WO2011038678A1 - Méthode et stylo testeur permettant de détecter la présence de microorganismes pathogènes par immunoréaction cellulaire en réponse à une stimulation antigénique - Google Patents

Méthode et stylo testeur permettant de détecter la présence de microorganismes pathogènes par immunoréaction cellulaire en réponse à une stimulation antigénique Download PDF

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Publication number
WO2011038678A1
WO2011038678A1 PCT/CN2010/077439 CN2010077439W WO2011038678A1 WO 2011038678 A1 WO2011038678 A1 WO 2011038678A1 CN 2010077439 W CN2010077439 W CN 2010077439W WO 2011038678 A1 WO2011038678 A1 WO 2011038678A1
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antigen
ifn
pathogenic microorganism
detecting
test
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PCT/CN2010/077439
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English (en)
Chinese (zh)
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杨吉
杨挥
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上海英伯肯医学生物技术有限公司
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Publication of WO2011038678A1 publication Critical patent/WO2011038678A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
    • G01N2333/05—Epstein-Barr virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria

Definitions

  • the invention relates to the field of molecular biology, immunology and protein detection technology. More specifically, it relates to a method for detecting a pathogenic microorganism by an antigen-stimulated cellular immune reaction; in addition, the present invention relates to a colloidal gold immunochromatographic test pen for detecting a pathogenic microorganism.
  • pathogenic microorganisms such as tuberculosis, AIDS, avian influenza, severe acute respiratory syndrome (SARS), hepatitis, malaria, anthracnose, pneumonia, hepatitis, mad cow disease, etc. Because it can seriously endanger personal health and is highly contagious, it poses a great threat to human health.
  • pathogenic microorganisms can lie in the body for a long time without morbidity or mild disease, it brings great obstacles to the accurate and timely diagnosis of the disease, and often can be diagnosed when the patient is very ill and the symptoms are obvious. , which delays the timing of treatment and is easy to infect others. Therefore, it is important to detect the presence of these pathogenic microorganisms in animals or humans as early as possible.
  • IFN- ⁇ release from sputum lymphocytes after activation is an important marker for cellular immune responses to intracellular pathogenic microbial antigens, and can be used to detect cellular immune responses and thereby indicate the presence of pathogenic microorganisms within the cells.
  • cytokines are released due to cellular immune responses, such as IFN- ⁇ , interleukin (IL), transforming growth factor. (transforming growth factor ⁇ , TGF- ⁇ ), etc., therefore, it is possible to reflect whether or not the pathogenic microorganism is present in the animal by detecting cytokines in the blood after stimulation.
  • cytokine-specific antibodies There are many methods for detecting cytokines in solution, and cytokine-specific antibodies are used.
  • the specific methods may be ELISA, chemiluminescence, immunofluorescence or antibody immunochromatography.
  • the technical problem to be solved by the present invention is to provide a method for detecting pathogenic microorganisms by antigen-stimulated cellular immune response, which is capable of rapidly and efficiently detecting pathogenic microorganisms in the latent period or early onset.
  • the present invention also provides a colloidal gold immunochromatographic test pen for detecting pathogenic microorganisms.
  • the present invention provides a novel method and test pen for detecting pathogenic microorganisms.
  • the main process is to use a functional fragment of pathogenic microorganisms or antigens specific antigen blood or blood cells are incubated for at least 6 hours, and IFN- Y detecting blood sample labeled with colloidal gold immunochromatography, IL TGF- ⁇ or other cytokines, thereby Determine whether the animal is infected with pathogenic microorganisms such as Mycobacterium tuberculosis, HIV, avian influenza virus, SARS coronavirus, hepatitis virus, Ebola virus, prion protein, and the like.
  • pathogenic microorganisms such as Mycobacterium tuberculosis, HIV, avian influenza virus, SARS coronavirus, hepatitis virus, Ebola virus, prion protein, and the like.
  • a method of detecting a pathogenic microorganism by an antigen-stimulated cellular immune response using one or more antigens or antigens specific to the pathogenic microorganism The fragment can be mixed with animal or human whole blood or blood cells and incubated at 37 ° C for at least 6 hours, and then the specific antibody is used to detect the cytokine released by the blood cells. It is detected that a higher concentration of cytokines indicates that there is no antigen stimulation.
  • Pathogenic microorganisms are provided.
  • pathogenic microorganisms are also called infectious pathogens, and are microorganisms capable of causing animal diseases, including prokaryotes, bacteria, viruses, fungi, and prions, which can be transmitted through the respiratory tract, digestive tract, skin, and blood.
