CN106008685A - Antigenic polypeptides capable of identifying Trxc antibodies and application of antigenic polypeptides - Google Patents

Abstract

The present invention discloses an antigen polypeptide recognized by Trxc antibody and its use, wherein the polypeptide active fragment of the antigen polypeptide is at least one of the following: 1) the sequence shown in SEQ ID NO: 1: SFATDVLSSNKPVLV; 2) the sequence shown in SEQ ID NO: 1 Amino acid derivative sequence formed by modification, substitution, deletion or addition of amino acid residues. Using the antigenic polypeptide recognized by the Trxc antibody of the present invention can effectively detect the level of the Trxc antibody in a sample, such as a blood sample (such as a serum sample) of a patient with tuberculosis or suspected tuberculosis.

Description

Antigen polypeptide recognized by Trxc antibody and use thereof

technical field

The present invention relates to the field of biomedicine. Specifically, the present invention relates to antigenic polypeptides recognized by Trxc antibodies and uses thereof. More specifically, the present invention relates to an antigenic polypeptide recognized by the Trxc antibody, the use of the antigenic polypeptide in preparing a kit for detecting the level of the Trxc antibody in serum, and a kit for detecting the level of the Trxc antibody in a sample.

Background technique

Tuberculosis is one of the three major infectious diseases in the world. The epidemiological investigation report released by the World Health Organization (WTO) shows that the pathology of Mycobacterium tuberculosis infection in my country accounts for 12% of the total number of cases in the world, ranking second in the world after India . The high incidence and prevalence of tuberculosis have seriously affected the social and economic development of our country. At present, the detection of tuberculosis is mainly used as the gold standard in clinical diagnosis of tuberculosis, such as sputum smear, sputum collection and sputum culture. The diagnostic methods are time-consuming and have low specificity or sensitivity.

The detection method based on serum immunology is one of the important auxiliary means for the diagnosis of tuberculosis at the present stage. The widely used antigen-specific r-interferon release assay (IGRA) includes commercial kits T-spot.TB and QFT-IT.

The antigen used by IGRA is the whole protein encoded by the RD (region of difference) gene of Mycobacterium tuberculosis, which is expensive to produce and expensive. The World Health Organization does not recommend that it be widely used as a screening test in low- and middle-income countries. Moreover, some studies have shown that in countries with high prevalence of tuberculosis, the specificity of IGRA diagnosis is reduced due to interference from environmental factors, and false positives are increased.

Therefore, the current research on tuberculosis serum immunological diagnosis and detection still needs to be strengthened.

Contents of the invention

The present invention has been accomplished based on the following findings of the inventors:

Mycobacterium tuberculosis membrane protein Trxc can cause human immune response and produce corresponding antibodies, and the expression level of serum anti-Trxc polyclonal antibody can reflect the individual's infection status of Mycobacterium tuberculosis. At present, the methods for detecting anti-Trxc antibodies in serum using ELISA and other western blot hybridization techniques all require pure Trxc protein as the recognition antigen. In addition, the preparation process of purified protein is cumbersome, time-consuming and labor-intensive, and the cost is high.

The present invention aims to solve at least one of the above-mentioned disadvantages of the prior art and provide a useful commercial alternative.

Thus, the present invention uses the technology of polypeptide microarray to screen and obtain the high-frequency epitope of Trxc protein antigen recognition, and provides a TRXC antibody that contains the core amino acid sequence and protective amino acid sequence of serum antibody recognizing Trxc protein and is coupled to carrier proteins such as KLH, etc. The identified antigenic polypeptides are based on the fact that the expression level of Trxc antibody in the serum of tuberculosis patients is significantly higher than that of healthy people. Using these antigenic polypeptides can effectively detect the level of Trxc antibody in the serum of the test subject by means of Western blot detection, and can be used for tuberculosis diagnosis.

