CN111521820B - Use of mycobacterium tuberculosis proteins in the preparation of a product for diagnosing tuberculosis latency infected persons and/or active tuberculosis - Google Patents
Use of mycobacterium tuberculosis proteins in the preparation of a product for diagnosing tuberculosis latency infected persons and/or active tuberculosis Download PDFInfo
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Abstract
The invention provides the use of products for detecting levels of anti-Rv 0389 antibodies, anti-Rv 1408 antibodies and anti-Rv 2097c antibodies in a sample in the preparation of products for the identification, diagnosis and/or screening of active tuberculosis. The method provided by the invention can be used for simultaneously detecting the serum antibody response levels of three different mycobacterium tuberculosis proteins, so that tuberculosis latent infection patients and active tuberculosis patients can be better identified and diagnosed, the sensitivity is higher, and the specificity is better.
Description
The present application is a divisional application of the invention patent application with the application number 2016112574947, the application date of which is 2016, 12 and 30.
Technical Field
The invention relates to the field of biological medicine, in particular to application of mycobacterium tuberculosis proteins in preparation of products for diagnosing tuberculosis latent infection and/or active tuberculosis.
Background
Tuberculosis caused by mycobacterium tuberculosis is still one of diseases seriously threatening human health, according to the latest report of world health organization, 1040 thousands of new cases of tuberculosis are worldwide in 2015, about 180 thousands of people die from tuberculosis, 91.8 thousands of people in China are new diseases, 3.8 thousands of people are dead, and the total number of diseases is the fourth place in the world. The prevention and treatment of tuberculosis is still a serious problem, both worldwide and in China, and particularly in some underdeveloped countries with poor health, the infection rate of tuberculosis is still high.
After the human body is infected with the mycobacterium tuberculosis, about 10% of people develop active tuberculosis, but 90% of people do not develop active tuberculosis, but the in-vivo tuberculosis is not completely cleared, but is hidden in the human body, which is called tuberculosis hidden infection, the people do not have any clinical symptoms, most of people do not develop the active tuberculosis in their life, and only 5% -10% of people can cause the occurrence of tuberculosis when the immunity of the human body is reduced. At present, about 1/3 of people in the whole world are infected with tubercle bacillus, and the number of people with latent infection is numerous, so that the people with latent infection are required to be monitored, and medicine treatment is timely given when the infection state changes, so that early detection and early treatment are realized, the worsening of the illness state can be prevented, the treatment time and the side effect caused by medicine treatment can be reduced, and the spreading of tuberculosis can be reduced. Therefore, detection and identification of two states after tuberculosis infection are very important for prevention and treatment of tuberculosis.
There are many methods for detecting tubercle bacillus infection at present, including tubercle bacillus skin test (TST), gamma-interferon release test (IGRA), traditional sputum acid fast bacteria smear, quick culture of mycobacterium, amplification of tuberculosis specific nucleic acid fragment (quantitative and qualitative), etc. TST is skin allergy, and the service time is long, is mainly used for outpatient screening, and although the test is simple and easy to implement, when the tuberculosis infection condition is detected, the same result appears in BCG inoculation, natural infection cannot be identified, the specificity is not strong, and in addition, tuberculosis latent infection and active tuberculosis cannot be identified. The gamma-interferon release assay (IGRA) is mainly used for detecting cellular immune responses, but can exclude the influence of bacillus calmette-guerin inoculation, and can not distinguish latent infection patients from active tuberculosis patients, and can not distinguish patients after treatment from symptomatic infection patients. The traditional sputum acid-fast bacteria smear, mycobacterium rapid culture and the like are mainly used for detecting active tuberculosis patients, have no meaning for judging the latent infection state, and can not find bacteriological evidence for active tuberculosis patients with atypical clinical symptoms. It would therefore be of great value to find a method that would accurately identify latent infection and active tuberculosis.
