阅读说明：本技术 蛋白标志物在诊断肠易激综合征的产品中的用途和用于诊断肠易激综合征的试剂盒 (Use of protein marker in product for diagnosing irritable bowel syndrome and kit for diagnosing irritable bowel syndrome ) 是由 郭元彪 潘逼然 赵聪 于 2018-09-05 设计创作，主要内容包括：本公开涉及一种蛋白标志物在诊断肠易激综合征的产品中的用途和用于诊断肠易激综合征的试剂盒。本公开利用配体联合检测样本中的Thymosinβ4蛋白,可以有效对肠易激综合征患者进行辅助诊断,有利于指导肠易激综合征患者的临床用药,使患者得到最佳的受益。(The invention utilizes ligand combination to detect Thymosin β 4 protein in a sample, can effectively carry out auxiliary diagnosis on patients with irritable bowel syndrome, is beneficial to guiding clinical medication of patients with irritable bowel syndrome, and enables the patients to obtain optimal benefit.)

1. Use of a ligand for detecting a protein marker in the manufacture of a product for diagnosing irritable bowel syndrome, wherein the protein marker is Thymosin β 4 protein.

2. The use of claim 1, wherein the ligand is a nucleic acid and/or a polypeptide comprising an antibody that is a human, chimeric, recombinant, humanized, monoclonal, antigen-binding fragment of a monoclonal, or polyclonal antibody.

3. The use according to claim 1, wherein detecting a protein marker is performed by:

1) obtaining a biological sample of a patient with irritable bowel syndrome, wherein the biological sample is one or more of an intestinal tissue sample, a plasma sample, an intestinal fluid sample and a stool sample;

2) determining the expression level of the protein marker in the biological sample.

4. Use according to claim 3, wherein the method of step 2) is an immunohistochemical method and/or an enzyme-linked immunosorbent assay method.

5. A kit for diagnosing irritable bowel syndrome, wherein the kit comprises a ligand for detecting a protein marker, which is Thymosin β 4 protein.

6. The kit of claim 5, wherein the ligand is a nucleic acid and/or a polypeptide comprising an antibody that is a human antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, a monoclonal antibody, an antigen-binding fragment of a monoclonal antibody, or a polyclonal antibody.

Technical Field

The present disclosure relates to the technical field of molecular biology and clinical detection, and in particular, to an application of a protein marker in a product for diagnosing irritable bowel syndrome and a kit for diagnosing irritable bowel syndrome by the protein marker.

Background

Irritable Bowel Syndrome (IBS) is a functional Bowel disorder with abdominal pain or abdominal discomfort with altered Bowel habits. The prevalence rate of IBS in China is about 5-6%, and the prevalence rate in the United states is as high as 22%. IBS has become a global disease and is of great concern because it seriously affects the quality of life of patients. To date, the research progress on IBS is still very limited, the etiology and pathogenesis of IBS are still poorly understood, the specific molecular mechanism of IBS onset is not yet elucidated, and the search for molecular markers closely related thereto and elucidation of specific mechanisms of action remain the current major research direction.

In 2011, the IBS experts in many countries release the IBS severity grading standard based on clinical symptoms, but objective diagnosis indexes such as morphology, inspection, imaging and endoscopy are lacked, and no index obtains the evaluation of absolute consistency of students all over the world.

In view of the above, there is an urgent need in the art to develop protein markers or marker combinations for IBS-assisted diagnosis, so as to accurately determine the severity of the disease and evaluate the therapeutic effect of the disease before clinical treatment.

Disclosure of Invention

The purpose of the present disclosure is to provide a use of a ligand of a detection protein marker for the preparation of a product for diagnosing irritable bowel syndrome and a kit for diagnosing irritable bowel syndrome.

In order to achieve the above objects, the present disclosure provides, in a first aspect, a use of a ligand for detecting a protein marker for the preparation of a product for diagnosing irritable bowel syndrome, wherein the protein marker is Thymosin β 4 protein.

Optionally, the ligand is a nucleic acid and/or a polypeptide, including an antibody that is a human antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, a monoclonal antibody, an antigen-binding fragment of a monoclonal antibody, or a polyclonal antibody.

Alternatively, detecting the protein marker is performed by:

1) obtaining a biological sample of a patient with irritable bowel syndrome, wherein the biological sample is one or more of an intestinal tissue sample, a plasma sample, an intestinal fluid sample and a stool sample;

2) determining the expression level of the protein marker in the biological sample.

Optionally, the method of step 2) is an immunohistochemical method and/or an enzyme-linked immunosorbent assay method.

In a second aspect of the present disclosure, a kit for diagnosing irritable bowel syndrome is provided, wherein the kit comprises a ligand for detecting a protein marker, and the protein marker is Thymosin β 4 protein.

