CN111122861A - Kit for detecting specific IgE of serum mycoplasma pneumoniae - Google Patents
Kit for detecting specific IgE of serum mycoplasma pneumoniae Download PDFInfo
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- CN111122861A CN111122861A CN202010066778.8A CN202010066778A CN111122861A CN 111122861 A CN111122861 A CN 111122861A CN 202010066778 A CN202010066778 A CN 202010066778A CN 111122861 A CN111122861 A CN 111122861A
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56933—Mycoplasma
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Abstract
The invention discloses a kit for detecting specific IgE of mycoplasma pneumoniae in serum. The kit consists of a coating antigen, a solid phase carrier, a positive quality control product, a negative quality control product, an enzyme-labeled anti-human IgE antibody, an antigen diluent, a sample dilution buffer, a detection antibody dilution buffer, a washing solution, a developing solution and a stop solution. The kit has the advantages of high sensitivity, strong specificity, low detection cost, small sample consumption, simple and convenient operation, no need of special instruments and equipment for experiment and result judgment and the like, provides a detection kit for detecting Mycoplasma pneumoniae IgE for clinic, and discusses the application of the detection kit in Mycoplasma pneumoniae pneumonia combined asthma diagnosis.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis immunodetection, in particular to a kit for detecting specific IgE in serum mycoplasma pneumoniae, which is a detection kit for quickly, simply, qualitatively and semi-quantitatively analyzing the level of the specific IgE in the serum of children with mycoplasma pneumoniae pneumonia and a preparation method thereof.
Technical Field
Asthma is one of the most common chronic non-infectious diseases, and the worldwide health organization 2014 reports global asthma estimating that 3.34 million people worldwide currently suffer from asthma. Globally with a clear diagnosis of asthma, the prevalence is about 4.3% (95% CI 4.2-4.4), placing a heavy burden on the family and society. Many recent research results show that Mycoplasma Pneumoniae (MP) infection is closely related to the development of asthma and to the development and exacerbation of asthma. Given that children are among the most susceptible populations to mycoplasma pneumoniae, taiwan scholars concluded that the corrected risk ratio for asthma in children compared to populations with no mycoplasma pneumoniae infection was 3.35 (95% CI,2.71-4.15) based on large data analysis by intra-island medical insurance. To evaluate the relationship between the two, we performed some prospective studies to find that 78 cases (52%) of 151 cases of children with asthma attack of 2-16 years, 43 cases (21%) of children with asthma diagnosed and acute attack of 206 cases, whereas the mycoplasma pneumoniae infection rate was only 5% and 4% in the asthma and healthy population in the stationary phase, see fig. 1A. It follows that mycoplasma pneumoniae infection is linked to the occurrence and onset of asthma. In another prospective study we found 77 recurrent asthma attacks out of 118 patients with MP-bearing asthma with a 65% incidence, while only 17 recurrent asthma attacks out of 108 patients without MP-bearing asthma with a 16% incidence, see fig. 1B. It is speculated that chronic infection with MP may be one of the causes of recurrent attacks in children with asthma.
The pathophysiological mechanisms by which mycoplasma pneumoniae causes asthma are not fully understood. Existing studies suggest that type I hypersensitivity reactions caused by mycoplasma pneumoniae infection play a role in the development of asthma. Our previous experimental studies show that mycoplasma pneumoniae infection can cause the imbalance of Th1/Th2 cells in infected people, so that Th0 cells are over-differentiated to Th2 cells, B cells are subjected to antibody class conversion, more IgE is produced, and asthma is mediated. In addition, our experimental results show that the serum total IgE levels of many MP pneumonia (MPP) patients are increased, and we further prove that the allergen corresponding to the increased IgE in the serum of the infant patients is the mycoplasma pneumoniae component. The specific experiment is as follows: the extracted mycoplasma pneumoniae bacterial protein is spotted on a cellulose membrane, MP pneumonia patient serum is added for incubation and washing, and then a secondary antibody of an anti-IgE antibody is added, if the serum contains the anti-MP IgE antibody, a positive strip appears (figure 2A), and if the serum does not contain the anti-MP IgE antibody, the positive strip does not appear (figure 2B). As a result, it was found that 35% of the serum of children with MP pneumonia had IgE antibodies against MP.
