Kit as well as preparation method and application thereof

Abstract

The invention discloses a kit and a preparation method and application thereof, and relates to the technical field of immunodetection analysis, the kit is used for detecting the content of soluble fms-like tyrosine kinase-1, the kit comprises: a first reagent comprising streptavidin magnetic beads and a first diluent; the second reagent comprises an enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and a second diluent; the third reagent comprises NHS-LC-biotin marked with a soluble fms-like tyrosine kinase-1 monoclonal antibody and a third diluent; a calibrator; and a light-emitting substrate. When the kit provided by the invention is used for detecting the content of soluble fms-like tyrosine kinase-1, the sensitivity is high, the stability and the repeatability are good, and the anti-interference capability is strong.

Description

technical field

[0001] The invention relates to the technical field of immunoassay and analysis, in particular to a kit and its preparation method and application. Background technique

[0002] sFlt-1 (soluble fms-like tyrosine kinase-1) is a rare spliced ​​form of vascular endothelial growth receptor-1 (fms-like tyrosinekinase receptor 1, Flt-1), which only retains the ligand-binding domain and does not have Tyrosine kinase activity, molecular weight 63.6KD, produced by trophoblast cells during pregnancy. Medical research at home and abroad has shown that sFlt-1 inhibits angiogenesis by affecting the effects of pro-angiogenic factors VEGF (vascular endothelial growth factor) and PIGF (placental growth factor); the expression of VEGF and PIGF in the placenta of PE (preeclampsia) patients is reduced , and deduced that the decreased expression of VEGF family proteins affected the differentiation and invasion of placental trophoblast cells, and even resulted in placental hypox...

Examples

preparation example Construction

[0070] The present invention further proposes a preparation method of the kit as described above, comprising the following steps:

[0071] S10. Streptavidin magnetic beads are added to the first diluent, mixed evenly, washed by magnetic separation, and then resuspended in the first diluent to obtain the first reagent.

specific Embodiment approach

[0073]Take 10 mg streptavidin magnetic beads, add 1 mL of the first dilution (50 mM 2-morpholineethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin300 (v / v), 0.9% NaCl ( w / v), 1% bovine serum albumin (w / v), 0.1% glycine (w / v), 0.5% trehalose (w / v) and 0.05% BSA-modified PEG (w / v), pH value of 6.5), magnetic separation and washing, repeated washing 3 times, resuspended in the first diluent, and diluted with the first diluent so that the concentration of streptavidin magnetic beads is 0.05mg / mL~0.2mg / mL, that is as the first reagent.

[0074] S20. After the soluble fms-like tyrosine kinase-1 monoclonal antibody is activated, mixed with the enzyme solution for reaction and purified, the enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody is prepared, and the enzyme-labeled soluble The fms-like tyrosine kinase-1 monoclonal antibody is uniformly mixed with the second diluent to obtain the second reagent.

[0075] Specifically, in the embodiment of the presen...

Embodiment 1

[0100] Example 1 Kit

[0101] The first reagent: streptavidin magnetic beads and the first diluent [50mM 2-morpholineethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin300 (v / v), 0.9% NaCl ( w / v), 1% bovine serum albumin (w / v), 0.1% glycine (w / v), 0.5% trehalose (w / v) and 0.05% BSA-modified PEG (w / v), pH Value is 6.5], the concentration of streptavidin magnetic beads is 0.05mg / mL, and the particle diameter of streptavidin magnetic beads is 1.5 μm;

[0102] Second reagent: enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and second diluent [50mM 2-morpholineethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin300 (v / v) v), 0.9% NaCl (w / v) and 1.5% bovine serum albumin (w / v), the pH value is 6.8], and the enzyme is alkaline phosphatase;

[0103] The third reagent: NHS-LC-biotin labeled with soluble fms-like tyrosine kinase-1 monoclonal antibody and the third dilution [50mM 2-morpholineethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin300 (v / v),...

technical field

[0001] The invention relates to the technical field of immunoassay and analysis, in particular to a kit and its preparation method and application. Background technique

[0002] sFlt-1 (soluble fms-like tyrosine kinase-1) is a rare spliced ​​form of vascular endothelial growth receptor-1 (fms-like tyrosinekinase receptor 1, Flt-1), which only retains the ligand-binding domain and does not have Tyrosine kinase activity, molecular weight 63.6KD, produced by trophoblast cells during pregnancy. Medical research at home and abroad has shown that sFlt-1 inhibits angiogenesis by affecting the effects of pro-angiogenic factors VEGF (vascular endothelial growth factor) and PIGF (placental growth factor); the expression of VEGF and PIGF in the placenta of PE (preeclampsia) patients is reduced , and deduced that the decreased expression of VEGF family proteins affected the differentiation and invasion of placental trophoblast cells, and even resulted in placental hypox...

Examples

preparation example Construction

[0070] The present invention further proposes a preparation method of the kit as described above, comprising the following steps:

[0071] S10. Streptavidin magnetic beads are added to the first diluent, mixed evenly, washed by magnetic separation, and then resuspended in the first diluent to obtain the first reagent.

specific Embodiment approach

[0073]Take 10 mg streptavidin magnetic beads, add 1 mL of the first dilution (50 mM 2-morpholineethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin300 (v / v), 0.9% NaCl ( w / v), 1% bovine serum albumin (w / v), 0.1% glycine (w / v), 0.5% trehalose (w / v) and 0.05% BSA-modified PEG (w / v), pH value of 6.5), magnetic separation and washing, repeated washing 3 times, resuspended in the first diluent, and diluted with the first diluent so that the concentration of streptavidin magnetic beads is 0.05mg / mL~0.2mg / mL, that is as the first reagent.

[0074] S20. After the soluble fms-like tyrosine kinase-1 monoclonal antibody is activated, mixed with the enzyme solution for reaction and purified, the enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody is prepared, and the enzyme-labeled soluble The fms-like tyrosine kinase-1 monoclonal antibody is uniformly mixed with the second diluent to obtain the second reagent.

[0075] Specifically, in the embodiment of the presen...

Embodiment 1

[0100] Example 1 Kit

[0101] The first reagent: streptavidin magnetic beads and the first diluent [50mM 2-morpholineethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin300 (v / v), 0.9% NaCl ( w / v), 1% bovine serum albumin (w / v), 0.1% glycine (w / v), 0.5% trehalose (w / v) and 0.05% BSA-modified PEG (w / v), pH Value is 6.5], the concentration of streptavidin magnetic beads is 0.05mg / mL, and the particle diameter of streptavidin magnetic beads is 1.5 μm;

[0102] Second reagent: enzyme-labeled soluble fms-like tyrosine kinase-1 monoclonal antibody and second diluent [50mM 2-morpholineethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin300 (v / v) v), 0.9% NaCl (w / v) and 1.5% bovine serum albumin (w / v), the pH value is 6.8], and the enzyme is alkaline phosphatase;

[0103] The third reagent: NHS-LC-biotin labeled with soluble fms-like tyrosine kinase-1 monoclonal antibody and the third dilution [50mM 2-morpholineethanesulfonic acid, 0.05% Tween 20 (v / v), 0.1% Proclin300 (v / v),...

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