  • the prokaryote includes a Plasmodium
  • the bacterium includes Mycobacterium tuberculosis causing tuberculosis, Bacillus anthracis causing anthrax, Streptococcus or Pseudomonas causing pneumonia, P.
  • pallidum causing syphilis, etc. including HIV, each Influenza virus, SARS coronavirus, hepatitis virus, Ebola virus, etc., fungi include yeast and the like.
  • Prion protein can cause mad cow disease and pruritus.
  • antigen refers to a specific antigen of a pathogenic microorganism, mainly a surface-specific protein, sugar or lipid of a pathogenic microorganism, and the specific antigen is a pathogenic microorganism-specific, and a pathogenic microorganism can be identified by these antigen-specific antibodies. And distinguish it from different pathogenic microorganisms. Antigens specific to different pathogenic microorganisms can also be distinguished from the animal's immune system and remembered, and later when these antigens re-enter the animal, they trigger an immune response.
  • the "functional fragment of an antigen” in the present invention means an antigen component or fragment containing all or part of an antigenic determinant of a pathogenic microorganism antigen.
  • the immune response of the animal's immune system is also triggered when the functional fragment of the antigen enters the animal.
  • the pathogenic microorganism is Mycobacterium tuberculosis
  • the antigen is Mycobacterium tuberculosis-specific antigen ESAT6, CFP10 or P4.
  • the pathogenic microorganism is HIV
  • the antigen is HIV-specific antigen GP120, GP41, GP36 or P24.
  • the pathogenic microorganism is an avian influenza virus
  • the antigen is avian influenza virus-specific antigen hemagglutinin (HA), neuraminidase (NA). Or proton channel M2.
  • the pathogenic microorganism is a SARS coronavirus
  • the antigen is a spike glycoprotein specific to a SARS coronavirus.
  • the cytokine released by the blood cells is gamma interferon IFNi, interleukin IL or transforming growth factor TGF-p.
  • the detection method may employ an ELISA (Enzyme-Linked Immunosorbent Assay), chemiluminescence, immunofluorescence or colloidal gold-labeled antibody immunochromatography.
  • ELISA Enzyme-Linked Immunosorbent Assay
  • chemiluminescence chemiluminescence
  • immunofluorescence colloidal gold-labeled antibody immunochromatography
  • the cytokine for detecting blood cell release using a specific antibody is immunochromatographic test pen using a colloidal gold-labeled IFN- ⁇ monoclonal antibody.
  • a test pen for detecting pathogenic microorganisms comprising a blood filter paper, a colloidal gold pad, a nitrocellulose membrane, a water absorbing filter paper, and a PVC bottom plate; the rubber on the colloidal gold pad is mouse anti-human a complex of monoclonal antibody colloidal gold of IFN- ⁇ having a test line and a control line on the nitrocellulose membrane, the test line being coated with IFN- ⁇ , and the control line coated with goat anti-mouse IgG Polyclonal antibody.
  • a test pen for detecting pathogenic microorganisms comprising a blood filter paper, a colloidal gold pad, a nitrocellulose membrane, a water absorbing filter paper and a PVC bottom plate; the colloidal gold pad is coated with colloidal gold-labeled mouse anti-human IFN a monoclonal antibody of ⁇ , having a test line and a control line on the nitrocellulose membrane, the test line coated with another monoclonal antibody against mouse IFN- ⁇ , which is coated on the control line Goat anti-mouse IgG polyclonal antibody.
  • a test pen for detecting pathogenic microorganisms comprising a blood filter paper, a colloidal gold pad, a nitrocellulose membrane, a water absorbing filter paper and a PVC bottom plate; the colloidal gold pad is coated with colloidal gold-labeled mouse anti-human IFN a monoclonal antibody of ⁇ and a monoclonal antibody against human IFN- ⁇ labeled with biotin, which has a test line and a control line, and the test line is coated with Streptavidin, which is coated with goat anti-mouse IgG Polyclonal antibody.
  • a test pen for detecting pathogenic microorganisms comprising a blood filter paper, a colloidal gold pad, a nitrocellulose membrane, a water absorbing filter paper and a PVC bottom plate; the colloidal gold pad is coated with a fluorescently labeled mouse anti-human IFN- a monoclonal antibody of ⁇ having a test line and a control line on the nitrocellulose membrane, the test line coated with another mouse monoclonal antibody against human IFN- ⁇ , the control line coated with a sheep Anti-mouse IgG polyclonal antibody.
