6.0 MES solution, vortex to mix, 15000rpm, 10min, 4℃ centrifugation, remove Supernatant, add 500μL of 50mmol / L pH6.0 MES solution, and reconstitute it by ultrasonic;

[0038] Activation: add 2μL of 25mg / mL NHS (50mmol / L pH6.0 MES configuration), vortex to mix, then add 10mg / mL EDC (50mmol / L pH6.0 MES configuration) 2μL, vortex to mix, shaker 250r / min, react at 37℃ for 15min;

[0039] Coupling: 15000rpm, 10min, centrifugation at 4℃, remove the supernatant, add 500μL ultrapure water, ultrasonic reconstitution, repeat centrifugation to reconstitute twice, use 50mmol / L pH8...

Embodiment 2

[0062] The specific preparation process of a time-resolved fluorescence immunoassay kit for cardiac myosin binding protein C (cMyBP-C) based on immunomagnetic beads of the present invention:

[0063] Step 1: Preparation of immunomagnetic beads

[0064] Pretreatment: Take 25μL of 1% solid content particle size 2μm surface carboxyl modified magnetic beads in a 2mL imported centrifuge tube, add 500μL of 20mmol / L pH6.0 MES solution, vortex to mix, 15000rpm, 10min, 4℃ centrifugation to remove Supernatant, add 500μL of 20mmol / L pH6.0 MES solution and reconstitute it by ultrasonic;

[0065] 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, shaker 250r / min, react at 37℃ for 15min;

[0066] Coupling: 15000rpm, 10min, 4℃ centrifugation, remove the supernatant, add 500μL ultrapure water, ultrasonic reconstitution, repeat centrifugation to reconstitute twice, use 20mmol / L pH8.0 HEPES ...

Embodiment 3

[0089] The specific preparation process of a time-resolved fluorescence immunoassay kit for cardiac myosin binding protein C (cMyBP-C) based on immunomagnetic beads of the present invention:

[0090] Step 1: Preparation of immunomagnetic beads

[0091] Pretreatment: Take 25μL of 1% solid content particle size 500nm surface hydroxyl modified magnetic beads in a 2mL imported centrifuge tube, add 500μL of 50mmol / L pH6.0 MES solution, vortex to mix, 15000rpm, 10min, 4℃ centrifugation to remove Supernatant, add 500μL of 50mmol / L pH6.0 MES solution, and reconstitute it by ultrasonic;

[0092] Activation: add 2μL of 25mg / mL NHS (50mmol / L pH6.0 MES configuration), vortex to mix, then add 10mg / mL EDC (50mmol / L pH6.0 MES configuration) 2μL, vortex to mix, shaker 100r / min, reaction at 30℃ for 30min;

[0093] Coupling: 15000rpm, 10min, centrifugation at 4℃, remove the supernatant, add 500μL ultrapure water, ultrasonic reconstitution, repeat centrifugation to reconstitute twice, use 50mmol / L pH8...

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PUM

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Abstract

The invention provides an immunomagnetic bead-based time-resolved fluorescence immunoassay kit of cardiac myosin binding protein C (cMyBP-C). The kit includes a calibrator, immunomagnetic beads, immunofluorescence microspheres, analysis buffering liquid and wash liquid. The magnetic beads are coupled with antibodies, and the time-resolved fluorescence microspheres are coupled with the antibodies;thus the two parts form immunomagnetic beads-cMyBP-C antigen-immunofluorescence microsphere complexes with cMyBP-C antigens in a sample in a reaction tube after shaking incubation; a time-resolved instrument is used to determine intensity of fluorescence emitted thereby under excitation of ultraviolet light; and comparison of a standard curve is carried out to determine the amount of the cMyBP-C antigens in the sample. The kit of the invention greatly shortens reaction time, and improves efficiency and sensitivity of detection.

Description

Technical field [0001] The invention belongs to the fields of bioanalytical chemistry and nano-biotechnology, and particularly relates to a time-resolved fluorescence immunoassay kit for cardiac myosin binding protein C (cMyBP-C) based on immunomagnetic beads. Background technique [0002] cMyBP-C is a structural protein of thick myocardial filaments, which is specifically present in myocardial cells. When acute myocardial infarction (AMI) occurs, cMyBP-C is dephosphorylated and released into the blood, which makes the blood cMyBP-C concentration rise sharply, which makes serum cMyBP-C have a good diagnostic specificity for AMI complicated by heart failure Sex. In AMI patients whose onset to hospitalization time is less than 4h, their serum cMyBP-C has been significantly increased, but serum cTnI is not significantly increased. ROC curve analysis shows that serum cMyBP-C and cTnI are differentially diagnosed for AMI patients whose onset to hospitalization time is less than 4h T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/58G01N33/533G01N33/536G01N33/68G01N21/64
CPCG01N21/6428G01N33/533G01N33/536G01N33/5434G01N33/582G01N33/585G01N33/68G01N2800/324
Inventor 孙康俊黄宝福潘为民王学锋
Owner 江苏美克医学技术有限公司
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