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Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof

A technology of nano-magnetic particles and chemiluminescence, which is applied in chemiluminescence/bioluminescence, analysis and measurement devices through chemical reactions of materials, and can solve problems such as poor process stability, cumbersome preparation process, and limited detection effect , to achieve the effect of cost advantage, high sensitivity, simple operation and time saving

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

Although the kit can realize rapid and accurate detection, the preparation process of the solid phase carrier coated with CA19-9 monoclonal antibody and the horseradish peroxidase-labeled CA19-9 monoclonal antibody is very cumbersome and the process The stability is not good, which limits its detection effect, especially the precision between analyzes and increases the cost

Method used

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  • Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof
  • Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof
  • Nanometer magnetic particle chemiluminescence detection kit for carbohydrate antigen CA19-9 as well as preparation method thereof and detecting method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1 The preparation of the first reagent

[0054] (1) Materials and instruments: Carbohydrate-associated antigen CA19-9 (CA19-9) monoclonal antibody preserved in phosphate buffer (purity over 95wt%, concentration 2mg / mL); fluorescein isothiocyanate (FITC ), sodium carbonate and other reagents should be chemically pure; the G-25 gel purification column was purchased from GE.

[0055] (2) Preparation steps:

[0056] ① Prepare 0.5mg / mL FITC solution with 0.1~0.2mol / L carbonate buffer solution with pH 9.0~10.0;

[0057] ② Add the FITC solution prepared in step ① to the antibody solution according to the ratio of CA19-9 antibody to FITC molecular ratio of 1:20, mix well, and let stand at room temperature for 12 hours to react to generate CA19-9 antibody-FITC conjugate;

[0058] ③Separate the reaction solution after step ② through a G-25 gel column to remove unreacted FITC to obtain a solution containing the CA19-9 antibody-FITC conjugate (ie FITC-labeled CA19-9 an...

Embodiment 2

[0060] Embodiment 2 Preparation of the second reagent

[0061] (1) Materials and instruments: CA19-9 monoclonal antibody preserved in phosphate buffer (purity over 95wt%, concentration 2 mg / mL); alkaline phosphatase preserved 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 are purchased from THERMO company, TRIS and other chemical reagents should be chemically pure; G-25 gel purification column is GE company's product.

[0062] (2) Preparation steps:

[0063] ①Take 1mg of CA19-9 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, gel with G-25 Desalt the column, collect the activated CA19-9 antibody, and store it at 2-8°C for later use;

[0064] ② Take 1.5 mg of ALP solution, add 10-20 μL of 5 mg / mL SMCC solution, let it stand at ...

Embodiment 3

[0067] The preparation of embodiment 3 magnetic separation reagents

[0068] (1) Materials and instruments:

[0069] 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.

[0070] 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;

[0071] 2-Morpholineethanesulfonic acid (MES), carbodiimide (EDC), TRIS, and other reagents should be of chemical purity.

[0072] (2) Preparation steps:

[0073] 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;

[0074] 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 chemiluminescence detection kit for a carbohydrate antigen CA19-9 as well as a preparation method thereof and a detecting method thereof. The kit comprises a solution, which contains a fluorescein labeled carbohydrate related antigen CA19-9, a suspension, which is coated with a fluorescein antibody, and a solution, which contains an alkaline phosphatase labeled carbohydrate related antigen CA19-9 antibody, wherein the alkaline phosphatase labeled carbohydrate related antigen CA19-9 antibody is obtained by connecting an alkaline phosphatase and the carbohydrate related antigen CA19-9 antibody through cross-linking agents SMCC and 2-IT. According to the invention, the carbohydrate related antigen CA19-9 can be quantitatively detected with lower cost, higher accuracy and higher precision.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a nano-magnetic particle chemiluminescence assay kit for carbohydrate-related antigen CA19-9 combined with immunomagnetic particle separation technology and chemiluminescence immunoassay technology, and a preparation method and detection method thereof. Background technique [0002] Carbohydrate antigen CA19-9 (Carbohydrate Antigen19-9) is a glycolipid on the cell membrane with a molecular weight of about 1000KD. CA19-9 was named after the 116NS19-9 monoclonal antibody obtained by Koprowski et al. in 1979 by immunizing mice with colon cancer cells and hybridizing with myeloma. It is the most sensitive marker for pancreatic cancer reported so far. [0003] CA19-9 is mainly used clinically in the following aspects: [0004] Gastrointestinal Malignant Tumor [0005] The levels of CA 19-9 in pancreatic cancer, hepatobiliary cancer, gastric cancer, and colorectal cancer were ...

Claims

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Application Information

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IPC IPC(8): G01N33/574G01N21/76
Inventor 于大为程晓蕾赵文姬
Owner SUZHOU HAOOUBO BIOPHARML
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