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 the problems affecting the detection sensitivity and accuracy, and achieve the effects of good accuracy, high coupling efficiency, simple operation and time-saving

Active Publication Date: 2015-04-15
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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Preparation of the first reagent

[0043] (1) Materials and equipment: Monoclonal antibody to tumor-associated antigen CA125 (CA125) stored in phosphate buffer (purity over 95wt%, concentration 2mg / mL); fluorescein isothiocyanate (FITC), sodium carbonate, etc. The reagents should be chemically pure; the 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 with pH 9.0~10.0;

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

[0047] ③The reaction solution after step ② is separated by a G-25 gel column to remove unreacted FITC to obtain a solution containing CA125 antibody-FITC conjugate (ie FITC-labeled CA125 antibody);

[0048] ④Dilute the solution containing ...

Embodiment 2

[0049] Example 2 Preparation of the second reagent

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

[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, store at 2-8°C for later use;

[0053] ②Take 1.5mg of ALP solution, add 10-20μL of 5mg / mL SMCC solution, let stand at room temperature for 30min, remove salt with ...

Embodiment 3

[0056] Example 3 Preparation of magnetic separation reagent

[0057] (1) Materials and instruments:

[0058] Magnetic particle suspension: magnetic particle content 5wt%, magnetic particle contains carboxyl group (COOH) active group, carboxyl group content per gram (g) magnetic particle (dry weight) is not less than 0.4 millimoles (mmol), with super paramagnetism, 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 90wt%, and a dilution titer of more than 1:1 million;

[0060] 2-Morpholine ethanesulfonic acid (MES), carbodiimide (EDC), TRIS and other reagents should be chemically pure.

[0061] (2) Preparation steps:

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

[0063] ②Add 2~4mg of anti-FITC antibody and suspend at room temperature for 30~60min;

[0064] ③Add 0.5~1mL of a freshly ...

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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 that combines 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. It has been reported that its sensitivity and specificity are between 85-95% and 75-90% at home and abroad. The levels of CA125 in patients with serous endometrial cancer, clear cell carcinoma, fallopian tube cancer and undifferentiated ovarian cancer can be significantly increased. A slight increase in the serum concentration of CA125 is also seen in 1% of healthy women and 3% to 6% of patie...

Claims

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Application Information

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Patent Type & Authority Patents(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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