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Kit for detecting anti-mullerian hormone (AMH) by establishing time-resolved fluorescence immunochromatographic assay

A technology of fluorescence immunochromatography and Mullerian hormone, which is applied in biological testing, material inspection products, etc., can solve the problems of long detection time, inability to automate, expensive chemiluminescence detection system, etc., and achieve high output and low cost Effect

Inactive Publication Date: 2018-11-23
CHANGCHUN HENGXIAO BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] Clinically, ELISA is commonly used to detect AMH in patient serum, which has high sensitivity and specificity, but the detection time is long and cannot be automated; chemiluminescence detection of AMH in serum can be detected automatically, but requires an expensive chemiluminescence detection system

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  • Kit for detecting anti-mullerian hormone (AMH) by establishing time-resolved fluorescence immunochromatographic assay
  • Kit for detecting anti-mullerian hormone (AMH) by establishing time-resolved fluorescence immunochromatographic assay
  • Kit for detecting anti-mullerian hormone (AMH) by establishing time-resolved fluorescence immunochromatographic assay

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Embodiment Construction

[0030] 1. Preparation of AMH calibrator

[0031] The kit for detecting AMH by TRFIA chromatography of the present invention needs purified AMH protein as a calibrator.

[0032]RNA was extracted from HGL5 cells, used to synthesize cDNA, and AMH DNA (nucleotide sequence 1-1683) was amplified by PCR. Select the insect cell expression vector pFastBac-Flag, which contains Polyhedrin promoter, and express exogenous protein in insect cells; this vector inserts 8 amino acid residues (DYKDDDDK) short peptide (Flag) DNA sequence for expressing Flag fusion protein; This vector carries a part of AcNPV DNA sequence and is used to produce gene recombinant insect virus (AcNPV) plasmid in HD10Bac bacteria. Cut AMH DNA with endonuclease NdeI / SalI, cut pFastBac-Flag with NdeI / XhoI, connect AMH DNA to pFastBac-Flag NdeI / XhoI site, and construct pFast-Flag-AMH vector; this vector DNA is transfected into HD10Bac bacteria, Generate recombinant Flag-AMH AcNPV plasmid; transduce the plasmid DNA int...

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Abstract

The invention provides a kit for detecting anti-mullerian hormone (AMH) by establishing a time resolution fluorescence immunochromatographic assay. The kit utilizes the long fluorescent lifetime characteristic of a rare earth element to delay measurement, i.e., a time-resolved fluoroimmunoassay (TRFIA) is combined with a lateral flow immunoassay (LFIA), and each step is optimized, so that AHM time-resolved fluoroimmunoassay quantitative determination is realized. The kit is composed of an AMH detecting test paper card and a time-resolved immunofluorescence (TRFIA) analyzer. A preparation method comprises the following steps: expressing AHM by insect cells, and purifying out AHM as a calibration product; enabling an AMH antibody to couple with a rare-earth element fluorescent microsphere; spraying an AMH antibody-fluorescent microsphere onto a combined cushion, spraying the AMH antibody onto a nitrocellulose film, assembling a test paper strip, and adding a housing to form the test paper card; adding the AMH calibration product to the AMH test paper card, detecting by the TRFIA analyzer, and establishing an automatic-display AMH concentration detecting system of the TRFIA analyzer for clinically detecting concentration of AMH in blood.

Description

technical field [0001] The invention relates to the field of gene recombinant protein preparation, expresses and purifies the natural structure Flag-AMH protein, and establishes a TRFIA process for measuring the fluorescence intensity on LFIA test strips. technical background [0002] Anti-Mullerian hormone (AMH) is a member of the transforming growth factor β superfamily. The human AMH encoding gene is located on the short arm of chromosome 19, with a size of 2.4-2.8 kb and 5 exons. AMH is a dimeric glycoprotein composed of two identical 70KDa subunits connected by disulfide bonds, with a molecular mass of 140KDa. [0003] AMH plays an important role in the development of gonad organs and is one of the important markers of gonad function in men and women. In men, AMH is mainly produced by Leydig cells of the testis, starting from embryogenesis and throughout life; during the development of male fetuses, AMH leads to the degeneration of Mullerian ducts and the formation of ...

Claims

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Application Information

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IPC IPC(8): G01N33/74
CPCG01N33/74
Inventor 张蕾李宝民关恒刘戈赵青史永丰刘宁李添伟隋宝珍仇淑园孙晓婷孙净张金玲黄景林高敏林林朱道林孙玉峰王贺
Owner CHANGCHUN HENGXIAO BIOTECH CO LTD