Based on the difference of host protective immune response caused after tubercle bacillus infection, the search of serum markers for distinguishing latent infection and active tuberculosis has good application prospect. The serological diagnosis of antigen-antibody reaction is focused on the ease and rapidity of sample acquisition, and some specific antigens of mycobacterium tuberculosis are currently used for serological diagnosis of tuberculosis infection, such as secreted protein antigen: antigen 85 complex antigens, i.e., ag85A, ag85B and Ag85C, etc.; lipoprotein antigen: a 16kDa,27kDa,38kDa antigen, etc.; glycolipid antigen: lipoarabinomannan antigen, tubercle bacillus saccharide lipoantigen, etc.; however, in practical use, the antigens have low positive detection rate and cross immunity with other mycobacteria, and have certain value in general investigation, treatment effect monitoring, prognosis judgment and recurrence prompting of high-risk groups, but cannot be used for the definite diagnosis of latent mycobacterium tuberculosis infection at present.
Therefore, it is very important to find a new method and a marker that can accurately diagnose tuberculosis latent infectors and active tuberculosis patients.
Disclosure of Invention
The invention aims to solve the technical problems that in the prior art, products for differential diagnosis of tuberculosis latent infected persons and/or active tuberculosis have low positive detection rate and latent mycobacterium tuberculosis infection cannot be differential diagnosed, and provides application of mycobacterium tuberculosis protein in preparation of products for diagnosis of tuberculosis latent infected persons and/or active tuberculosis.
In order to solve the technical problems, the technical scheme provided by one aspect of the invention is as follows:
use of a product for detecting the level of an anti-Rv 0389 antibody, an anti-Rv 1408 antibody and/or an anti-Rv 2097c antibody in a sample for the preparation of a product for the identification, diagnosis and/or screening of latent tuberculosis infection and/or active tuberculosis.
In the technical scheme of the invention, any product for detecting the level of the anti-Rv 0389 antibody, the anti-Rv 1408 antibody and/or the anti-Rv 2097c antibody in a sample can be used for preparing a product for identifying, diagnosing and/or screening latent tuberculosis infection and/or active tuberculosis.
Preferably, the product for detecting the level of the anti-Rv 0389 antibody, the anti-Rv 1408 antibody and/or the anti-Rv 2097c antibody in the sample contains an Rv0389 protein, an Rv1408 protein and/or an Rv2097c protein.
More preferably, the Rv0389 protein is shown in SEQ ID No.1, or one or more amino acid residues in the amino acid sequence shown in SEQ ID No.1 are modified to have the same function as the protein shown in SEQ ID No. 1; the Rv1408 protein is shown in SEQ ID NO.2, or one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2 are modified to have the same function as the protein shown in SEQ ID NO. 2; the Rv2097c protein is shown as SEQ ID NO.3, or one or more amino acid residues in the amino acid sequence shown as SEQ ID NO.3 are modified to have the same function as the protein shown as SEQ ID NO. 3.
The term "engineering" as described above may use any prior art method of engineering proteins, including, but not limited to, substitution and/or addition and/or deletion of amino acid residues, to form derivatives of the original protein.
Preferably, the assay may be any suitable assay known in the art, but preferably, the assay may be an enzyme-linked immunosorbent assay, an agglutination assay, a precipitation assay, an E-garland assay, a small phagocytosis assay, an immunofluorescence assay, a colloidal gold immunochromatography assay, a spot gold immunodiafiltration assay and/or an electrochemiluminescence assay. More preferably, in one embodiment of the invention, the method of detection is an enzyme-linked immunosorbent assay.
Preferably, the sample may be any suitable sample obtained from the subject to be tested. But preferably the sample comprises serum, plasma, saliva, urine, hydrothorax and/or cerebrospinal fluid. More preferably, in one embodiment of the invention, the sample is serum.
