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Golgi protein gp73 latex-enhanced immunoturbidimetric detection kit and preparation method thereof

A technology of kits and latex microspheres, which is applied in the field of medical immunology in vitro diagnosis, and achieves the effects of high stability, good experimental repeatability and high sensitivity

Inactive Publication Date: 2016-08-17
ZHONGSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] There is no report on the detection of Golgi protein gp73 in human serum by PETIA method

Method used

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  • Golgi protein gp73 latex-enhanced immunoturbidimetric detection kit and preparation method thereof
  • Golgi protein gp73 latex-enhanced immunoturbidimetric detection kit and preparation method thereof
  • Golgi protein gp73 latex-enhanced immunoturbidimetric detection kit and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043]Example 1 Preparation of latex microspheres coupled with gp73 antibody of the present invention (1)

[0044] 1. Reagent preparation and standards

[0045] Preparation of recombinant protein gp73: for the use of serum-free medium, secreted and expressed by CHO cells, the expressed recombinant protein gp73 (amino acid sequence is as shown in SEQ ID NO.1) was subjected to nickel affinity chromatography and ion exchange chromatography, and the target protein It is a single band on the SDS-PAGE gel, and the purity is above 98%.

[0046] Preparation of gp73 antibody: the polyclonal antibody obtained by immunizing New Zealand white rabbits with purified recombinant protein gp73, the serum titer reached 1:16 by agarose diffusion test, and the antiserum was purified by gp73 antigen.

[0047] 100nm carboxylated polystyrene latex microspheres (SWA04): purchased from Zhejiang Tianke High-tech Development Co., Ltd.

[0048] All other reagents were of domestic analytical grade or re...

Embodiment 2

[0056] Example 2 The gp73 latex-enhanced immune turbidimetric detection kit of the present invention

[0057] The kit includes: reagent R1, reagent R2 and gp73 protein standard. Reagent R1 is a buffer solution containing preservatives and stabilizers, which is mainly used to dilute the sample to be tested; reagent R2 is mainly latex microspheres coupled with gp73 antibody; the gp73 protein standard product is recombinant protein gp73. The gp73 protein in the sample or standard product is diluted with reagent R1 and mixed with reagent R2. The gp73 protein interacts with the gp73 antibody, causing latex microspheres to coagulate, resulting in changes in the light transmission or light scattering intensity of the solution. The light transmission within a certain range The intensity or light scattering intensity is linearly related to gp73 protein concentration.

[0058] Reagent R1, reagent R2 and gp73 standard are preferably:

[0059] Preparation of Reagent R1

[0060] Prepare...

Embodiment 3

[0065] Embodiment 3 Sensitivity and precision detection of the inventive method

[0066] 1. Instrument

[0067] Hitachi automatic biochemical analyzer 7080.

[0068] 2. Operation method

[0069] Set up Take 50 μL of standard, 100 μL of R1 solution and 150 μL of R2 solution. The detection wavelength was set at 500nm, and after the instrument was zeroed with distilled water, the absorbance values ​​of 5 gradients of the standard were measured and repeated 3 times. 3. Results

[0070] Data fitting curve see figure 1 . It can be seen from the figure that the method of the present invention has high sensitivity, the minimum detection limit is as low as 25ng / mL, and it is still in a good linear range; the linear range is wide.

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Abstract

The invention relates to a latex-enhanced immunoturbidimetry detection kit of Golgi protein gp73 and a preparation method thereof, and provides latex microspheres coupled with gp73 antibody. The preparation method comprises the following steps: (a) catalyzing N-hydroxysuccinimide to react with carboxyl of carboxyl polystyrene latex microspheres with the particle size being 90-110nm by utilizing DCC, reacting for 8-12 minutes at the temperature of 24-26 DEG C, and carrying out centrifugal treatment to remove supernatant; and (b) taking gp73 antibody solution, precooling to not more than 4 DEG C, adding the gp73 antibody solution into activated carboxyl polystyrene latex microspheres prepared in the step (a), carrying out ultrasonic treatment for 4-6 minutes at 110-130W, and reacting for 25-35 minutes under the temperature of 3-5 DEG C. The invention also provides a kit containing the latex microspheres. The latex microspheres coupled with gp73 antibodies is used for detecting the content of the gp73 protein in a sample, is high in linearity range, high in sensitivity, precision and accuracy, good in repeatability and simple in operation, and can be used for diagnosis of related diseases on clinic.

Description

technical field [0001] The invention relates to the technical field of medical immune in vitro diagnosis, in particular to a Golgi protein gp73 latex-enhanced immune turbidimetric detection kit and a preparation method thereof. Background technique [0002] Golgi protein gp73 (gp73, Golm1, Golph2) is a Golgi protein with an apparent molecular weight of 73kD. Furin protease can recognize gp73 and cut off the N-terminal 55 amino acids (including the cytoplasmic region, transmembrane region and 10 amino acids of the coiled-coil domain in the Golgi), which enables Golph2 to be secreted into the blood (Traffic 2007; 8:1415- 1423.). [0003] The content of gp73 in the serum of patients with acute and chronic hepatitis, liver cirrhosis and liver cancer is significantly higher than that of healthy people. The sensitivity, specificity and accuracy of gp73 for the early diagnosis of primary liver cancer reached 72%, 95% and 86.2% respectively (China Oncology Clinic, 2013; v40, Issue...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 叶青海郭磊张博张巨波李小强
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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