阅读说明：本技术 一种人嗜酸性粒细胞阳离子蛋白和髓过氧化物酶检测试剂盒及其应用 (Human eosinophil cationic protein and myeloperoxidase detection kit and application thereof ) 是由 殷丽 于 2019-08-14 设计创作，主要内容包括：本发明属于生物技术领域,具体涉及一种人嗜酸性粒细胞阳离子蛋白和髓过氧化物酶检测试剂盒及其应用。本发明的两种试剂盒采用的是“双抗体夹心一步法”的反应模式,可以同时对病人血清及鼻分泌物样本中的人嗜酸性粒细胞阳离子蛋白和髓过氧化物酶分子进行非常专一的定量及定性检测。它既有效地利用了化学发光技术原理,又在酶联免疫分析的基础上应用酶催化发光底物,提高了检测的灵敏度,同时也具有稳定性高、特异性好、准确性佳、操作简单及无放射性污染的优点,可为支气管哮喘、鼻窦炎、鼻炎等炎症疾病的临床诊断提供更为特异、快速、可靠的依据。(The invention belongs to the technical field of biology, and particularly relates to a human eosinophil cationic protein and myeloperoxidase detection kit and application thereof. The two kits of the invention adopt a reaction mode of a double-antibody sandwich one-step method, and can simultaneously carry out very specific quantitative and qualitative detection on human eosinophilic granulocyte cationic protein and myeloperoxidase molecules in patient serum and nasal secretion samples. The method not only effectively utilizes the technical principle of chemiluminescence, but also applies an enzyme-catalyzed luminescent substrate on the basis of enzyme-linked immunoassay, improves the detection sensitivity, has the advantages of high stability, good specificity, good accuracy, simple operation and no radioactive pollution, and can provide a more specific, rapid and reliable basis for clinical diagnosis of inflammatory diseases such as bronchial asthma, nasosinusitis, rhinitis and the like.)

1. A human eosinophil cationic protein and myeloperoxidase detection kit is characterized by comprising the following two types:

A) chemiluminescence immunoassay kit (CLIA) and chemochromic immunoassay kit (ELISA) of human eosinophil cationic protein and myeloperoxidase, which comprises the following components:

① human eosinophil cationic protein and human myeloperoxidase standard;

② a vector coated with a human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody;

③ labeled human eosinophil cationic protein monoclonal antibody and human myeloperoxidase monoclonal antibody, ③ labeled antibody and ② coating antibody are paired antibodies which respectively form a double-anti-sandwich structure with human eosinophil cationic protein and human myeloperoxidase, and no cross reaction exists between the human eosinophil cationic protein paired antibody and the human myeloperoxidase paired antibody;

④ substrate for color development;

B) an immunochromatographic assay kit (colloidal gold) for human eosinophil cationic protein and myeloperoxidase, comprising:

① sample loading pad adhered to one end of the bottom plate;

② a colloidal gold pad containing a labeled human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody, which is tightly pressed against the loading pad;

③ nitrocellulose NC membrane tightly pressed with one end of the colloidal gold pad, the nitrocellulose NC membrane is coated with a detection line T and a quality control line C which are separated from each other, the detection line T is coated with a human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody which are matched with the ② labeled antibody, the quality control line C is coated with a goat anti-mouse IgG antibody, the coated antibody on the detection line T and the labeled antibody in ② are matched antibodies which respectively form a double anti-sandwich structure with the human eosinophil cationic protein and the human myeloperoxidase, and no cross reaction exists between the human eosinophil cationic protein matched antibody and the human myeloperoxidase matched antibody

④ and a sample sucking pad closely pressed with the nitrocellulose NC film.

2. The kit for detecting cationic protein and myeloperoxidase of human eosinophils according to claim 1, wherein the preparation method of the vector coated with monoclonal antibody against cationic protein and monoclonal antibody against human eosinophils of A) comprises: and (2) taking a carbonate buffer solution as a solvent, uniformly mixing the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody with the solvent, loading the mixture on a carrier, cleaning the carrier, sealing the carrier by adopting a sealing solution, and drying to obtain the carrier coated with the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody.

3. The kit of claim 1, wherein said carrier is a microplate, magnetic particles, plastic tubes or plastic beads.

4. The kit for detecting cationic protein and myeloperoxidase of human eosinophils according to claim 1, wherein said enzyme for labeling in A) is alkaline phosphatase or horseradish peroxidase.

