CN107091925B

CN107091925B - Kit for detecting periodontitis related protein

Info

Publication number: CN107091925B
Application number: CN201710489328.8A
Authority: CN
Inventors: 孟玲
Original assignee: Zhejiang Yuan Kangrui Biological Technology Co ltd
Current assignee: Zhejiang Yuan Kangrui Biological Technology Co.,Ltd.
Priority date: 2017-06-24
Filing date: 2017-06-24
Publication date: 2020-06-23
Anticipated expiration: 2037-06-24
Also published as: CN107091925A

Abstract

The invention specifically discloses an antibody chip and a kit for detecting periodontitis related protein, comprising an antibody chip, a periodontal disease related protein standard mixture and a biotin-labeled periodontal disease related protein detection antibody mixture; and fluorescein Cy 3-labeled streptavidin; the antibody chip takes a slide glass treated by a hydrophilic reagent containing alkyl glycoside as a solid phase carrier, and a specific antibody mixture of a plurality of periodontal disease-related proteins is spotted on the solid phase carrier under the conditions of 4-20 ℃ and 20-29% of humidity. The antibody chip kit overcomes the defects of complex operation, single detection index, low sensitivity and the like in the prior art, and has the advantages of low price, convenience, sensitivity, accuracy, high flux, small sample consumption, popularization in common laboratories, large scale and the like.

Description

Kit for detecting periodontitis related protein

Technical Field

The invention relates to the technical field of biological detection, in particular to a kit for detecting periodontitis related protein.

Background

Periodontitis is a chronic inflammation of the periodontal supporting tissue caused primarily by local factors. The age of onset is more common after age 35 years. If gingivitis is not treated timely, inflammation can diffuse from the gingiva to the deep layer to the periodontal ligament, alveolar bone and cementum, and periodontitis can develop. Because early stage has no obvious subjective symptoms and is easy to ignore, the symptoms are serious and even the teeth can not be kept. Therefore, the propaganda and education must be strengthened to make the patient see a doctor early and treat the disease in time. The early symptoms of periodontitis are not obvious, and patients often have the manifestations of secondary gingival bleeding or halitosis, similar to the symptoms of gingivitis. During examination, swelling, soft and dark red of gingival margin, gingival papilla and attached gingiva can be seen, and bleeding is easy to occur during probing.

Gingival Crevicular Fluid (GCF) refers to the fluid that permeates from the gingival connective tissue into the gingival sulcus through the epithelium and associated epithelium in the sulcus. Healthy patients have very little gingival crevicular fluid, and the outflow of gingival crevicular fluid is proportional to the degree of inflammation at the site. The gingival crevicular fluid is primarily derived from serum, while the other components are derived from serum, adjacent periodontal tissue and bacteria, respectively. The gingival crevicular fluid comprises various electrolytes, proteins, glucose, enzymes and the like, also contains various biological macromolecules such as leucocytes, exfoliated epithelial cells and the like, the biological macromolecules can be used as markers for detecting periodontitis, and the proteins of the gingival crevicular fluid can be used as ideal pathological markers for detecting periodontitis.

The existing detection method for periodontitis can only judge the disease condition from pathological forms by comprising observation and X-ray detection, and the judgment of the disease condition is accompanied by the subjective factors of doctors, so the detection result is not objective. Therefore, there is an urgent need to develop a method for rapidly detecting periodontitis using biomarkers.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention provides a kit for detecting periodontitis-related protein, so as to solve the above-mentioned technical problems.

A kit for detecting periodontitis related protein comprises an antibody chip, a periodontal disease related protein standard mixture and a biotin-labeled periodontal disease related protein detection antibody mixture; and fluorescein Cy 3-labeled streptavidin; the antibody chip takes a slide glass treated by a hydrophilic reagent containing alkyl glycoside as a solid phase carrier, and a specific antibody mixture of a plurality of periodontal disease-related proteins is spotted on the solid phase carrier under the conditions of 4-20 ℃ and 20-29% of humidity.

