阅读说明：本技术 检测脂***甘露聚糖的组分及其试剂盒 (Component for detecting lipoarabinomannan and kit thereof ) 是由 盛盼龙 朱绍荣 于 2019-08-06 设计创作，主要内容包括：一种检测脂阿拉伯甘露聚糖的组分,其为SEQ ID No 1所示的多肽,或SEQ ID No2所示的多肽和SEQ ID No 3所示的多肽分别与SEQ ID No 1所示的多肽形成组合物,再与结核分枝杆菌脂阿拉伯甘露聚糖抗原共包被,实现结核分枝杆菌的检测。本发明采用脂阿拉伯甘露聚糖抗原和多肽(或组合物)共包被的方式,实现了对结核杆菌抗体的特异性检测,并具有较高的灵敏度,适合于体外检测。(A component for detecting the lipoarabinomannan is a polypeptide shown in SEQ ID No. 1, or a polypeptide shown in SEQ ID No2 and a polypeptide shown in SEQ ID No3 respectively form a composition with a polypeptide shown in SEQ ID No. 1, and then are coated with a lipoarabinomannan antigen of mycobacterium tuberculosis to realize the detection of the mycobacterium tuberculosis. The invention adopts the mode of coating the lipoarabinomannan antigen and the polypeptide (or the composition) together, realizes the specificity detection of the tubercle bacillus antibody, has higher sensitivity and is suitable for in vitro detection.)

1. A mycobacterium tuberculosis lipoarabinomannan antibody detection component is characterized by comprising a polypeptide shown as SEQ ID No 1.

2. The mycobacterium tuberculosis lipoarabinomannan antibody detection component of claim 1, further comprising a polypeptide shown as SEQ ID No2 and a polypeptide shown as SEQ ID No3, and forming a composition with the polypeptide shown as SEQ ID No1, wherein the polypeptide shown as SEQ ID No1, the polypeptide shown as SEQ ID No2 and the polypeptide shown as SEQ ID No3 are in a molar ratio of 3: 2-5: 3.

3. A mycobacterium tuberculosis lipoarabinomannan antibody detection composition is characterized in that a polypeptide and lipoarabinomannan antigen are coated together for improving the sensitivity and specificity of lipoarabinomannan antibody detection, wherein the polypeptide is selected from a polypeptide shown in SEQ ID No1, or a polypeptide shown in SEQ ID No1, a mixture of the polypeptide shown in SEQ ID No2 and the polypeptide shown in SEQ ID No3 according to the molar ratio of 3: 2-5: 3.

4. The Mycobacterium tuberculosis lipid arabinomannan antibody assay composition of claim 3, wherein the amount of the Mycobacterium tuberculosis lipid arabinomannan antigen coated on the assay reaction plate is from 0.5 μ g/well to 2 μ g/well.

5. The Mycobacterium tuberculosis lipid arabinomannan antibody assay composition of claim 3, wherein the amount of the Mycobacterium tuberculosis lipid arabinomannan antigen coated on the assay plate is 1 μ g/well.

6. The Mycobacterium tuberculosis lipoarabinomannan antibody assay composition of claim 3, wherein the polypeptide is coated at a concentration of 0.5 μ g/well to 1 μ g/well on the assay plate.

7. A kit for detecting a Mycobacterium tuberculosis lipid arabinomannan antibody, comprising a sample treatment solution, a Mycobacterium tuberculosis lipid arabinomannan antigen, a Mycobacterium tuberculosis lipid arabinomannan antibody detection component according to any one of claims 1 to 2, and a sample diluent, wherein the Mycobacterium tuberculosis lipid arabinomannan antigen and the Mycobacterium tuberculosis lipid arabinomannan antibody detection component according to any one of claims 1 to 2 are coated on a detection plate.

8. The kit for detecting the antibody against the Mycobacterium tuberculosis lipoarabinomannan of claim 7, wherein the concentration of the blocking agent added to the sample diluent is 10 μ g/ml to 100 μ g/ml.

