阅读说明：本技术 一种抗合成酶综合征oj自身抗体的检测方法与应用 (Detection method and application of anti-synthetase syndrome OJ autoantibody ) 是由 赵洪军 周亚欧 宁旺斌 刘思佳 于 2019-10-11 设计创作，主要内容包括：本发明公开了一种抗合成酶综合征OJ自身抗体的检测方法与应用,具体涉及生物技术领域,具体包括有以下步骤：步骤一：取人体肺内活细胞作为细胞悬液,采用催化抗体作为抗体催化剂,将抗体催化剂添加至细胞悬液内部。本发明通过采用催化抗体作为抗体催化剂,有效的提高了OJ自身抗体的活跃性,使其更加易于检测,并由荧光染色的抗体结合微粒与OJ自身抗体相互结合,并利用抗体结合微粒吸附膜与抗体结合微粒之间的吸附作用,使得OJ自身抗体更加有效的被检测出来,提高了OJ自身抗体检测的准确性和高效率性,改变了以往的单一的荧光标记以及放射性法检测,避免了发生检测失误的状况,利于肌炎症患者的早期诊断,具备较高的诊断性能。(The invention discloses a detection method and application of an anti-synthetase syndrome OJ autoantibody, and particularly relates to the technical field of biology, and the detection method specifically comprises the following steps: the method comprises the following steps: taking live cells in human lung as cell suspension, adopting catalytic antibody as antibody catalyst, and adding the antibody catalyst into the cell suspension. According to the invention, the catalytic antibody is used as an antibody catalyst, so that the activity of the OJ autoantibody is effectively improved and the OJ autoantibody is easier to detect, the fluorescent dyed antibody binding particles are combined with the OJ autoantibody, and the adsorption effect between the antibody binding particle adsorption film and the antibody binding particles is utilized, so that the OJ autoantibody is more effectively detected, the accuracy and high efficiency of the detection of the OJ autoantibody are improved, the previous single fluorescent labeling and radioactive detection are changed, the condition of detection errors is avoided, the early diagnosis of myositis patients is facilitated, and the diagnosis performance is higher.)

1. A method for detecting an anti-synthetase syndrome OJ autoantibody is characterized by comprising the following steps:

the method comprises the following steps: taking living cells in human lung as a cell suspension, adopting a catalytic antibody as an antibody catalyst, adding the antibody catalyst into the cell suspension, slightly shaking the cell suspension, and standing for half an hour in an appropriate temperature environment;

step two: preparing antibody binding particles, placing the antibody binding particles in a fluorescent antibody suspension, stirring, placing the dyed antibody binding particles at a proper temperature, preserving for half an hour, pouring the dyed antibody binding particles into a cell suspension, and stirring by adopting an airflow method to combine the antibody binding particles with an OJ autoantibody in the cell suspension;

step three: preparing an antibody-bound particle adsorption film, wherein an antigen which is mutually bound with the antibody-bound particles is used as a raw material, and the content of each pore in the antibody-bound particle adsorption film is 100 ul;

step four: slowly inverting the cell suspension in the process of the step on the antibody-bound particle adsorption film, enabling the cell suspension to sequentially pass through the plurality of layers of antibody-bound particle adsorption films, and manually shaking the plurality of layers of antibody-bound particle adsorption films when the cell suspension is poured so that the cell suspension can be comprehensively adsorbed on the antibody-bound particle adsorption films;

step five: the multilayer antibody-bound microparticle-adsorbed membrane was slowly inverted, the antibody-bound microparticles attached thereto were poured out, and the operation and observation were performed under a microscope, and the OJ autoantibodies were determined from the fluorescently labeled antibody-bound microparticles.

2. The method of claim 1 for detecting autoantibodies against the synthetase syndrome OJ, wherein the autoantibodies comprise: in the first step, the cell suspension is diluted to an appropriate concentration of 100ug/ml by carbonate buffer solution, and after standing for ten minutes, the supernatant is discarded, and the lower sediment solution is taken out.

3. The method of claim 1 for detecting autoantibodies against the synthetase syndrome OJ, wherein the autoantibodies comprise: and a magnetic stirrer is required in the stirring process in the step two, wherein the magnetic stirrer is used for centrifuging for 5 minutes at 1000r/min, and then the stirred liquid is kept still.

4. The method of claim 1 for detecting autoantibodies against the synthetase syndrome OJ, wherein the autoantibodies comprise: the antibody binding particle adsorption membranes in the third step are provided with three layers, each layer of antibody binding particle adsorption membrane is arranged in a funnel shape, and when the cell suspension is inverted, the cell suspension needs to be slowly poured around the antibody binding particle adsorption membranes distributed in the funnel shape in a rotating mode.

