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One-step homogeneous phase D-dimer detection kit and application thereof

A detection kit and dimer technology, which is applied in the field of one-step homogeneous D-dimer detection kits, can solve the problems of poor label stability, heterogeneous reaction, large intra-batch and inter-batch variation, etc. Accuracy, specificity, and easy operation

Inactive Publication Date: 2016-09-28
NANJING PERLONG MEDICAL EQUIP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RIA has high detection sensitivity, but it has gradually withdrawn from the field of clinical testing due to the radioactive hazards of markers, poor stability of markers, and difficult disposal of waste; ELISA method uses horseradish peroxidase (HRP) Or alkaline phosphatase to label antibodies, and catalyze the substrate to produce color changes. It has the characteristics of simple operation and long stable period of reagents. However, the detection sensitivity of ELISA method is low. At present, it is mainly used in infectious disease screening and other detection sensitivity requirements. Lower items; GICA has the advantages of simple operation and fast detection speed, but also has the disadvantages of low sensitivity, unstable reagents, poor repeatability, and difficult to quantify
CLIA has the advantages of simple operation, fast detection speed, and high-throughput detection, but it also has the disadvantages of heterogeneous reaction, long detection time, poor actual stability, and large intra-assay and inter-assay variability.

Method used

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  • One-step homogeneous phase D-dimer detection kit and application thereof
  • One-step homogeneous phase D-dimer detection kit and application thereof
  • One-step homogeneous phase D-dimer detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Kit

[0041] Sources of raw materials and reagent formulations in the experiment:

[0042]

[0043]

[0044] 1. Anti-D-dimer antibody coupled luminescent microspheres, taking aldehyde-based luminescent microspheres as an example:

[0045] 1) Add 0.1 mg of antibody to an ultrafiltration tube, centrifuge for 10 minutes, wash with HEPES buffer (pH7.4) for 6 times, and dilute the antibody to 1 mg / ml for use. Take 1 mg of luminescent microspheres, wash twice with HEPES buffer (pH7.4), centrifuge, and remove the supernatant.

[0046] 2) Add the antibody solution to the luminescent microspheres (purchased from Platinum Elmer) and resuspend.

[0047] 3) Add 1.25 μl of 10% Tween20.

[0048] 4) Add 10 μl 400mM NaCNBH 3 , add HEPES buffer to make up the volume to 200 μl.

[0049] 5) Shaking reaction at 37°C for 24-48 hours.

[0050] 6) Blocking: Prepare 65 mg / ml CMO solution with 800 mM NaOH. Add 10 μl of CMO solution to the reaction system.

[0051] 7) Shaki...

Embodiment 2

[0072] Application of embodiment 2 kit

[0073] The method for using the kit comprises the following steps:

[0074] 1) Add the sample, anti-D-dimer antibody-coupled luminescent microspheres and anti-D-dimer antibody-coupled photosensitive microspheres into the reaction well of the kit, and mix and react for 5-60 minutes;

[0075] 2) The reaction wells are irradiated with excitation light, and the luminescence of each reaction well is measured to obtain the light signal value.

[0076] The reaction wells of this kit can be microwell plates, microfluidic reagent plates, reaction cups, reaction tubes, etc.

[0077] Kit methodology evaluation of the present invention:

[0078] 1. Linear

[0079] Prepare D-dimer standard solutions with concentrations of 0ng / ml, 100ng / ml, 500ng / ml, 2500ng / ml, 10000ng / ml, 50000ng / ml, and 100000ng / ml. Add 5 μl of standard substance, 20 μl of luminescent microspheres coupled with anti-D-dimer antibody (final concentration 10 μg / ml), and 20 μl of p...

Embodiment 3

[0097] It is basically the same as Example 1, except that the luminescent microspheres used are carboxyl luminescent microspheres, and the photosensitive microspheres used are carboxyl photosensitive microspheres;

[0098] After verification by the methodology of Example 2, the linearity, accuracy, precision, analytical sensitivity, specificity and correlation are basically the same as those of Example 1.

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Abstract

The invention provides a one-step homogeneous phase D-dimer detection kit and application thereof to detecting the D-dimer content. The kit comprises a D-dimer antibody coupling resisting light-emitting microballoon test solution (1), a D-dimer antibody coupling resisting light-sensitive microballoon test solution (2) and reaction holes capable of receiving and emitting light. Compared with the prior art, the kit has the advantages of being convenient to operate, fast in detection, high in sensitivity, good in accuracy and the like and is high in specificity. The kit is applied to monitoring the hypercoagulable state and thrombotic diseases, and the accuracy of monitoring the hypercoagulable state and the thrombotic diseases can be improved.

Description

technical field [0001] The invention belongs to the field of detection kits, in particular to a one-step homogeneous D-dimer detection kit and its application. Background technique [0002] The fibrinolytic system is the most important anticoagulant system in the human body. In the process of fibrinolysis, after thrombin hydrolyzes fibrinogen, it releases fibrin peptides (A and B) successively, and the remaining soluble fibrin monomers form stable cross-linked fibrin under the action of factor Ⅻa, During the degradation of cross-linked fibrin by plasmin, the released fragments are further degraded into the smallest fragment D-dimer. In pathological conditions, the dynamic balance of coagulation and fibrinolysis is disrupted, and the tendency of coagulation is enhanced, thereby increasing the degradation products of fibrin, resulting in an increase in the content of D-dimer. An increase in the level of D-dimer indicates that fibrin thrombus formation and fibrinolysis occur ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/68
CPCG01N33/577G01N33/68G01N2800/32
Inventor 李明明张春东黄宝福淳林
Owner NANJING PERLONG MEDICAL EQUIP
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