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Treponema pallidum igm antibody biotin avidin ELISA kit and preparation method thereof

An ELISA technology for Treponema pallidum, applied in measuring devices, instruments, scientific instruments, etc., can solve the problems of low sensitivity and specificity of serological detection, inability to culture Treponema pallidum in vitro, clinical missed diagnosis and high rate of misdiagnosis , to improve the sensitivity and specificity, shorten the window period, and achieve the effect of accurate results

Active Publication Date: 2016-01-06
ZHONGSHAN HOSPITAL XIAMEN UNIV
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  • Abstract
  • Description
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Problems solved by technology

[0004] (1) Etiological detection: In the early stage of Treponema pallidum infection, when the syphilis antibody has not yet been produced or the content is low, dark field microscopy to search for Treponema pallidum has become the earliest laboratory diagnostic method, but it is easily affected by the sampling technique, sampling site, and Treponema pallidum in the sample. Affected by many factors such as content, topical medication and delivery time, the sensitivity is low
[0005] (2) Antibody detection test: Treponema pallidum cannot be cultured in vitro, and the diagnosis mainly depends on laboratory examination. The sensitivity and specificity of the existing serological detection are not high, and any detection method has defects. high
[0006] 用于梅毒血清学检验的抗原主要以重组抗原TPN15、TPN17、TPN37、TPN44.5、TPN47为主([2]Lin,L.R.,Z.G.Fu,etal."Developmentofacolloidalgold-immunochromatographyassaytodetectimmunoglobulinGantibodiestoTreponemapallidumwithTPN17andTPN47."Diagnosticmicrobiologyandinfectiousdisease.2010.68(3) :193-200.), most of these antigens are derived from the outer membrane of Treponema pallidum, but it cannot be distinguished whether it is caused by pathogenic Treponema

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  • Treponema pallidum igm antibody biotin avidin ELISA kit and preparation method thereof

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preparation example Construction

[0057] The preparation method of described Treponema pallidum IgM antibody biotin avidin ELISA kit comprises the following steps:

[0058] 1) Extract the outer membrane proteins of Treponema pallidum Nichol and Reiter strains for two-dimensional electrophoresis, transfer them to nitrocellulose membranes, and perform immunoblotting experiments with syphilis serum to obtain characteristic immunoblotting spots, and take corresponding protein spots for mass spectrometry analysis . Find out the outer membrane proteins with high immunogenicity of Nichol strain and Reiter strain respectively, and find out the highly immunogenic outer membrane protein unique to Nichol strain but lacking in Reiter strain;

[0059] 2) Find the corresponding gene in the whole genome sequence of Treponema pallidum, design primers and construct the corresponding expression vector, study the effect of different induction conditions on the expression of the recombinant target protein in E. coli, obtain the b...

Embodiment 1

[0075] The preparation method of described Treponema pallidum IgM antibody biotin avidin ELISA kit comprises the following steps:

[0076] (1) Immunoproteomics study of outer membrane protein:

[0077] The outer membrane proteins of Treponema pallidum Nichol and Reiter strains were extracted for two-dimensional electrophoresis, then transferred to nitrocellulose membranes, and immunoblotting experiments were performed with syphilis serum to obtain characteristic immunoblotting spots, and corresponding protein spots were taken for mass spectrometry analysis. Find out the outer membrane proteins with higher immunogenicity of Nichol strain and Reiter strain respectively, and find out the outer membrane protein with high immunogenicity unique to Nichol strain but lacking in Reiter strain.

[0078] (2) Cloning and purification of candidate proteins

[0079] Find the corresponding gene in the whole genome sequence of Treponema pallidum, design primers and construct the correspondin...

Embodiment 2

[0105] The Treponema pallidum antibody in clinical specimens detected by the Treponema pallidum IgM antibody biotin avidin enzyme-linked immunoassay kit is given below:

[0106] (1) Specimen processing: Serum: 5 mL of venous blood, placed in a 37°C water bath for 30 minutes, centrifuged at 3000 g for 10 minutes, and the supernatant was used as a test sample for later use.

[0107] (2) Adding samples: add 100 uL of the sample and 50 uL of biotin-labeled anti-human IgM specific fragment μ chain monoclonal antibody to the reaction wells, and simultaneously serve as blank, negative and positive control wells. Incubate at 37°C for 1h.

[0108] (3) Washing: wash five times with washing buffer and then buckle dry.

[0109] (4) Enzyme addition: 100 μL of horseradish peroxidase-labeled avidin was added to each well, and incubated at 37° C. for 30 min.

[0110] (5) Washing: wash five times with washing buffer and then buckle dry.

[0111] (6) Color development: add 50 μL each of colo...

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Abstract

The invention relates to a treponema pallidum IgM antibody biotin avidin enzyme-linked immune detection kit and a preparation method thereof, which relates to the syphilis detection. The kit comprises a concentrated washing liquid bottle, a negative and positive control bottle, a biotin mark antihuman IgM specific fragment mum chain monoclonal antibody bottle, a horseradish peroxidase mark avidin bottle, a TMB coloured solution bottle A, a coloured solution bottle B, a reaction stopping solution bottle, a micropore plate coated with recombinant antigen. The treponema pallidum has infectious standard strain Nichol strain and treponema pallidum non-infectious standard strain Reiter strain, by using a proteomics method, the treponema pallidum proteomics can be separated through dimensional electrophoresis, protein with strong immunogenicity can be identified by different patient or infectious animal serum, and then an antigen marker can be searched. The antigen marker is used for syphilis confirmation test, so that diagnosis sensitivity and specificity can be increased, a window stage is shortened. The treponema pallidum antibody can be sensitively detected and enables quantitative measurement, and has the advantages of cheap price, simple operation and accurate result.

Description

technical field [0001] The invention relates to the detection of syphilis, in particular to a treponema pallidum IgM antibody biotin avidin ELISA kit and a preparation method thereof. Background technique [0002] Syphilis is a sexually transmitted disease caused by Treponema pallidum. In recent years, the incidence rate in China has remained high. The prevention and control of syphilis has become one of the main tasks of public health services in my country. [0003] The laboratory diagnosis methods of syphilis are mainly as follows ([1] Lin Lirong, Yang Bo, Pan Xitao, etc. Selection of serological detection methods for syphilis in patients with potential blood-borne transmission. Chinese Journal of Hospital Infection. 2010, 20(10): 1491-1494): [0004] (1) Etiological detection: In the early stage of Treponema pallidum infection, when the syphilis antibody has not yet been produced or the content is low, dark field microscopy to search for Treponema pallidum has become th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577
CPCG01N33/571G01N33/6803
Inventor 童曼莉林丽蓉刘莉莉张惠林杨天赐张长弓
Owner ZHONGSHAN HOSPITAL XIAMEN UNIV
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