Based on the basic research, an MP specificity IgE dot enzyme immunoassay kit and a preparation method thereof are established, and laboratory basis is provided for clinical MPP and asthma early diagnosis.
Disclosure of Invention
The application aims to provide a kit for detecting specific IgE of Mycoplasma Pneumoniae (MP), which mainly comprises a coating antigen, a solid-phase carrier, a positive quality control product, a negative quality control product, an enzyme-labeled anti-human IgE antibody, an antigen diluent, a sample dilution buffer solution, a labeled antibody dilution buffer solution, a washing solution, a substrate color developing agent and a stop solution, wherein the signal detection method is a light color developing method.
The coating antigen is mycoplasma pneumoniae thallus total protein. The mycoprotein is used as the antigen protein by culturing mycoplasma pneumoniae and extracting and quantifying the mycoprotein.
The positive quality control product is mixed serum of MPP patients with high mycoplasma pneumoniae specificity IgE concentration and is quantitatively used as the positive quality control product, and the negative quality control product is normal human serum or fetal bovine serum.
The enzyme-labeled anti-human IgE antibody is horse radish peroxidase-labeled anti-human IgE.
The coating antigen (mycoplasma pneumoniae bacterial total protein) is fixed on a solid phase carrier, and the preferred solid phase carrier comprises: a nitrocellulose membrane.
The antigen diluent is 1 XPBS PH7.4, 163mM NaCL, 1% TritonX-100; the sample dilution buffer solution is 0.01M PBS PH7.4, 10% newborn bovine serum; the labeled antibody dilution buffer is prepared by adding 0.01M PBS containing 0.35% Tween2 to 100ml of 2% glycerol and 1M D-glucose; the washing solution is as follows: 1 XPBS PH7.4, 163mM NaCl, 1% TritonX-100, 10% glycerol; the substrate color developing agent is TMB, AMPPD and 4-MUP; the stop solution is: 2MH2SO4。
According to the invention, the serum is a mammalian (human) serum.
According to the invention, the signal detection method used comprises a light colour development method.
The method for fixing the coating antigen (mycoplasma pneumoniae thallus total protein) is a direct coating method: the antigen is directly fixed on the nitrocellulose membrane by non-covalent bond or chemical combination or physical adsorption.
According to the invention, through a large number of clinical and molecular mechanism researches at the early stage, the Mycoplasma Pneumoniae (MP) infection is closely related to the occurrence of asthma and closely related to the development and the deterioration of the asthma, the total serum IgE level of mycoplasma pneumoniae pneumonia children is higher, and the allergen corresponding to the serum IgE of the children is proved to be the mycoplasma pneumoniae thallus component. Therefore, whether MP specific IgE exists in serum or not is beneficial to early diagnosis, guidance of clinical treatment, prevention of repeated attack and prognosis judgment of asthma children patients caused by mycoplasma pneumoniae.
The specific innovation points of the invention can be summarized as follows:
(1) at present, only kits for detecting IgG and IgM levels in serum after MP infection are available at home and abroad, the kits are used for diagnosing mycoplasma pneumoniae infection, kits for detecting mycoplasma pneumoniae specificity IgE are not available, and the project fills up the blank at home and abroad.
(2) The kit disclosed by the invention relates to qualitative and semi-quantitative analysis of specific IgE of mycoplasma pneumoniae of human serum, wherein a dot enzyme immunoassay method is simple and convenient to operate, the sample dosage is small, the reagent dosage is reduced by about 10 times compared with that of the traditional ELISA, and the detection cost is reduced; the adsorption capacity of the NC membrane is nearly 100%, trace antigens can be completely adsorbed, the sensitivity is high, the specificity is strong, in addition, the NC membrane adsorbing the antigens or the antibodies can be stored for a long time (the NC membrane can be stored for half a year at the temperature of-20 ℃), and the activity of the NC membrane is not influenced, so that the stability of the kit is ensured.
Drawings
Figure 1 MP infection induces asthma development and causes recurrent attacks of asthma.
FIG. 2 IgE antibodies against MP detected in serum of children with MP pneumonia.
FIG. 3 is a technical route for extracting and quantifying MP mycoprotein.
FIG. 4 is a schematic diagram of the operation of detecting MP-specific IgE by the dot enzyme immunoassay method.
