Preparation method of Treponema pallidum total antibody biotin avidin ELISA kit

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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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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  • Preparation method of Treponema pallidum total antibody biotin avidin ELISA kit

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preparation example Construction

[0052] The preparation method of the Treponema pallidum total antibody biotin avidin enzyme-linked immunoassay kit comprises the following steps:

[0053] 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 protein 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; find out in the whole genome sequence of Treponema pallidum Corresponding to the gene, design primers and construct corresponding expression vectors, according to the influence of different induction conditions on the expression of the recombinant target protein in E. co...

Embodiment 1

[0066] The Treponema pallidum total antibody biotin avidin ELISA kit of the present invention consists of an outer packaging box 1, a concentrated washing liquid bottle 2, a negative control bottle 3, a positive control bottle 4, and a biotin-labeled recombinant antigen bottle 5 A bottle of horseradish peroxidase-labeled avidin 6, a bottle of TMB chromogenic solution A 7 , a bottle of chromogenic solution B 8 , a bottle of reaction termination solution 9 , and a microwell plate 10 coated with recombinant antigen.

[0067] The preparation method of the Treponema pallidum total antibody biotin avidin enzyme-linked immunoassay kit comprises the following steps:

[0068] 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 ma...

Embodiment 2

[0092] The following shows the Treponema pallidum antibody in the clinical specimens of patients detected by the Treponema pallidum total antibody biotin avidin enzyme-linked immunoassay kit:

[0093] (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.

[0094] (2) Adding samples: add 100uL of the sample and 50uL of biotin-labeled recombinant antigen to the reaction wells, and use them as blank, negative and positive control wells at the same time. Incubate at 37°C for 1h.

[0095] (3) Washing: Wash 5 times with washing buffer and then buckle dry.

[0096] (4) Enzyme addition: Add 100 μL of avidin-labeled horseradish peroxidase solution to each well, and incubate at 37° C. for 30 min.

[0097] (5) Washing: wash 5 times with washing buffer and then buckle dry.

[0098] (6) Color development: 50 μL each of TMB color development solut...

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Abstract

The invention relates to a treponema pallidum total 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 recombination antigen 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 antibodies, in particular to a biotin avidin enzyme-linked immunoassay kit for treponema pallidum total antibody 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/531
CPCG01N33/571
Inventor 林丽蓉童曼莉刘莉莉张惠林杨天赐张长弓
Owner ZHONGSHAN HOSPITAL XIAMEN UNIV
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