  • the test pen for detecting IFN- ⁇ shown in Fig. 1 includes a plastic cap 1, a water absorbing stick 2, a blood filter paper 3, a colloidal gold pad 4, a test wire (T wire) 5, a nitrocellulose membrane (NC film) 6, and control Line (C line) 7, absorbent filter paper 8, plastic lower cover 9, desiccant 10, plastic upper cover 11, viewing window 12.
  • the test strip for detecting IFN- ⁇ shown in Fig. 2 includes a blood filter paper 3, a colloidal gold pad 4, a T line 5, an NC film 6, a C line 7, a water absorbing filter paper 8, and a PVC bottom plate 13.
  • the rubber on the colloidal gold pad 4 is a complex of mouse monoclonal antibody colloidal gold against human IFN- ⁇ , and the NC membrane 6 is coated with T-line 5 (containing IFN- ⁇ ) and C-line 7 (including goat anti- Mouse IgG polyclonal antibody).
  • the test principle of the test pen is: After the sample is added to the water absorption rod, the IFN- ⁇ in the sample moves to the colloidal gold pad with the solution and combines with the gold standard IFN- ⁇ monoclonal antibody to form an antigen-antibody complex, if the sample The IFN- ⁇ is not enough to allow the gold-labeled IFN- ⁇ monoclonal antibody to recombine with the antigen IFN- ⁇ , and these monoclonal antibodies can bind to the IFN- ⁇ at the T line as the solution moves to the T line.
  • the T line is red; on the other hand, if the IFN- ⁇ in the sample is excessive and the gold IFN- ⁇ monoclonal antibody can no longer bind to the antigen IFN- ⁇ , these monoclonal antibodies cannot move with the T line when the solution moves to the T line. The IFN- ⁇ binds and the T line will appear without color.
  • the gold-labeled IFN- ⁇ monoclonal antibody that did not bind to the T line continued to move to the C line with the solution, and binded to the goat anti-mouse polyclonal antibody at the C line to make the C line red, suggesting that the mouse is resistant to human IFN- ⁇ .
  • Monoclonal antibodies can be recognized by goat anti-mouse polyclonal antibodies.
  • the C line when the C line is red, if the T line is also red, the concentration of IFN- ⁇ in the sample solution is low, and the patient is not pathogenic; the C line is red and the T line has no color. The concentration of IFN- ⁇ in the sample solution is high, and the patient in the test contains pathogenic microorganisms.
  • IFN- ⁇ , IL, TGF- ⁇ , etc. thereby detecting whether or not the pathogenic microorganism is present in the animal by detecting these cytokines.
  • This method can increase specificity by selecting specific protein components (antigens or functional fragments thereof). Because it is not necessary to detect the antigen of the pathogenic microorganism or the antibody produced in the body, it is only necessary to detect the blood cell reaction, so the presence of the pathogenic microorganism in the body can be detected in the latent period or early onset of the pathogenic microorganism, the diagnosis time can be shortened, and the patient can be treated in time, as soon as possible. Rehabilitation.
  • the invention detects a highly infectious pathogenic microorganism such as Mycobacterium tuberculosis, HIV, avian influenza virus and SARS coronavirus by detecting a cellular immune reaction, and can detect microorganisms in the latent period or early stage of the microorganism, unlike the existing main direct
  • the serological method for detecting antigens or antibodies can be detected after a large number of pathogenic microorganisms or a strong humoral immune response, so that pathogenic microorganisms can be detected as early as possible, and time for the patient's early treatment is obtained. Moreover, the detection time is very short and takes only about 20 hours.
  • the method is also very simple, which greatly reduces manpower and material resources and saves costs. Further, the present invention significantly improves the specificity and sensitivity of the detection method by utilizing antigens specific to various pathogenic microorganisms or functional fragments thereof.
  • FIG. 1 is a schematic view showing the structure of a test pen for detecting IFN- ⁇ by colloidal gold-labeled IFN- ⁇ monoclonal antibody immunochromatography
  • Fig. 2 is a schematic view showing the structure of a test strip for detecting IFN-? by colloidal gold-labeled IFN-? monoclonal antibody immunochromatography in the present invention.
  • reference numerals 1-13 illustrate: 1-plastic hat; 2-water absorbing rod; 3-filter paper; 4-colloidal gold pad; 5-test line (T line); Nitrocellulose membrane (NC membrane); 7- Control line (C line); 8-water filter paper; 9-plastic cover; 10-drying agent; 11-plastic cover; 12-view window; 13-PVC Base plate. detailed description
  • colloidal gold was prepared by trisodium citrate reduction method (HAuCl 4 , trisodium citrate purchased from Sigma), and the size of colloidal gold particles was about 40 nm.
