Method for detecting nephrotic syndrome hemorrhagic fever antibody

A technology for renal syndrome and hemorrhagic fever, which is applied in the field of specific IgM/IgG antibodies and pathogens of renal syndrome hemorrhagic fever, can solve the problems of false positives, large volume of enzyme plate, inconvenient portability, poor sensitivity, etc.

Inactive Publication Date: 2006-08-02
天津市卫生防病中心 +2
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Problems solved by technology

From a methodological point of view, the IFA method has strong specificity and relatively poor sensitivity, and is often used for confirmation experiments; the ELISA method adopts the enzymatic cascade amplification effect, and the sensitivity is improved, but the capture method is required to exclude IgM antibodies when detecting IgM antibodies. Interference with other components in the blood (such as rheumatoid factor, etc.), otherwise false positives are likely to occur
And both need certain instruments and equipment, it is difficult to popularize, and the existing ELISA method uses the imported microtiter plate to be bulky and not easy to carry, and the cost is high, which undoubtedly affects the primary medical units to use this method for early stage Therefore, it is imperative to study a detection method that improves the sensitivity of the test, is simple to operate, portable, fast, sensitive, specific, and easy to promote, so as to be suitable for the diagnosis and disease monitoring of HFRS at the grassroots level.

Method used

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  • Method for detecting nephrotic syndrome hemorrhagic fever antibody
  • Method for detecting nephrotic syndrome hemorrhagic fever antibody

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Experimental program
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Effect test

Embodiment 1

[0036] 1. Preparation and labeling of hantavirus S gene recombinant expression protein:

[0037] 1. Virus cultivation and RNA extraction:

[0038] The Z10 and L99 virus strains were routinely inoculated into Vero-E6 cells, and the degree of cell infection was checked by indirect immunofluorescence (IFA). Cells were harvested when more than 75% of the cells were infected. For the extraction of total cellular RNA, Qiagen's RNeasy Mini Kit was used, and the extraction was performed according to the instructions.

[0039] 2. Use RT-PCR technology to obtain the S gene encoding the target protein:

[0040] The total RNA extracted from infected cells was used as a template for reverse transcription to synthesize cDNA, and the RNA Kit (AMV) kit from TaKaRa Company was used to operate according to the instructions. Reverse transcription products were directly used for PCR amplification. The reaction conditions were: pre-denaturation at 94°C for 2 minutes; 30 cycles at 94°C for 50 s...

Embodiment 2

[0050] One, the preparation and labeling of Hantavirus S gene recombinant protein: (same as embodiment 1)

[0051] Two, the preparation of immunomagnetic microspheres: (with embodiment 1)

[0052] 3. Methods for detecting specific IgM antibodies in patients with hemorrhagic fever with renal syndrome:

[0053] Add 1 mL of the serum of the patient to be tested diluted 1:100 in PBS to the centrifuge tubes containing 15 μL of immunomagnetic microspheres sensitized by goat anti-human IgM (μ chain), respectively, 1 mL of the negative and positive controls, and shake at 37 ° C for 1 hour , collect and fix the microspheres outside the wall of the centrifuge tube with the magnetic field of a magnet, discard the reaction solution, and wash with PBS repeatedly for 3 times. The enzyme-labeled recombinant protein antigen was determined by adding 1mL of biotin-labeled expressed protein diluted 100 times in PBS to the experimental centrifuge tube, and shaking at 37°C for 1 hour. After wash...

Embodiment 3

[0055] One, the preparation and labeling of Hantavirus S gene recombinant protein: (same as embodiment 1)

[0056] Two, the preparation of immunomagnetic microspheres: (with embodiment 1)

[0057] 3. Methods for detecting specific IgM antibodies in patients with hemorrhagic fever with renal syndrome:

[0058] Add 1 mL of the serum of the patient to be tested diluted 1:100 in PBS to the centrifuge tubes containing 15 μL of immunomagnetic microspheres sensitized by goat anti-human IgM (μ chain), respectively, 1 mL of the negative and positive controls, and shake at 37 ° C for 1 hour , collect and fix the microspheres outside the wall of the centrifuge tube with the magnetic field of a magnet, discard the reaction solution, and wash with PBS repeatedly for 3 times. For color determination of enzyme-labeled recombinant protein antigens, 1 mL of biotin-labeled expressed protein diluted 100 times in PBS was added to an experimental centrifuge tube, and the reaction was shaken at 37...

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Abstract

The present invention provides a method for detecting hemorrhagic fever with renal syndrome (HFRS) antibody, belonging to the field of biological technology. It relates to a technique capable of utilizing immunomagnetic trapping technique and antigen protein of enzyme-labelled recombinant virus to detect antibody of HFRS due to Hantanvirus. Said method includes the following steps: purifying S gene recombinant protein of Hantanvirus representative strain Z10 and L99 expressed by pET prokaryotic expression system, labeling biotin or horseradish peroxidase, making magnetic microsphere be connected with sheep anti-human IgM (mu chain) or sheep anti-human IgG antibody and making it be sensitized to obtain immunomagnetic microsphere for detecting specific IgM/IgG antibody of seoul type or Hantan type of Hantanvirus and creating the quick and sensitive immunomagnetic microphere ELISA method for detecting HFRS specific IgM/IgG antibody in blood serum sample.

Description

Technical field: [0001] The present invention relates to the preparation and labeling of Hantavirus (HV) S gene recombinant protein and the preparation of immunomagnetic microspheres, the pathogen of hemorrhagic fever with renal syndrome (HFRS). Immunomagnetic microsphere-ELISA method to detect specific IgM / IgG antibodies in serum samples of patients with hemorrhagic fever with renal syndrome. Belongs to the field of biotechnology. technical background: [0002] my country is the country most seriously affected by hemorrhagic fever with renal syndrome. The number of cases each year accounts for more than 90% of the total reported in the world, and the fatality rate is extremely high. Attention at home and abroad. Most of the studies on the diagnosis and prevention of the disease are carried out by Chinese scholars. The disease has an acute onset, complicated clinical manifestations, prominent kidney damage, and no specific drug, which is seriously harmful. Therefore, early...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/543G01N33/531
Inventor 王撷秀陈锦英白钢杨东靖
Owner 天津市卫生防病中心
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