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Carbohydrate antigen CA19-9 assay kit and detection method using the same

A carbohydrate antigen and kit technology, applied in the direction of biological testing, material inspection products, etc., can solve the problems of high price, unfavorable promotion and use, etc.

Inactive Publication Date: 2017-11-24
北京健安生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, domestic CA19-9 clinical detection is mainly based on imported reagents, while foreign imported reagents are expensive, which is not conducive to popularization and use. Therefore, it is very important to develop a CA19-9 assay kit with high sensitivity and high accuracy.

Method used

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  • Carbohydrate antigen CA19-9 assay kit and detection method using the same
  • Carbohydrate antigen CA19-9 assay kit and detection method using the same
  • Carbohydrate antigen CA19-9 assay kit and detection method using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] The various buffers involved in the kit preparation process and detection process are as follows:

[0078] 1. Preparation of magnetic bead buffer, prepare 1L, the specific operation steps are as follows:

[0079] (1). Weigh 800 g of purified water, 7.27 g of Tris, 0.6 g of PROCLIN 3000, and 4.38 g of methyl cellulose ether into a beaker with a capacity of 1 L, and stir thoroughly for 2 hours until completely dissolved.

[0080] (2). Weigh 3.15g of Tween-20, 1.12g of neomycin sulfate, 35mg of tetracycline, and 5.18g of bovine serum albumin (BSA) in the solution obtained in step (1), stir for 1h until completely dissolved, and adjust the pH meter to measure Its pH value is adjusted with dilute HCl and dilute NaOH solution so that its pH value is 8.0±0.05.

[0081] (3). Quantify the solution obtained in step (2) to 1000g, verify the pH value, filter it with a 0.2μm filter into a 1L glass bottle, label it and store it in a cold storage at 2-8°C. The validity period is one ...

Embodiment 2

[0102] Implementation 2: Preparation of Carbohydrate Antigen CA19-9 Assay Kit

[0103] 1. Preparation of magnetic separation reagent R1:

[0104] (1). Use a pipette gun to take 250 μL of magnetic beads into a 5 mL glass vial, absorb on a magnetic plate, discard the supernatant, measure 500 μL of 100 mM MES pH 5.0 buffer solution and wash twice; add 1250 μL of buffer solution, mix well; ultrasonically disperse for 20 s , power 40% (4 times x 5s / time).

[0105] (2). Weigh 20mg of Sulfo-NHS into a centrifuge tube, add 800μL buffer solution 100mM MES pH 5.0, the concentration is 25mg / ml, add 575μL to the solution obtained in step (1). (Use within 2 minutes after preparation)

[0106] (3). Weigh 10 mg of EDC, add 500 μL buffer solution 100 mM MES pH 5.0, the concentration is 20 mg / ml, add 250 μL to the solution obtained in step (2). (Use within 2 minutes after preparation)

[0107] (4). Add buffer solution 100mM MES pH 5.0 to 2.5ml (supplement 425μL) to the solution obtained in...

Embodiment 3

[0137] Implementation 3. The steps of measuring CA19-9 with the carbohydrate antigen CA19-9 assay kit

[0138] The carbohydrate antigen CA19-9 assay kit prepared by the present invention is used to measure CA19-9, and the detection steps are as follows:

[0139] (1). Adding samples: Take 9 test tubes and add 30 μL each of CA19-9 calibrator, quality control product and sample to be tested.

[0140] (2). Add 60 μL of magnetic separation reagent R1 and 60 μL of labeling reagent R2 to the test tubes in step (1), mix with a multi-tube screw mixer for 30 seconds, and incubate in a 37°C water bath for 15 minutes.

[0141] (3). After the incubation, put the test tube on the magnetic separator and let it stand for adsorption for 3 minutes, discard the supernatant, and pat the bottom of the magnetic separator to separate the supernatant as clean as possible.

[0142] (4). Add 300 μL of lotion to the test tube obtained in step (3), place it on a multi-tube spiral mixer to mix, then plac...

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Abstract

The invention discloses a carbohydrate antigen CA19-9 assay kit. The kit comprises a magnetic separation reagent R1, a labeling reagent R2, a CA19-9 calibration product and a CA19-9 quality control product. The invention also provides a preparation method of the kit. The magnetic separation reagent R1 in the kit is prepared through coupling a CA19-9 monoclonal antibody to the surface of a carboxyl magnetic bead. The labeling reagent R2 is prepared through coupling a luminescent substance acridinium ester to a paired CA19-9 monoclonal antibody. The reactions occur under nearly homogeneous conditions. Through use of an excitation solution, the reaction product antibody-antigen-antibody sandwich complex can immediately release photons at 430 nm and can release strong photons without use of a luminescent enhancer and a catalyst so that luminescence detection can be directly carried out. Through optimizing the experimental processes of magnetic bead coating with an antibody and antibody labeling with acridinium ester, the stability and the detection sensitivity of the kit are greatly improved. The kit has a wide linear detection range, is easy to operate and has a fast detection rate.

Description

technical field [0001] The invention belongs to the field of biological immune in vitro diagnosis of medical equipment, and mainly relates to the preparation of a kit for measuring the content of carbohydrate antigen 19-9 in blood and a detection method thereof. Background technique [0002] CA199 is a mucin-type glycoprotein tumor marker. It is a glycolipid on the cell membrane and exists in the form of salivary mucin in serum with a molecular weight of 3 to 5 million. CA199 is expressed in the stomach, intestine, liver and pancreas of the fetus, while it is only expressed in a small amount in the pancreas, liver, gallbladder and lung of normal adults. Studies have shown that the sensitivity and specificity of elevated serum CA199 in patients with cholangiocarcinoma are 88.15% and 92% (<37U / mL), respectively. Therefore, the detection of serum CA199 has a high value in the diagnosis of cholangiocarcinoma patients. In addition, CA199 has a certain value in the identificat...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 李冉冉于猛张静田聪会邱笑违董飒英
Owner 北京健安生物科技有限公司
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