CN110865187A - Method for calculating average spot area of ESAT-6 antigen hole based on enzyme-linked immunosorbent assay kit - Google Patents
Method for calculating average spot area of ESAT-6 antigen hole based on enzyme-linked immunosorbent assay kit Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- 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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
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- G01N2333/7156—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for interferons [IFN]
Abstract
The invention discloses a method for calculating the average spot area of an ESAT-6 antigen hole based on an enzyme-linked immunosorbent assay kit, which comprises the steps of firstly, adding collected peripheral blood mononuclear cell suspension to be detected into a micropore culture plate coated with IFN-gamma antibody; adding ESAT-6 protein liquid into the peripheral blood mononuclear cell suspension to be tested for culture, and adding IFN-gamma antibody enzyme-labeled working solution into the corresponding micropores for incubation; adding a substrate into each hole, incubating at room temperature in a dark place, and drying; calculating the average spot area of the spot forming cells in the microplate using a spot counter; the method of the invention detects the release of IFN-gamma by peripheral blood mononuclear cells under the condition of the stimulation of tuberculosis specific antigen ESAT-6 through ELISPOT technology and guides doctors to carry out differential diagnosis on active tuberculosis patients and latent tuberculosis patients through the average spot area of spots formed by color development.
Description
Technical Field
The invention relates to the field of active tuberculosis, in particular to a method for calculating the average spot area of an ESAT-6 antigen hole based on an enzyme-linked immunosorbent assay kit.
Background
Tuberculosis is one of serious diseases threatening human health, mainly causes lung diseases, and has high morbidity and mortality. Historically, tuberculosis has been prevalent throughout the world, taking hundreds of millions of people away from life. Antituberculosis drugs have been discovered since the 50 s of the 20 th century, and the prevalence of tuberculosis has been controlled to a certain extent. In recent years, the prevalence of tuberculosis is rising due to the neglect of the tuberculosis prevention and treatment work in some countries, the increase of mobile population, the spread of AIDS infected persons, the continuous emergence of drug-resistant tuberculosis strains and other factors. Tuberculosis is still one of the first 10 causes of death in the world, and the world health organization estimates about 1000 thousands of new tuberculosis patients in the world in 2017, and the number of deaths is about 157 thousands. The latent tuberculosis infection in the world is about 17 hundred million, and the latent tuberculosis infection rate is 23 percent. Therefore, the world health organization has announced that "global tuberculosis emergency" is entered, and 3 months and 24 days per year are defined as "worldwide tuberculosis prevention days", and a plan for stopping tuberculosis in 2030 is made.
In the face of such severe situations, we should take measures early. The early diagnosis and effective treatment of tuberculosis are the central importance of the tuberculosis prevention and treatment work, and have important clinical significance for controlling the spread of mycobacterium tuberculosis. At present, most hospitals in China diagnose active tuberculosis according to direct smear microscopy, a mycobacterium tuberculosis culture technology and a molecular detection means such as Gene-Xpert MTB/RIF. The tubercle bacillus smear detection is simple and easy to implement, has high accuracy, but has low positive rate. The culture and detection result of the mycobacterium tuberculosis has high reliability, but the time consumption is long, and the clinical requirement cannot be met. Gene-Xpert detection has high specificity and short time consumption, but the detection sensitivity in a few bacteria sample can not meet the requirement. In addition, tuberculin skin test (PPD) experiments are the main basis for diagnosing tubercle bacillus infection at present in China. The method is simple and inexpensive, but has the greatest disadvantage that the antigen used in the PPD test is a crude antigen mixture of Mycobacterium tuberculosis, and many nontuberculous mycobacteria and Bacillus Calmette-Guerin (BCG) have common antigen components, resulting in poor specificity of the method for diagnosing tuberculosis. China is a tuberculosis highly epidemic area, and because BCG is widely inoculated, the false positive rate of a PPD experiment is high, and the diagnosis can be assisted only according to the strength of the reaction of the skin of the PPD experiment in clinic.
