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Magnetic particle chemiluminescence immune assay kit of tumor marker AFP (alpha fetal protein) and detection method thereof

A chemiluminescence immunoassay and detection method technology, which is applied in the field of magnetic particle chemiluminescence immunoassay kits, can solve the problems of insufficient sensitivity of ELISA detection, and achieve the effect of simple pretreatment process, high sensitivity, and convenient and easy detection method

Inactive Publication Date: 2014-09-10
GUANGXI DOCTOR HAIYI INFORMATION TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved in the present invention is to provide a kind of AFP quantitative determination kit and its detection method, adopt this kit to carry out AFP detection to have higher sensitivity and specificity, easier to operate, shorter detection time, can overcome existing The disadvantage of using ELISA detection in the technology is not sensitive enough

Method used

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  • Magnetic particle chemiluminescence immune assay kit of tumor marker AFP (alpha fetal protein) and detection method thereof
  • Magnetic particle chemiluminescence immune assay kit of tumor marker AFP (alpha fetal protein) and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] 1. Operating procedures for preparing magnetic bead buffer: Take the preparation of 1L as an example:

[0040] 1. According to the preparation amount, select a suitable container and add 700ml of pure water;

[0041] 2. Weigh 6.02g of Tris and 0.99g of NaN3 in a container, mix well, and stir at room temperature for 2 hours;

[0042] 3. Check the pH of the solution, it should be around 10;

[0043] 4. Adjust the pH value to 8.0;

[0044] 5. Measure 2.76ml of Tween-20; weigh 0.99g of neomycin sulfate; 0.03g of tetracycline; 4.97g of BSA in a container, stir for 1 hour, and adjust the pH value to 8.0±0.05;

[0045] 6. Set the volume to 1L, adjust the pH value to 8.0±0.05;

[0046] 7. Filter with a 0.22um filter and store at 4°C.

[0047] 2. Preparation process of magnetic particle reagent

[0048] Activate ferric oxide microspheres with a diameter of 0.1 micron with glutaraldehyde, mix at room temperature for 4 hours, wash with 0.01mol / L PBS pH7.4 buffer solution th...

Embodiment 2

[0050] 1. Coupling of alkaline phosphatase AP and anti-AFP monoclonal antibody

[0051]5mg / mL APF monoclonal antibody, add 20mmol / L activator N-succinimide 3-(2-pyridine dimercapto)propionic acid [SPDP] dimethyl sulfoxide solution 20μL, room temperature for 45 minutes; through Sephadex G25 Remove the activator from the column and collect the protein peak; add 500 μL 24 mg / mL dithiothreitol (DTT) 0.01 mol / L PBS pH7.4 solution to each ml of activated antibody solution, mix well, and place at room temperature for 30 minutes; pass through Sephadex G25 column removes free dithiothreitol and collects protein peaks.

[0052] Dissolve alkaline phosphatase in 2mmol / L EDTA 20mmol / L Tris-HCl pH8.0 solution, the concentration is 5 mg / mL; add 0.10mg / mL Traunt reagent (sulfhydryl activating reagent) and let stand at room temperature for 1 hour; pass through Sephadex G25 column Free activator is removed and protein peaks are collected.

[0053] The activated antibody and activated alkaline...

Embodiment 3

[0058] Preparation of calibrators / quality controls:

[0059] 1. The highest point: the highest concentration point is X, and the concentration of the target point is A, B, C, D, E, F. When preparing a V-volume solution, the volumes of raw materials to be added are as follows: Table 1

[0060]

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Abstract

The invention relates to a magnetic particle chemiluminescence immune assay kit of tumor marker AFP (alpha fetal protein) and a detection method of the magnetic particle chemiluminescence immune assay kit, which belongs to the technical field of the immune detection and analysis. An AFP monoclonal antibody marked by fluorescein isothiocyanate (FITC) and a monoclonal antibody marked by alkaline phosphatase (AP) are combined with an antigen to form a sandwiched immune compound of an FITC marked antibody-antigen-AP marked antibody, and the sandwiched immune compound is similar to a sandwich structure. Then the magnetic particles which are connected with the anti-FITC antibody are added, the specific binding of the anti-FITC antibody and the FITC enables antigen-antibody complex to be connected onto the magnetic particles, the magnetic particles are directly precipitated in an external magnetic field, and the compound formed in the immune reaction manner can be separated from other non-combined substances without centrifuging. According to the kit, the chemiluminescence is combined with the magnetic particles, a reaction system which is approximate to the homogenous phase is provided, and compared with the prior art, the kit has the advantages of high sensitivity, wide linear range, high speed and the like; moreover, the product cost is greatly reduced, and the application prospect on the aspects such as the clinical inspection is promising.

Description

technical field [0001] The invention belongs to the technical field of immune detection and analysis, and relates to a magnetic particle chemiluminescent immunoassay kit for detecting the content of tumor marker AFP in serum and a detection method thereof. Background technique [0002] Tumor markers (tumor mark, TM) refer to substances secreted or shed by tumor cells into body fluids or tissues, or substances produced by the host in response to parasites in the body and entered into body fluids or tissues. Most of the commonly used clinical tumor markers are non-specific markers. These markers also appear in benign tumors and normal tissues, but their levels are significantly increased when tumors occur. Since such markers are not tumor-specific, they are also called broad-spectrum tumor markers. [0003] Alpha-fetoprotein (α-Fetoprotein, AFP) is a single multimer peptide bond glycoprotein, containing 590 amino acids, with a molecular weight of about 70KD, and belongs to th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N21/76
CPCG01N33/574G01N21/76G01N33/54326G01N33/6893G01N2800/7028
Inventor 李永锋陈国勇傅汝毅陶玲云蓝文苑
Owner GUANGXI DOCTOR HAIYI INFORMATION TECH
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