5. The method of claim 1 for detecting autoantibodies against the synthetase syndrome OJ, wherein the autoantibodies comprise: in the fourth step, the antibody binding particles pass through the pores of the adsorption film of the antibody binding particles and are adsorbed with each other.

6. The method of claim 1 for detecting autoantibodies against the synthetase syndrome OJ, wherein the autoantibodies comprise: and in the fifth step, the screened OJ autoantibody and antibody binding particles are placed on a glass slide.

7. Use of a method of detecting autoantibodies to synthetase syndrome OJ according to any of claims 1-6, for detecting the status of autoantibodies to OJ in a patient's body.

Technical Field

The invention relates to the technical field of biology, in particular to a detection method and application of an anti-synthetase syndrome OJ autoantibody.

Background

Anti-synthetase syndrome is a clinical disease caused by the dysfunction of self myositis specific antibody. Since Nishikai discovered anti-Jo-1 antibody for the first time in 1980 and confirmed that the antibody is closely related to myositis, anti-aminoacyl-tRNA synthetase antibodies such as PL-7, PL-12, OJ and EJ were discovered in succession. They are "myositis-specific autoantibodies" with anti-SRP, Mi-2 and anti-Mas antibodies. Their positive rate in the serum of DM and PM patients is about 25% -40%. These antibody-positive patients, regardless of the type of antibody, exhibited essentially identical clinical manifestations, i.e., all had the so-called "anti-synthetase syndrome" (anti-Jo-1 antibody syndrome).

Of the above 5 anti-synthetase antibodies found to date, the anti-Jo-1 antibody is most common in myositis, with a positive rate of 15% to 30%, 25% to 45% in idiopathic polymyositis, and generally less than 10% in dermatomyositis, while the other 4 anti-synthetase antibodies were no more than 5% in myositis, but were more positive than polymyositis.

These 5 antibodies are not normally present in the same patient but are equally present with other autoantibodies such as anti-ds-DNA, Sm, SSA or SSB antibodies, although Gelpi et al found 1 patient with anti-Jo-1 and anti-OJ antibodies simultaneously, DM and PM patients with anti-synthetase antibodies were ill-weighted, poor-prognosis and were highlighted as pulmonary interstitial lesions with a positive rate of up to 40% to 75% far greater than 10% to 20% of anti-synthetase negatives, e.g. Oddis et al studied PM/DM patients since 1975 at the university of pittsburgh hospital, found 18 patients positive for anti-Jo-1 antibodies, 12 of which had pulmonary interstitial lesions accounting for 67%, and 79 of which had only 15 (15%) of those negative for anti-Jo-1 antibodies. Many patients are diagnosed with "lung infection" prior to a visit, and with prolonged use of antibiotics in large quantities, symptoms are not effectively controlled. Some patients even experience respiratory failure.

The incidence rates of arthritis and Raynaud phenomenon of DM and PM patients with anti-synthetase antibody are 57% -100% and 60% respectively. Although arthritis is considered to be mild by detecting anti-Jo-1 antibody at home, symptoms of arthritis and the degree of deformity are considered to be serious by foreign reports, such as a group of 29 patients with positive anti-synthetase antibody reported by Maggnerie, 1/3 with polyarthritis, and 4 with joint erosion. Oddis et al reported 21 anti-Jo-1 positive patients, 12 with polyarthritis, 4 with deformities of the hands or wrists, and 1 with erosion of MCP and ulna. In addition, the symptoms associated with anti-synthetase antibodies include "mechanical 'shand", fever, leukocytosis, serositis, xeroderma and seropositive RF in the serum, which manifests as hyperkeratosis, cracking and pigmentation of the skin on the radial and palmar sides of the patient's fingers.

The invention patent of patent application publication No. CN 106680503A discloses a method for screening and identifying an autoantibody in ECH1 and application thereof, which comprises the following steps: performing two-dimensional electrophoresis on a lung cancer cell line H1299 whole protein extract, performing Western blotting on the lung cancer cell line H1299 whole protein extract and 5 cases of lung cancer patient serum and 5 cases of normal human serum, screening meaningful tumor autoantigens, and performing comparison analysis to further analyze only protein spots appearing in the patient serum; after cutting and digesting the protein points of interest on the SDS-PAGE gel, analyzing the protein points by adopting an LC-MS/MS mass spectrometry analysis method, and detecting and identifying the protein points as ECH 1; further verifying whether ECH1 self antibody can be used as a lung cancer diagnosis marker. The research of the invention finds that the ELISA method is used for detecting the serum ECH1 autoantibody, so that the lung cancer patients can be accurately distinguished from normal people and chronic lung disease patients, and the method can be used for early diagnosis of the lung cancer.