The release test of the cell immune mediated mycobacterium tuberculosis gamma-interferon (IFN-gamma) adopts an enzyme linked immunosorbent assay (ELISA) or an enzyme linked immunosorbent assay (ELISPOT) in recent years, and diagnoses latent and active tuberculosis infection by quantitatively detecting IFN-gamma released by peripheral blood mononuclear cells of a detected person under the stimulation of specific antigen of mycobacterium tuberculosis. The principle of the method is that Th1 type secretion factor IFN-gamma is closely related to the content of tubercle bacillus antigen in vivo. T cells sensitized with Mycobacterium tuberculosis antigen produce high levels of IFN-gamma when they encounter the same antigen again, and thus are used for diagnosis of tuberculosis infection. Based on this principle, Oxford Immunotechnology in England has developed TSPOT-TB diagnostic kits that use early antigen target 6(ESAT-6) of tubercle bacillus specific antigen, culture filtrate protein 10(CFP-10) to stimulate mononuclear cells in the peripheral blood of a subject and detect IFN-. gamma.levels to diagnose tuberculosis infection. The experiment is not influenced by BCG and nontuberculous mycobacteria, and the detection sensitivity and specificity are high. Unfortunately, the method based on the interferon-gamma release assay, while enabling rapid diagnosis of mycobacterium tuberculosis infection, cannot be used to distinguish between active and latent tuberculosis infection.
Patients with active tuberculosis should be treated as early as possible. However, China is a big tuberculosis country, and a large number of latent tuberculosis infected people exist, and the latent tuberculosis infected people may have no clinical symptoms for all the life and do not need any treatment. Due to the high sensitivity of the gamma-interferon release test, except for active tuberculosis infectors, the method is also applied to detect latent tuberculosis infectors, thereby bringing difficulty to clinical medication. This is also an important reason why the gamma-interferon release test for tuberculosis diagnosis is difficult to be popularized in China. If a detection method capable of distinguishing active tuberculosis infection from latent tuberculosis infection can be developed, the method has important significance for diagnosis and treatment of tuberculosis in China.
At present, a method for detecting the average spot area of an ESAT-6 antigen hole based on an enzyme-linked immunospot (ELISPOT) kit so as to be applied to the field of active tuberculosis is not developed in China.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for calculating the average spot area of an ESAT-6 antigen hole based on an enzyme-linked immunosorbent assay (ELISPOT) kit; the method is based on an ELISPOT kit to detect that peripheral blood mononuclear cells of an active tuberculosis patient are induced by a tuberculosis specific antigen ESAT-6, and then the average spot area of antigen holes is calculated; the method is used in the field of active tuberculosis.
In order to realize the aim, the method for calculating the average spot area of the ESAT-6 antigen hole based on the ELISA spot test kit comprises the following steps:
1) adding the collected peripheral blood mononuclear cell suspension to be detected into a micropore culture plate coated with IFN-gamma antibody; adding ESAT-6 protein liquid into the peripheral blood mononuclear cell suspension to be detected; wherein the volume ratio of the ESAT-6 protein liquid to the peripheral blood mononuclear cell suspension to be detected is 1: 1-3;
2) placing the micropore culture plate in an incubator for culture;
3) taking out the micropore culture plate in the step 2), removing culture solution supernatant in the pores, washing with PBS buffer solution, and drying the liquid in the pores;
4) preparing IFN-gamma antibody enzyme-labeled working solution by using PBS buffer solution;
5) adding IFN-gamma antibody enzyme-labeled working solution into the corresponding micropores, and incubating for 1 hour at the temperature of 2-8 ℃;
6) discarding the liquid, washing with PBS buffer solution, and drying the liquid in the hole;
7) adding substrate into each hole (placing at room temperature in advance for balancing for more than 1 hour), and incubating at room temperature in a dark place;
8) discarding the liquid, flushing with deionized water to terminate the reaction, drying the liquid in the holes, and drying;
9) the average spot area of the spot forming cells within the microplate was calculated using a spot counter.