  • the anti-IFN- ⁇ monoclonal antibody (Abeam) was labeled with colloidal gold at a ratio of 1 mg antibody/1500D colloidal gold at pH 8.4. After 30 minutes, an appropriate amount of BSA was added under the conditions of pH 7.4, and concentrated by centrifugation. The IFN- ⁇ antibody gold probe was obtained.
  • the gold probe was diluted with a colloidal gold film diluent (0.1 M TAPS buffer, 20% sucrose, 3. 75% BSA, pH 8.0) to 0D2.0.
  • a colloidal gold film diluent 0.1 M TAPS buffer, 20% sucrose, 3. 75% BSA, pH 8.0
  • the excess liquid was taken out and squeezed, and laid on a special grid; dried overnight at room temperature in a relative humidity of 20%.
  • the IFN- ⁇ antibody colloidal gold pad was obtained and used.
  • the IFN-Y (Abeam) was diluted to 0.12 mg/ml, 0.22 ⁇ m by filtration in a solution of pH 7.4 in 10 mM PBS.
  • the goat anti-mouse IgG polyclonal antibody (Xiamen Bosheng Biotechnology Co., Ltd.) was diluted to 2. Omg/ml, 0.22 ⁇ m by filtration in a 10 ⁇ m PBS solution of ⁇ 7.4.
  • the relative humidity requirement of the working environment of the workshop is less than 30%.
  • Fig. 1 and Fig. 2 take a 60 ⁇ X 300 ⁇ plastic sheet (backing), and affix the coated NC film 6 at the center position.
  • the T line 5 of the NC film is facing downward; at the NC film T
  • a colloidal gold pad 4 is attached to the lower portion of one side of the wire 5 and partially covers the lower edge of the NC film 6 to ensure that the two have an overlap of 1 to 2 inches;
  • a blood filter paper 3 is attached to the lower side of the colloidal gold pad 4 (Ahl strom Fi Ltration company) partially covers the lower edge of the colloidal gold pad 4;
  • a piece of absorbent filter paper 8 (Ahlstrom Filtration) is attached to one end of the PVC substrate 13 (mi ll ipore) without the filter paper 3, and a partial coverage of the NC film is ensured.
  • the relative humidity of the workshop working environment during cutting and assembly is less than 30%.
  • the large card was cut into a 6 mm wide bio-test chip test strip using a Matrix 2501 cutter, and a 6 mm test strip, a water-absorbing rod 2 (Filtrona Fibertec), and a desiccant 10 (Sud-Chemie).
  • the plastic parts including the plastic part cap 1, the plastic part lower cover 9 and the plastic part upper cover 11) are assembled into a test pen, and the test pen is sealed with an aluminum foil package.
  • blood samples are collected. Take 1 ml of blood from the patient and add it to the sterile test tube containing heparin (Hong Kong Advanced Technology Worker 4k Co., Ltd.) as a negative control tube; then take 1 ml of blood from the patient and add heparin and Mycobacterium tuberculosis antigens (ESAT6 and CFP10, Hong Kong Advanced Technology Industry Limited)
  • the company's sterile test tube is used as a measuring tube; the patient's 1 ml of blood is taken, and heparin-containing, cytokines (such as plant lectin, phytohemagglutinin, Hong Kong Advanced Technology Co., Ltd.) are added as positive control tubes.
  • the 3 tubes of blood were fully oscillated separately. The test was started within 12 hours. In the second step, the sample is kept warm. After collecting the blood samples, the three tubes were incubated at 37 ° C for 6-24 hours in 12 hours. The third step is sample testing. The ⁇ sample was added dropwise to the sorbent gold immunochromatographic test pen (the box containing the colloidal gold immunochromatographic test strip for detecting IFN- ⁇ shown in Fig. 2) shown in Fig. 1 after 10 minutes, 10 minutes later.
  • the sorbent gold immunochromatographic test pen the box containing the colloidal gold immunochromatographic test strip for detecting IFN- ⁇ shown in Fig. 2
  • the test line 5 position A red line also appeared, indicating that the concentration of IFN- ⁇ in the sample was low; conversely, if a red line appeared at the position of the control line 7, the red line was not present at the position of the test line 5, indicating that the concentration of IFN- ⁇ in the sample was high.
