WO2011038678A1

WO2011038678A1 - Method and test pen for detecting pathogenic microorganism by cell immunoreaction in response to antigen stimulation

Info

Publication number: WO2011038678A1
Application number: PCT/CN2010/077439
Authority: WO
Inventors: 杨吉; 杨挥
Original assignee: 上海英伯肯医学生物技术有限公司
Priority date: 2009-09-29
Filing date: 2010-09-29
Publication date: 2011-04-07
Also published as: CN102033129A

Abstract

A method and a test pen for detecting a pathogenic microorganism by cell immunoreaction in response to antigen stimulation. Firstly, Immunocytes having contacted said microorganism in blood are stimulated by the pathogenic microorganism-specific antigen or functional segments thereof. Then, the immunocytes secrete cytokinin, for example, interferonγ (IFN-γ).Finally, whether said pathogenic microorganism exists in animal is judged by measuring the quantity of IFN-γ. In the invention, the pathogenic microorganism at latent or early disease stage can be detected quickly and effectively by measuring the quantity of IFN-γ in blood using colloidal gold immunochromatographic method.

Description

Method for detecting pathogenic microorganisms by antigen-stimulated cellular immune reaction and test pen

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.

Background technique

Many diseases caused by infection with 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. In addition, because many of these 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.

At present, most commonly used methods are to detect antibodies, antigens, DNA or RNA of pathogenic microorganisms in body fluids, but it is difficult to detect due to the low content of initial pathogenic microorganisms, and taking samples to culture and amplifying microorganisms takes a long time and the process is complicated. Not only is it easy to make human error, but it also delays the optimal treatment time, so it is necessary to develop a rapid, simple and effective detection method to detect diseases caused by such pathogenic microorganisms.

It is well known that the main function of the immune system in animals is to eliminate infectious agents such as pathogenic microorganisms in animals. In the process of clearing the infectious agent, humoral immunity dependent on B lymphocytes and cellular immunity dependent on T lymphocytes are essential. Intracellular viruses or bacteria are relatively far from humoral immunity, but processed and presented proteins derived from these microorganisms promote T lymphocyte activation. Correspondingly, for intracellular pathogenic microorganisms (Mycobacterium tuberculosis, HIV, influenza virus) Obtaining resistance depends on T lymphocytes and gamma interferon (interferon γ, IFN-γ), and the synergy of IFN-γ and other cytokines is critical to gain resistance to intracellular pathogenic microbial infections. Therefore, IFN-γ 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.

For immune cells that have been exposed to pathogenic microorganisms, such as sputum lymphocytes, when stimulated again by the antigen of the pathogenic microorganism, a large number of cytokines are released due to cellular immune responses, such as IFN-γ, interleukin (IL), transforming growth factor. (transforming growth factor β, TGF-β), 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.

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.

Summary of the invention

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. To this end, the present invention also provides a colloidal gold immunochromatographic test pen for detecting pathogenic microorganisms.

Using the principles of cellular immune responses, 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-β 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.

In one aspect of the invention, there is provided 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.

In the present invention, "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. More specifically, 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.

In the present invention, "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. When the pathogenic microorganism enters the animal and is recognized and remembered by the animal's immune system, the immune response of the animal's immune system is also triggered when the functional fragment of the antigen enters the animal.

Preferably, the pathogenic microorganism is Mycobacterium tuberculosis, and the antigen is Mycobacterium tuberculosis-specific antigen ESAT6, CFP10 or P4.

Preferably, the pathogenic microorganism is HIV, and the antigen is HIV-specific antigen GP120, GP41, GP36 or P24.

Preferably, the pathogenic microorganism is an avian influenza virus, and the antigen is avian influenza virus-specific antigen hemagglutinin (HA), neuraminidase (NA). Or proton channel M2.

Preferably, the pathogenic microorganism is a SARS coronavirus, and the antigen is a spike glycoprotein specific to a SARS coronavirus. Hemagglutinin-acetyl esterase glycoprotein or M protein .

The cytokine released by the blood cells is gamma interferon IFNi, interleukin IL or transforming growth factor TGF-p.

Preferably, the specific antibody is a monoclonal antibody to IFN-γ.

