Treponema pallidum IgM antibody Dot-ELISA detection method

A technology of treponema pallidum and detection method, which is applied in the direction of measuring devices, biological tests, material inspection products, etc., can solve the problems of serological detection sensitivity, low specificity, high clinical missed diagnosis and misdiagnosis rate, and low sensitivity, so as to avoid Antigen denaturation, extended storage time, and wide application range

Inactive Publication Date: 2016-05-04
SHENZHEN RES INST OF XIAMEN UNIV +1
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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

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  • Treponema pallidum IgM antibody Dot-ELISA detection method

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Experimental program
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Effect test

Embodiment 1

[0028] see figure 1 , the dot-ELISA detection method of described Treponema pallidum specific IgM antibody, comprises the steps:

[0029] (1) Preparation of Treponema pallidum-specific recombinant antigen:

[0030] Using gene cloning technology, PCR amplifies the DNA encoding Treponema pallidum antigen, inserts it into Escherichia coli for expression, and obtains Treponema pallidum-specific recombinant antigens TPN17 and TPN47.

[0031] (2) Preparation of nitrocellulose membrane coated with recombinant antigen:

[0032] Use a circular puncher with a diameter of 6 mm to press on the smooth surface of nitrocellulose to form a circular hole as the spotting site; use a micropipette to take 0.5 μL of the antigen mixture and add it to the nitrocellulose membrane. In the center of the imprint, dry at room temperature; immerse the nitrocellulose membrane after spotting in 5% skimmed milk powder, and seal at room temperature for 30 minutes; place the sealed nitrocellulose membrane in...

Embodiment 2

[0049] The dot-ELISA detection method for Treponema pallidum-specific IgM antibodies is given below to detect Treponema pallidum antibodies in clinical samples of patients:

[0050](1) Test specimen processing:

[0051] Take 5 mL of human venous blood, put it in a water bath at 37°C for 30 minutes, centrifuge at 3000 g for 10 minutes, and use the supernatant as a test sample for later use.

[0052] (2) Sample addition:

[0053] Add 50 μL of the sample to be tested to each well of the polystyrene micro-reaction plate wells that have been placed in the rapid diagnosis membrane, and incubate at 37 °C for 10 min.

[0054] (3) Nitrocellulose membrane washing:

[0055] Add 350μL of 0.01mmol / L pH7.4 phosphate buffer to fully wash 3 times, each time for 2min, and discard the liquid after each washing.

[0056] (4) Add 50 μL of horseradish peroxide-labeled anti-human μ chain monoclonal antibody, and incubate at 37° C. for 10 min.

[0057] (5) 350μL 0.01mmol / L pH7.4 phosphate buffer...

Embodiment 3

[0062] The stability test of the dot-ELISA detection method for Treponema pallidum-specific IgM antibody is given below:

[0063] (1) Appearance inspection: The white coated reaction film is smooth, clean, free of pollution spots and cracks, the adhesive tape has no glue opening, and no cutting oblique phenomenon.

[0064] (2) Coincidence rate of positive samples: 50 copies of positive reference sera with different titers positive for Treponema pallidum IgM antibody were tested by dot-ELISA detection method for Treponema pallidum-specific IgM antibody, and the positive coincidence rate was calculated. The positive reference serum was determined by clinical specimens determined by the TPPA (Fuji Corporation, Japan) method.

[0065] (3) Negative specimen coincidence rate: use 50 negative reference sera to test, and calculate the positive coincidence rate. The negative reference serum was determined by clinical specimens determined by the TPPA (Fuji Corporation, Japan) method. ...

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Abstract

The invention discloses a treponema pallidum IgM antibody Dot-ELISA detection method, and relates to syphilis specificity IgM antibody detection. The method comprises the steps that a nitrocellulose membrane coated with a recombinant antigen is prepared; an anti-human gamma chain monoclonal antibody is prepared; horse radish peroxidase labeling is carried out on an anti-human mu chain monoclonal antibody; the nitrocellulose membrane is washed; the anti-human mu chain monoclonal antibody labeled by horse radish peroxidase is added for incubation; phosphate buffer is adopted for washing, and developing is carried out; a phosphate buffer scrubbing solution is added for rinsing, and liquid is abandoned; results are judged, wherein the result is positive if spots on the nitrocellulose membrane are brown, and the result is negative if spots are colorless. The nitrocellulose membrane coated with the recombinant antigen prolongs the preservation time of the coating antigen, independent preservation of a single sample can be achieved, and antigen degeneration caused by multigelation is avoided; by applying the spot enzyme linked immunosorbent assay to detecting the syphilis specificity IgM antibody, no special instrument is needed, large-scale screening can be achieved, the application range is wide, and practicability is high.

Description

technical field [0001] The invention relates to the detection of syphilis-specific IgM antibodies, in particular to a Dot-ELISA detection method for Treponema pallidum IgM antibodies. 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 Applications(China)
IPC IPC(8): G01N33/68G01N33/569G01N33/545
CPCG01N33/68G01N33/545G01N33/56911G01N2333/20
Inventor 杨天赐童曼莉张惠林林丽蓉刘莉莉
Owner SHENZHEN RES INST OF XIAMEN UNIV
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