However, the above technical solutions still have many disadvantages in practical use, and the activity of the studied OJ autoantibody is reduced after being isolated, so that the OJ autoantibody is difficult to be effectively detected, and the detection efficiency and accuracy are reduced.

Disclosure of Invention

In order to overcome the above defects in the prior art, embodiments of the present invention provide a method for detecting an anti-synthetase syndrome OJ autoantibody and an application thereof, wherein a catalytic antibody is used as an antibody catalyst, so that the activity of the OJ autoantibody is effectively improved and the OJ autoantibody is easier to detect, and the OJ autoantibody is more effectively detected by combining a fluorescent-dyed antibody binding particle with the OJ autoantibody and utilizing the adsorption effect between an antibody binding particle adsorption film and an antibody binding particle, so that the detection accuracy and high efficiency of the OJ autoantibody are improved, the previous detection by a single fluorescent label and a radioactive method is changed, the condition of detection errors is avoided, the early diagnosis of a myositis patient is facilitated, and the method has high diagnostic performance.

In order to achieve the purpose, the invention provides the following technical scheme: a method for detecting an anti-synthetase syndrome OJ autoantibody specifically comprises the following steps:

the method comprises the following steps: taking living cells in human lung as a cell suspension, adopting a catalytic antibody as an antibody catalyst, adding the antibody catalyst into the cell suspension, slightly shaking the cell suspension, and standing for half an hour in an appropriate temperature environment;

step two: preparing antibody binding particles, placing the antibody binding particles in a fluorescent antibody suspension, stirring, placing the dyed antibody binding particles at a proper temperature, preserving for half an hour, pouring the dyed antibody binding particles into a cell suspension, and stirring by adopting an airflow method to combine the antibody binding particles with an OJ autoantibody in the cell suspension;

step three: preparing an antibody-bound particle adsorption film, wherein an antigen which is mutually bound with the antibody-bound particles is used as a raw material, and the content of each pore in the antibody-bound particle adsorption film is 100 ul;

step four: slowly inverting the cell suspension in the process of the step on the antibody-bound particle adsorption film, enabling the cell suspension to sequentially pass through the plurality of layers of antibody-bound particle adsorption films, and manually shaking the plurality of layers of antibody-bound particle adsorption films when the cell suspension is poured so that the cell suspension can be comprehensively adsorbed on the antibody-bound particle adsorption films;

step five: the multilayer antibody-bound microparticle-adsorbed membrane was slowly inverted, the antibody-bound microparticles attached thereto were poured out, and the operation and observation were performed under a microscope, and the OJ autoantibodies were determined from the fluorescently labeled antibody-bound microparticles.

In a preferred embodiment, in step one, the cell suspension is diluted with carbonate buffer solution to a suitable concentration of 100ug/ml, and after standing for ten minutes, the supernatant is discarded and the sediment is removed.

In a preferred embodiment, a magnetic stirrer is used for the stirring in the second step, wherein the magnetic stirrer is centrifuged at 1000r/min for 5 minutes, and then the stirred liquid is allowed to stand.

In a preferred embodiment, the antibody-binding particle-adsorbing membrane in step three is provided with three layers, each layer of antibody-binding particle-adsorbing membrane is arranged in a funnel shape, and when the cell suspension is inverted, the cell suspension needs to be slowly poured around the funnel-shaped antibody-binding particle-adsorbing membrane in a rotating manner.

In a preferred embodiment, the antibody-binding particles in step four adsorb to each other when passing through the pores of the antibody-binding particle adsorption film.

In a preferred embodiment, the selected OJ autoantibodies and antibody binding particles are placed on a slide glass in step five.

In a preferred embodiment, the use of an autoantibody against the synthetase syndrome OJ is also included: for detecting the condition of anti-OJ autoantibodies in the body of a patient, for detecting the condition of anti-OJ autoantibodies in the body of a patient.

The invention has the technical effects and advantages that:

the invention effectively improves the activity of the OJ autoantibody by adopting the catalytic antibody as an antibody catalyst, so that the OJ autoantibody is easier to detect, the fluorescent dyed antibody combined particles are combined with the OJ autoantibody, the adsorption effect between the antibody combined particle adsorption film and the antibody combined particles is utilized, so that the OJ autoantibody is more effectively detected, the accuracy and the high efficiency of the detection of the OJ autoantibody are improved, the previous single fluorescent labeling and radioactive detection are changed, the condition of detection error is avoided, the early diagnosis of a myositis patient is facilitated, the invention has higher diagnosis performance, can be widely applied to the antibody detection of other diseases, improves the practicability and the applicability of the invention, and has higher popularization value.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.