Therefore, in the first aspect of the present invention, the present invention provides an antigenic polypeptide recognized by the Trxc antibody. According to an embodiment of the present invention, its polypeptide active fragment is at least one of the following:

1) The sequence shown in SEQ ID NO: 1: SFATDVLSSNKPVLV;

2) An amino acid derivative sequence formed by modifying, replacing, deleting or adding amino acid residues to the amino acid sequence shown in SEQ ID NO:1.

The inventors have surprisingly found that using the antigenic polypeptide recognized by the above-mentioned Trxc antibody of the present invention, the Trxc antibody level in the serum of the subject can be effectively detected by means of immunoblotting detection, and then based on whether the expression level of the Trxc antibody in the serum of the subject is higher than that of healthy people Significantly increased, it can effectively diagnose tuberculosis and determine whether the test subject is a suspected tuberculosis patient. Among them, the serum Trxc antibody expression level of tuberculosis patients was significantly higher than that of healthy people. This technical solution replaces the traditional use of pure protein with a synthetic polypeptide sequence, which can achieve the same diagnostic value and has higher economic benefits. Moreover, according to the embodiments of the present invention, the above-mentioned antigen polypeptide Trxc antibody of the present invention has good recognition specificity, high detection efficiency of Trxc antibody level, simple preparation, low cost, easy industrial production, and is conducive to mass promotion and application.

Further, based on the antigenic polypeptide recognized by the above-mentioned Trxc antibody, the Trxc antibody level in the serum of the subject can be effectively detected by means of immunoblotting detection. In the second aspect of the present invention, the present invention provides the Trxc antibody recognized above. Use of the antigen polypeptide in the preparation of a kit for detecting the level of Trxc antibody in serum.

According to an embodiment of the present invention, the kit is used for the diagnosis of tuberculosis. Among them, based on the fact that the serum Trxc antibody expression level of tuberculosis patients is significantly higher than that of healthy people, it can effectively diagnose tuberculosis in the test subjects.

In the third aspect of the present invention, the present invention provides a kit for detecting the level of Trxc antibody in a sample. According to an embodiment of the present invention, the kit includes: the antigen polypeptide recognized by the aforementioned Trxc antibody. According to the embodiments of the present invention, the kit can effectively detect the level of Trxc antibody in the sample, and based on the significantly higher expression level of Trxc antibody in the serum of tuberculosis patients than that of healthy people, it can effectively diagnose tuberculosis in the test subjects.

Specifically, according to an embodiment of the present invention, the antigen polypeptide recognized by the Trxc antibody is provided in at least one of the following forms:

1) a 96-well plate, the antigen polypeptide recognized by the Trxc antibody is coated in the wells of the 96-well plate;

2) microspheres, the microspheres are coupled to the antigen polypeptide recognized by the Trxc antibody.

Furthermore, according to some specific examples of the present invention, the kit further includes: a positive control sample, wherein the positive control sample is an antibody against Trxc protein. Therefore, when the kit is used for Trxc antibody level detection and tuberculosis diagnosis, the results are more reliable.

According to an embodiment of the present invention, it further includes: a standard sample, the standard sample is an equal volume mixture of multiple positive Trxc antibody sera. Therefore, when the kit is used for Trxc antibody level detection and tuberculosis diagnosis, the results are more authentic and reliable.

According to some specific examples of the present invention, the standard sample is a gradient dilution product of an equal-volume mixture of sera positive for the expression of the multiple Trxc antibodies, and the ratio of the gradient dilution is from 1:50 to 1:2000. Therefore, when the kit is used to detect the Trxc antibody level and diagnose tuberculosis, the results are highly reliable.

According to an embodiment of the present invention, the concentration of the antigen polypeptide recognized by the Trxc antibody is 1 μg/ml.

According to an embodiment of the present invention, the sample is serum.

Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

detailed description

Embodiments of the present invention are described in detail below, and the embodiments are intended to explain the present invention, but should not be construed as limiting the present invention.

Antigenic polypeptide recognized by Trxc antibody

In the first aspect of the present invention, the present invention provides an antigenic polypeptide recognized by the Trxc antibody. According to an embodiment of the present invention, its polypeptide active fragment is at least one of the following:

1) The sequence shown in SEQ ID NO: 1: SFATDVLSSNKPVLV;

2) An amino acid derivative sequence formed by modifying, replacing, deleting or adding amino acid residues to the amino acid sequence shown in SEQ ID NO:1.