The detection method may employ an ELISA (Enzyme-Linked Immunosorbent Assay), chemiluminescence, immunofluorescence or colloidal gold-labeled antibody immunochromatography.

Preferably, the cytokine for detecting blood cell release using a specific antibody is immunochromatographic test pen using a colloidal gold-labeled IFN-γ monoclonal antibody.

In another aspect of the present invention, there is provided 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-γ having a test line and a control line on the nitrocellulose membrane, the test line being coated with IFN-γ, and the control line coated with goat anti-mouse IgG Polyclonal antibody.

Preferably, a test pen for detecting pathogenic microorganisms is provided, 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 γ, having a test line and a control line on the nitrocellulose membrane, the test line coated with another monoclonal antibody against mouse IFN-γ, which is coated on the control line Goat anti-mouse IgG polyclonal antibody.

Preferably, a test pen for detecting pathogenic microorganisms is provided, 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 γ and a monoclonal antibody against human IFN-γ 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.

Preferably, a test pen for detecting pathogenic microorganisms is provided, 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 γ having a test line and a control line on the nitrocellulose membrane, the test line coated with another mouse monoclonal antibody against human IFN-γ, the control line coated with a sheep Anti-mouse IgG polyclonal antibody.

The test pen for detecting IFN-γ 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-γ 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-γ, and the NC membrane 6 is coated with T-line 5 (containing IFN-γ) 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-γ in the sample moves to the colloidal gold pad with the solution and combines with the gold standard IFN-γ monoclonal antibody to form an antigen-antibody complex, if the sample The IFN-γ is not enough to allow the gold-labeled IFN-γ monoclonal antibody to recombine with the antigen IFN-γ, and these monoclonal antibodies can bind to the IFN-γ at the T line as the solution moves to the T line. The T line is red; on the other hand, if the IFN-γ in the sample is excessive and the gold IFN-γ monoclonal antibody can no longer bind to the antigen IFN-γ, these monoclonal antibodies cannot move with the T line when the solution moves to the T line. The IFN-γ binds and the T line will appear without color. The gold-labeled IFN-γ 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-γ. Monoclonal antibodies can be recognized by goat anti-mouse polyclonal antibodies. In summary, when the C line is red, if the T line is also red, the concentration of IFN-γ 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-γ in the sample solution is high, and the patient in the test contains pathogenic microorganisms.

The method utilizes a memory function of an animal or a human blood cell that has been exposed to a pathogenic microorganism, and then When exposed to the protein component of the pathogenic microorganism, it can be stimulated to release cytokines, such as

IFN-γ, IL, TGF-β, 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. DRAWINGS

1 is a schematic view showing the structure of a test pen for detecting IFN-γ by colloidal gold-labeled IFN-γ 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.

In Figures 1 and 2, 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

The present invention is further illustrated by the following examples and comparative experiments:

Example 1 Preparation of Colloidal Gold Immunochromatographic Test Pen for Detection of IFN-γ

The preparation method of the test pen shown in Figure 1 is as follows:

1. Preparation of colloidal gold: 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.

2. Preparation of anti-IFN-γ antibody colloidal gold pad

The anti-IFN-γ 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-γ 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.

After fully immersing the glass fiber (Millipore) in the IFN-γ colloidal gold film solution of 0D2.0, 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-γ antibody colloidal gold pad was obtained and used.

3. Preparation of IFN-γ nitrocellulose membrane (NC membrane)

The IFN-Y (Abeam) was diluted to 0.12 mg/ml, 0.22 μm 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 μm by filtration in a 10 μm PBS solution of ρΗ7.4.

Using the IFN-γ solution on the NC membrane (Mil lipore) T-line (test line) spray point while using the goat anti-mouse IgG polyclonal antibody solution on the NC membrane C line (control line) spray point to form a test on the membrane Line and control line, and spray the finished nitrocellulose membrane at room temperature in a relative humidity of 20% overnight at room temperature for use.

4. Preparation of mucosa large card:

The relative humidity requirement of the working environment of the workshop is less than 30%. As shown in Fig. 1 and Fig. 2, take a 60 匪 X 300 匪 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. 6 upper edge; put the affixed large card into a plastic bag and store it in a low-humidity storage room with a relative humidity of less than 20%.