9. The kit for detecting the antibody against the mycobacterium tuberculosis lipoarabinomannan according to claim 7, wherein the sample treatment solution is an acidic solution selected from one or more of perchloric acid, hydrochloric acid, dilute nitric acid, dilute sulfuric acid and trifluoroacetic acid, and is used for treating the sample to be detected and controlling the final pH value of the sample to be detected to be 6.0-8.0.

10. The kit for detecting the antibody against the mycobacterium tuberculosis lipoarabinomannan of claim 7, wherein the kit further comprises a coated plate, an enzyme conjugate working solution, a substrate developing solution A, a substrate developing solution B, a washing solution, a negative and positive control and a stop solution.

Technical Field

The invention relates to a polypeptide auxiliary agent, in particular to a sensitizer for improving detection sensitivity and specificity, which is used for detecting lipoarabinomannan, a detection composition and a detection kit.

Background

Tuberculosis is an infectious disease that has plagued humans for thousands of years, and it is estimated that the adult population in europe 1/4 of the nineteenth century dies from tuberculosis. By the time of the fifties of the twentieth century tuberculosis has almost been eradicated by industrialized countries due to improvements in standards of living in the twentieth century and the discovery of antibiotics. However, tuberculosis remains rampant as a recurrent infectious disease. Currently, it is estimated that one of three people in the world population is infected with mycobacterium tuberculosis (latent tuberculosis). The total number of new-onset tuberculosis patients in the world is said to be nine hundred and forty-one million, and two million of them are estimated to die of the disease. Currently, approximately 95% of active tuberculosis (active tuberculosis) patients live in developing countries, with 99% of the people who die from tuberculosis concentrating in developing countries.

The LAM antigen tubercle bacillus survives and breeds in the genus host through a unique cell wall structure. The cell wall of tubercle bacillus contains various components, such as: porin (Porin), polysaccharides, Lipids (Lipids), mycolic acid (mycolic acid), phosphatidylinositol mannosides (PIM), Lipomannans (LM), Lipoarabinomannans (LAM). Wherein LAM is the major component of the cell wall of mycobacteria, up to 15 mg/g total weight of bacteria. LAM broadly affects the immune system of the infected host. It has been reported that LAM isolated from mycobacterium tuberculosis and leprosy causes inhibition of T cell proliferation, interference with interferon-mediated macrophage activation, elimination of toxic free oxygen radicals, inhibition of protein kinase activity and gene-encoded Interleukins (IL) -2 and IL-5 and granulocyte synthesis/macrophage colony stimulation of human T cell factor, induction of macrophage immediate early gene expression, promotion of monocyte tumor necrosis factor production and complement activation. Research results show that the LAM serving as a genus specific antigen has stronger immunogenicity and immunoregulation function, participates in a plurality of processes of tubercle bacillus infection, and is beneficial to timely detecting tuberculosis morbidity (tuberculosis) and other mycobacterial infections.

Enzyme-Linked ImmunoSorbent Assay (ELISA) is the most widely used technique in Enzyme immunoassay. The basic method is to adsorb known antigen or antibody on the surface of solid phase carrier (polystyrene micro reaction plate), to make enzyme-labeled antigen-antibody reaction proceed on the solid phase surface, to wash away the free component in the liquid phase by washing method, to detect the absorbance by enzyme-linked immunoassay detector, the method can not only determine the nature, but also determine the quantity of the antigen and antibody of various pathogens, the technique has wide application in medical inspection, scientific research, environmental monitoring and food safety.

The reagent is usually provided in a complete kit form, and is matched with a special enzyme-linked immunosorbent assay instrument, the result is judged according to the detected absorbance value, the enzyme used in the reagent is generally horseradish peroxidase (HRP), and the enzyme has high and stable specific activity and small molecular weight, and is easy to prepare pure enzyme, so the reagent is most commonly used. HRP can be used as a substrate for peroxidaseThrough H₂O₂And a luminol system, which fluoresces under the action of HRP (horse radish peroxidase). Therefore, the method is most widely applied to clinical diagnosis.