The inventors have surprisingly found that using the antigenic polypeptide recognized by the above-mentioned Trxc antibody of the present invention, the Trxc antibody level in the serum of the subject can be effectively detected by means of immunoblotting detection, and then based on whether the expression level of the Trxc antibody in the serum of the subject is higher than that of healthy people Significantly increased, it can effectively diagnose tuberculosis and determine whether the test subject is a suspected tuberculosis patient. Among them, the serum Trxc antibody expression level of tuberculosis patients was significantly higher than that of healthy people. Moreover, according to the embodiments of the present invention, the above-mentioned antigen polypeptide Trxc antibody of the present invention has good recognition specificity, high detection efficiency of Trxc antibody level, simple preparation, low cost, easy industrial production, and is conducive to mass promotion and application.

It should be noted that the above polypeptides all come from the positions corresponding to the full-length sequence of the Trxc protein (for example, from the protein public resource database uni-prot). In addition, the term "polypeptide" used herein refers to an oligomer obtained by connecting at least two amino acids through peptide bonds, wherein the number of amino acid residues contained in the polypeptide is not particularly limited. According to some embodiments of the invention, the polypeptide may contain 15 amino acids. Of course, those skilled in the art can understand that the above polypeptides can also be chemically modified to increase the antigenicity of the polypeptides and help the polypeptides to coat the bottom of the ELISA plate. According to the embodiments of the present invention, using the polypeptide having the above amino acid sequence can effectively detect the TRXC antibody level in a sample, such as a blood sample (such as a serum sample) of a patient with tuberculosis or suspected tuberculosis. The inventors found that the above-mentioned polypeptide obtained from the complete sequence of the Trxc protein can mimic the antigenic epitope of Trxc, and can react specifically with the TRXC antibody in the blood sample. Therefore, it can be effectively used to detect the level of the TRXC antibody in the blood sample of the subject, by Therefore, it can be effectively used to screen the serum of suspected tuberculosis patients, and provides a new detection method for the diagnosis of tuberculosis.

The above-mentioned antigenic polypeptides can be obtained by chemical synthesis, or by genetic engineering techniques, which are well known in the industry. Those skilled in the art can understand that the above-mentioned polypeptides can be synthesized effectively by conventional synthetic methods, so as to replace the biosynthetic way of recombinant expression.

According to the embodiments of the present invention, the types of samples that can be detected by the above polypeptides are not particularly limited. According to an embodiment of the present invention, a preferred sample is a blood sample from a tuberculosis or suspected tuberculosis patient. Thus, the polypeptide can be used to detect the TRXC antibody level in blood samples of patients with tuberculosis or suspected tuberculosis, so as to determine whether the patient has tuberculosis, provide auxiliary means for tuberculosis detection, or provide early screening for tuberculosis. In addition, by detecting the Trxc antibody level in the blood of the subject, the curative effect of the treatment can be evaluated, monitored and analyzed for the patient.

The method of using the polypeptide of the present invention to detect the level of Trxc antibody in a sample is not particularly limited, including but not limited to ELISA, liquid phase chip, solid phase chip and other immunohybridization techniques. According to an embodiment of the present invention, the level of Trxc antibody in a sample can be detected by means of the polypeptide of the present invention by enzyme-linked immunosorbent assay (ELISA). Thus, the efficiency of using the polypeptide to detect the Trxc antibody level in blood samples of patients with tuberculosis or suspected tuberculosis can be further improved. As an example, the following methods can be used to detect the Trxc antibody level in blood samples of tuberculosis or suspected tuberculosis patients by using polypeptides:

First, dilute the polypeptide to 1 μg/ml with pH 9.6 carbonate buffer solution (NaCO ₃ 0.159g, NaHCO ₃ 0.293g, add water to 100ml, pH value 9.6), add 100μl/well to 96-well enzyme In the wells of the target plate, seal the plate with the sealing film overnight at 4°C.