5. Test chip cutting, assembly and packaging

The relative humidity of the workshop working environment during cutting and assembly is less than 30%.

As shown in Figure 1, 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). According to the test pen assembly step, 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.

Example 2 Detection of Mycobacterium tuberculosis by detecting IFN-γ in human blood

In the first step, 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 ΙΟΟμΙ sample was added dropwise to the sorbent gold immunochromatographic test pen (the box containing the colloidal gold immunochromatographic test strip for detecting IFN-γ shown in Fig. 2) shown in Fig. 1 after 10 minutes, 10 minutes later. If the red line appears at the position of the control line 7, the test line 5 position A red line also appeared, indicating that the concentration of IFN-γ 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-γ in the sample was high. The fourth step, the result is interpreted. For the method for detecting Mycobacterium tuberculosis in a human body, when the concentration of IFN-γ in the negative control is low (red line appears at the test line position) and the concentration of IFN-γ in the positive control is high (the red line is not present at the test line position), if the measuring tube is used The concentration of IFN-γ 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-γ 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. If the IFN-γ concentration in the negative control is high (the red line is not present at the test line position) or the IFN-γ concentration in the positive control is low (the red line appears at the test line position), the blood sample itself is too high in the IFN-γ 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 results of blood samples from 10 patients showed that 7 cases contained Mycobacterium tuberculosis, and 3 cases did not contain M. tuberculosis, which was consistent with clinical diagnosis.

The IFN-γ 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-γ 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. -γ 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-γ The standard curve was used to analyze the concentration of IFN-γ.

Those skilled in the art will recognize that 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-γ in human blood In the first step, 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 迸Technical Industry Co., Ltd. is used as a measuring tube in a sterile test tube; 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. Second, 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 ΙΟΟμΙ sample was added to the test stick of the test IFN-γ shown in Fig. 1 (the cartridge contains the colloidal gold immunochromatographic test strip for detecting IFN-γ 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-γ 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-γ in the sample is high. The fourth step, the result is interpreted. For the method for detecting HIV in human body, when the concentration of IFN-γ in the negative control is low (red line appears at the test line position) and the concentration of IFN-γ in the positive control is high (the red line is not present at the test line position), if the measurement tube is in the tube The lower concentration of IFN-γ (red line at the test line) suggests that there is no HIV in the patient; conversely, if the concentration of IFN-γ in the measuring tube is high (the red line does not appear at the test line), the patient is prompted to have HIV. If the IFN-γ concentration in the negative control is high (the red line is not present at the test line position) or the IFN-γ concentration in the positive control is low (the red line appears at the test line position), the blood sample itself is too high in the IFN-γ 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 results of blood samples from 10 patients showed that 4 cases contained HIV and 6 cases did not contain HIV, which was consistent with the clinical diagnosis.

Example 4 Detection of avian influenza virus by detecting IFN-γ in human blood

In the first step, 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 In the sterile test tube of the Industrial Co., Ltd., 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. In the second step, 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 ΙΟΟμΙ sample was added to the test stick of the test IFN-γ shown in Fig. 1 (the cartridge contains the colloidal gold immunochromatographic test strip for detecting IFN-γ 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-γ 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-γ in the sample is high. Fourth, the result was interpreted. For the method for detecting avian influenza virus in human body, when the concentration of IFN-γ in the negative control is low (red line appears at the test line position) and the concentration of IFN-γ in the positive control is high (the red line is not present at the test line position), if the measurement is performed The concentration of IFN-γ 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-γ 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. If the IFN-γ concentration in the negative control is high (the red line is not present at the test line position) or the IFN-γ concentration in the positive control is low (the red line appears at the test line position), the blood sample itself is too high in the IFN-γ baseline or blood cells in the blood. There was a problem and did not respond to cell division. In both cases, the test failed and was retested.

The results of blood samples from 10 patients showed that 3 cases contained avian influenza virus and 7 cases did not contain avian influenza virus, which was consistent with clinical diagnosis.