  • the fourth step the result is interpreted.
  • the concentration of IFN- ⁇ in the negative control is low (red line appears at the test line position) and the concentration of IFN- ⁇ in the positive control is high (the red line is not present at the test line position)
  • the concentration of IFN- ⁇ is low (red line appears at the test line position), indicating that there is no tubercle bacillus in the patient; conversely, if the concentration of IFN- ⁇ in the measuring tube is high (the red line is not present at the test line position), the patient is prompted to have Mycobacterium tuberculosis.
  • the blood sample itself is too high in the IFN- ⁇ baseline or blood cells in the blood. There was a problem and it did not respond to the cell division. In both cases, the test failed and it was retested.
  • the IFN- ⁇ in the sample can also be measured by ELISA (enzyme l inked immunosorbent assay).
  • the method was as follows: the wells of the ELISA plate were coated with IFN- ⁇ monoclonal antibody (abeam), and then the sample solution was added, and the plate was incubated at room temperature for 2 hours, and then the horseradish peroxidase HRP-conjugated IFN was added.
  • - ⁇ monoclonal antibody (abeam) after 2 hours incubation at room temperature, wash the plate, add HRP substrate (abeam) reaction, and place the plate on Bio-Rad microplate reader at 450 nm, according to IFN- ⁇
  • the standard curve was used to analyze the concentration of IFN- ⁇ .
  • cytokines such as IFN-? can also be detected by antibodies such as chemiluminescence, immunofluorescence, and the like.
  • This method can not only detect tubercle bacilli in tuberculosis patients, but also detect tubercle bacilli that are latent in untreated patients.
  • the method overcomes the shortcomings of the rapid rapid amplification of phage, the sensitivity and specificity of the serological diagnosis method, reduces the diagnosis process to about 20 hours, and improves the sensitivity and specificity to meet the rapid The need to diagnose tuberculosis is convenient for timely treatment of patients.
  • Example 3 Detection of HIV by detecting IFN- ⁇ in human blood
  • blood samples are collected. Take 1 ml of blood from the patient, add it to the sterile test tube containing heparin (Hong Kong Advanced Technology Industry Co., Ltd.) as a negative control tube; then take 1 ml of blood from the patient, add heparin, HIV antigen (GP120, GP41, GP36 and P24, Hong Kong ⁇ Technical Industry Co., Ltd.
  • heparin Hong Kong Advanced Technology Industry Co., Ltd.
  • HIV antigen GP120, GP41, GP36 and P24, Hong Kong ⁇ Technical Industry Co., Ltd.
  • cytokines such as plant lectin, phytohemagglutinin, Hong Kong Advanced Technology Industrial Co., Ltd.
  • the 3 tubes of blood were fully oscillated separately.
  • the test was started within 12 hours.
  • the sample is kept warm.
  • After collecting the blood sample 3 tubes were placed at 37 within 12 hours.
  • C is kept for 6-24 hours.
  • the third step is sample testing.
  • the ⁇ sample was added to the test stick of the test IFN- ⁇ shown in Fig. 1 (the cartridge contains the colloidal gold immunochromatographic test strip for detecting IFN- ⁇ in Fig.
  • Example 4 Detection of avian influenza virus by detecting IFN- ⁇ in human blood
  • blood samples are collected. Take 1 ml of blood from the patient, add heparin (Hong Kong Advanced Technology Co., Ltd.; as a negative control tube in a sterile test tube; then take 1 ml of blood from the patient, add heparin, avian influenza virus antigen (hemagglutinin HA and neuraminidase) NA, Hong Kong advanced technology
  • the measuring tube is used; the patient's 1 ml of blood is taken, and heparin-containing, cytokines (such as plant lectin, phytohemagglutinin, Hong Kong Advanced Technology Industrial Co., Ltd.) are added as positive control tubes.
  • the 3 tubes of blood were fully oscillated separately.
  • the test was started within 12 hours.
  • the sample is kept warm.
  • 3 tubes were placed at 37 within 12 hours.
  • C is kept for 6-24 hours.
  • the third step is sample testing.
  • the ⁇ sample was added to the test stick of the test IFN- ⁇ shown in Fig. 1 (the cartridge contains the colloidal gold immunochromatographic test strip for detecting IFN- ⁇ in Fig. 2), and the position of the control line 7 was 10 minutes later.
