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Latex enhanced turbidimetric immunoassay kit for diagnosing gastric diseases or gastric cancer, preparation method thereof and application

A technology of latex enhancement and immune turbidimetry, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of high cost of manpower and material resources

Active Publication Date: 2012-08-22
BEIJING MOKOBIO LIFE SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gastric cancer screening began in Japan in the 1960s, when gastric cancer was highly common. At that time, a step-by-step screening method of gastrointestinal double-contrast radiography-gastroscopy-pathological examination was adopted, which cost a lot of manpower and material resources.

Method used

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  • Latex enhanced turbidimetric immunoassay kit for diagnosing gastric diseases or gastric cancer, preparation method thereof and application
  • Latex enhanced turbidimetric immunoassay kit for diagnosing gastric diseases or gastric cancer, preparation method thereof and application
  • Latex enhanced turbidimetric immunoassay kit for diagnosing gastric diseases or gastric cancer, preparation method thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] The extraction and purification of pepsinogen I and pepsinogen II of embodiment 1

[0056] (1) Collect surgically resected gastric tissue, rinse with phosphate buffer, separate gastric mucosa, blot dry, weigh, add phosphate buffer, mash and homogenate, centrifuge, collect supernatant, precipitate with phosphate buffer The solution was resuspended, centrifuged again, and the supernatant was combined to obtain the pepsinogen crude extract;

[0057] (2) Add the pepsinogen crude extract to the equilibrated DEAE-52 chromatographic column, elute the chromatographic column with PBS buffer until the absorbance is 0 at 280nm, and continue to elute the chromatographic column with PBS buffer column, combined elution peaks to obtain active pepsinogen;

[0058] (3) Take the peak of active protease, load it on the gel chromatography column, and use 50mmol containing 0.05mol / LNa 2 SO 4 elution with PBS, flow rate 3.0ml / min, pressure 8Kpa, the elution curve shows 4 protein elution p...

Embodiment 2

[0061] Embodiment 2 Preparation of anti-pepsinogen I or anti-pepsinogen II monoclonal antibody

[0062] (1) Preparation of fused cells: 50-100 μg of human pepsinogen I or human pepsinogen II prepared in Example 1 was mixed with Freund's complete adjuvant in equal volumes to subcutaneously inject 8-week-old female BALB / C mice After an interval of 2-3 weeks, the mice were boosted with the same dose of antigen for 2-3 times, and the tail vein blood was taken 3-4 days after the last immunization for detection. When the titer reached more than 4000, it was ready for fusion; the immunized After the mice were sacrificed, the mice were subjected to local disinfection and laparotomy, and the spleen was taken under aseptic conditions to prepare a spleen cell suspension; 2-3 days before fusion, each bottle of Sp2 / 0 myeloma cells was transferred to 4 bottles of cells, and the number of cells in the logarithmic The cells in the growth phase are used for fusion; after counting the splenocyt...

Embodiment 3

[0067] The assembly of embodiment 3 kits (taking PG II as example)

[0068] 1 buffer configuration

[0069] 1.1 Preparation of diluent (R1)

[0070] Weigh the reagents in the table below and put them into a clean container, add purified water and mix well, measure the pH value to 7.4, and set the volume to 1000ml.

[0071] Reagent Amount of liquid per liter Disodium phosphate 2.88g Sodium dihydrogen phosphate 2.4g Sodium chloride 116.88g PEG-6000 2g BSA 10.0g Tween-20 0.5ml

[0072] PC-300 0.5ml Add purified water to 1000ml

[0073] 1.2 Preparation of standard diluent

[0074] Weigh the above reagents into a clean container, add purified water to dissolve and mix, measure the pH value to 7.4, and set the volume to 1000ml.

[0075] Reagent Amount of liquid per liter Disodium phosphate 1.44g Potassium dihydrogen phosphate 1.36g Sodium chloride 8g ...

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Abstract

The invention discloses a latex enhanced turbidimetric immunoassay kit for diagnosing gastric diseases or gastric cancer, a preparation method thereof and application. Components of the kit include diluent, blank solution and a latex reagent of an antibody of pepsinogen I or an antibody of pepsinogen II and can further include a calibrator and quality control serum. The latex reagent contains nanoparticles coupled with the antibody of the pepsinogen I or the antibody of the pepsinogen II, and the particle sizes of the nanoparticles are different. The examination sensitivity cannot meet requirements when latex with the single particle size lower than 100nm is used for examination, the linear range is small when latex with the single particle size of about 200nm is used for examination, and accordingly the examination sensitivity and the linear range cannot meet requirements when latex with the single particle size is used for examination. After composite latex of the kit is used for marking, the examination sensitivity can be improved, the linear examination range can be broadened, and the latex enhanced turbidimetric immunoassay kit has the advantages of fast examination, high sensitivity and specificity, good accuracy and the like in terms of gastric disease or gastric cancer examination.

Description

technical field [0001] The present invention relates to a kit for in vitro diagnosis, in particular to a latex enhancement for diagnosing gastric disease or early gastric cancer by measuring the concentration of pepsinogen I and pepsinogen II in a subject and comparing it with a set cut-off value The immunoturbidimetric kit, the invention also relates to the preparation method and application of the kit, which belongs to the field of diagnosis or detection of gastric disease or gastric cancer. Background technique [0002] Pepsinogen (PG) is a precursor of aspartic acid protease, a single-chain polypeptide with a molecular mass of 42kDa, including three disulfide bonds, and an isoelectric point of 3.7. Human PG can be divided into 7 components according to its electrophoretic mobility, and the immunogenicity of the 1-5 components that move faster to positive is similar, called pepsinogen I (PG I), which is mainly composed of the principal cells of gastric glands and Secrete...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/573G01N33/574
Inventor 金鑫
Owner BEIJING MOKOBIO LIFE SCI CO LTD
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