Another aspect of the present invention is to provide a marker composition for identifying, diagnosing and/or screening for latent tuberculosis infection and/or active tuberculosis, the marker composition consisting of an anti-Rv 0389 antibody, an anti-Rv 1408 antibody and/or an anti-Rv 2097c antibody;
wherein, the Rv0389 is shown in SEQ ID NO.1, or one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1 are modified to have the same function as the protein shown in SEQ ID NO. 1; the Rv1408 is shown as SEQ ID NO.2, or one or more amino acid residues in the amino acid sequence shown as SEQ ID NO.2 are modified to have the same function as the protein shown as SEQ ID NO. 2; the Rv2097c is shown as SEQ ID NO.3, or one or more amino acid residues in the amino acid sequence shown as SEQ ID NO.3 are modified to have the same function as the protein shown as SEQ ID NO. 3.
In another aspect, the invention provides the use of a marker composition as described above for the preparation of a product for the identification, diagnosis and/or screening of latent tuberculosis infection and/or active tuberculosis.
In another aspect of the invention there is provided a kit for identifying, diagnosing and/or screening for latent tuberculosis infection and/or active tuberculosis, the kit comprising an Rv0389 protein, an Rv1408 protein and/or an Rv2097c protein.
Preferably, the Rv0389 protein is shown in SEQ ID NO.1, or one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1 are modified to have the same function as the protein shown in SEQ ID NO. 1; the Rv1408 protein is shown in SEQ ID NO.2, or one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2 are modified to have the same function as the protein shown in SEQ ID NO. 2; the Rv2097c protein is shown as SEQ ID NO.3, or one or more amino acid residues in the amino acid sequence shown as SEQ ID NO.3 are modified to have the same function as the protein shown as SEQ ID NO. 3.
The Rv0389 protein, rv1408 protein and/or Rv2097c protein contained in the above kit may be in any form, including but not limited to direct protein samples, and forms of immobilizing proteins on a carrier such as a detection chip.
The kit comprises at least kit instructions in addition to the Rv0389 protein, the Rv1408 protein and/or the Rv2097c protein.
Criteria for the results are described in the specification: and (3) using three proteins for combined detection, if at least 2 antibodies in the anti-Rv 0389 antibody, the anti-Rv 1408 antibody and/or the anti-Rv 2097c antibody are positive in the detection result of the sample to be detected, judging that the sample to be detected is positive in active tuberculosis, otherwise, judging that the sample to be detected is latent tuberculosis infection.
In another aspect, the invention provides the use of a kit as described above for the preparation of a product for the identification, diagnosis and/or screening of latent tuberculosis infection and/or active tuberculosis.
The beneficial effects of the invention are as follows:
the invention can detect the response level of three different mycobacterium tuberculosis protein serum antibodies at the same time, thereby being capable of better distinguishing and diagnosing tuberculosis latent infection patients and active tuberculosis patients, and having higher sensitivity and better specificity.
Drawings
FIG. 1 is a statistical chart showing ELISA detection of expression levels of anti-Rv 0389 antibody, anti-Rv 1408 antibody and anti-Rv 2097c antibody in serum of tuberculosis latent infected person (n=93) and active tuberculosis patient (n=92)
FIG. 2 is a ROC graph of antibodies corresponding to the combined detection of three antigens;
DESCRIPTION OF THE SEQUENCES
SEQ ID NO.1 is the amino acid sequence of the Rv0389 protein in the example of the present invention;
SEQ ID NO.2 is the amino acid sequence of the Rv1408 protein in the embodiment of the invention;
SEQ ID NO.3 is the amino acid sequence of the Rv2097c protein in the examples of the present invention.
Detailed Description
The invention discloses application of a product for detecting the level of an anti-Rv 0389 antibody, an anti-Rv 1408 antibody and/or an anti-Rv 2097c antibody in a sample in preparing a product for identifying, diagnosing and/or screening latent tuberculosis infection and/or active tuberculosis. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is to be particularly pointed out that all similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the invention and that the relevant person can make modifications and appropriate alterations and combinations of what is described herein to make and use the technology without departing from the spirit and scope of the invention.
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art.
In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail with reference to specific embodiments.
Example 1: sample collection and processing
The sample detected by the invention is serum, the collection method is that blood of a subject is collected on an empty stomach, a common biochemical test tube without anticoagulant is used, the sample is stood at room temperature and naturally solidified, and is centrifuged for 10 minutes at a speed of 3000 rpm Zhong Changwen, the serum is collected and split-packed in a freezing tube, and the freezing tube is placed in a low-temperature refrigerator at-80 ℃ for preservation and is used for experiments.
