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Diagnostic kit for in-vitro quantitative determination of GP73 (Golgi Protein 73) with latex-enhanced immunoturbidimetry

An immunoturbidimetric and latex-enhancing technology, applied in the field of medical immunology in vitro diagnosis, can solve the problems of unusable ELISA kits, labor-intensive and other problems, and achieve the effects of low cost, simple and rapid use, and high titer.

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

ELISA kits cannot be used on fully automatic biochemical instruments and require a lot of manpower, so it is a good direction to develop latex-enhanced immunoturbidimetric assay kits

Method used

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  • Diagnostic kit for in-vitro quantitative determination of GP73 (Golgi Protein 73) with latex-enhanced immunoturbidimetry
  • Diagnostic kit for in-vitro quantitative determination of GP73 (Golgi Protein 73) with latex-enhanced immunoturbidimetry
  • Diagnostic kit for in-vitro quantitative determination of GP73 (Golgi Protein 73) with latex-enhanced immunoturbidimetry

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Preparation of goat anti-human GP73 polyclonal antibody by affinity purification

[0032] Preparation of goat anti-human GP73 polyclonal antibody:

[0033] Take healthy immunized animals and immunize with GP73 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 GP73 antigen was immobilized on a HITRAPNHS-active HP column (from AmershamPharmaciaBiotech);

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

Embodiment 2

[0038] Embodiment 2: Preparation of GP73R2 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 R2...

Embodiment 3

[0044] Embodiment 3: Preparation of GP73R1 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 GP73 (Golgi Protein 73) content with a latex-enhanced immunoturbidimetry. The kit comprises a GP73 R1 reagent, a GP73 R2 reagent and a GP73 calibrator, wherein the GP73 R1 reagent comprises an electrolyte, a coagulant, a stabilizer, a surfactant, a preservative and a buffer solution; the GP73 R2 reagent comprises human GP73 resistant polyclonal antibody coated latex particles, an electrolyte, a stabilizer, a surfactant, a preservative and a buffer solution; the GP73 calibrator comprises a preservative, an electrolyte, a stabilizer, a GP73 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 GP73 (Golgi glycoprotein-73, GolgiProtein73) in human serum by using a latex-enhanced immune turbidimetric method, which belongs to the field of in vitro medical immunodiagnosis. Background technique [0002] GP73 (Golgi Glycoprotein-73, GolgiProtein73) is also known as type II Golgi membrane protein, Type II Golgimembraneprotein, Golph2), because Golph2 polyclonal antiserum can specifically recognize protein molecules with a size of 7.3×104 in human epithelial cell lysates, Therefore, this protein is also called GP73. GP73 is a transmembrane protein present in the Golgi apparatus with a molecular weight of 45KDa. However, GP73 undergoes a large amount of glycosylation in serum, and its molecular weight is about 73KDa in serum detection. Since GP73 is rich in acidic amino acids (5.5% aspartic acid, 12.2% glutamic acid), its isoelectric point PI = 4.72. GP73 is strongly hydrophilic but has hy...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6893G01N33/57484
Inventor 朱钰峰周亚萍曹利民
Owner ENZYMAKER LABCHANGZHOU CO LTD
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