Vascular endothelial growth factor chemiluminescence immunoassay kit and preparation method thereof

A chemiluminescent immunoassay and detection kit technology, which is applied in the field of kits, can solve the problems of being greatly affected by human factors, low degree of automation, and long detection time, and achieve unattended, high degree of automation, and short detection time. Effect

Inactive Publication Date: 2019-08-02
DIRUI MEDICAL TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The purpose of the present invention is to provide a vascular endothelial growth factor in order to solve the problems of the existing methods for detecting vascular endothelial growth factor, which are cumbersome to operate, long in detection time, low in automation, greatly influenced by human factors, low in sensitivity and poor in repeatability. Chemiluminescent immunoassay kit and preparation method thereof

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  • Vascular endothelial growth factor chemiluminescence immunoassay kit and preparation method thereof
  • Vascular endothelial growth factor chemiluminescence immunoassay kit and preparation method thereof
  • Vascular endothelial growth factor chemiluminescence immunoassay kit and preparation method thereof

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preparation example Construction

[0045] The present invention also provides a preparation method of the chemiluminescent immunoassay kit for vascular endothelial growth factor, comprising the following steps:

[0046] Step 1, R1 reagent preparation

[0047] After mixing the streptavidin magnetic particle solution and TBST solution, place it on a magnetic separator until the supernatant is free of turbidity, discard the supernatant, keep the magnetic particles, wash with buffer Ⅰ and put them in buffer Ⅰ Prepare the solid-phase reagent with the required mass percentage concentration, which is the R1 reagent, and store at 2-8°C;

[0048] Wherein, the concentration of the streptavidin magnetic particle solution is preferably 50-100 mg / ml, and the source is commercially available, such as selected from Agilent Corporation, product number PL6827-1006; the volume ratio of the streptavidin magnetic particle solution to the TBST solution Preferably (0.5~1):(5~10); the mixing time is preferably 10~15min; generally re...

Embodiment 1

[0062] Step 1, R1 reagent preparation

[0063] Take 0.5mL (50mg) of streptavidin magnetic particle solution with a concentration of 100mg / mL, add 10mL of TBST solution and mix well for 10min, place on a magnetic separator until the supernatant is free of turbidity, discard the supernatant, and keep the magnetic Particles, after repeated washing with buffer Ⅰ for 3 times, make a solid-phase reagent with a mass percentage concentration of 0.05% magnetic particles in buffer Ⅰ, which is the R1 reagent, and store at 2-8°C. Buffer Ⅰ contains 50mM MES , 0.05% Tween and 0.05% Proclin300, pH6.5 buffer.

[0064] Step 2, R2 reagent preparation

[0065] Put 250μg of antibody into a centrifuge tube, make sure that the antibody is at the bottom of the centrifuge tube (centrifuge at room temperature for 20s), then add PBS buffer solution, mix well, add 5μl 2mg / mL acridinium ester DMF solution after mixing, and centrifuge Centrifuge for 0.5 min at room temperature. Seal the centrifuge tube...

Embodiment 2

[0069] Step 1, R1 reagent preparation

[0070] Take 0.72 milliliters (72 mg) of streptavidin magnetic particle solution with a concentration of 100 mg / ml, add 15 mL of TBST solution and mix well for 15 minutes, then place it on a magnetic separator until the supernatant is free of turbidity, discard the supernatant, and keep Take the magnetic particles, wash with buffer Ⅰ repeatedly for 3 times, and then prepare a solid-phase reagent with a mass percentage concentration of 0.072% magnetic particles in buffer Ⅰ, which is the R1 reagent, and store at 2-8°C. Buffer Ⅰ contains 100 mMPBS, 0.1% Tween-20 and 0.1% Proclin300, pH 7.2 buffer.

[0071] Step 2, R2 reagent preparation

[0072] Put 500μg of antibody into a centrifuge tube, make sure that the antibody is at the bottom of the centrifuge tube (centrifuge at room temperature for 30s), then add TRIS washing solution, mix well, add 15μl 2.5mg / mL acridinium ester DMF solution after mixing, use The centrifuge was centrifuged at roo...

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Abstract

The invention relates to a vascular endothelial growth factor chemiluminescence immunoassay kit and a preparation method thereof, belonging to the technical field of kits. The existing method of detecting vascular endothelial growth factor is complex to operate, takes long detection time, is low in automation, is greatly affected by human factors, has low sensitivity, and has low repeatability. The invention solves the mentioned problems. The kit comprises a reagent R1, a reagent R2, and a reagent R3, wherein the reagent R1 comprises streptavidin magnetic particles and buffer solution I, the reagent R2 comprises vascular endothelial growth factor monoclonal antibody marked by a chemiluminescence marker and buffer solution II, and the reagent R3 comprises vascular endothelial growth factormonoclonal antibody marked by a coupling marker and buffer solution III. The kit is simple to operate, takes short detection time, is high in automation, is not affected by human factors, and has highsensitivity, good repeatability, good interference resistance, wide detection range, and high thermal stability.

Description

technical field [0001] The invention belongs to the technical field of kits, and in particular relates to a vascular endothelial growth factor chemiluminescent immunoassay kit and a preparation method thereof. Background technique [0002] Vascular endothelial growth factor (VEGF) is a highly glycosylated basic protein with a molecular weight of 45KD. It has two identical subunits formed by disulfide bonds. The isoelectric point is 8.5. It has strong heat and acid resistance. . The human VEGF gene has five different isoforms, namely VEGF206, VEGF189, VEGF165, VEGF145 and VEGF121. Three VEGF receptors (VEGFR-1, VEGFR-2, VEGFR-3) located on vascular endothelial cells are high-affinity binding sites for VEGF, all of which are transmembrane receptors. Studies have shown that VEGF receptors have a high affinity with VEGF in vitro. [0003] VEGF is the most important cytokine regulating neovascularization. Studies have shown that VEGF plays an irreplaceable role in the develop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/532G01N33/543
CPCG01N33/532G01N33/54326G01N33/6803
Inventor 李磊李冬梅孙成艳高威何浩会
Owner DIRUI MEDICAL TECH CO LTD
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