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A kind of c-peptide monoclonal antibody cross-linked magnetic particle and its preparation method and detection kit including it

A monoclonal antibody and detection kit technology, applied in the field of in vitro diagnostic medical testing, can solve problems such as insufficient sensitivity, different BSA specificities, and non-specific binding.

Active Publication Date: 2016-05-04
SHANGHAI KEHUA BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the diluent for diluting the C-peptidase reagent is required in the currently used C-peptide detection kit, but the diluent also affects the stability of the C-peptidase reagent
At present, the C-peptide detection kits in the prior art have disadvantages such as non-specific binding, insufficient sensitivity, weak anti-interference, and poor stability of detection results.
The existing magnetic particles are directly washed and blocked with some irrelevant proteins (such as BSA) after cross-linking, but there are batch-to-batch differences in BSA, and the specificity of different BSAs is also different. Direct blocking with BSA often cannot fully achieve the ideal. The closing effect of

Method used

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  • A kind of c-peptide monoclonal antibody cross-linked magnetic particle and its preparation method and detection kit including it
  • A kind of c-peptide monoclonal antibody cross-linked magnetic particle and its preparation method and detection kit including it
  • A kind of c-peptide monoclonal antibody cross-linked magnetic particle and its preparation method and detection kit including it

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preparation example Construction

[0059] Correspondingly, the present invention also provides a method for preparing the above-mentioned C-peptide monoclonal antibody cross-linked magnetic particles, the method comprising the following steps:

[0060] a) cross-linking the C-peptide monoclonal antibody to the magnetic particles; and

[0061] b) Pretreating the C-peptide monoclonal antibody cross-linked magnetic particles obtained in step a) with a non-ionic surfactant.

[0062] The C-peptide monoclonal antibody cross-linked magnetic particle of the present invention can be used to detect C-peptide or prepare a C-peptide detection kit, thereby significantly improving the sensitivity of C-peptide detection.

[0063] The term "nonionic surfactant" as used herein has the meaning commonly understood by those of ordinary skill in the art, ie, a surfactant that does not generate ions in aqueous solution. Nonionic surfactants do not dissociate when dissolved in water, and the lipophilic groups in their molecules are r...

Embodiment 1

[0135] Example 1. Preparation of C-peptide monoclonal antibody cross-linked magnetic particles

[0136] 1) Take 10 mg of 1.0 μm magnetic particles whose active functional groups are carboxyl groups, and wash them twice with 50 mM MES buffer at pH 6.0;

[0137] 2) Remove the supernatant after magnetic suction, add 0.5mL of 50mM pH6.0 MES buffer and mix well, then add 0.5mL of 25mg / mL carbodiimide (EDC) solution and mix well;

[0138] 3) react at room temperature for 30 minutes;

[0139] 4) Remove the supernatant after magnetic suction, and wash twice with 50mM pH6.0 MES buffer;

[0140] 5) Add 0.05 mg of the C-peptide monoclonal antibody obtained in Example 1, then dilute to 1 mL with 50 mM MES buffer at pH 6.0, and mix well;

[0141] 6) React overnight at 37°C;

[0142] 7) Wash twice with a solution containing 50 mM pH7.8 Tris-HCl, 150 mM NaCl, and 1% BSA.

[0143] 8) Add 2 mL of a solution containing 50 mM pH7.8 Tris-HCl, 150 mM NaCl, and 1% BSA, and mix well.

[0144] 9...

Embodiment 2

[0148] Example 2. Pretreatment of C-peptide magnetic particle cross-linked product

[0149] 1) the nonionic surfactant PE6400 and PE6200, diluted with purified water to 0.1% (mass percentage concentration)

[0150] 2) After the procedure in Example 1.6), magnetically absorb and remove the supernatant.

[0151] 3) Add C-peptide magnetic particle cross-linked product to 0.1% PE6400 solution or 0.1% In the PE6200 solution, the final concentration of the cross-linked magnetic particles is 10mg / mL; or add the cross-linked C-peptide magnetic particles to 0.1% PE6400 solution and 0.1% In the mixed solution of PE6200 solution (volume ratio: 1:1), the final concentration of the cross-linked magnetic particles is 10mg / mL, and mix at 15-30°C for 90 minutes;

[0152] 4) Continue the procedure of Example 1.7) and subsequent steps.

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Abstract

The invention discloses a C-peptide monoclonal antibody cross-linked with magnetic particles, a preparation method thereof and a C-peptide test kit including the same. The C-peptide monoclonal antibody is pretreated with a non-ionic surfactant after cross-linked with the magnetic particles. The invention further discloses a C-peptide enzyme conjugate diluent with trehalose for improving the stability of the enzyme conjugate. The C-peptide test kit has the advantages of high sensitivity, good specificity, low drug interfere and good stability in results, thereby being applicable to clinical diagnosis and screening of diabetes widely.

Description

technical field [0001] The invention relates to the field of in vitro diagnostic medical testing. Specifically, the present invention relates to a C-peptide monoclonal antibody cross-linked magnetic particle, a preparation method thereof and a C-peptide detection kit comprising the same. Background technique [0002] C-peptide is a part of proinsulin synthesized by β islet cells in the pancreas. Before it is secreted from islet cells, proinsulin mostly exists in the form of an 86-molecule amino acid polypeptide, including two cystine-linked polypeptides, one is Insulin with 51 amino acid molecules is the A chain of 21 amino acids and the B chain of 30 amino acids, and the other is the C peptide of 30 amino acid molecules and two 2 peptides. [0003] C-peptide and insulin are secreted in equal amounts, but the concentration of C-peptide in the blood is much higher than that of insulin. The main reasons are as follows: (1) C-peptide is different from insulin, it is not metab...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/553
CPCG01N33/531G01N33/553G01N33/577G01N33/68
Inventor 陈超李基彭波孙小禁
Owner SHANGHAI KEHUA BIO ENG
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