The samples used in the present invention are divided into two groups: a group of latent infections and active tuberculosis patients; the latent infection group refers to a patient without clinical complaints, and the TST experiment and the T-SPOT experiment are positive and the chest X-ray film is negative; the active tuberculosis patient group refers to the tuberculosis patients which are positive through smear or culture and are not or just beginning systematic chemical drug treatment; both groups were excluded from diabetes, HIV infection and other autoimmune diseases.
The latent infection group specimen used in the invention is from epidemiological investigation projects of epidemiology research laboratory of Beijing tuberculosis chest tumor research institute, the active tuberculosis patient group specimen is from inpatients in each disease area of Beijing tuberculosis hospital affiliated to the university of the capital medical science, the subject is approved by the ethics committee of Beijing tuberculosis chest hospital/Beijing tuberculosis chest tumor research institute of the university of the capital medical science, and all the study objects are informed consent.
Example 2: detection of samples using ELISA experiments
1. Various buffers, dilutions, reaction solutions and termination solutions required to establish ELSIA experiments:
(1) Coating buffer (pH 9.6,0.05mol/L carbonate buffer):
Na 2 CO 3 1.59g，NaHCO 3 2.93g of double distilled water is added to 1000ml, and the pH is adjusted to 9.6;
(2) PBS solution at pH 7.4:
NaCl 8.0g，KCl 0.2g，KH 2 PO 4 0.2g，Na 2 HPO 4 ·12H 2 O 2.9g，
adding double distilled water to 1000ml, and regulating pH to 7.4;
(3) PBST wash at ph 7.4:
tween-20 is added into 1000ml of PBS solution for 0.5ml, and the pH is adjusted to 7.4;
(4) Blocking solution (PBS solution ph7.4 containing skimmed milk powder):
5g of skimmed milk powder was added to 100ml of PBS solution;
(5) Substrate buffer: 0.2M NaH 2 PO 4 (28.4g/L)25.7ml，
0.1M citric acid (19.2 g/L) 24.3ml, and distilled water 50ml.
(6) TMB (tetramethylbenzidine) use solution TMB (10 mg/5ml absolute ethanol) 0.5ml substrate
10ml of buffer (pH 5.5) 0.75% H 2 O 2 32μl；
(7) Termination liquid (2M H) 2 SO 4 )：21.32ml H 2 SO 4 ，178.68ml H 2 O。
(8) And (2) secondary antibody: horseradish peroxidase (HRP) -labeled goat anti-human IgG;
(9) 96-well elisa plate; nunc Incorporated (Thermomo, gebenhagen, denmark)
2. The detection step comprises:
(1) Coating: the Rv0389, rv1408 and Rv2097c antigens were dissolved in coating buffer (5 ug/ml), respectively; 100 ul/well was added to the 96-well ELISA plate at 4deg.C overnight;
(2) Washing the plate: PBST washing solution 300 ul/hole for 3min, washing 3 times;
(3) Closing: blocking the solution, 300 ul/hole, and incubating for 1h at room temperature;
(4) Washing the plate: 300 ul/hole of washing liquid, 3min, 3 times of washing;
(5) Adding serum: the serum to be tested is diluted according to the ratio of 1:400, 100 ul/hole is incubated for 1h at room temperature;
(6) Washing the plate: 300 ul/hole of washing liquid, 3min, 5 times of washing;
(7) Adding enzyme-labeled secondary antibodies: 100 ul/well of freshly diluted enzyme-labeled antibody (1:30000), incubated for 1h at room temperature;
(8) Washing the plate: 300 ul/hole of washing liquid, 3min, 5 times of washing;
(9) Color development: freshly prepared TMB color development solution, 100 ul/hole, incubate at room temperature in the dark for 5-10min;
(10) And (3) terminating: termination liquid 2M H 2 SO 4 50 ul/well;
(11) And (3) measuring: the microplate reader was adjusted to 450nm and the OD of each well was measured after zeroing the first blank PBS control well.