FIG. 5 is a schematic diagram showing the determination of MP-specific IgE by dot-enzyme immunoassay.
FIG. 6520 shows the positive rate of MPP-specific IgE in the serum of children patients.
Detailed Description
The invention is further described below with reference to the following figures and specific examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.
1. The examples illustrate that: the experimental principle is that antigen (mycoplasma pneumoniae thallus total protein) is adsorbed on a solid phase carrier to be used as coating antigen, then positive quality control substances or a serum sample to be detected are added for incubation, enzyme-labeled secondary antibodies are added for antigen-antibody reaction, and finally positive and negative results are judged by naked eyes by utilizing a light chromogenic method so as to achieve the purpose of qualitatively and semi-quantitatively analyzing mycoplasma pneumoniae specificity IgE in human serum.
1. Antigen: mycoprotein extracted and quantified after mycoplasma pneumoniae culture is used as an antigen.
1.2. Solid phase carrier: the solid phase carrier selected by the invention comprises the following components: a nitrocellulose membrane.
1.3. Antigen (mycoplasma pneumoniae bacterial total protein) immobilization method: the antigen coating solid phase carrier method of the invention comprises a direct coating method: the antigen is bound to the nitrocellulose membrane by non-covalent bond or physical adsorption.
1.4. Positive quality control product and negative quality control product: the positive quality control material selected by the invention is the mixed serum of a plurality of MPP patients with higher concentration of specific IgE containing mycoplasma pneumoniae and is quantitatively used as the positive quality control material, and the negative quality control material is normal human serum or fetal bovine serum.
1.5. Labeled antibody and substrate developer: the labeled antibody selected by the invention is a goat anti-human IgE antibody labeled by Horse Radish Peroxidase (HRP); the developer is tetramethyl benzidine (TMB).
1.6. Signal detection: and (3) a light color development method.
2. The components and the preparation of the solution and the conventional reagent used in the invention.
2.1. Nitrocellulose membrane (NC) (commercially available).
2.2.PBS PH7.4:KCL 2.7mM、NaCL137mM、Na2HPO4 8.1mM、KH2PO4 1.5mM；
2.3.5% BSA blocking solution: 5g of bovine serum albumin was weighed and dissolved in 100mL of 0.01M PBS.
2.4. Antigen dilution: 1x PBS pH7.4, 163mM NaCL, 1% TritonX-100.
2.5. Sample dilution buffer 0.01M PBS pH7.4, newborn bovine serum 10%.
2.6. Labeled antibody dilution buffer: 0.01M PBS containing 0.35% Tween2 was added to 100ml of 2% glycerol, 1M D-glucose, and filtered under reduced pressure through a 0.22 μ M filter.
2.7. Washing liquid: 1 XPBS PH7.4, 163mM NaCL, 1% TritonX-100, 10% glycerol.
TMB color developer: 50mM imidazole buffer pH5, 7.5mM PEG3350, 2.94 mM urea hydrogen peroxide, 1.6mM TMMB.
2.9. Stopping liquid: 2M H2SO4。
2.10.10% SDS-PAGE.
a.5% concentrated glue preparation:
b.10% separating glue preparation:
example 1 preparation of Total protein of Mycoplasma pneumoniae Strain
1.1 Mycoplasma pneumoniae culture: MP standard strain (ATCC15531, available from MBL, USA) was cultured in Mycoplasma pneumoniae broth, based on Mycoplasma pneumoniae broth CM403 (Oxiod, Hampshire, UK), supplemented with Mycoplasma pneumoniae selection medium feed G SR59 (Oxiod, Hampshire, UK), 0.5% glucose and 0.002% phenol red at 37 ℃ in a 5% C02 incubator overnight.