Next, discard the liquid in the well, 0.1% TBS-T washing buffer (the TBS-T solution containing 0.1% Tween-20 is prepared as NaCl 8.7g, Tris 1.21g, add deionized water to 1000ml, pH 7.5, Then add Tween-20 1ml) to wash the microtiter plate 5 times, 3min each time.

Then, add TBS-T blocking buffer containing 5% BSA and 0.05% Tween-20 (its preparation method is: NaCl 0.87g, Tris 0.121g, add deionized water to 100ml, pH 7.5, then add Tween -20 0.5ml, BSA5g), 300μl per well, and incubate at 37°C for 1h after sealing with the sealing film.

Next, add standards and samples to be tested. The serum to be tested was diluted with the above-mentioned TBS-T blocking buffer containing 5% BSA and 0.05% Tween-20, and the dilution ratio was 1:100. Gradual dilution of the serum used as a standard, the dilution ratio is from 1:50 to 1:2000. Discard the liquid in the wells completely, add 100 μl of the serum dilution solution of the sample to be tested in each well, and set up 2 positive control wells in each plate, add known positive serum, and each plate Set up 2 blank control wells, add 0.1% TBS-T washing buffer. Incubate at 37°C for 2 hours after sealing the plate. The liquid in the well was discarded, and the microtiter plate was washed 5 times, each time for 3 minutes.

Next, carry out the second antibody immune reaction. Use the TBS-T blocking buffer containing 5% BSA and 0.05% Tween-20 to dilute the horseradish peroxidase-labeled goat anti-human IgG at a dilution ratio of 1:5000, add 100 μl to each well, and seal the plate 37 Incubate at ℃ for 1h. The liquid in the well was discarded, and the microtiter plate was washed 5 times, each time for 3 minutes.

Next, add substrate to develop color. According to the instructions of conventional ELISA kits (such as Kangwei Century TMB Chromogenic Kit), add chromogenic substrate, add 100 μl per well, develop color at room temperature for 10 minutes, and add 50 μl 2M H ₂ SO ₄ per well to terminate the reaction.

Next, measure the optical density OD value under the condition of 492nm wavelength of microplate reader.

Finally, based on the standard curve fitted between the obtained optical density OD value and the concentration of the standard substance, the relative concentration of the corresponding test sample is obtained, and then compared with a predetermined reference value to determine whether the test sample is from a patient with tuberculosis.

According to an embodiment of the present invention, the values obtained in parallel experiments with the blood samples of diagnosed cancer patients and healthy people (healthy volunteers) can be used as predetermined reference values.

Assuming that the relative concentration of the Trxc antibody in the sample to be tested is higher than X, the serum to be tested is the serum of a suspected tuberculosis patient. X is obtained by the inventor through experimental calculation, and the specific method is to detect the relative concentration at the optimal point of the receiver operating curve (ROC curve) of the relative concentration of serum antibodies of tuberculosis patients and healthy people in the population. According to the embodiment of the present invention, using 40 tuberculosis patient serum samples and 80 healthy human serum samples, the X value of the antigen polypeptide shown in the sequence SEQ ID NO: 1 is calculated to be 5.3.

Application of the antigen polypeptide recognized by TRXC antibody

In the second aspect of the present invention, the present invention provides the use of the aforementioned antigen polypeptide recognized by the Trxc antibody in the preparation of a kit for detecting the level of the Trxc antibody in serum.

As mentioned above, the Trxc antibody level in the serum of the test subject can be effectively detected by using the prepared kit and the antigenic polypeptide recognized by the Trxc antibody included in it by means of immunoblotting detection, so as to effectively diagnose tuberculosis. Therefore, according to an embodiment of the present invention, the kit is used for the diagnosis of tuberculosis. Among them, based on the fact that the serum Trxc antibody expression level of tuberculosis patients is significantly higher than that of healthy people, it can effectively diagnose tuberculosis in the test subjects.