5. The human eosinophil cationic protein and myeloperoxidase detection kit of claim 1, wherein the chromogenic substrate in said chemiluminescent immunoassay kit of A) is luminol, isoluminol, (adamantane) -1, 2-dioxyethane, 3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane, CSPD or CDP-Star; the chromogenic substrate in the chemical chromogenic immunoassay kit is 3,3',5,5' -tetramethyl benzidine or diaminobenzidine.

6. The kit for detecting cationic protein and myeloperoxidase of human eosinophils according to claim 1, wherein said colloidal gold pad of B) is prepared by the following steps: preparing a colloidal gold solution by a chloroauric acid-trisodium citrate reduction method, adding a marked human eosinophilic granulocyte cationic protein monoclonal antibody and a marked human myeloperoxidase monoclonal antibody into the colloidal gold solution, stirring for 2 hours at room temperature, adding bovine serum albumin with the final concentration of 1 percent and 1 percent polyethylene glycol 20000, sealing for 20 minutes, centrifuging, discarding supernatant, redissolving by using colloidal gold working solution, and uniformly paving the solution on a glass fiber membrane or non-woven fabric to prepare a colloidal gold pad.

7. Use of the eosinophil cationic protein and myeloperoxidase detection kit of any of claims 1 ~ 6 for the simultaneous detection of eosinophil cationic protein and myeloperoxidase products.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a human eosinophil cationic protein and myeloperoxidase detection kit and application thereof.

Background

Allergic Rhinitis (AR) and Chronic Rhinosinusitis (CRS) are the most common diseases in ear, nose, throat, head and neck surgery. Often the diagnosis depends on the symptoms and signs of the patient. The cytological examination of nasal secretion is used as an important auxiliary diagnosis method, is beneficial to the accurate diagnosis and individualized treatment of rhinitis patients, and has not wide application although being developed clinically because of complicated operation and lack of uniform standard. In our previous nasal secretion cytology studies, it can be determined that eosinophils and neutrophils account for the absolute majority (> 98%) of nasal secretions, and that nasal secretions of patients with AR are mainly characterized by eosinophils, and nasal secretions of patients with CRS are mainly characterized by neutrophils. The accuracy of the results is affected because the sampling process of the nasal cytology examination is unstable and can be affected and restricted by the condition of the patient at the time of the visit (such as the secretion amount is too small, the patient is not matched, etc.). In contrast, nasal secretions are more easily and stably obtained. Eosinophil Cationic Protein (ECP) is a basic protein, is an important index reflecting the activation amount of eosinophils, and is one of the markers of clinical examination of allergic patients. Myeloperoxidase (MPO) is a heme protease containing a heme prosthetic group and is also the most abundant pro-inflammatory enzyme stored in neutrophils, reflecting the activation of neutrophils.

At present, no product for judging the nature of nasal inflammation exists in clinic, no competitive product exists, and even no product exists. The judgment of the nasal cavity inflammation and the adjustment of the treatment are carried out by doctors completely depending on experience. ECP and MPO have wide and important application value clinically; the nasal cavity local inflammation is accurately judged, so that the rhinitis is accurately treated, and the treatment effect is objectively evaluated.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a human eosinophil cationic protein and myeloperoxidase detection kit and application thereof.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

a kit for detecting cationic protein and myeloperoxidase of human eosinophils comprises the following two types:

A) chemiluminescence immunoassay kit (CLIA) and chemochromic immunoassay kit (ELISA) of human eosinophil cationic protein and myeloperoxidase, which comprises the following components:

① human eosinophil cationic protein and human myeloperoxidase standard;

② a vector coated with a human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody;

③ labeled human eosinophil cationic protein monoclonal antibody and human myeloperoxidase monoclonal antibody, ③ labeled antibody and ② coating antibody are paired antibodies which respectively form a double-anti-sandwich structure with human eosinophil cationic protein and human myeloperoxidase, and no cross reaction exists between the human eosinophil cationic protein paired antibody and the human myeloperoxidase paired antibody;

④ substrate for color development;

B) an immunochromatographic assay kit (colloidal gold) for human eosinophil cationic protein and myeloperoxidase, comprising:

① sample loading pad adhered to one end of the bottom plate;