  • a red line appears, and a red line appears at the position of the test line 5, indicating that the concentration of IFN- ⁇ in the sample is low; conversely, if a red line appears at the position of the control line 7, the red line is not present at the position of the test line 5, indicating that the concentration of IFN- ⁇ in the sample is high. Fourth, the result was interpreted.
  • the concentration of IFN- ⁇ in the negative control is low (red line appears at the test line position) and the concentration of IFN- ⁇ in the positive control is high (the red line is not present at the test line position)
  • the concentration of IFN- ⁇ in the tube is low (red line appears at the test line position), indicating that there is no avian influenza virus in the patient; conversely, if the concentration of IFN- ⁇ in the measuring tube is high (the red line is not present at the test line position), the patient is indicated to have avian flu virus.
  • blood samples are collected. Take 1 ml of blood from the patient, add it to the sterile test tube containing heparin (Hong Kong Advanced Technology Industry Co., Ltd.) as a negative control tube; then take 1 ml of blood from the patient and add heparin, SARS coronavirus antigen (spike glycoprotein and erythrocyte lectin acetyl) Esterase glycoprotein, Hong Kong Advanced Technology Industries Co., Ltd.) used as a measuring tube in a sterile test tube; then took 1 ml of blood from the patient and added heparin and mitogen (such as plant lectin, Phytohemagglutinin, Hong Kong Xianhao Technology Industry Co., Ltd.) as a positive control tube.
  • heparin Hong Kong Advanced Technology Industry Co., Ltd.
  • SARS coronavirus antigen spike glycoprotein and erythrocyte lectin acetyl Esterase glycoprotein, Hong Kong Advanced Technology Industries Co., Ltd.
  • the 3 tubes of blood were fully oscillated separately.
  • the test was started within 12 hours.
  • the sample is kept warm.
  • 3 tubes were placed at 37 within 12 hours.
  • C is kept for 6-24 hours.
  • the third step is sample testing.
  • the ⁇ sample was added to the test stick of the test IFN- ⁇ shown in Fig. 1 (the cartridge contains the colloidal gold immunochromatographic test strip for detecting IFN- ⁇ in Fig. 2), and the position of the control line 7 was 10 minutes later.
  • a red line appears, and a red line appears at the position of the test line 5, indicating that the concentration of IFN- ⁇ in the sample is low; conversely, if a red line appears at the position of the control line 7, the red line is not present at the position of the test line 5, indicating that the concentration of IFN- ⁇ in the sample is high.
  • the fourth step the result is interpreted.

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Abstract

La présente invention concerne une méthode et un stylo testeur permettant de détecter un microorganisme pathogène par immunoréaction cellulaire en réponse à une stimulation antigénique. D'abord, des immunocytes ayant été en contact avec ledit microorganisme dans le sang sont stimulés par l'antigène spécifique du microorganisme pathogène ou par des segments fonctionnels de celui-ci. Ensuite, les immunocytes sécrètent une cytokine, par exemple de l'interféron γ (IFN-γ). Enfin, pour savoir si ledit microorganisme pathogène est présent chez l'animal, on procède à une mesure de la quantité d'IFN-γ présente. Selon l'invention, le microorganisme, même à un stade latent ou précoce de la maladie, peut être détecté rapidement et efficacement en mesurant la quantité d'IFN-γ présente dans le sang et ce, par immunochromatographie avec de l'or colloïdal.
PCT/CN2010/077439 2009-09-29 2010-09-29 Méthode et stylo testeur permettant de détecter la présence de microorganismes pathogènes par immunoréaction cellulaire en réponse à une stimulation antigénique WO2011038678A1 (fr)

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CN200910057981.2 2009-09-29
CN2009100579812A CN102033129A (zh) 2009-09-29 2009-09-29 用抗原刺激的细胞免疫反应来检测病原微生物的方法及测试笔

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EP2687848A1 (fr) * 2012-07-16 2014-01-22 Lionex GmbH État d'infection de tuberculose chez un individu
CN105259354B (zh) * 2015-11-13 2017-05-10 夏晶 结核T细胞释放γ‑干扰素检测试剂盒及其使用方法
CN105548566A (zh) * 2015-12-25 2016-05-04 合肥安为康医学检验有限公司 一种快速检测结核分枝杆菌的方法
CN105717307B (zh) * 2016-03-16 2017-07-04 四川大学华西第二医院 一种精子质量评估的试剂盒及其使用方法
CN112691117A (zh) * 2020-06-02 2021-04-23 中国人民解放军军事科学院军事医学研究院 肝素钠用于新型冠状病毒SARS-CoV-2感染的新用途
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