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Human insulin monoclonal antibody crosslinking magnetic particle as well as preparation method thereof and human insulin detection kit comprising human insulin monoclonal antibody crosslinking magnetic particle

A monoclonal antibody and detection kit technology, applied in the field of in vitro diagnostic medical testing, can solve the problems of poor repeatability of insulin kits, unfavorable promotion of insulin detection, poor clinical application prospects, etc.

Active Publication Date: 2014-09-03
SHANGHAI KEHUA BIO ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although there are already registered and approved insulin chemiluminescence immunoassay assay kits in the Chinese market, most of them are imported reagents with high cost, and the preparation methods and related materials of the kits are kept as confidential information and have not been made public, which is not conducive to Promotion of Insulin Testing in Community Primary Hospitals
Domestic insulin kits have poor repeatability, difficult to control batch-to-batch differences, low sensitivity, high cross-reaction rate, and poor clinical application prospects

Method used

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  • Human insulin monoclonal antibody crosslinking magnetic particle as well as preparation method thereof and human insulin detection kit comprising human insulin monoclonal antibody crosslinking magnetic particle
  • Human insulin monoclonal antibody crosslinking magnetic particle as well as preparation method thereof and human insulin detection kit comprising human insulin monoclonal antibody crosslinking magnetic particle
  • Human insulin monoclonal antibody crosslinking magnetic particle as well as preparation method thereof and human insulin detection kit comprising human insulin monoclonal antibody crosslinking magnetic particle

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preparation example Construction

[0052] Correspondingly, the present invention also provides a method for preparing the above-mentioned human insulin monoclonal antibody cross-linked magnetic particles, the method comprising the following steps:

[0053] a) treating the human insulin monoclonal antibody with a nonionic surfactant; and

[0054] b) cross-linking the human insulin monoclonal antibody treated in step a) with magnetic particles.

[0055] The human insulin monoclonal antibody cross-linked magnetic particles of the present invention can be used to detect human insulin or prepare a human insulin detection kit, thereby significantly improving the sensitivity of human insulin detection.

[0056] 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 roughly the...

Embodiment 1

[0124] Example 1. Pretreatment of human insulin monoclonal antibody

[0125] 1) Dilute the human insulin antibody (BM1, Roche) with 10mM pH7.40 PBS solution to 2mg / ml;

[0126] 2) the nonionic surfactant LF221 and F68, respectively diluted to 1% (mass percentage concentration) with purified water;

[0127] 3) Mix human insulin antibody with 1% LF221 or 1% F68 is mixed according to the volume ratio of 9:1; or human insulin antibody and 1% LF221, 1% For F68, mix according to the volume ratio of 8:1:1, and mix for 30 minutes at 2-8°C;

[0128] 4) Place the treated insulin antibody in 10mM pH7.40 PBS solution, and dialyze at 37°C for 4-8 hours;

[0129] 5) Take out the dialysate and keep the insulin antibody at 2-8°C for use.

Embodiment 2

[0130] Example 2. Preparation of Human Insulin Monoclonal Antibody Crosslinked Magnetic Particles

[0131] 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;

[0132] 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;

[0133] 3) react at room temperature for 30 minutes;

[0134] 4) Remove the supernatant after magnetic suction, and wash twice with 50mM MES buffer at pH 6.0;

[0135] 5) Add 0.05 mg of insulin monoclonal antibody obtained in Example 1, then dilute to 1 mL with 50 mM MES buffer at pH 6.0, and mix well;

[0136] 6) React overnight at 37°C;

[0137] 7) Wash twice with a solution containing 50 mM pH7.8 Tris-HCl, 150 mM NaCl, and 1% BSA.

[0138] 8) Add 2 mL of a solution containing 50 mM pH7.8 Tris-HCl, 150 mM NaCl, and 1% BSA, and mix thoroughly.

[01...

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Abstract

The invention discloses a human insulin monoclonal antibody crosslinking magnetic particle as well as a preparation method thereof and a human insulin detection kit comprising the human insulin monoclonal antibody crosslinking magnetic particle. Before the human insulin monoclonal antibody is cross-linked with the magnetic particles, the human insulin monoclonal antibody is preprocessed by utilizing a nonionic surface active agent. The human insulin detection kit has the advantages of high detection sensitivity, strong specificity, small medicine interference, good result stability and the like; the human insulin monoclonal antibody crosslinking magnetic particle can be widely suitable for clinical auxiliary diagnosis and screening of diabetes.

Description

technical field [0001] The invention relates to the field of in vitro diagnostic medical testing. Specifically, the present invention relates to a human insulin monoclonal antibody cross-linked magnetic particle, a preparation method thereof and a human insulin detection kit comprising the same. Background technique [0002] Insulin is a protein hormone composed of 51 amino acids, which is composed of two peptide chains, ie, chain A (containing 21 amino acids) and chain B (containing 30 amino acids), connected by disulfide bonds, with a relative molecular weight of 5808. Insulin is divided into three major regions in molecular conformation: the molecular core, the hydrophobic region on the molecular surface and the polar region on the molecular surface. The molecular core is composed of hydrophobic residues, including most of the branched-chain amino acid residues and four cysteine ​​residues of A6, A11, A20, and B19. [0003] Insulin has two main uses after secretion. On...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531
CPCG01N33/54333G01N33/74
Inventor 陈超彭波孙小禁李基
Owner SHANGHAI KEHUA BIO ENG
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