Diagnostic kit for in-vitro quantitative determination of GPI (Glucose-6-phosphateisomerase) with latex-enhanced immunoturbidimetry

An immunoturbidimetric and latex-enhanced technology, applied in disease diagnosis, measurement devices, instruments, etc., can solve the problems of cumbersome steps, unfavorable large-scale detection, time-consuming and labor-intensive, etc., and achieve the effect of easy and fast use.

Inactive Publication Date: 2016-01-06
ENZYMAKER LABCHANGZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the ELISA method has low requirements for equipment, the steps are cumbersome, time-consuming and labor-intensive, which is not conducive to large-scale detection

Method used

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  • Diagnostic kit for in-vitro quantitative determination of GPI (Glucose-6-phosphateisomerase) with latex-enhanced immunoturbidimetry
  • Diagnostic kit for in-vitro quantitative determination of GPI (Glucose-6-phosphateisomerase) with latex-enhanced immunoturbidimetry
  • Diagnostic kit for in-vitro quantitative determination of GPI (Glucose-6-phosphateisomerase) with latex-enhanced immunoturbidimetry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Preparation of goat anti-human GPI polyclonal antibody by affinity purification

[0032] Preparation of goat anti-human GPI polyclonal antibody:

[0033] Take healthy immunized animals and immunize with GPI antigen. For the first time, mix the antigen with BCG and Freund’s adjuvant, inject the animal’s cervical lymph nodes and subcutaneous muscle tissue, once every two weeks, and immunize 4-5 times in total. The amount of antigen depends on Animal weight gain and loss, blood collection two weeks after the last immunization, animal anti-human serum was preliminarily separated by secondary precipitation with saturated ammonium sulfate.

[0034] Purification of goat anti-human polyclonal antibody:

[0035] First, the separation column used for affinity purification was prepared: 10 mg of highly purified GPI antigen was immobilized on a HITRAPNHS-active HP column (from AmershamPharmaciaBiotech);

[0036] Next, sample loading and elution: Dilute the initially se...

Embodiment 2

[0038] Embodiment 2: Preparation of GPIR2 reagent

[0039] The process is divided into three steps: antibody cross-linking, latex washing, latex suspension

[0040] Antibody cross-linking: Dilute 0.5 mg of the antibody prepared above with 5 ml of 0.1M phosphate buffer (pH7.8), add surface-modified carboxyl groups, polystyrene latex solution (from JSR) with a diameter of 120 nm, and then add 5 mg of EDAC. React at 37°C for 6 hours, add 0.5ml of 0.1M glycine buffer (pH8.5) and stir for 1 hour to terminate the reaction.

[0041] Latex cleaning: Centrifuge to remove the supernatant to remove excess antibodies, cross-linking agents and by-products of cross-linking reactions, etc., and the bottom sediment is the coated latex. Wash 3 times with 20 ml of 50 mM glycine-NaOH buffer.

[0042] Latex suspension: Add 20ml of the same glycine-NaOH buffer solution to the above precipitation, ultrasonic treatment for 4min, and mix well to obtain a milky white latex suspension, which is the R...

Embodiment 3

[0044] Example 3: Preparation of GPIR1 reagent

[0045] Add 0.9% sodium chloride, 0.2% Tween 20, 5% PEG-6000, 0.2% BSA, 0.5% EDTA, 0.1% sodium azide to 50mM TRIS-HCL buffer at pH 7.2, and stir evenly to obtain R1 reagent.

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Abstract

The invention relates to a kit for determination of human GPI (Glucose-6-phosphateisomerase) content with a latex-enhanced immunoturbidimetry. The kit comprises a GPI R1 reagent, a GPI R2 reagent and a GPI calibrator, wherein the GPI R1 reagent comprises an electrolyte, a coagulant, a stabilizer, a surfactant, a preservative and a buffer solution; the GPI R2 reagent comprises human GPI resistant polyclonal antibody coated latex particles, an electrolyte, a stabilizer, a surfactant, a preservative and a buffer solution; the GPI calibrator comprises a preservative, an electrolyte, a stabilizer, a GPI antigen and a buffer solution. The kit is high in specificity, high in sensitivity, uniform, stable, rapid, convenient and applicable to various biochemical analyzers.

Description

technical field [0001] The invention relates to a kit for measuring the content of GPI (Glucose-6-phosphate isomerase, Glucose-6-phosphateisomerase) in human body fluids (including serum and joint effusion) by using a latex-enhanced immune turbidimetric method, which belongs to medical immunology in vitro diagnosis field. Background technique [0002] Glucose-6-phosphate isomerase (GPI or G6PI) is an important enzyme in the process of glycolysis and gluconeogenesis. GPI deficiency can cause non-spherical hemolytic anemia in humans. Its concentration in the serum and joint fluid of patients with rheumatoid arthritis (RA) was significantly increased, and its concentration was also found to be significantly increased in various cancers such as gastrointestinal cancer, thymus cancer, and kidney cancer. [0003] GPI is widely present in the cytoplasm of various cells. Current studies have found that there are 24 isomers of GPI, and their structural characteristics are very simil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573
CPCG01N33/573G01N2800/102
Inventor 朱钰峰周亚萍曹利民
Owner ENZYMAKER LABCHANGZHOU CO LTD
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