The performance indexes of such kits generally include: assay sensitivity, clinical sensitivity, specificity, precision, accuracy, linear range, stability, and the like. Only if the comprehensive requirements of the indexes are met at the same time, the selling license can be obtained through registering the application on the market.

An enzyme linked immunoassay kit typically comprises the following components: enzyme-linked immunosorbent assay plate, coating antigen (antibody), enzyme-labeled antibody and luminescent substrate. The preparation process comprises the following steps: coating the coated antigen (antibody) on an enzyme-linked immunoassay plate; the luminescence enzyme used is labeled to the antibody.

In the preparation process of the kit, the operation steps of coating the antigen or antibody on the ELISA plate have an important influence on the sensitivity of the kit, and usually, to increase the sensitivity of the kit, an engineer will choose two main solutions, one is to increase the coating concentration, and the other is to increase the marker concentration. In most cases, the two solutions can effectively improve the sensitivity of the test strip. However, in some cases, it is difficult to achieve the goal of increased sensitivity by both of these solutions. In order to meet the performance index requirements of the kit, other technical means are required to improve the sensitivity, so that the development goal can be realized.

Disclosure of Invention

The invention aims to provide a mycobacterium tuberculosis lipoarabinomannan antibody detection sensitizer.

Another objective of the invention is to provide a mycobacterium tuberculosis lipoarabinomannan antibody detection composition.

Still another object of the present invention is to provide a kit for detecting antibodies against mycobacterium tuberculosis lipoarabinomannan.

Still another object of the present invention is to provide a pretreatment method for detecting a mycobacterium tuberculosis lipoarabinomannan antibody, in which a lipoarabinomannan antigen is coated in combination with a polypeptide composition by sample pretreatment, for high sensitivity and high specificity in detection of mycobacterium tuberculosis.

In the present invention, the terms "lipoarabinomannan", "lipoarabinomannan antigen" or "antigen of the present invention" are used interchangeably and refer to a glycan derived from mycobacterium tuberculosis that constitutes the cell wall. The lipoarabinomannan antigen can be prepared, isolated and purified by conventional methods and has a molecular weight generally greater than 37 kD.

A detection component of mycobacterium tuberculosis lipoarabinomannan antibody is polypeptide shown in SEQ ID No1, and the polypeptide and lipoarabinomannan antigen are coated together for improving the sensitivity and specificity of lipoarabinomannan antibody detection.

The mycobacterium tuberculosis lipoarabinomannan antibody detection component is also added with a polypeptide shown in SEQ ID No2 and a polypeptide shown in SEQ ID No3 to form a composition with the polypeptide shown in SEQ ID No1, wherein the polypeptide shown in SEQ ID No1, the polypeptide shown in SEQ ID No2 and the polypeptide shown in SEQ ID No3 are in a molar ratio of 3: 2-5: 3.

On the detection reaction plate, the coating amount of the mycobacterium tuberculosis lipoarabinomannan antigen is 0.5 mu g/hole to 2 mu g/hole, and the preferable examples are that: 1 μ g/well.

The amount of polypeptide coated on the assay plate is 0.5. mu.g/well to 1. mu.g/well. Such as: the coating amount of the polypeptide shown in SEQ ID No1 is 0.5 mu g/pore-1 mu g/pore, and the coating amount is as follows: the coating amount of the polypeptide shown in SEQ ID No1 and the polypeptide shown in SEQ ID No2 is 0.5 mu g/hole to 1 mu g/hole; the following steps are repeated: the coating amount of the polypeptide shown in SEQ ID No1 and the polypeptide shown in SEQ ID No3 is 0.5 mu g/hole to 1 mu g/hole

A method for pretreating a sample to be tested, wherein a sample treatment solution is an acidic solution, such as: perchloric acid, hydrochloric acid, dilute nitric acid, dilute sulfuric acid, trifluoroacetic acid and the like, treating the sample to be detected, and controlling the final pH value of the sample to be detected to be 6.0-8.0.

