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Blood vessel expanding type vasodilator-stimulated phosphoprotein detection kit

A detection kit and vasodilation technology, applied in the field of molecular immunology, can solve problems such as cumbersome operation, high cost, and long reaction time

Pending Publication Date: 2018-12-11
BEIJING PERGRANDE BIOTECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] At present, the mainstream product for VASP detection is Stago’s PLT VASP / P2Y12, which needs to be detected by flow cytometry. Flow cytometry and operation have high requirements for personnel and machines, resulting in relatively expensive costs. Stago also provides reagents for detecting VASP. box, but the reaction time is long and the operation is cumbersome

Method used

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  • Blood vessel expanding type vasodilator-stimulated phosphoprotein detection kit
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  • Blood vessel expanding type vasodilator-stimulated phosphoprotein detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Obtaining of monoclonal antibodies

[0048] 1. Animal immunization

[0049] In order to produce VASP monoclonal antibody, 6-week-old female Balb / c mice weighing about 20 g were selected. For the initial immunization, 20-50 μg of VASP peptide (Ser239 phosphorylated) and VASP plus complete Freund's adjuvant were injected subcutaneously at multiple points. On the 14th and 28th days, the second and third immunization doses were the same as above, plus Freund's incomplete adjuvant was injected intraperitoneally, and the immunization was boosted 3 days before fusion, with a suitable dose of 20-50 μg. After 3 days, the spleen was taken for fusion. Generally, the preparation of monoclonal antibodies of murine origin can refer to the method described in the "Antibodies" manual (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp.726, 1988) or Kohler and The technique for preparing hybridomas described by Milste...

Embodiment 2

[0062] Example 2. Preparation of Monoclonal Antibody

[0063] The two preserved hybridoma cells were divided into 1×10 7 The concentration of cells was injected into the peritoneal cavity of BALB / c mice, and the ascites were collected 10 days later. Purify the two monoclonal antibodies separately by the following steps:

[0064] 1. The collected ascites was centrifuged at 2500 rpm, and the supernatant was taken. Add an equal volume of PBS (pH7.4) and 1 / 2 volume of saturated ammonium sulfate, and let stand at 4°C for 30 minutes;

[0065] 2. Centrifuge at 3000rpm, 4°C for 20min;

[0066] 3. Remove precipitation, add an equal volume of saturated ammonium sulfate to the supernatant, and let it stand for 30 minutes;

[0067] 4. Centrifuge at 3000rpm, 4°C for 20min;

[0068] 5. Take the precipitate, add 5ml normal saline and 5ml saturated ammonium sulfate and let stand for 30min;

[0069] 6. Centrifuge at 3000rpm, 4°C for 20min;

[0070] 7. Add 5ml normal saline and 5ml 0.02M...

Embodiment 3

[0077] Embodiment 3. Preparation of detection reagent

[0078] 1. Preparation of magnetic particles coupled with VASP antibody:

[0079] Shake the magnetic particles well, take them out and put them into a vial, wash the magnetic particles with 0.05M MES (10.67g MES purified water to 1000ml), and add EDC to activate at room temperature for 30 minutes after cleaning;

[0080] According to the amount of magnetic particles, according to the ratio (mass ratio) of magnetic particles and antibodies (mass ratio) 10:1, 20:1 and 40:1, add VASP antibody respectively, and couple the centrifuge tube overnight at room temperature;

[0081] The reaction solution was sucked out by magnetic separation and washed by adding PBST (5.8g disodium hydrogen phosphate dodecahydrate, 0.59g sodium dihydrogen phosphate dihydrate, 9g sodium chloride, 0.5ml Tween-20, purified water to 1000ml) for 2 times, Magnetic separation sucks out the lotion;

[0082] Then add magnetic particle diluent (5.8g disodiu...

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Abstract

The invention relates to a blood vessel expanding type vasodilator-stimulated phosphoprotein detection kit which comprises magnetic particles coated with VASP monoclonal antibodies, an enzyme workingsolution containing marked VASP-P monoclonal antibodies, a lysate and a reaction substrate. The kit is fast in detection speed, simple and convenient to operate, high in sensitivity, and good in specificity.

Description

technical field [0001] The application belongs to the field of molecular immunology, and specifically relates to a detection kit for vasodilator-stimulated phosphoprotein (hereinafter referred to as VASP), a VASP monoclonal antibody and its application. Background technique [0002] With the development of my country's society and economy, the incidence of cardiovascular and cerebrovascular diseases is increasing year by year, and its high disability rate and high mortality rate pose a serious threat to people's health and bring a heavy burden to society. [0003] A large number of clinical trials and medical research have shown that cardiovascular and cerebrovascular diseases have common risk factors and pathophysiological mechanisms, among which platelet activation is a key link in the pathogenesis and disease progression. Therefore, active antiplatelet therapy has become one of the core strategies to prevent and treat cardiovascular diseases. [0004] By inhibiting plate...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/68G01N33/535
CPCG01N33/577G01N33/535G01N33/6893G01N2800/32
Inventor 于晖李雨心谷明星代晓曼
Owner BEIJING PERGRANDE BIOTECH DEV
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