3. Result analysis and decision criteria:
drawing a ROC curve by using SPSS22.0 software, obtaining the specificities and sensitivities corresponding to different critical points according to coordinate values, and then calculating about dengue indexes (about dengue indexes = specificities + sensitivities-1), wherein the OD values of the critical points of the three antigens are respectively 0.291 for Rv0389, 0.283 for Rv1408 and 0.308 for Rv2097 c;
the results are shown in figure 1, and the results show that all three proteins have the effect of distinguishing latent tuberculosis infection from active tuberculosis.
Determination criteria:
and (3) using three proteins for combined detection, if at least 2 antibodies in the anti-Rv 0389 antibody, the anti-Rv 1408 antibody and/or the anti-Rv 2097c antibody are positive in the detection result of the sample to be detected, judging that the sample to be detected is positive in active tuberculosis, otherwise, judging that the sample to be detected is latent tuberculosis infection.
Example 3: specificity and sensitivity of methods for differential diagnosis of active tuberculosis patients and latent infectors using ELISA
92 parts of patient serum with clinical diagnosis of active tuberculosis and 93 parts of serum of latent infectious agents; the ELISA method in examples 1 and 2 was used to determine whether the test subjects were positive for active tuberculosis by the criteria of example 2, and the results are shown in FIG. 2, with an analytical specificity of 86.0% and a sensitivity of 71.7%. In FIG. 2, the abscissa 1-specificity represents the false positive rate, and the ordinate sensitivity represents the true positive rate.
The results prove that the invention detects the serum antibody response level of three different mycobacterium tuberculosis proteins, can better diagnose tuberculosis latent infection patients and active tuberculosis patients in a differential mode, and has higher sensitivity and better specificity.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
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<120> use of Mycobacterium tuberculosis proteins for the preparation of a product for the diagnosis of tuberculosis latent infected persons and/or active tuberculosis
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Claims (4)
1. Use of a product for detecting levels of an anti-Rv 0389 antibody, an anti-Rv 1408 antibody and an anti-Rv 2097c antibody in a sample for the preparation of a product for the identification, diagnosis and/or screening of active tuberculosis;
the sample is serum.
2. A use according to claim 1, wherein the products for detecting levels of anti-Rv 0389 antibodies, anti-Rv 1408 antibodies and anti-Rv 2097c antibodies in a sample comprise Rv0389 protein, rv1408 protein and Rv2097c protein.
3. The use according to claim 2, wherein the Rv0389 protein is shown in SEQ ID No. 1; the Rv1408 protein is shown in SEQ ID NO. 2; the Rv2097c protein is shown as SEQ ID NO. 3.
4. The use according to any one of claims 1 to 3, wherein the method of detection comprises an enzyme-linked immunosorbent assay, an agglutination assay, a precipitation assay, an E-garland assay, a small phagocytosis assay, an immunofluorescence assay, a colloidal gold immunochromatography assay, a spot gold immunodiafiltration assay and/or an electrochemiluminescence assay.
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| CN201611257494.7A CN108267588B (en) | 2016-12-30 | 2016-12-30 | Application of mycobacterium tuberculosis protein in preparing product for diagnosing latent tuberculosis infected person and/or active tuberculosis |
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| JP4623825B2 (en) * | 1999-12-16 | 2011-02-02 | 協和発酵バイオ株式会社 | Novel polynucleotide |
| EP2196473A1 (en) * | 2001-07-04 | 2010-06-16 | Health Protection Agency | Mycobacterial antigens expressed during latency |
| US7282491B2 (en) * | 2002-12-06 | 2007-10-16 | Cornell Research Foundation, Inc. | Prokaryotic proteasomal proteases of Mycobacterium tuberculosis (MTB) as targets for antibiotic therapy |
| WO2006029248A2 (en) * | 2004-09-04 | 2006-03-16 | Haper Laboratories Llc | Hollow fiber technique for in vivo study of cell populations |
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