Drawings

Fig. 1 is a schematic structural view of the present invention.

Detailed Description

The invention is further described with reference to the following examples.

A kit for detecting periodontitis related protein, as shown in figure 1, comprises an antibody chip, a periodontal disease related protein standard mixture, a biotin-labeled periodontal disease related protein detection antibody mixture; and fluorescein Cy 3-labeled streptavidin; the antibody chip takes a slide glass treated by a hydrophilic reagent containing alkyl glycoside as a solid phase carrier, and a specific antibody mixture of a plurality of periodontal disease-related proteins is spotted on the solid phase carrier under the conditions of 4-20 ℃ and 20-29% of humidity.

Preferably, the periodontitis-associated protein detection antibody cocktail is a cocktail of antibodies specific for fibronectin receptor (FN), laminin receptor (LN), lymphocyte function-related molecule-1 (LFA-1), clear connexin receptor (LRP), endothelial leukocyte adhesion molecule-1 (ELAM-1), platelet α -granulin (GMP-140), mucosal vascular adhesion (HCAM), and peripheral lymph node vascular adhesion (LAM-1).

The kit for detecting periodontitis-associated protein according to the present invention is suitable for the combination of antibodies specific to 8 periodontitis-associated proteins, fibronectin receptor (FN), laminin receptor (LN), lymphocyte function-associated molecule-1 (LFA-1), vitronectin receptor (LRP), endothelial leukocyte adhesion molecule-1 (ELAM-1), platelet α -granulin (GMP-140), mucosal vascular adhesion (HCAM) and peripheral lymph node vascular adhesion (LAM-1).

Preferably, the hydrophilic reagent is a deionized water solution containing 0.03-0.5% of alkyl glycoside, 0.2-0.8% of glycerol and 0.06-0.1% of polyethylene glycol 6000 by mass percent; the method for treating the glass slide by using the hydrophilic reagent comprises the steps of soaking the glass slide in the hydrophilic reagent for 6-15 minutes, and freeze-drying at 4 ℃. Preferably, the periodontitis-related protein detection antibody mixture is spotted on a solid phase carrier under the conditions of 4-8 ℃ and 20-25% of humidity.

Preferably, the periodontitis-associated protein detection antibody mixture is spotted on the solid phase carrier at 4 ℃ and 22% humidity.

Preferably, the hydrophilic reagent is a deionized water solution containing 0.3% of alkyl glycoside, 0.2% of glycerol and 0.08% of polyethylene glycol 6000 by mass; the method for treating the glass slide by using the hydrophilic reagent comprises the steps of soaking the glass slide in the hydrophilic reagent for 10 minutes, and freeze-drying at 4 ℃.

According to the invention, through comparing various methods for processing the glass slide, the method discovers that the background of the glass slide processed by the hydrophilic reagent containing the alkyl glycoside is low, the detection sensitivity is high, more than ten periodontal disease related proteins can be detected at one time, and the sensitivity of the detected periodontal disease related proteins can reach the ELISA detection sensitivity of a single protein. The antibody chip kit overcomes the defects of complex operation, single detection index, low sensitivity and the like in the prior art, and has the advantages of low price, convenience, sensitivity, accuracy, high flux, small sample consumption, popularization in common laboratories, large scale and the like.

The sensitivity of antibodies specific to different periodontal disease-related proteins in the on-chip detection system of the present invention is different, and therefore, if some antibodies against periodontal disease-related proteins with low sensitivity are combined with some antibodies against periodontal disease-related proteins with high sensitivity, omission of periodontal disease-related proteins with low sensitivity is caused.

Experimental examples antibody chip vector screening

Screening of antibody chip carriers: the conventional antibody chip mostly uses a nitrocellulose membrane as a carrier, and the nitrocellulose membrane is of a multilayer structure, so that the washing difficulty of the chip is high, the background of the chip is high, and the result fluctuation is large. Meanwhile, the nitrocellulose membrane is fragile and not easy to operate on a large scale, and is not generally used for large-scale clinical samples. The traditional slide is cheap and has low background, which brings breakthrough to the wide application of the diagnosis and research fields. We screened slides that were not treated by the active method on the market, and the effect of the slide spotting was unstable, both for the aldehyde and the amino groups. The temperature and the active reagent components on the surface of the slide are obtained through a large amount of screening, and the key for determining the sample application effect of the slide is.

