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Tubular magnetic particle chemiluminescence immunoassay quantitative kit for detecting CD47 as well as preparation method and application of tubular magnetic particle chemiluminescence immunoassay quantitative kit

A chemiluminescence immunoassay and CD47 technology, applied in the field of immunoassay medicine, can solve the problem of less research on the in vitro detection method of CD47 protein, and achieve the effects of expanding the area of ​​the solid phase carrier, sufficient signal amplification, rapid enrichment and separation

Pending Publication Date: 2021-06-22
山东省大健康精准医疗产业技术研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are few studies on in vitro detection methods of CD47 protein, or they are only in scientific research, so it is urgent to develop a kit that can quickly, sensitively and accurately detect the expression level of CD47 protein

Method used

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  • Tubular magnetic particle chemiluminescence immunoassay quantitative kit for detecting CD47 as well as preparation method and application of tubular magnetic particle chemiluminescence immunoassay quantitative kit
  • Tubular magnetic particle chemiluminescence immunoassay quantitative kit for detecting CD47 as well as preparation method and application of tubular magnetic particle chemiluminescence immunoassay quantitative kit
  • Tubular magnetic particle chemiluminescence immunoassay quantitative kit for detecting CD47 as well as preparation method and application of tubular magnetic particle chemiluminescence immunoassay quantitative kit

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Effect test

Embodiment 1

[0054] A tubular magnetic particle chemiluminescence immunoassay kit for detecting CD47, including magnetic particle complex, sample diluent, enzyme conjugate, luminescent liquid A, luminescent liquid B, 20× concentrated detergent, CD47 standard gradient solution, Reaction tube and instruction manual;

[0055] Wherein, the magnetic particle complex is a magnetic particle-immunoglobulin binding molecule-CD47 antibody complex; the enzyme conjugate is a CD47 antibody-neutravidin-biotin-horseradish peroxidase complex The CD47 antibody is a biotin-labeled CD47 monoclonal antibody; the immunoglobulin binding molecule is staphylococcal protein A.

[0056] The formula of the sample diluent is: sterilized 1L purified water containing 8g of NaCl, NaH 2 PO 4 2H 2 O 0.2g, NaH 2 PO 4 2H 2 O 2.5g, casein sodium salt 3.5g, Proclin300 1.0mL.

[0057] The luminescent liquid A contains luminol 4mmol / L, p-iodophenol 0.5mmol / L, FeCl 3 15μmol / L, dioxane 3mL / L, Tris-HCl buffer 5mmol / L, pH ...

Embodiment 2

[0066] A method for preparing a tubular magnetic particle chemiluminescence immunoassay kit for detecting CD47, comprising the following steps:

[0067] S1: Preparation of magnetic particle-immunoglobulin binding molecule-CD47 antibody complex

[0068] Place 500 μg of carboxyl magnetic particles in MES (50 mM, pH 5) buffer, then add 120 μg of EDC and 120 μg of Sulfo-NHS, incubate with shaking at room temperature for 45 min, centrifuge and wash, add 120 μg of staphylococcal protein A and incubate for 3.5 h, then Wash by centrifugation, add 120 μg of CD47 monoclonal antibody and incubate for 3.5 hours with the magnetic particle-immunoglobulin binding molecule to obtain the magnetic particle-immunoglobulin binding molecule-CD47 antibody complex;

[0069] S2: Preparation of CD47 antibody-neutravidin-biotin-horseradish peroxidase complex

[0070] 1) Preparation of CD47 antibody-neutravidin

[0071]Dissolve 40 mg of neutravidin in 100 mL of 0.1 mol / L phosphate buffer, add 0.6 mL o...

Embodiment 3

[0079] The application of the tubular magnetic particle chemiluminescence immunoassay kit described in the embodiment in the detection of CD47, the detection method is as follows:

[0080] (1) Equilibrate the sample to be tested and the reagents in the kit at 20-25°C for 25±5min, dilute the washing solution 20 times with distilled water and set aside;

[0081] (2) Set standard wells and sample wells to be tested, add 50 μL magnetic particle complex suspension to the sample wells to be tested and standard wells respectively, then add 50 μL sample to be tested and 50 μL CD47 standard substance gradient solution respectively, and then Add 50 μL of sample diluent, then incubate at 37°C and shake for 10 minutes;

[0082] (3) Place the reaction tube on the magnetic separator and let it stand for 10 seconds, discard the liquid, remove the reaction tube from the magnetic separator, rinse the magnetic particle complex with detergent, perform magnetic separation after washing, and disca...

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Abstract

The invention relates to a tubular magnetic particle chemiluminescence immunoassay quantitative kit for detecting CD47 as well as a preparation method and application thereof. The kit comprises a magnetic particle-immunoglobulin binding molecule-CD47 antibody compound, a CD47 antibody-neutral avidin-biotin-horse radish peroxidase compound, a sample diluent, a luminescent solution A, a luminescent solution B, a detergent, a CD47 standard substance gradient solution, a reaction tube and an operation instruction. The invention also provides a preparation method and application of the tubular magnetic particle chemiluminescence immunoassay quantitative kit. According to the tubular magnetic particle chemiluminescence immunoassay quantitative kit for detecting the CD47 as well as the preparation method and application of the tubular magnetic particle chemiluminescence immunoassay quantitative kit of the present invention, the magnetic separation technology, the chemiluminescence detection technology and the immunological method are combined, such that the detection precision and the detection stability of the obtained magnetic particle chemiluminescence immune quantitative kit are substantially improved. The kit has good stability and can be stably stored at 2-8 DEG C for at least one year.

Description

technical field [0001] The invention relates to the technical field of immunoanalysis medicine. Specifically, the invention provides a tube-type magnetic particle chemiluminescence immunoassay kit for detecting CD47, its preparation method and application. Background technique [0002] CD47 is a member of the immunoglobulin superfamily. It was originally discovered due to the combination and co-purification of integrin ανβ3 protein, so it is also known as integrin-associated protein (integrin associated protein, IAP), with a relative molecular mass of 50kD. The structure of CD47 includes: A single Ig V-like N-terminus, five highly hydrophobic extended transmembrane segments and a short alternatively spliced ​​carboxyl segment cytoplasmic C-terminus, which is widely expressed on the surface of different tissue cells, such as hematopoietic cells (erythrocytes, lymphocytes) cells, platelets, etc.), non-hematopoietic cells (placenta, liver and brain cells, etc.) and tumor cells,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/58G01N33/543G01N33/535G01N21/76
CPCG01N33/57423G01N33/57488G01N33/54326G01N33/535G01N33/581G01N21/76G01N2333/70503
Inventor 高丽鹤欧兰香武建伟陈振寇宗阳张绍明
Owner 山东省大健康精准医疗产业技术研究院
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