Further, in the step 1), the volume ratio of the ESAT-6 protein liquid to the peripheral blood mononuclear cell suspension to be detected in the micropore culture plate is 1: 2.
And further stillIn the step 2), the culture conditions of the incubator are as follows: the temperature is 35-37 ℃, and CO is2The concentration is 5%; the culture time is 16-20 hours.
Furthermore, in the step 5), the volume ratio of the IFN-gamma antibody enzyme-labeled working solution to the ESAT-6 protein solution is 1: 1.
Still further, in the step 7), the volume ratio of the substrate to the ESAT-6 protein solution is 1: 1.
The articles (the micropore culture plate coated with mouse anti-human IFN-gamma monoclonal antibody and the mouse anti-human IFN-gamma monoclonal antibody marked by alkaline phosphatase) used in the kit can be purchased from the market and directly used.
The principle of the invention is as follows:
according to the invention, specific effector cells in a patient infected with active tuberculosis and latent tuberculosis can be rapidly activated when being induced by tuberculosis specific antigen ESAT-6 (early antigen target 6) again, inflammatory cytokine IFN-gamma (gamma-interferon) is secreted, the specific effector cells can be captured by a micropore plate coated with IFN-gamma antibody, a substrate is added to enable a bottom plate to develop color to form spots, and then a spot counter is used for calculating the average spot area of the spot forming cells. Since the mean spot area of ESAT-6 is significantly different between active and latent tuberculosis infected patients, a physician-assistant can be used to distinguish active from latent tuberculosis infection.
The invention has the beneficial effects that:
the method of the invention detects the release of IFN-gamma by peripheral blood mononuclear cells under the condition of the stimulation of tuberculosis specific antigen ESAT-6 by an ELISPOT technology and the average spot area of spots formed by color development, thereby guiding doctors to carry out differential diagnosis on active tuberculosis patients and latent tuberculosis patients; the method provided by the invention is used for assisting a doctor to distinguish active tuberculosis infection and latent tuberculosis infection by calculating the average spot area of tuberculosis specific spots, wherein the sensitivity of diagnosing the active tuberculosis infection is 55%, and the specificity is more than 90%. In addition, the invention applies the ELISPOT-based method principle, thereby having the advantages of rapidness, economy and the like, and the method has important significance for early diagnosis of tuberculosis.
Drawings
FIG. 1 is a graph showing the distribution of the average spot area under ESAT-6 induction;
FIG. 2 is a representative plaque plot of active tuberculosis patients versus latent tuberculosis controls;
FIG. 3 is a graph of the working characteristics of subjects diagnosed with active tuberculosis versus latent tuberculosis.
Detailed Description
The present invention is described in further detail below with reference to specific examples so as to be understood by those skilled in the art.
Example 1
The method for calculating the average spot area of the ESAT-6 antigen hole based on the ELISA spot test kit comprises the following steps:
1) adding the collected peripheral blood mononuclear cell suspension to be detected into a micropore culture plate; adding ESAT-6 protein liquid into the peripheral blood mononuclear cell suspension to be detected; wherein the volume ratio of the ESAT-6 protein liquid to the peripheral blood mononuclear cell suspension to be detected is 1: 2;
2) placing the micropore culture plate at the temperature of 35-37 ℃ and CO2Culturing for 16-20 hours in an incubator with the concentration of 5%;
3) taking out the micropore culture plate in the step 2), removing culture solution supernatant in the pores, washing with PBS buffer solution, and drying the liquid in the pores;
4) preparing IFN-gamma antibody enzyme-labeled working solution by using PBS buffer solution;
5) adding the prepared IFN-gamma antibody enzyme-labeled working solution into the corresponding micropores according to the volume ratio of 1:1 of the substrate to the ESAT-6 protein solution, and incubating for 1 hour at the temperature of 2-8 ℃;
6) discarding the liquid, washing with PBS buffer solution, and drying the liquid in the hole;
7) adding the substrate into each hole according to the volume ratio of the substrate to the ESAT-6 protein solution of 1:1 (placing the substrate at room temperature in advance for balancing for more than 1 hour), and incubating the substrate at room temperature in a dark place;
8) discarding the liquid, washing with deionized water to terminate the reaction, drying the liquid in the holes, drying the culture plate in a drying oven at 50 ℃ for 1 hour;
9) the average spot area of the spot forming cells within the microplate was calculated using a spot counter. The raw materials in the reagent kit for the enzyme-linked immunosorbent spot test can be purchased from the market; based on the method, in the actual working process, a doctor is guided to judge whether active tuberculosis infection exists or not according to the average spot area:
if the average spot area of the detection hole is more than or equal to 16 multiplied by 10-3mm2If so, judging the result as active tuberculosis infection; if the average spot area of the detection holes is less than 16 multiplied by 10-3mm2And judging the result to be no active tuberculosis infection.
