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Method and reagent box for inspecting mycelian protein antibody of white candida

A technology of Candida albicans and kits, applied in the direction of immunoglobulin, chemical instruments and methods, measuring devices, etc., can solve the problems of complex protein extraction and purification process and difficult preparation, and achieve good clinical application effect and good repeatability , the specific effect of the detection method

Inactive Publication Date: 2007-01-10
NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The detection of Candida albicans mycelial protein antibody requires P47 protein (SDS-PAGE electrophoresis shows a relative molecular weight of 47000, the same below) as a detection reagent, but the extraction and purification process of this protein is complicated and difficult to prepare, so the preparation method needs to be improved

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] 1.1 Preparation of natural Candida albicans mycelial protein P47

[0065] Candida albicans C1 strain (introduced from the Institute of Dermatology, Chinese Academy of Sciences) was inoculated in RPMI1640 culture medium containing 15% calf serum, cultured with shaking at 37°C for 7 hours, and more than 95% of the bacteria were transformed into hyphae phase under a microscope , centrifuge at 3000×g for 30 minutes to collect the bacteria, wash with normal saline for 3 times, prepare bacterial suspension with distilled water containing 1mmol / L phenylmethanesulfonyl fluoride, ultrasonically crush the wall in an ice bath, and centrifuge at 3000×g for 30 minutes Cell debris was removed, the supernatant was salted out with 70% saturated ammonium sulfate, and dialyzed with PBS to desalt. Mannose and mannoprotein were removed by Con A-Sepharose 4B affinity chromatography column, and the protein with a relative molecular weight of 47,000 was collected to obtain P47 protein, which ...

Embodiment 2

[0075] The ELISA kit for detecting Candida albicans mycelial protein antibody consists of:

[0076] 1) Coating antigen: the amino acid sequence is any one of the mimic antigens of SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3, or an equal mixture of the three;

[0077] 2) Solid phase support: polystyrene plate microwell reaction plate, used for coating the above-mentioned mock antigen;

[0078] 3) Enzyme-labeled antibody: use enzyme-labeled anti-human IgG or enzyme-labeled anti-human IgM;

[0079] 4) Negative control serum;

[0080] 5) Enzyme-labeled antibody diluent: PBS;

[0081] 6) Substrate solution: Solution A: 0.06% carbamide peroxide, 0.01M pH4.5 citric acid buffer;

[0082] Solution B: 0.06% TMB, 0.01M pH4.5 citrate buffer;

[0083] 7) Washing solution: PBS, containing 0.01% Tween-20;

[0084] 8) Stop solution: 1M sulfuric acid.

Embodiment 3

[0086] The ELISA kit for detecting Candida albicans mycelial protein antibody consists of:

[0087] 1) Coating antigen: Candida albicans mycelial protein P47 antigen;

[0088] 2) Solid phase support: polystyrene plate microwell reaction plate, used for coating the above-mentioned mycelial protein P47 antigen;

[0089] 3) Enzyme-labeled antibody: use enzyme-labeled anti-human IgG or enzyme-labeled anti-human IgM;

[0090] 4) Negative control serum;

[0091] 5) Enzyme-labeled antibody diluent, PBS;

[0092] 6) Substrate solution: Solution A: 0.06% carbamide peroxide, 0.01M pH4.5 citric acid buffer;

[0093] Solution B: 0.06% TMB, 0.01M pH4.5 citrate buffer;

[0094] 7) Washing solution: PBS, containing 0.01% Tween-20;

[0095] 8) Stop solution: 1M sulfuric acid.

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Abstract

The invention discloses a method and a reagent box for inspecting a candida albicans hypase protein antibody and diagnosing attack infection of candida albicans. The method performs filtering by using a monoclonal antibody of anti-candida albicans hypase protein P47 in a bacteriophage surface display peptide library, prepares simulated antigen or candida albicans hypase protein P47 natural antigen by expanding and purifying, then uses the antigen peridiuming a solid support, adds a biologic sample to be inspected to incubate and wash, adds a marked second antibody for human antibodies to incubate and wash, detects existing of an immune combined composition in an incubated mixture. The reagent box includes an ELISA reagent box and a colloid gold reagent box. The candida albicans hypase protein antibody can be inspected by usign the method simply and in high speed and efficiency; the reagent box has a low cost, convenient usage and high specificity.

Description

technical field [0001] The invention relates to a detection method for Candida albicans invasive infection, in particular to a method and a kit for detecting Candida albicans mycelial protein antibody. Background technique [0002] Candida albicans (C.albi an), also known as Candida albicans, often exists in the human mouth, throat, intestinal tract, vaginal mucosa, etc., and is an important conditional pathogenic bacteria. blood disease, severe malnutrition, etc.), or when the excessive use of antibiotics leads to dysbiosis, the bacteria can cause serious systemic invasive infection. The laboratory diagnosis of Candida albicans invasive infection is relatively difficult, because the bacterium is an opportunistic pathogen that inhabits many parts of the human body, often causing superficial infections, and it is difficult to judge whether it is pathogenic when the clinical isolation is positive: In addition, many deep samples Can not be collected, can not be isolated by c...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/531G01N33/543C07K7/04C07K16/00G01N33/532G01N33/535
Inventor 李芳秋邵海枫
Owner NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
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