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Derivatized Method for Accurate Quantification of C-Peptide in Serum for Non-Disease Diagnostic Purposes

A technology for disease diagnosis and derivatization, applied in the field of biochemistry, it can solve problems such as differences in size and physical and chemical properties, and limit the reliable binding ability of antibodies, so as to reduce the dosage and simplify the detection process.

Active Publication Date: 2020-08-18
NAT INST OF METROLOGY CHINA
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Problems solved by technology

Since magnetic particles can be prepared using different synthetic methods, their size and physicochemical properties can vary widely, which limits the prediction of reliable binding capacity of antibodies

Method used

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  • Derivatized Method for Accurate Quantification of C-Peptide in Serum for Non-Disease Diagnostic Purposes
  • Derivatized Method for Accurate Quantification of C-Peptide in Serum for Non-Disease Diagnostic Purposes
  • Derivatized Method for Accurate Quantification of C-Peptide in Serum for Non-Disease Diagnostic Purposes

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Embodiment Construction

[0027] 1. Materials

[0028] Ethyl orthosilicate (99.9%, Alfa Aesar, USA), immunomagnetic beads (Thermo Fisher Scientific, Dynabeads MyOne Tosylactivated, USA), serum (provided by China Aerospace General Hospital), C-peptide (Nanjing KingScript Biotechnology Co., Ltd. ), C-peptide antibody (Hytest, Finland, clone#7E10), BSA (National Institute of Metrology), slide glass (Thermo Fisher, USA), succinic acid (ACROS, Belgium, 99%), p-toluenesulfonic acid ( ACROS, Belgium).

[0029] 2. Coupling efficiency of immunomagnetic beads

[0030] The liquid after the incubation of the magnetic beads and the antibody was filtered and injected directly, and the concentrations of p-toluenesulfonic acid standards were accurately prepared by weighing method to be 5.132, 10.264, 20.528 and 41.056 μg / g, and the standards were injected into the liquid phase under the same conditions For detection, measure three times to take the average value of the integral area, and fit a linear curve.

[0031...

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Abstract

The invention discloses a method for accurately quantifying C peptide in serum without derivatization, which aims to improve the source efficiency of C peptide extraction and further improve the quantification limit of the method without derivatization treatment. A composite material of porous PVDF template geometric constraint immunomagnetic beads is prepared by taking aims of increasing contactarea of antibody and antigen and determining a contact direction of antigen and the magnetic beads as a starting point. The prepared PVDF template dispersed immunomagnetic bead composite material is applied to C peptide quantitative limit investigation, and compared with undispersed immunomagnetic beads, the quantification limit is reduced to 0.05ng from 0.3ng on the column after the PVDF templatedispersed immunomagnetic bead composite material is used for extracting pure C peptide products; compared with a C peptide potential reference measurement program published by National Measurement Institute of Japan, the target of quantifying the C peptide in the serum of the normal subject can be reached without derivatization, the detection flow is simplified, and the dosage of the magnetic bead and the antibody is reduced at the same time.

Description

technical field [0001] The invention relates to the technical field of biochemistry, in particular to a derivatization method for accurately quantifying C-peptide in serum. Background technique [0002] Measuring the concentration of C-peptide can know the function of islet cells, which is of great significance to the diagnosis and treatment of diabetes. The commercial immunoassay method of C-peptide is to measure the concentration of C-peptide through C-peptide immunoreactivity, but the detection value is usually high. Isotope dilution mass spectrometry can distinguish between endogenous and exogenous proteins, so that the target substance can be directly and accurately determined. Japan Metrology Institute modified the N-terminal amino group of C peptide with 6-aminoquinolyl-N-hydroxysuccinimidyl carboxylate (AQC). The aminoquinolyl group can increase the ionization efficiency by adding a positive charge to the peptide. Chemical modification and magnetic bead immuno-extr...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/88G01N33/543G01N33/532
CPCG01N30/88G01N33/532G01N33/54326G01N2030/045G01N2030/8822
Inventor 宋德伟职承瑶李红梅李志林王馨雪马凌云刘健仪朱文
Owner NAT INST OF METROLOGY CHINA
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