Example 2
140 active tuberculosis patients and 138 latent tuberculosis controls were tested using the method described above.
Case selection
All patients and controls were from the affiliated college medical college of science and technology, huazhong. Inclusion criteria for both groups of people were as follows:
patients with active tuberculosis: has clinical symptoms of fever, night sweat or emaciation, and the mycobacterium tuberculosis is Gene-Xpert positive.
Latent tuberculosis controls: has no clinical symptoms such as fever, night sweat or emaciation, and no active tuberculosis imaging performance, but has positive gamma-interferon release test (QuantiFERON).
(II) a detection method:
1. isolation of peripheral blood mononuclear cells from a subject
1) Extracting 4-6 ml of blood, adding the blood into a sterile anticoagulation blood collection tube, and taking the blood, reversing and uniformly mixing to obtain anticoagulation blood;
2) taking out a 15ml sterile centrifuge tube, adding 4ml anticoagulation blood and 4ml 1640 cell culture solution to obtain 8ml blood dilution, and mixing well;
3) another 15ml of sterile centrifuge tube is taken, 4ml of peripheral blood mononuclear cell separation liquid is added, then 8ml of diluted blood is carefully added to the upper layer of the centrifuge tube to form an obvious interface, and the centrifuge tube is centrifuged for 22min at 1000g under the condition of room temperature;
4) after centrifugation, a layer of obvious cloud-like mononuclear cells can be seen, and the layer of cells are sucked into another sterile centrifuge tube;
5) adding 9-10ml of 1640 cell culture solution into a centrifugal tube filled with mononuclear cells, uniformly mixing by blowing, and centrifuging for 7min at 600g at room temperature;
6) discarding the supernatant, adding 9-10ml 1640 cell culture solution, uniformly mixing by blowing, and centrifuging for 7min at 350g under the room temperature;
7) discarding the supernatant, adding 700. mu.l of culture medium to resuspend the cells, taking 10. mu.l of the resuspended cells, adding the cells into a blood counting plate, counting under a microscope, and diluting the cells to 2.5X 10 according to the counting result6Cells/ml for use;
2. cell induction
1) Adding 100 mul of peripheral blood mononuclear cell suspension to be detected into a micropore culture plate;
2) adding 50 μ l of ESAT-6 protein solution into the above well;
3) placing the micro-pore culture plate at 37 deg.C and 5% CO2Culturing for 16-20 hours in the incubator;
3. detection of
1) Taking out the microporous culture plate in the cell induction step 3), discarding culture solution supernatant in the pores, washing for 4 times by using 200 mu l of PBS buffer solution, and drying the liquid in the pores;
2) preparing IFN-gamma antibody enzyme-labeled working solution by PBS buffer solution according to the ratio of 1: 200;
3) adding 50 μ l of enzyme-labeled working solution into the hole, and incubating for 1 hour at 2-8 ℃;
4) discarding the liquid, washing 4 times with 200 μ l PBS buffer solution, and drying the liquid in the hole;
5) add 50. mu.l substrate per well (put in advance at room temperature and equilibrate for more than 1 hour), incubate for 7 minutes at room temperature in the dark;
6) discarding the liquid, flushing with deionized water to terminate the reaction, and drying the liquid in the holes;
7) drying the culture plate in a drying oven at 50 ℃ for 1 hour;
8) the average spot area of the spot forming cells in the microplate was measured using a spot counter.
