Latex-enhanced immunoturbidimetric kit for vascular endothelial growth factor and application of kit

A growth factor and vascular endothelium technology, applied in the field of latex-enhanced immune turbidimetric kits, can solve problems such as inability to accurately detect VEGF content, and achieve the effects of improving utilization and detection sensitivity, reducing interference, and reducing detection interference.

Inactive Publication Date: 2018-01-16
上海原科实业发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In order to solve the problem that the latex-enhanced immune turbidimetric analysis technology in the prior art cannot accurately detect the conte...

Method used

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  • Latex-enhanced immunoturbidimetric kit for vascular endothelial growth factor and application of kit
  • Latex-enhanced immunoturbidimetric kit for vascular endothelial growth factor and application of kit
  • Latex-enhanced immunoturbidimetric kit for vascular endothelial growth factor and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Preparation of Human VEGF Antibody

[0068] Antibodies were obtained by immunizing Japanese white rabbits with human VEGF antigen. The specific method is: subcutaneous multi-point immunization on the back of rabbits, the first immunization dose is 200 μg / rabbit, once a month, the immunization dose is 100 μg / rabbit, a total of 4 times, and the final booster immunization dose is 200 μg / rabbit, and the immune serum is collected after 7 days. The antiserum was purified with protein G and finally stored in phosphate buffer at pH 7.4, and the antibody titer was identified by enzyme-linked immunosorbent assay.

Embodiment 2

[0069] Example 2 Coupling of human VEGF antibody to latex microspheres

[0070] Wash 100mg / ml latex microspheres (particle size: 100nm) (purchased from Millipore, model is Fluorescent Microspheres - F-XC 030) twice with 10ml MES buffer; after the second wash, resuspend in 10ml MES buffer For microspheres, adjust the concentration of microspheres to 1%, ultrasonically or stir to resuspend the microspheres evenly; add 350 μL NHS (2-4.5mg / ml) and EDC (2.5-5mg / ml) 25°C) for 15-30 minutes; take 50mM MOPS buffer (pH7.0) to wash twice and resuspend the microspheres in 5ml 15mM MES buffer; dissolve human VEGF antibody in 5ml MOPS buffer, and the microspheres The suspension was mixed with the antibody solution, and reacted at room temperature for 2-6 hours; rinsed, resuspended in 10ml phosphate buffer, mixed slowly for 30 minutes, ultrasonically dispersed the microspheres, and stored at 4°C.

Embodiment 3

[0071] Example 3 Composition of Vascular Endothelial Growth Factor Latex Enhanced Immunoturbidity Kit

[0072] Composition of reagent R1:

[0073] 3-(N-morpholino)propanesulfonic acid buffer 0.06 mol / L, pH 7-8.5

[0074] Polyethylene glycol p-isooctylphenyl ether 45 mmol / L

[0075] BSA 1% (w / v)

[0076] Surfactant 0.1% (w / v)

[0077] Glycine 0.15% (w / v)

[0078] Preservative 0.1% (w / v).

[0079] Composition of reagent R2:

[0080] Phosphate buffer 0.15mol / L, pH 7.2-7.4

[0081] Latex Microspheres Coated with Vascular Endothelial Growth Factor Antibody 1% (w / v)

[0082] BSA 1% (w / v)

[0083] Surfactant 0.2% (w / v)

[0084] Trehalose 1% (w / v)

[0085] Sucrose 2.5% (w / v)

[0086] PEG6000 0.18% (w / v)

[0087] Mannitol 0.03% (w / v)

[0088] Preservative 0.1% (w / v)

[0089] Preparation of calibrator:

[0090] Dissolve the human VEGF protein in the diluent, the diluent is composed of 0.1 mol / L, PBS buffer solution with pH 7.2-7.4, BSA with a mass concentration of 0.5%, a...

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Abstract

The invention discloses a latex-enhanced immunoturbidimetric kit for a vascular endothelial growth factor and an application of the kit. The kit comprises a reagent R1, a reagent R2 and a vascular endothelial growth factor calibrator, wherein the reagent R1 includes 3-(N-morpholinyl)propanesulfonic acid buffer, polyethylene glycol p-isooctyl phenyl ether, BSA, glycine, a surfactant and an antiseptic, and the reagent R2 comprises a phosphate buffer, latex microspheres coated with a vascular endothelial growth factor antibody, BSA, a surfactant, trehalose, mannitol, sucrose, PEG 6000, and an antiseptic. The kit allows a Fc segment of a VEFG antibody to be coupled with polystyrene latex particles through a directional coupling method. The coating effect is good, the antibody is not easy to beseparated from latex particles, and the utilization rate and detection sensitivity of the antibody are greatly improved.

Description

technical field [0001] The invention relates to the technical field of a latex-enhanced immune turbidimetric kit, in particular to a vascular endothelial growth factor latex-enhanced immune turbidimetric kit and an application thereof. Background technique [0002] Vascular Endothelial Growth Factor (VEGF) is the most effective pro-angiogenic growth factor and the most powerful vascular permeability factor found so far, so it is also called vascular permeability factor (VPF). [0003] VEGF is a mitogen that mainly acts on vascular endothelial cells. It is a homodimeric glycoprotein composed of two identical polypeptide chains through a disulfide bond, with a molecular weight of 36-46kDa. The human VEGF gene is located in band 2, region 1, short arm of chromosome 6 (6p12.3). The gene consists of 8 exons and 7 introns. After transcriptional splicing, 5 different transcripts can be produced. VEGF121, VEGF145, VEGF165, VEGF185, VEGF206 and other at least 5 kinds of protein form...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543
Inventor 王连升
Owner 上海原科实业发展有限公司
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