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Kit for detecting knubble type M2 pyruvate kinase and preparation method thereof

A pyruvate kinase and kit technology, applied in the fields of biomedicine and medical testing, can solve the problems of inability to achieve rapid detection, cumbersome operation, etc., and achieve the effects of stable method, easy portability and storage, and rapid detection.

Inactive Publication Date: 2008-07-23
FUDAN UNIV SHANGHAI CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method still has the disadvantages of relatively cumbersome operation, repeated washings, and the need to read with a microplate reader, which cannot achieve the purpose of a single rapid detection

Method used

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  • Kit for detecting knubble type M2 pyruvate kinase and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Antigens were purified by salting out crude extraction and chromatography:

[0035] Remove the excised human liver cancer tissue block, remove fat and connective tissue, add homogenization buffer containing 0.5mmol / L 1,6-diphosphate fructose and 10mmol / L β-mercaptoethanol (20mmol / Ltris-HCl pH7.5 Buffer, 100mmol / LKCl, 5mmol / LMgSO4, 1mmol / LEDTA), homogenate under low temperature conditions, centrifuge at 15000rpm for 20min, take the supernatant, and use the segmental salting-out method for pretreatment: separate with 45-75% ammonium sulfate Precipitate, obtain the crude extract, and then dialyze and balance with normal saline and phosphate buffer solution respectively, then use a phosphocellulose column (treated with acid and alkali and equilibrated with 10mmol / L phosphate buffer) to load and combine, and use a solution containing 0.5mmol / L Phosphoenolpyruvate was eluted with a linear concentration gradient in elution buffer (10-140mmol / L phosphate buffer), and after conc...

Embodiment 2

[0044] The antigen was purified by crude salting-out extraction and chromatography: the preparation was the same as in Example 1.

[0045] Take out 0.3 mg of the tumor-type M2 pyruvate kinase antigen from the refrigerator, rewarm, and dilute to 1 ml with normal saline. New Zealand male rabbits, weighing about 2.5kg, were used for the immunization experiment. The mixture of Freund's complete adjuvant and fusion protein antigen was injected subcutaneously in both hind paws of the rabbit at 1 mg / ml / only. For the initial immunization, Freund's complete adjuvant and antigen were fully Mix until it becomes a "water-in-oil" antigen emulsion. After 2 weeks, 0.5ml of Freund's incomplete adjuvant antigen mixture was injected into the swollen popliteal lymph nodes of both hind limbs, and a total of 1mg of Freund's incomplete adjuvant antigen mixture was injected at multiple points on the back at the 6th and 8th weeks Two weeks after the injection, 1 mg of antigen was used subcutaneously...

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Abstract

The invention relates to a test kit for detecting tumor M2 pyruvate kinase and a relative preparation method, belonging to biological medicine and medical test technical field. The inventive test kit comprises a bottom plate (1), a colorful particle conjugate pad (3), a test sample pad (4), a water absorption pad (5) and a porous filter membrane for reaction, wherein the conjugate pad is fixed with anti-tumor M2 pyruvate kinase antibody marked by colorful particles, the porous filter membrane is arranged with a test region (a) and a reference region (b), the test region covers anti-tumor M2 pyruvate kinase antibody, the reference region covers anti-mouse IgG or antigen solution. The invention can process semi-quantitatively analyze and measure tumor M2 pyruvate kinase in 10min. The test kit is portable and easy to store, with simple operation, stable method, non additional tester and enzyme and the application for quickly and simply detecting tumor M2 pyruvate kinase on solid membrane medium.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and medical testing, and relates to immunochemical detection technology, in particular to a kit for detecting tumor-type M2 pyruvate kinase, and more specifically to an immunoassay labeled with colored particles for detecting tumor-type M2 pyruvate kinase. Chromatography kit and preparation method thereof. Background technique [0002] According to reports, the morbidity and mortality of malignant tumors are increasing year by year. The main way to reduce the mortality of malignant tumors is early diagnosis and early treatment. At present, tumor diagnosis mainly relies on imaging examination, cytology, biochemistry and immunology tests. The latter is the main examination of serum tumor markers (tumor markers, TM). Due to the relatively high sensitivity and specificity of TM, it is more economical, less painful during the examination, and suitable for the detection of large quantities of spec...

Claims

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Application Information

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IPC IPC(8): G01N33/574G01N33/573
Inventor 卢仁泉郭林
Owner FUDAN UNIV SHANGHAI CANCER CENT
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