Therefore, in the third aspect of the present invention, the present invention provides a kit for detecting the level of Trxc antibody in a sample. According to an embodiment of the present invention, the kit includes: the antigen polypeptide recognized by the aforementioned Trxc antibody. According to the embodiments of the present invention, the kit can effectively detect the level of Trxc antibody in the sample, and based on the significantly higher expression level of Trxc antibody in the serum of tuberculosis patients than that of healthy people, it can effectively diagnose tuberculosis in the test subjects.

Specifically, according to an embodiment of the present invention, the antigen polypeptide recognized by the Trxc antibody is provided in at least one of the following forms:

1) a 96-well plate, the antigen polypeptide recognized by the Trxc antibody is coated in the wells of the 96-well plate;

2) microspheres, the microspheres are coupled to the antigen polypeptide recognized by the Trxc antibody.

Furthermore, according to some specific examples of the present invention, the kit further includes: a positive control sample, wherein the positive control sample is an antibody against Trxc protein. Therefore, when the kit is used for Trxc antibody level detection and tuberculosis diagnosis, the results are more reliable.

According to an embodiment of the present invention, it further includes: a standard sample, the standard sample is an equal volume mixture of multiple positive Trxc antibody sera. Therefore, when the kit is used for Trxc antibody level detection and tuberculosis diagnosis, the results are more authentic and reliable. The number of cases of positive sera, that is, the number of patient sources of the serum, is not particularly limited, and the "multiple cases" may also be one case. According to some specific examples of the present invention, the standard sample is an equal volume mixture of sera positive for Trxc antibody expression.

According to some specific examples of the present invention, the standard sample is a gradient dilution product of an equal-volume mixture of sera positive for the expression of the multiple Trxc antibodies, and the ratio of the gradient dilution is from 1:50 to 1:2000. Among them, the gradient dilution is for the convenience of subsequent drawing of the standard curve. Therefore, when the kit is used to detect the Trxc antibody level and diagnose tuberculosis, the results are highly reliable.

According to an embodiment of the present invention, the concentration of the antigen polypeptide recognized by the Trxc antibody is 1 μg/ml.

According to an embodiment of the present invention, the sample is serum.

According to some embodiments of the present invention, the kit may include: a 96-well plate, and the wells of the 96-well plate are coated with the antigen polypeptide recognized by the aforementioned TRXC antibody. Further, the kit can further include: a positive control sample, the positive control sample is an antibody against Trxc protein, or/additionally can further include: a standard sample, the standard sample is a serum that is positive for Trxc antibody expression through a gradient Diluted combination. According to some specific examples of the present invention, the standard sample is a gradient dilution product of an equal-volume mixture of sera positive for the expression of the multiple Trxc antibodies, and the ratio of the gradient dilution is from 1:50 to 1:2000. According to an embodiment of the present invention, the concentration of the antigen polypeptide recognized by the Trxc antibody is 1 μg/ml. According to an embodiment of the present invention, the sample is serum.

According to an embodiment of the present invention, the sample is a blood sample from a tuberculosis or suspected tuberculosis patient. Thus, the polypeptide can be used to detect the Trxc antibody level in blood samples of patients with tuberculosis or suspected tuberculosis, so as to determine whether the patient has tuberculosis, provide auxiliary means for tuberculosis detection, or monitor the progress of tuberculosis. In addition, the kit can also be used to predict the patient's response to the treatment method by detecting the Trxc antibody level in the blood of the subject.