4. The experimental results are as follows:
the mean spot area under ESAT-6 stimulation for both groups of subjects is shown in FIG. 1 below:
if the average spot area of the detection hole is more than or equal to 16 multiplied by 10-3mm2If so, judging the result as active tuberculosis infection; if the average spot area of the detection holes is less than 16 multiplied by 10-3mm2And judging the result to be no active tuberculosis infection.
The mean spot area was greater than or equal to 16X 10 in 140 selected patients with active tuberculosis-3mm2In 77 cases, the sensitivity detected by using the method reaches 53 percent;
out of 138 cases of the latent tuberculosis control, 95 cases of the latent tuberculosis control have average spot area smaller than 16 multiplied by 10-3mm2Only 9 cases gave results greater than 16X 10-3mm2The specificity detected by the method reaches 93 percent.
Representative plots of active tuberculosis patients versus latent tuberculosis controls are shown in FIG. 2 below, and the working characteristic curves of subjects who are differentially diagnosed as active tuberculosis patients versus latent tuberculosis controls are shown in FIG. 3 below.
The results show that the method for calculating the average spot area of the spot forming cells in the microporous plate has high accuracy and strong specificity, and can accurately assist doctors in detecting the active tuberculosis infection; the experimental operation is simple, and the result can be reported in 24 hours, which shows that the method can be used as a means for auxiliary diagnosis of active tuberculosis.
Other parts not described in detail are prior art. Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (5)
1. A method for calculating the average spot area of an ESAT-6 antigen hole based on an enzyme-linked immunosorbent assay kit is characterized in that: the method comprises the following steps:
1) adding the collected peripheral blood mononuclear cell suspension to be detected into a micropore culture plate coated with IFN-gamma antibody; adding ESAT-6 protein liquid into the peripheral blood mononuclear cell suspension to be detected; wherein the volume ratio of the ESAT-6 protein liquid to the peripheral blood mononuclear cell suspension to be detected is 1: 1-3;
2) placing the micropore culture plate in an incubator for culture;
3) taking out the micropore culture plate in the step 2), removing culture solution supernatant in the pores, washing with PBS buffer solution, and drying the liquid in the pores;
4) preparing IFN-gamma antibody enzyme-labeled working solution by using PBS buffer solution;
5) adding IFN-gamma antibody enzyme-labeled working solution into the corresponding micropores, and incubating for 1 hour at the temperature of 2-8 ℃;
6) discarding the liquid, washing with PBS buffer solution, and drying the liquid in the hole;
7) adding substrate into each hole, and incubating at room temperature in a dark place;
8) discarding the liquid, flushing with deionized water to terminate the reaction, drying the liquid in the holes, and drying;
9) the average spot area of the spot forming cells within the microplate was calculated using a spot counter.
2. The method for calculating the average spot area of ESAT-6 antigen wells based on the ELISA spot test kit of claim 1, wherein the method comprises the steps of: in the step 1), the volume ratio of the ESAT-6 protein liquid to the peripheral blood mononuclear cell suspension to be detected in the micropore culture plate is 1: 2.
3. The method for calculating the average spot area of ESAT-6 antigen wells based on the ELISA spot test kit of claim 1, wherein the method comprises the steps of: in the step 2), the culture conditions of the incubator are as follows: the temperature is 35-37 ℃, and CO is2The concentration is 5%; the culture time is 16-20 hours.
4. The method for calculating the average spot area of ESAT-6 antigen wells based on the ELISA spot test kit of claim 1, wherein the method comprises the steps of: in the step 5), the volume ratio of the IFN-gamma antibody enzyme-labeled working solution to the ESAT-6 protein solution is 1: 1.
5. The method for calculating the average spot area of ESAT-6 antigen wells based on the ELISA spot test kit of claim 1, wherein the method comprises the steps of: in the step 7), the volume ratio of the substrate to the ESAT-6 protein liquid is 1: 1.
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