② a colloidal gold pad containing a labeled human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody, which is tightly pressed against the loading pad;

③ nitrocellulose NC membrane tightly pressed with one end of the colloidal gold pad, the nitrocellulose NC membrane is coated with a detection line T and a quality control line C which are separated from each other, the detection line T is coated with a human eosinophil cationic protein monoclonal antibody and a human myeloperoxidase monoclonal antibody which are matched with the ② labeled antibody, the quality control line C is coated with a goat anti-mouse IgG antibody, the coated antibody on the detection line T and the labeled antibody in ② are matched antibodies which respectively form a double anti-sandwich structure with the human eosinophil cationic protein and the human myeloperoxidase, and no cross reaction exists between the human eosinophil cationic protein matched antibody and the human myeloperoxidase matched antibody

④ and a sample sucking pad closely pressed with the nitrocellulose NC film.

In the above scheme, the preparation method of the vector coated with the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody in a) comprises: and (2) taking a carbonate buffer solution as a solvent, uniformly mixing the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody with the solvent, loading the mixture on a carrier, cleaning the carrier, sealing the carrier by adopting a sealing solution, and drying to obtain the carrier coated with the human eosinophil cationic protein monoclonal antibody and the human myeloperoxidase monoclonal antibody.

In the scheme, the carrier is a microporous plate, magnetic particles, a plastic tube or plastic beads.

In the above scheme, the enzyme for labeling in A) is alkaline phosphatase or horseradish peroxidase.

In the above scheme, the chromogenic substrate in the chemiluminescent immunoassay kit of a) is luminol, isoluminol, (adamantane) -1, 2-dioxyethane, 3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane, CSPD or CDP-Star; the chromogenic substrate in the chemical chromogenic immunoassay kit is 3,3',5,5' -tetramethyl benzidine or diaminobenzidine.

In the above scheme, the preparation process of the colloidal gold pad in B) is: preparing a colloidal gold solution by a chloroauric acid-trisodium citrate reduction method, adding a marked human eosinophilic granulocyte cationic protein monoclonal antibody and a marked human myeloperoxidase monoclonal antibody into the colloidal gold solution, stirring for 2 hours at room temperature, adding bovine serum albumin with the final concentration of 1 percent and 1 percent polyethylene glycol 20000, sealing for 20 minutes, centrifuging, discarding supernatant, redissolving by using colloidal gold working solution, and uniformly paving the solution on a glass fiber membrane or non-woven fabric to prepare a colloidal gold pad.

The eosinophil cationic protein and myeloperoxidase detection kit is applied to synchronous detection of eosinophil cationic protein and myeloperoxidase products.

The invention has the beneficial effects that: the two kits of the invention adopt a reaction mode of a double-antibody sandwich one-step method, and can simultaneously carry out very specific quantitative and qualitative detection on human eosinophilic granulocyte cationic protein and myeloperoxidase molecules in patient serum and nasal secretion samples. The chemiluminescence immunoassay kit (CLIA) and the chemiluminescence immunoassay kit (ELISA) of the human eosinophil cationic protein and the myeloperoxidase not only effectively utilize the principle of chemiluminescence technology, but also apply enzyme-catalyzed luminescence substrates on the basis of enzyme-linked immunoassay, improve the sensitivity of detection, and simultaneously have the advantages of high stability, good specificity, good accuracy, simple operation and no radioactive pollution. The immunochromatography detection kit (colloidal gold) for the human eosinophil cationic protein and the myeloperoxidase has accurate, stable and reliable detection result; is particularly suitable for the health requirements of the public at the basic level and the vast people in rural areas, and can provide more specific, rapid and reliable basis for the clinical diagnosis of inflammatory diseases such as bronchial asthma, nasosinusitis, rhinitis and the like.

Drawings

FIG. 1 is a diagram showing the results of the immunochromatographic assay kit (colloidal gold) for human eosinophil cationic protein and myeloperoxidase in example 2.

FIG. 2 is a line graph of the standards in the kit prepared in example 3.

FIG. 3 shows the correlation between the ECP immunofluorescence assay kit (CLIA) of the present invention and ECP immunofluorescence assay kit (FI) of Thermo Fisher Scientific in the United states, showing that the ECP content in30 samples was measured, and the correlation reached r-0.9757 (P < 0.0001).

Detailed Description

In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.