In yet another aspect of the present invention, the present invention also proposes a kit for detecting the level of TRXC antibody in a sample. According to an embodiment of the present invention, the kit includes: the antigen polypeptide recognized by the aforementioned Trxc antibody. Thus, using the embodiment of the present invention, the level of the Trxc antibody in the sample can be detected effectively by means of the antigenic polypeptide recognized by the Trxc antibody according to the present invention. According to an embodiment of the present invention, the kit may further include: reagents suitable for ELISA detection. Thus, the polypeptide according to the present invention can be used to detect the Trxc antibody in the sample through the ELISA detection method, so that the efficiency of using the polypeptide to detect the Trxc antibody level in the blood sample of the tuberculosis or suspected tuberculosis patient can be further improved. According to an embodiment of the present invention, the polypeptide is arranged in a 96-well plate, for example, the wells of the 96-well plate can be coated, for example, it can be performed by conventional treatment such as using a coating solution and a blocking solution. Thus, high-throughput detection can be conveniently performed. The efficiency of using the polypeptide to detect the Trxc antibody level in blood samples of patients with tuberculosis or suspected tuberculosis can be further improved. The kit can also contain other reagents, such as washing buffer, sample dilution buffer, TMB substrate solution, reaction stop solution, horseradish peroxidase-labeled secondary antibody, positive control samples, and standard samples. Wherein, the positive control sample is an antibody against Trxc protein, and the standard sample is a combination of serially diluted serum with positive expression of Trxc antibody. Alternatively, the standard sample is a gradient dilution product of an equal-volume mixture of sera positive for the multiple cases of Trxc antibody expression.

According to an embodiment of the present invention, the samples are blood samples from tuberculosis or suspected tuberculosis patients and healthy volunteers.

In addition, the kit can also be used to predict the patient's response to the treatment method by detecting the Trxc antibody level in the blood of the subject.

The present invention is explained below through specific examples. It should be noted that the following examples are only illustrative and do not limit the present invention in any way. In addition, all instruments, materials, etc. used in the following examples are commercially available, and operations that are not clearly indicated in the following examples can be performed by conventional operating methods for those skilled in the art.

Example 1

1. Preparation of polypeptides

1. Preparation of Peptide Library

The amino acid sequence of Trxc was obtained from the protein public resource database uni-prot.

2. In situ synthesis of Trxc protein peptide library to prepare peptide array

According to the length of protein and peptide amino acid residues and the length of overlapping sequences, the peptide synthesizer ASP SL (Germany, intavis company) MutilPep synthesis control program is set, and a specific amino acid is added to the activated nitrocellulose in each synthesis cycle according to the program control Synthetic site on membrane surfaces and catalyzes peptide bond formation between amino acids. After the peptide chip was synthesized, it was sealed and stored at -20°C for future use.

2. Screening of peptides related to tuberculosis (screening of antigenic epitopes)

1. Hydration and Western Blotting of Peptide Chips

Immerse the peptide chip prepared in the preceding step 1 (herein referred to as "peptide chip 1") in 40 ml of 100% ethanol, and shake it on a shaker for 5 minutes. Then, the polypeptide chip was immersed in 40 ml of 75% ethanol and shaken on a shaker for 5 minutes. Next, immerse the peptide chip in 40ml of 50% ethanol and shake it on a shaker for 5 minutes. Next, add 150ml PBS and soak for 30 minutes. Finally, immerse the peptide chip in 40ml of 5% skim milk powder/PBS-T solution, and incubate at room temperature for 3 hours to block.

Dilute the serum samples involved in the following primary screening and identification experiments with 5% skimmed milk powder/PBS-T solution 1:1000 to prepare an immune reaction solution, put the peptide chip into the hybridization bag, and add the reaction solution at 0.1ml/cm2 , Remove all air bubbles in the bag, seal with a film sealing machine, and shake gently overnight at 4°C.

After the serum immune reaction is over, cut off the hybridization bag, discard the reaction solution, wash the peptide chip 3 times with 40ml of PBS-T, 15 minutes each time, put the peptide chip into the hybridization bag, add 1:2000 diluted goat anti-human IgG Solution, add secondary antibody reaction solution at 0.1ml/cm2, seal the hybridization bag, shake gently at room temperature for 2 hours, wash the membrane 3 times with 40ml PBS-T, 15 minutes each time.

Next, ECL development and exposure were carried out, and the exposure results were analyzed with the AlphaView software system.