A kit for detecting a mycobacterium tuberculosis lipoarabinomannan antibody comprises a sample treatment solution, a mycobacterium tuberculosis lipoarabinomannan antigen, a component and a sample diluent, wherein the mycobacterium tuberculosis lipoarabinomannan antigen and the component are coated on a detection plate.

Blocking agents are added to the sample dilution, such as: the HIER-E-014, HIER-R-001, HIER-M-004, HIER-M-005 and HIER-E-001 are purchased from Fengcong biological products Ltd, the concentration is preferably 10 mu g/ml to 100 mu g/ml, and the HIER-E-001 is preferably selected.

The kit also comprises a coating plate, an enzyme conjugate working solution, a substrate color development solution A, a substrate color development solution B, a washing solution, a negative and positive control and a stop solution.

Enzyme conjugate working solution: na (Na)₂HPO₄.12H₂O 2.9g/L、NaCl 8g/L、KCl 0.2g/L、KH₂PO₄0.2g/L, 2.0g/L of horseradish peroxidase protective agent, 500ml/L of calf serum, 500ml/L of goat serum, 1g/L of thimerosal and a proper amount of anti-TB-HRP.

Positive control: na (Na)₂HPO₄.12H₂O 2.9g/L、NaCl 8g/L、KCl 0.2g/L、KH₂PO₄0.2g/L, 150ml/L ethylene glycol, 250ml/L negative serum, 1g/L merthiolate and positive serum.

Negative control: na (Na)₂HPO₄.12H₂O 2.9g/L、NaCl 8g/L、KCl 0.2g/L、KH₂PO₄0.2g/L and 150ml/L of ethylene glycol; thimerosal 1g/L and negative serum 250 ml/L.

Washing liquid: NaCl 160 g/L; na (Na)₂HPO₄.12H₂O 58g/L；KCl 4g/L；KH₂PO₄4g/L and Tween-2010 ml/L.

Substrate color developing solution A: 8g/L of citric acid, 0.004g/L of 8-hydroxyquinoline, 2.4ml/L of glacial acetic acid and 0.7g/L of carbamide peroxide.

Substrate color developing solution B: 2.1g/L of citric acid, 0.38g/L of ethylene diamine tetraacetic acid and 0.4g/L of TMB.

Stopping liquid: concentrated sulfuric acid 110 ml/L.

Sample diluent: tris Base 2.4g/L, NaCl 17.8.8 g/L, casein sodium 5g/L, bovine serum albumin 2g/L, Tween-201 ml/L, Triton X-1002 ml/L, goat serum 50ml/L, thimerosal 0.4g/L and blocking agent.

According to the scheme, the lipoarabinomannan antigen is verified to be an antigen which is specific to mycobacterium tuberculosis and is suitable for a combined enzyme immunization method after pretreatment.

The scheme of the invention adopts a mode of coating the lipoarabinomannan antigen and the polypeptide (or the composition) together, realizes the specificity detection of the tubercle bacillus antibody, has higher sensitivity, and is suitable for in vitro detection.

The scheme of the invention also applies the pretreatment method of the serum sample to the enzyme-linked immunosorbent assay detection method for combined coating of the lipoarabinomannan antigen and the polypeptide (or the composition), skillfully utilizes the chemical characteristics of the lipopolysaccharide of the LAM antigen, and successfully detects the antibody of the lipoarabinomannan antigen in the sample by the pretreatment of the serum sample and the use of the polypeptide (or the composition). Compared with the existing mycobacterium tuberculosis detection method, the scheme of the invention has the advantages of obviously improving the aspects of simplicity, rapidity, sensitivity and/or specificity and the like.

Detailed Description

The technical solution of the present invention is described in detail below. Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.