Example 5 Detection of SARS coronavirus by detecting IFN-γ in human blood

In the first step, 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. 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 sample, 3 tubes were placed at 37 within 12 hours. C is kept for 6-24 hours. The third step is sample testing. The ΙΟΟμΙ sample was added to the test stick of the test IFN-γ shown in Fig. 1 (the cartridge contains the colloidal gold immunochromatographic test strip for detecting IFN-γ 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-γ 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-γ in the sample is high. The fourth step, the result is interpreted. For the method for detecting SARS coronavirus in human body, when the concentration of IFN-γ in the negative control is low (red line appears at the test line position) and the concentration of IFN-γ in the positive control is high (the red line is not present at the test line position), if the measurement is performed The concentration of IFN-γ in the tube is low (red line appears at the test line position), indicating that there is no SARS coronavirus in the patient; conversely, if the concentration of IFN-γ in the measuring tube is high (the red line is not present at the test line position), the patient is informed that there is SARS in the body. Coronavirus. If the IFN-γ concentration in the negative control is high (the red line is not present at the test line position) or the IFN-γ concentration in the positive control is low (the red line appears at the test line position), the blood sample itself is too high in the IFN-γ 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 results of blood samples from 10 patients showed that 2 cases contained SARS coronavirus and 8 cases did not contain SARS coronavirus, which was consistent with clinical diagnosis.

Claims

Rights request

A method for detecting a pathogenic microorganism by an antigen-stimulated cellular immune reaction, characterized in that the method uses a functional fragment of one or more antigens or antigens specific to the pathogenic microorganism to mix with the whole blood or blood cells of the animal or human and After incubation at 37 ° C for at least 6 hours, specific antibodies were used to detect cytokines released by blood cells, and higher concentrations of cytokines were detected to indicate pathogenic microorganisms when compared to no antigenic stimulation.

The method for detecting pathogenic microorganisms by antigen-stimulated cellular immune reaction according to claim 1, wherein the pathogenic microorganism is Mycobacterium tuberculosis, and the antigen is Mycobacterium tuberculosis-specific antigen ESAT6, CFP10 or P4.

The method for detecting a pathogenic microorganism by antigen-stimulated cellular immune reaction according to claim 1, wherein the pathogenic microorganism is HIV, and the antigen is HIV-specific antigen GP120, GP41, GP36 or P24.

The method for detecting a pathogenic microorganism by an antigen-stimulated cellular immune reaction according to claim 1, wherein the pathogenic microorganism is an avian influenza virus, and the antigen is an avian influenza virus-specific antigen hemagglutinin HA. , neuraminidase NA or proton channel M2.

The method for detecting a pathogenic microorganism by antigen-stimulated cellular immune reaction according to claim 1, wherein the pathogenic microorganism is a SARS coronavirus, and the antigen is a SARS coronavirus-specific antigen spike glycoprotein. , hemagglutinin acetylesterase glycoprotein or M protein.

6. A method for detecting a pathogenic microorganism of claim cellular immune responses with antigen stimulation in claim 1, wherein said blood cells release cytokines gamma interferon IFN- _Y, IL interleukin or of TGF -p.

The method for detecting a pathogenic microorganism by an antigen-stimulated cellular immune reaction according to any one of claims 1 to 5, wherein the specific antibody is a monoclonal antibody against IFN-γ.

8. The antigen-stimulated cellular immune response according to claim 1 for detecting pathogenic microbes The method of the method, characterized in that the method of detecting uses ELISA, chemiluminescence, immunofluorescence or antibody immunochromatography.

The method for detecting a pathogenic microorganism by an antigen-stimulated cellular immune reaction according to claim 1, wherein the cytokine released by the specific antibody to detect blood cells is a colloidal gold-labeled IFN-γ monoclonal antibody. Antibody immunochromatographic test pen.

10. A colloidal gold immunochromatographic test pen for detecting a pathogenic microorganism, comprising: a blood filter paper, a colloidal gold pad, a nitrocellulose membrane, a water absorbing filter paper, and a PVC bottom plate; the gelatin gold pad is covered with a mouse a composite of a monoclonal antibody colloidal gold against human IFN-γ, the nitrocellulose membrane containing a test line and a control line, the test line coated with IFN-γ, the control line coated with sheep anti-small Mouse IgG polyclonal antibody.