2. Preliminary screening of antigenic epitopes

The peripheral blood serum of 10 tuberculosis patients was mixed in equal amounts to prepare a tuberculosis mixed serum pool, and the peripheral blood serum of 10 PPD negative volunteers was mixed in equal amounts to prepare a healthy mixed serum pool. The above-mentioned serum pool and the polypeptide chip were immunohybridized according to the above-mentioned method, and according to the results of the immune reaction, two different polypeptide sites were selected that were positive in serum of tuberculosis patients and negative in serum of healthy volunteers, namely Trxc protein in tuberculosis serum. antigen recognition epitope.

3. Antigen peptide screening with diagnostic value for tuberculosis

According to the preparation method described in Example 1, a polypeptide chip (herein referred to as "polypeptide chip 2") comprising the above two differential polypeptides and a control was prepared, and immunized with serum from 100 cases of tuberculosis patients and 90 cases of healthy people. In the reaction, one high-frequency site of the Trxc antigen epitope (sequence shown in SEQ ID NO: 1: SFATDVLSSNKPVLV) was screened out, which has the value of distinguishing tuberculosis patients from healthy people.

3. ELISA test

The polypeptide (sequence shown in SEQ ID NO: 1) obtained by screening in step 2 was used as an antigen-coated 96-well plate, and the following samples were tested for Trxc antibody by ELISA method: 82 cases of tuberculosis patient serum and 38 cases of healthy control serum .

The specific detection method is as follows:

Firstly, the antigenic polypeptide was synthesized according to the sequence of amino acid residues in the sequence listing using the polypeptide synthesizer MultiPep RS.

Next, dilute the polypeptide to 1 μg/ml with pH 9.6 carbonate buffer solution (NaCO ₃ 0.159g, NaHCO ₃ 0.293g, add water to 100ml, pH value 9.6), add 100μl/well to 96-well enzyme label Wells of the plate were sealed overnight at 4°C.

Next, discard the liquid in the well, 0.1% TBS-T washing buffer (the TBS-T solution containing 0.1% Tween-20 is prepared as NaCl 8.7g, Tris 1.21g, add deionized water to 1000ml, pH 7.5, Then add Tween-20 1ml) to wash the microtiter plate 5 times, 3min each time.

Then, add TBS-T blocking buffer containing 5% BSA and 0.05% Tween-20 (its preparation method is: NaCl 0.87g, Tris 0.121g, add deionized water to 100ml, pH 7.5, then add Tween -20 0.5ml, BSA5g), 300μl per well, and incubate at 37°C for 1h after sealing with the sealing film.

Next, add standards and samples to be tested. The standard product is an equal-volume mixture of peripheral blood serum from 10 tuberculosis patients randomly selected. The serum to be tested was diluted with the above-mentioned TBS-T blocking buffer containing 5% BSA and 0.05% Tween-20, and the dilution ratio was 1:100. The serum mixture used as the standard was serially diluted from 1:50 to 1:2000. Discard the liquid in the wells completely, add 100 μl of the serum dilution solution of the sample to be tested in each well, and set up 2 positive control wells in each plate, add known positive serum, and each plate Set up 2 blank control wells, add 0.1% TBS-T washing buffer. Incubate at 37°C for 2 hours after sealing the plate. The liquid in the well was discarded, and the microtiter plate was washed 5 times, each time for 3 minutes.

Next, carry out the second antibody immune reaction. Use the TBS-T blocking buffer containing 5% BSA and 0.05% Tween-20 to dilute the horseradish peroxidase-labeled goat anti-human IgG at a dilution ratio of 1:5000, add 100 μl to each well, and seal the plate 37 Incubate at ℃ for 1h. The liquid in the well was discarded, and the microtiter plate was washed 5 times, each time for 3 minutes.

Next, add substrate to develop color. According to the instructions of the conventional ELISA kit (Kangwei Century TMB Chromogenic Kit), add chromogenic substrate, add 100 μl per well, develop color for 10 min at room temperature in the dark, and add 50 μl 2M H ₂ SO ₄ per well to terminate the reaction.

Next, measure the optical density OD value under the condition of 492nm wavelength of microplate reader.

Finally, based on the standard curve fitted between the obtained optical density OD value and the concentration of the standard substance, the relative concentration of the corresponding test sample is obtained, and then compared with a predetermined reference value to determine whether the test sample is from a patient with tuberculosis.

