Nanometer magnetic particle chemiluminiscence determination kit for antigen CA125 relating to tumor, as well as preparation method and determining method of same

A tumor-associated antigen, CA125 technology, applied in the biological field, can solve problems affecting detection sensitivity and accuracy, and achieve good accuracy, low production cost, and wide detection range

Active Publication Date: 2013-04-17
SUZHOU HAOOUBO BIOPHARML
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, in the actual immunoassay, due to the large amount of impurities contained in the sample to be tested, which affects the detection sensitivity and accuracy to a certain extent, the target analyte can be quickly separated and purified from the complex sample matrix. It is one of the problems faced by clinical laboratory workers

Method used

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  • Nanometer magnetic particle chemiluminiscence determination kit for antigen CA125 relating to tumor, as well as preparation method and determining method of same
  • Nanometer magnetic particle chemiluminiscence determination kit for antigen CA125 relating to tumor, as well as preparation method and determining method of same
  • Nanometer magnetic particle chemiluminiscence determination kit for antigen CA125 relating to tumor, as well as preparation method and determining method of same

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 The preparation of the first reagent

[0043] (1) Materials and instruments: Tumor-associated antigen CA125 (CA125) monoclonal antibody preserved in phosphate buffer (purity over 95wt%, concentration 2mg / mL); fluorescein isothiocyanate (FITC), sodium carbonate, etc. Reagents should be chemically pure; G-25 gel purification column was purchased from GE.

[0044] (2) Preparation steps:

[0045] ① Prepare 0.5mg / mL FITC solution with 0.1~0.2mol / L carbonate buffer solution with pH 9.0~10.0;

[0046] ② Add the FITC solution prepared in step ① to the antibody solution according to the ratio of CA125 antibody to FITC molecular ratio of 1:20, mix well, and let it stand at room temperature for 12 hours to react to generate CA125 antibody-FITC conjugate;

[0047] ③Separate the reaction liquid after step ② through G-25 gel column, remove unreacted FITC, and obtain a solution containing CA125 antibody-FITC conjugate (ie, FITC-labeled CA125 antibody);

[0048] ④ Dilut...

Embodiment 2

[0049] Embodiment 2 Preparation of the second reagent

[0050] (1) Materials and instruments: CA125 monoclonal antibody preserved in phosphate buffer (purity over 95wt%, concentration 2 mg / mL); alkaline phosphatase preserved in phosphate buffer (ALP solution, ALP purity of about 99 %, the specific activity is about 1500U / mg, and the concentration is 10mg / mL); the cross-linking agent SMCC, 2-IT are purchased from THERMO company, TRIS and other chemical reagents should be chemically pure; G-25 gel purification column is a product of GE company .

[0051] (2) Preparation steps:

[0052] ①Take 1mg of CA125 antibody, add 2-4μL of 10mg / mL coupling agent 2-IT solution, let stand at room temperature for 20min, add 10μL of 0.1mol / L glycine solution, let stand at room temperature for 5min, remove with G-25 gel column salt, collect the activated CA125 antibody, and store it at 2-8°C for later use;

[0053] ② Take 1.5 mg of ALP solution, add 10-20 μL of 5 mg / mL SMCC solution, let it st...

Embodiment 3

[0056] The preparation of embodiment 3 magnetic separation reagents

[0057] (1) Materials and instruments:

[0058] Suspension of magnetic particles: the content of magnetic particles is 5wt%, and the magnetic particles contain carboxyl (COOH) active groups. The carboxyl group content per gram (g) of magnetic particles (dry weight) is not less than 0.4 millimoles (mmol), with superparamagnetism, The diameter is between 0.5-2μm.

[0059] Anti-FITC antibody: It can be a polyclonal antibody or a monoclonal antibody, with a purity of more than 90% by weight and a dilution titer of more than 1:1 million;

[0060] 2-Morpholineethanesulfonic acid (MES), carbodiimide (EDC), TRIS, and other reagents should be of chemical purity.

[0061] (2) Preparation steps:

[0062] ①Take 100mg of magnetic particle suspension, magnetically separate to remove the supernatant, and resuspend in 10mL of 0.05mol / L, pH 4.5~5 MES buffer;

[0063] ②Add 2~4mg of anti-FITC antibody and suspend at room te...

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Abstract

The invention relates to a nanometer magnetic particle chemiluminiscence determination kit for antigen CA125 relating to tumor, and the preparation method and the determining method of the kit. The kit comprises solution of tumor related antigen CA125 antibody containing fluorescein labels, suspending liquid of magnetic particles wrapped by fluorescent antibody, and solution of tumor related antigen CA125 antibody containing alkaline phosphatase labels. According to the invention, quantitative determination to the antigen CA125 relating to tumor can be performed with lower cost and higher degree of accuracy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a nano-magnetic particle chemiluminescence assay kit for tumor-associated antigen CA125 combined with immunomagnetic particle separation technology and chemiluminescence immunoassay technology, and a preparation method and detection method thereof. Background technique [0002] Tumor-associated antigen CA125 (Carbohydrate Antigen, CA125) is a high molecular weight glycoprotein associated with epithelial ovarian cancer. CA125 is a marker of epithelial ovarian cancer and endometrial cancer. Its sensitivity and specificity are reported to be 85-95% and 75-90%, respectively. The content of CA125 in patients with serous endometrial carcinoma, clear cell carcinoma, fallopian tube carcinoma and undifferentiated ovarian carcinoma can be significantly increased. A slight increase in the serum concentration of CA125 is also seen in 1% of healthy women, 3%-6% of benign ovarian disea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/532G01N21/76
Inventor 于大为程晓蕾赵文姬
Owner SUZHOU HAOOUBO BIOPHARML
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