A suitable carrier for an antibody chip was explored using the following protocol for slide treatment in 3:

the group A is an amination slide, and the purchased commercial slide is not processed;

group B is polylysine slide: adding polylysine diluted by PBS into a common glass slide, soaking for 0.5-1 hour, and then cleaning with pure water to prepare a polylysine glass slide;

group C is slides treated with hydrophilic reagents: soaking a common glass slide in a hydrophilic reagent for 3-5 minutes, and airing at room temperature; the hydrophilic reagent is a deionized water solution containing 0.03-0.5% of alkyl glycoside, 0.2-0.8% of glycerol and 0.06-0.1% of polyethylene glycol 6000 in percentage by mass; the method for treating the glass slide by using the hydrophilic reagent comprises the steps of soaking the glass slide in the hydrophilic reagent for 6-15 minutes, and freeze-drying at 4 ℃.

The spotting effect of various slides is different, 3 groups of slides are combined with the antibody, then the secondary antibody and the substrate are added, and the sensitivity screening results under different temperature and humidity conditions are shown in the following table 1, wherein the index of the sensitivity shown in the table 1 is the lowest detection line with the unit ng/ml.

TABLE 1

As can be seen from the data in Table 1, the sensitivity of the treatment methods in group C was higher than that of group A and group B in 3 different treatment humidities at 4 ℃, 8 ℃ and 16 ℃, and it was found that the treatment methods in group C were very suitable for the preparation of the antibody chip of the present invention.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A kit for detecting periodontitis related protein is characterized by comprising an antibody chip, a periodontal disease related protein standard mixture and a biotin-labeled periodontal disease related protein detection antibody mixture; and fluorescein Cy 3-labeled streptavidin; the antibody chip takes a slide glass treated by a hydrophilic reagent containing alkyl glycoside as a solid phase carrier, and a specific antibody mixture of various periodontal disease related proteins is spotted on the solid phase carrier under the conditions of 4-20 ℃ and 20-29% of humidity;

the hydrophilic reagent is a deionized water solution containing 0.03-0.5% of alkyl glycoside, 0.2-0.8% of glycerol and 0.06-0.1% of polyethylene glycol 6000 in percentage by mass; the method for treating the glass slide by using the hydrophilic reagent comprises the steps of soaking the glass slide in the hydrophilic reagent for 6-15 minutes, and freeze-drying at 4 ℃;

the periodontitis-associated protein detection antibody mixture is a mixture of specific antibodies of fibronectin receptor (FN), laminin receptor (LN), lymphocyte function-related molecule-1 (LFA-1), clear connexin receptor (LRP), endothelial leukocyte adhesion molecule-1 (ELAM-1), platelet α -granula membrane protein (GMP-140), mucosal vascular adhesion (HCAM) and peripheral lymph node vascular adhesion (LAM-1).

2. The kit for detecting a periodontitis-associated protein according to claim 1, wherein the periodontitis-associated protein detection antibody mixture is spotted on the solid phase carrier at 4 to 8 ℃ and 20 to 25% humidity.

3. The kit for detecting a periodontitis-associated protein according to claim 1, wherein the periodontitis-associated protein detection antibody mixture is spotted on the solid phase carrier at 4 ℃ under a humidity of 22%.

4. The kit for detecting periodontitis-associated protein according to claim 1, wherein the hydrophilic reagent is a deionized water solution of 0.3% of alkyl glycoside, 0.2% of glycerol, and 0.08% of polyethylene glycol 6000 by mass; the method for treating the glass slide by using the hydrophilic reagent comprises the steps of soaking the glass slide in the hydrophilic reagent for 10 minutes, and freeze-drying at 4 ℃.