1.2. And (3) extracting and quantifying total protein of the thallus: the MP culture broth cultured in 1.1 overnight was collected in a centrifuge tube to extract total protein of MP cells by the following steps: (1) centrifuging the tube in a high-speed refrigerated centrifuge at 12000rpm for 30 minutes; (2) discarding the supernatant, washing the precipitate with PBS for 5 times, resuspending in a vortex mixer, and transferring to an EP tube; (3) centrifuging the EP tube obtained in the step 2 for 30 minutes in a high-speed refrigerated centrifuge at 12000 rpm; (4) discarding the supernatant, adding lysis buffer (composed of phenylmethylsulfonyl fluoride: radioimmunoprecipitation assay buffer 20 μ L:1.3 mL) into the EP tube of step 3, thoroughly beating with a pipette, thoroughly lysing at-60 deg.C for 30min and 37 deg.C for 10min, and thoroughly mixing in a vortex mixer; (5) repeating the operation step 4 once; (6) placing the EP tube obtained in the step 5 in a high-speed refrigerated centrifuge at 12000rpm, centrifuging for 30 minutes, and collecting supernatant fluid, namely MP mycoprotein; (7) the MP mycoprotein concentration collected in step 6 was detected by BCA protein quantification kit and stored at-80 ℃ for further use, see FIG. 3.
Example 2 preparation of Positive quality control
2.1. Extraction of mycoplasma pneumoniae specific IgE: through preliminary experiments, a plurality of positive serums with high content of mycoplasma pneumoniae specific IgE are screened and mixed together, the IgE in the mixed serum is quantified according to a German Mediwiss allergen in-vitro detection system, and the mixed serum is stored in a refrigerator at minus 80 ℃ for later use.
Example 3 establishment of Spot enzyme Immunity kit for detecting MP-specific IgE
3.1. The preparation of the antigen-coated nitrocellulose membrane and the detection of the serum sample comprise the following steps:
3.1.1. coating: directly dropping 5 μ l of antigen solution diluted with antigen diluent on nitrocellulose membrane, and drying in 37 deg.C incubator for 30 min;
3.1.2. and (3) sealing: placing the coated antigen NC membrane in a plate groove, adding 100 μ l of 5% BSA blocking solution, blocking in a 37 deg.C incubator for 10min, discarding the blocking solution, and washing with washing solution for 2 times;
3.1.3. antigen incubation: adding 10 μ l of IgE standard or serum diluted with sample diluent into the reaction tank, performing positive control and negative control, mixing, placing in a 37 deg.C wet box, reacting for 30min, and setting 3 repeat holes for each sample;
3.1.4. and (3) secondary antibody incubation: discarding the liquid in the tank, washing with washing solution for 3 times, each time for 1min, adding 20 μ l alkaline phosphatase-labeled goat anti-human IgE diluted with antibody diluent, mixing well, and reacting at room temperature for 30 min;
3.1.5. and (3) color development and naked eye judgment results: discarding liquid in the tank, washing with washing solution for 3 times, 1min each time, adding 500 μ l of developer A/B liquid, reacting at room temperature for 10min, adding 500 μ l of 2M H2SO4 stop solution, mixing completely, discarding liquid in the tank after 1min, washing with washing solution for 3 times, 1min each time, taking out the test strip, drying the membrane strip with a blower, and performing macroscopic qualitative judgment after natural drying: negative reaction (no brown spots); + -: suspicious positive (hidden visibility of brown spots); +: positive reaction (brown spots are clearer); ++: strong positive reactions (dark brown spots) (explained the same below) are shown in FIGS. 4 and 5.
Example 4 evaluation of test System Performance
4.1. The sensitivity of the kit is used for detecting MP specific IgE in 118 parts of patient serum with positive MP mycoprotein ingredient skin tests and 98 parts of MP mycoprotein ingredient skin test negative healthy examinee serum, and the result shows that 115 parts of serum MP specific IgE in 118 parts of asthma children carrying MP are positive, and 98 parts of serum MP specific IgE of healthy examinees are negative, namely the sensitivity of the kit reaches 97.5 percent and meets the technical requirements of the kit.
4.2. The specificity of the kit is adopted to detect the positive serum of respiratory syncytial virus, adenovirus, influenza A/B virus, chlamydia pneumoniae and legionella pneumophila of several common children respiratory pathogens of 100 parts respectively, and the detection is repeated three times per part to evaluate the specificity. The results show that the MP specific IgE in the serum of the pathogens are all negative, which indicates that the specificity of the method for detecting the MP specific IgE by using the kit is very high, and the table 1 shows.