Optionally, the ligand is a nucleic acid and/or a polypeptide, including an antibody that is a human antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, a monoclonal antibody, an antigen-binding fragment of a monoclonal antibody, or a polyclonal antibody.

Through the technical scheme, the Thymosin β 4 protein in the sample is jointly detected by using the ligand, so that the irritable bowel syndrome patient can be effectively diagnosed.

Additional features and advantages of the disclosure will be set forth in the detailed description which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:

FIG. 1 is a graph of the characterization of intestinal tissue samples from IBS rats and IBS patients by IHC method, wherein A is IBS rat, B is IBS patient and Control represents Control group.

Fig. 2 shows the expression level of Thymosin β 4 protein in serum, intestinal juice and stool samples of IBS patients measured by ELISA, wherein a is a serum sample and P is 0.014, B is an intestinal juice sample and P is 0.011, C is a stool sample and P is 0.001, and NC represents a control group.

Detailed Description

The following detailed description of specific embodiments of the present disclosure is provided in connection with the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

In a first aspect of the present disclosure, there is provided a use of a ligand for detecting a protein marker for the preparation of a product for diagnosing irritable bowel syndrome, wherein the protein marker is Thymosin β 4 protein.

The Thymosin β 4 protein is referred to in NCBI database under the reference number NP-066932, and the Thymosin β 4 gene is referred to in NCBI database under the reference number 7114.

According to the present disclosure, the Thymosin β 4 protein has the following characteristics:

relatively high levels in intestinal tissue samples of irritable bowel syndrome patients;

relatively high levels in intestinal fluid samples of irritable bowel syndrome patients;

relatively low levels in stool samples from irritable bowel syndrome patients;

relatively low levels in plasma samples of irritable bowel syndrome patients;

in accordance with the present disclosure, the ligand is one that can specifically bind to Thymosin β 4 protein for detection of the protein, and can be, for example, a nucleic acid and/or a polypeptide, wherein the polypeptide comprises an antibody, such as a human antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, a monoclonal antibody, an antigen-binding fragment of a monoclonal antibody, or a polyclonal antibody.

According to the present disclosure, detecting a protein marker is performed by:

1) obtaining a biological sample of a patient with irritable bowel syndrome, wherein the biological sample is one or more of an intestinal tissue sample, a plasma sample, an intestinal fluid sample and a stool sample;

2) determining the expression level of the protein marker in the biological sample.

Wherein, the method of the step 2) is an immunohistochemical method and/or an enzyme-linked immunosorbent assay method.

The presence of a protein marker can be quantitatively detected, for example, using immunoassay methods. The immunoassay typically involves incubating the biological sample with an antibody and detecting the bound antibody by techniques well known to those skilled in the art, such as immunohistochemical assays (IHC) or enzyme-linked immunosorbent assay (ELISA).

In one embodiment of the present disclosure, the method of preparing a sample, the IHC method and the positive judgment criteria are as follows:

(1) preparing a sample:

a biopsy sample or surgical tissue obtained by enteroscopy within 30min is put into neutral formalin fixing solution for fixing overnight, washed by running water, dehydrated, transparent and waxed to prepare a tissue wax block. The diagnosis of IBS diseases is based on clinical symptoms according to the ROME III criteria. Each patient selected 3 intestinal tissues, made paraffin wax blocks and prepared 4 μm sections.

(2) The specific detection method of IHC is as follows:

placing the front side of the slice into a 65 ℃ baking oven to bake for 40min to enable the sample to be better attached, soaking the slice into xylene washing cylinders I, II and III, respectively placing the slices into a 25 ℃ constant temperature water bath for 10min, transferring the slices into 100%, 85% and 75% ethanol for gradient hydration for 3min respectively, then transferring the slices into PBS buffer solutions I, II and III, placing the slices into a 3% hydrogen peroxide washing cylinder for 15min every 5min, and sealing endogenous peroxidase. Soaking in heated sodium citrate buffer solution, and heating in microwave oven with slow fire for 20 min. Immersing in PBS buffer solution I, II, III for 3min each time. The range of the tissue section is drawn along the edge of the tissue section by PAP Pen for immunohistochemistry fossil, the diluted primary antibody solution or antibody working solution is spread on the surface of the tissue in the drawn range by a 200 mu L microsyringe and is placed in a wet box, and the tissue is incubated in a constant temperature incubator at 37 ℃ for 2h or overnight in a refrigerator at 4 ℃. Excess primary antibody was removed on a paper towel, and the tissue chip was immersed in PBS buffers I, II, III in sequence for 3min each time. Adding reagent II in the two-step immunohistochemical detection kit to ensure that the tissue surface within the drawn range is fully paved and placed in a wet box, and incubating for 15min in a constant temperature incubator at 37 ℃. Excess reagent was removed on a paper towel, and the tissue chip was immersed in PBS buffer I, II, III in sequence for 3min each time. Adding reagent III (Polyperoxidase-anti-mouse/rabbitt IgG) in the two-step immunohistochemical detection kit, ensuring that the tissue surface within the drawn range is fully paved, placing the tissue surface in a wet box, and incubating for 30min in a constant-temperature incubator at 37 ℃. Excess reagent was removed on a paper towel, and the tissue chip was immersed in PBS buffer I, II, III in sequence for 3min each time. Taking a proper amount of reagent from the DAB kit, and uniformly mixing according to the proportion of 1mLDAB diluent to 50 mu LDAB concentrated solution; spreading the diluted DAB solution on the surface of the tissue within the drawn range by using a 200 mu L microsyringe for developing, and keeping the developing time of each slice consistent; observing the color development effect under a mirror until more obvious contrast is formed inside the same slice and among different slices, removing the redundant DAB solution on a paper towel and placing the paper towel in distilled water; the development time was 60s, and the development time was different between different antibodies. Spreading hematoxylin staining solution on the surface of the tissue array within the range to be drawn rapidly by a 1000-mu-L microsyringe, and immediately flushing the surface by slow flowing water after 10 s; the stained chip was placed in 1% ammonia water. The slices are sequentially immersed in 75%, 85% and 100% ethanol for 3min for gradient dehydration. Immersing in xylene washing jar, removing the handwriting drawn by PAP Pen immunohistochemical fossil wax Pen for 10min, air drying the slice in ventilation place, and sealing the slice with quick-drying sealing agent.

(3) Positive judgment standard:

20 high-power fields of immunohistochemically stained cells were counted, and the staining intensity was integrated as: no dyeing is divided into 0, weak dyeing is divided into 1, medium dyeing is divided into 2, and strong dyeing is divided into 3; the integral of the dyeing area is as follows: the coloring range is less than or equal to 10% and is 0, more than 10% -25% is 1, more than 25% -50% is 2, more than 50% -75% is 3, and more than 75% is 4. If the sum of the two integrals is more than or equal to 3 points, the positive result is judged, and if the sum is less than 3 points, the negative result is judged.

In another embodiment of the present disclosure, the sample preparation method and the ELISA detection method are as follows:

(1) plasma (serum) sample processing procedure:

plasma (serum) samples of irritable bowel syndrome patients were collected (for comparison, normal human samples were collected at the same time), centrifuged at 1000g for 15 minutes, and the supernatant was aspirated for subsequent testing.

(2) The intestinal fluid sample treatment process comprises the following steps:

intestinal fluid samples of irritable bowel syndrome patients are collected under enteroscopy (for comparison, normal person samples can be collected at the same time), centrifuged for 15 minutes at 1000g, and the supernatant is sucked for subsequent detection.

(3) Treatment process of the fecal sample:

the patient with irritable bowel syndrome automatically collects a stool sample (for comparison, a normal person sample can be collected at the same time), the stool sample is sent to a laboratory within 1 hour, 50mg of the stool sample is added into a PE tube by using an inoculating loop, an extraction buffer solution (49 times of the net weight of the sample) is added into the tube, and the tube is tightly covered; placing the sample tube into a porous vortex mixer, and fully oscillating for 30 minutes at the highest speed to ensure that the sample becomes homogenate; transferring the homogenate to a 2ml microcentrifuge tube, and centrifuging for 5 minutes at 3000 g; the supernatant was transferred to a clean tube and used directly for the subsequent detection of ELISA experiments.

(4) The specific detection procedure of the ELISA experiment is as follows:

100 μ L of standard or plasma (serum) and fecal treated samples were added to the wells, incubated at 37 ℃ for 2 hours, and the wells were drained without washing. mu.L of biotin-labeled antibody was added and incubated at 37 ℃ for 1 hour. The plates were washed three times for 2 minutes each. 100 μ L of HRP-biotin was added and incubated at 37 ℃ for 1 hour. The plates were washed five times for 2 minutes each. Add 90. mu.L of TMB substrate, incubate at 37 ℃ for 15-30 minutes, then add 50. mu.L of stop buffer. Readings were taken on a 450nm microplate reader.

In a second aspect of the present disclosure, a kit for diagnosing irritable bowel syndrome is provided, wherein the kit comprises a ligand for detecting a protein marker, and the protein marker is Thymosin β 4 protein.

In accordance with the present disclosure, the ligand is one that can specifically bind to Thymosin β 4 protein for detection of the protein, and can be, for example, a nucleic acid and/or a polypeptide, wherein the polypeptide comprises an antibody, such as a human antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, a monoclonal antibody, an antigen-binding fragment of a monoclonal antibody, or a polyclonal antibody.

The present disclosure is further illustrated in detail by the following examples.