Assuming that the relative concentration of the Trxc antibody in the sample to be tested is higher than X, the serum to be tested is the serum of a suspected tuberculosis patient. X is the relative concentration at the optimum point of the Receiver Operating Curve (ROC curve) for detecting the serum antibody relative concentrations of tuberculosis patients and healthy individuals in the population. Based on the serum samples of 40 tuberculosis patients and 40 healthy people, the X value corresponding to each antigen polypeptide was calculated, wherein the X value of the antigen polypeptide shown in the sequence SEQ ID NO: 1 was 5.3.

Take this as the cutoff value (predetermined reference value), and the test results are as follows:

sample type Number of cases Antibody positive rate tuberculosis 40 26/40 (65.0%) healthy control 80 33/80 (41.3%)

The comparison of the number of positive and negative cases is as follows:

antibody level Number of TB patients Number of healthy volunteers Positive (>5.3) 26(a, ie true positive) 33(b, ie false positive) Negative (<5.3) 14(c, ie false negative) 47(d, ie true negative)

Sensitivity, specificity, and negative positive predictive value are summarized below:

The purified protein of Trxc was used as an antigen to detect the serum antibody concentration, and the detection results were as follows:

sample type Number of cases Antibody positive rate tuberculosis 40 34/40 (85.0%) healthy control 80 48/80 (60.0%)

The comparison of the number of positive and negative cases is as follows:

antibody level Number of TB patients Number of healthy volunteers Positive (>5.3) 34 (a, ie true positive) 48(b, ie false positive) Negative (<5.3) 6(c, ie false negative) 32(d, ie true negative)

Sensitivity, specificity, and negative positive predictive value are summarized below:

sensitivity specificity positive predictive value negative predictive value Purified protein 85.0% 40.0% 41.5% 84.2%

The results demonstrated that, in contrast, polypeptide antigens had the same diagnostic value as whole protein antigens.

Thus, it is proved that the antigenic polypeptide recognized by the Trxc antibody of the present invention can be effectively used for auxiliary diagnosis of tuberculosis.

In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications, substitutions and modifications can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the invention is defined by the claims and their equivalents.

Claims (10)

1. An antigen polypeptide recognized by a Trxc antibody, characterized in that its polypeptide active fragment is at least one of the following: 1) The sequence shown in SEQ ID NO: 1: SFATDVLSSNKPVLV; 2) An amino acid derivative sequence formed by modifying, replacing, deleting or adding amino acid residues to the amino acid sequence shown in SEQ ID NO:1. 2. the purposes of the antigenic polypeptide that the Trxc antibody recognition described in claim 1 is in the test kit of preparation detection serum Trxc antibody level. 3. The use according to claim 2, characterized in that the kit is used for tuberculosis diagnosis. 4. A test kit for detecting Trxc antibody level in a sample, characterized in that, comprising: The antigenic polypeptide recognized by the Trxc antibody of claim 1. 5. The kit according to claim 4, wherein the antigen polypeptide recognized by the Trxc antibody is provided in at least one of the following forms: 1) a 96-well plate, the antigen polypeptide recognized by the Trxc antibody is coated in the wells of the 96-well plate; 2) microspheres, the microspheres are coupled to the antigen polypeptide recognized by the Trxc antibody. 6. The kit according to claim 5, further comprising: a positive control sample, the positive control sample being an antibody against Trxc protein. 7 . The kit according to claim 5 , further comprising: a standard sample, which is an equal-volume mixture of sera with positive Trxc antibody expression. 8. test kit according to claim 7, is characterized in that, described standard sample is the gradient dilution product of the equivolume mixture of the positive serum of described multiple routine Trxc antibody expression, and the ratio of described gradient dilution is from 1: From 50 to 1:2000. 9. The kit according to claim 5, wherein the concentration of the antigen polypeptide recognized by the Trxc antibody is 1 μg/ml. 10. The kit according to claim 4, wherein the sample is serum.