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Latex-enhanced immunoturbidimetry detection kit of Golgi protein gp73 and preparation method thereof

A technology of kits and latex microspheres, applied in the field of medical immunology in vitro diagnosis, to achieve the effects of good experimental repeatability, high accuracy and precision, and high stability

Inactive Publication Date: 2015-04-29
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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  • Latex-enhanced immunoturbidimetry detection kit of Golgi protein gp73 and preparation method thereof
  • Latex-enhanced immunoturbidimetry detection kit of Golgi protein gp73 and preparation method thereof
  • Latex-enhanced immunoturbidimetry detection kit of Golgi protein gp73 and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0044] 1. Reagent preparation and standard

[0045] Preparation of recombinant protein gp73: In order to adopt serum-free medium, it is secreted and expressed by CHO cells. The expressed recombinant protein gp73 (amino acid sequence shown in SEQ ID NO. 1) is subjected to nickel affinity chromatography and ion exchange chromatography. It is a single band on SDS-PAGE gel with a purity of more than 98%.

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

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

[0048] All other reagents are of domestic analytical grade or of biologi...

Embodiment 2

[0056] Example 2 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 containing preservatives and stabilizers, mainly used to dilute the sample to be detected; Reagent R2 is mainly latex microspheres coupled with gp73 antibody; gp73 protein standard is recombinant protein gp73. The gp73 protein in the sample or standard is diluted with reagent R1 and mixed with reagent R2. The gp73 protein and gp73 antibody act to cause latex microspheres to agglomerate, resulting in changes in the light transmission or light scattering intensity of the solution, and light transmission within a certain range. Intensity or light scattering intensity is linearly related to gp73 protein concentration.

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

[0059] Preparation of Reagent R1

[0060] Prepare 50mM Tris-HCl buffer, pH 7.2. Add NaCl, NaN 3 , ...

Embodiment 3

[0065] Example 3 Sensitivity and precision detection of the method of the present invention

[0066] 1. Instrument

[0067] Hitachi automatic biochemical analyzer 7080.

[0068] 2. Operation method

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

[0070] Data fit curve see figure 1 . It can be seen from the figure that the sensitivity of the method of the present invention is high, the minimum detection limit is as low as 25ng / mL, and it is still within 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 in vitro diagnosis of medical immunity, in particular to a Golgi protein gp73 latex-enhanced immune turbidimetric detection kit and a preparation method thereof. Background technique [0002] The Golgi protein gp73 (gp73, Golm1, Golph2) is a Golgi protein with an apparent molecular weight of 73 kD. Furin protease recognizes gp73 and cleaves off the N-terminal 55 amino acids (including the cytoplasmic domain, the transmembrane domain, and the 10 amino acids in the intra-Golgi coiled-coil domain), which enables Golph2 to be secreted into the blood (Traffic 2007;8:1415- 1423.). [0003] Serum levels of gp73 in patients with acute and chronic hepatitis, liver cirrhosis and liver cancer were significantly higher than those in healthy controls. 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...

Claims

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Application Information

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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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