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Syphilis cardiolipin and specific antibody IgG (Immunoglobulin G) immunoblotting kit and preparation method thereof

An immunoblotting and specific technology, applied in the field of reagent kits, can solve the problems of high price and achieve the effect of low cost, wide application, simple and fast detection

Inactive Publication Date: 2011-06-01
ZHONGSHAN HOSPITAL XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The commercially available Western-blot reagents are all imported reagents, which are expensive

Method used

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  • Syphilis cardiolipin and specific antibody IgG (Immunoglobulin G) immunoblotting kit and preparation method thereof
  • Syphilis cardiolipin and specific antibody IgG (Immunoglobulin G) immunoblotting kit and preparation method thereof
  • Syphilis cardiolipin and specific antibody IgG (Immunoglobulin G) immunoblotting kit and preparation method thereof

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preparation example Construction

[0036] The preparation method of described syphilis-specific IgG antibody and cardiolipin IgG antibody immunoblotting kit comprises the following steps:

[0037] 1) Preparation of TPN15, TPN17, TPN37, TPN44.5, TPN47 syphilis-specific recombinant antigens

[0038] Using gene cloning technology, PCR amplifies DNA encoding Treponema pallidum antigen, inserts it into Escherichia coli for expression, and obtains TPN15, TPN17, TPN37, TPN44.5, TPN47 syphilis-specific recombinant antigens.

[0039] 2) Preparation of cardiolipin antigen

[0040] The pig myocardial tissue is quick-frozen at low temperature, air-dried, ground into powder, extracted with methanol and chloroform, and the extract is eluted through a silica gel chromatography column to extract cardiolipin antigen with a purity of more than 95%. .

[0041] 3) Spotting on nitrocellulose membrane

[0042] TPN15, TPN17, TPN37, TPN44.5, TPN47 recombinant antigens and cardiolipin antigens were coated on the nitrocellulose membr...

Embodiment 1

[0070] Paste the nitrocellulose membrane on the surface of the carrier plate, the syphilis-specific IgG antibody and cardiolipin IgG antibody detection line and the control line are set on the nitrocellulose membrane in turn; the syphilis-specific IgG antibody and cardiolipin IgG antibody detection line are coated TPN15, TPN17, TPN37, TPN44.5, TPN47 syphilis-specific recombinant antigens and cardiolipin antigens, coated with human IgG antibodies at the control line, cut into strips with a strip cutter, and enzyme-labeled anti-human IgG specific fragments The γ-chain monoclonal antibody and its substrate are assembled together into a syphilis-specific IgG antibody and a cardiolipin IgG antibody immunoblotting kit.

[0071] 1) The sample was diluted 1:50 with 0.01mol / L PBS buffer.

[0072] 2) Blocking: 5% skimmed milk powder (prepared with 0.01 mol / LPBST buffer) was used as the blocking solution, and incubated at room temperature (18-25° C.) on a rocking table for 30 min.

[00...

Embodiment 2

[0081] Similar to Example 1, the difference is that the nitrocellulose membrane only consists of TPN15, TPN17, TPN37, TPN44.5 syphilis-specific recombinant antigens and cardiolipin antigens, and does not contain TPN47 syphilis-specific recombinant antigens. Result judgment is identical with embodiment 1.

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Abstract

The invention discloses a syphilis cardiolipin and specific antibody IgG (Immunoglobulin G) immunoblotting kit and a preparation method thereof, relating to a kit. The kit is provided with a vector plate, a nitrocellulose membrane, a syphilis specific IgG antibody and cardiolipin IgG antibody detecting line and a contrast line, wherein the detecting line and the contrast line are arranged on the nitrocellulose membrane in sequence; a syphilis specific recombination antigen and a cardiolipin antigen are coated at the syphilis specific IgG antibody and cardiolipin IgG antibody detecting line, and a human IgG antibody is coated at the contrast line; and a horse radish peroxidase is marked with an antihuman gamma chain antibody. The preparation method comprises the following steps of: preparing the syphilis specific recombination antigen firstly and then preparing the cardiolipin antigen; then sampling the nitrocellulose membrane; preparing the antihuman IgG specific fragment gamma chain monoclonal antibody; and after marking the horse radish peroxidase with the antihuman IgG specific fragment gamma chain monoclonal antibody, preparing the immunoblotting kit. The syphilis cardiolipin and specific antibody IgG immunoblotting kit can be used for the detection of a syphilis specific IgG antibody and a cardiolipin IgG antibody in specimens, such as whole blood, serum, blood plasma, cerebrospinal fluid, and the like.

Description

technical field [0001] The invention relates to a kit, in particular to a immunoblotting kit for detecting syphilis-specific IgG antibody and cardiolipin IgG antibody and a preparation method thereof. Background technique [0002] Syphilis (Syphilis) is a sexually transmitted disease caused by Treponema pallidum (TP), and its pathogen is Treponema pallidum, which belongs to the family Treponemaceae. Treponema pallidum is mainly transmitted through sexual contact, blood transfusion, wound or placenta. Treponema pallidum enters the blood from the lymph nodes near the infected area, spreads throughout the body, and affects almost all tissues and organs of the body. The World Health Organization (WHO) once predicted optimistically: "Due to the availability of highly sensitive detection methods and efficient treatment options, syphilis is a sexually transmitted disease that can be successfully controlled through public health measures." Regrettably, syphilis is still a worldwid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/571G01N33/558G01N33/532
Inventor 张忠英刘莉莉林丽蓉杨天赐
Owner ZHONGSHAN HOSPITAL XIAMEN UNIV
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