TABLE 1 detection results of six pathogen positive sera
| Pathogen type | Positive results |
| Respiratory | 0/100 |
| | 0/100 |
| | 0/100 |
| | 0/100 |
| | 0/100 |
| | 0/100 |
4.3. Stability the prepared membrane strip coated with the antigen is placed in a drying oven at 60 ℃ and heated for 0 day, 2 days, 6 days and 10 days, and positive serum and negative serum are respectively detected three times, and the stability of the detection system is evaluated. The kit is heated at high temperature at 60 ℃ for 0 day, 2 days, 6 days and no obvious change is seen in the serum detection result after 10 days. The acceleration of 6 days at 60 ℃ is approximately equal to two years of storage at normal temperature, so that the kit can be stably stored for about two years at least.
Example 5 clinical application of kit for detecting MPP infant serum MP specificity IgE
5.1 detection rate of MPP infant serum MP specific IgE
The kit (the product appearance is shown in figure 2) is used for detecting the positive rate of MPP and asthma patient serum MP specific IgE diagnosed in our hospital from 2016 (3) to 2017 (3), and the result shows that 182 patient serum MP specific IgE in 520 MPP patients are positive, and the positive rate reaches 35%.
Claims (9)
1. A kit for detecting specific IgE of serum mycoplasma pneumoniae is characterized by comprising a coating antigen, a solid phase carrier, a positive quality control product, a negative quality control product, an enzyme-labeled anti-human IgE antibody, an antigen diluent, a sample dilution buffer, a detection antibody dilution buffer, a washing solution, a developing solution and a stop solution.
2. The kit for detecting serum mycoplasma pneumoniae specific IgE according to claim 1, wherein the coating antigen is mycoplasma pneumoniae bacterial total protein.
3. The kit for detecting serum mycoplasma pneumoniae specific IgE according to claim 1, wherein the solid phase carrier is nitrocellulose membrane.
4. The kit of claim 1, wherein the coating antigen is immobilized on a solid support.
5. The detection kit according to claim 4, wherein the coated antigen is immobilized directly on the solid support by chemical bonding, physical adsorption or non-covalent bonding.
6. The kit for detecting serum mycoplasma pneumoniae specific IgE according to claim 1, wherein the positive quality control product is a mixed serum of several mycoplasma pneumoniae pneumonia patients with higher mycoplasma pneumoniae specific total IgE concentration and is quantitatively used as the positive quality control product, and the negative quality control product is normal human serum or fetal bovine serum.
7. The kit for detecting serum mycoplasma pneumoniae specific IgE according to claim 1, wherein the enzyme-labeled anti-human IgE antibody is horseradish peroxidase-labeled goat anti-human IgE.
8. The kit for detecting serum mycoplasma pneumoniae specific IgE according to claim 1, wherein the antigen diluent is 1x PBS PH7.4, 163mM NaCL, 1% triton x-100; the sample dilution buffer solution is 0.01M PBS PH7.4, 10% newborn bovine serum; the labeled antibody dilution buffer is prepared by adding 0.01M PBS containing 0.35% Tween2 to 100ml of 2% glycerol and 1M D-glucose; the washing solution is as follows: 1 XPBS PH7.4, 163mM NaCL, 1% TritonX-100, 10% glycerol; the color developing agent is TMB; the stop solution is: 2M H2SO4。
9. The kit for detecting serum mycoplasma pneumoniae specific IgE according to claim 1, wherein the detection serum is mammalian serum.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111875706A (en) * | 2020-07-16 | 2020-11-03 | 广州康盛生物科技股份有限公司 | Single-domain antibody of anti-human IgE protein and application thereof |
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| CN105759034A (en) * | 2016-04-01 | 2016-07-13 | 山东德诺生物科技有限公司 | Mycoplasma pneumoniae detection kit |
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2020
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| EP0278340B1 (en) * | 1987-02-05 | 1993-08-04 | Yehudith Naot | Mycoplasma membrane antigens and their use |
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| CN202433382U (en) * | 2011-12-22 | 2012-09-12 | 北京海瑞祥天生物科技有限公司 | Solid phase carrier for enzyme-linked immunosorbent assay and kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111875706A (en) * | 2020-07-16 | 2020-11-03 | 广州康盛生物科技股份有限公司 | Single-domain antibody of anti-human IgE protein and application thereof |
| CN111875706B (en) * | 2020-07-16 | 2021-03-30 | 广州康盛生物科技股份有限公司 | Single-domain antibody of anti-human IgE protein and application thereof |
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