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Enzyme-linked immunosorbent reagent kit and method for preparing and using same for pepsin detection

An enzyme-linked immunosorbent reagent and pepsin technology, applied in the field of clinical diagnosis, can solve the problems of single structure, inconvenience, and no detection of pepsin by chemiluminescence enzyme-linked immunosorbent technology, and can improve the convenience of the kit. Accuracy, avoid the effect of complete separation

Active Publication Date: 2017-10-17
中国人民解放军总医院第六医学中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there is no related technology for pepsin detection using chemiluminescent ELISA, and there is no chemiluminescent ELISA kit for pepsin detection.
[0006] In addition, the structure of the traditional enzyme-linked reaction kit is relatively simple and single, which has certain inconvenience during the test operation.

Method used

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  • Enzyme-linked immunosorbent reagent kit and method for preparing and using same for pepsin detection
  • Enzyme-linked immunosorbent reagent kit and method for preparing and using same for pepsin detection
  • Enzyme-linked immunosorbent reagent kit and method for preparing and using same for pepsin detection

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preparation example Construction

[0066] A preparation method of an enzyme-linked immunosorbent kit, comprising the following steps:

[0067] Step 1. Preparation of pepsin monoclonal antibody, extracting pepsin in human gastric mucosal secretions, immunizing BALB / c mice with purified pepsin, and preparing the pepsin monoclonal antibody by hybridoma fusion technology;

[0068] Step 2, the preparation of the microplate 6, after diluting the obtained pepsin monoclonal antibody, adding it to the microwell 7 for coating, to obtain the microplate 6;

[0069] Step 3, the preparation of enzyme-labeled pepsin antibody, preparing a horseradish peroxidase solution, mixing the pepsin monoclonal antibody with the horseradish peroxidase solution, and then adding NaBH to the obtained reaction solution 4 The solution is then subjected to ultrafiltration, and the ultrafiltration retentate is retained to obtain the enzyme-labeled pepsin antibody.

[0070] In the preparation method of described ELISA kit,

[0071] In step 1, p...

Embodiment 1

[0080] Preparation Process:

[0081] Step 1, preparation of pepsin monoclonal antibody. Use ion exchange chromatography and gel filtration technology to separate and purify pepsin in human gastric juice, immunize BALB / c mice with purified pepsin, prepare monoclonal antibody against human pepsin by hybridoma fusion technology, and use protein affinity Chromatography and gel filtration techniques are used to purify monoclonal antibodies; enzyme-linked immunosorbent techniques are used to screen highly specific diagnostic monoclonal antibodies.

[0082] Step 2, preparation of the enzyme-labeled antibody in the kit. Weigh horseradish peroxidase and dissolve it in ultrapure water to make a solution, let it stand overnight at 3°C ​​to fully dissolve it; take the above solution the next day, and add freshly prepared NaIO to it 4 Aqueous solution, after mixing, place in the refrigerator to avoid light and stir. The reaction solution was taken out and put into a dialysis bag, and th...

Embodiment 2

[0088] Preparation Process:

[0089] Step 1, preparation of pepsin monoclonal antibody. Use ion exchange chromatography and gel filtration technology to separate and purify pepsin in human gastric juice, immunize BALB / c mice with purified pepsin, prepare monoclonal antibody against human pepsin by hybridoma fusion technology, and use protein affinity Chromatography and gel filtration techniques are used to purify monoclonal antibodies; enzyme-linked immunosorbent techniques are used to screen highly specific diagnostic monoclonal antibodies.

[0090] Step 2, preparation of the enzyme-labeled antibody in the kit. Weigh horseradish peroxidase and dissolve it in ultrapure water to prepare a solution, let it stand overnight at 4°C to fully dissolve it; take the above solution the next day, and add freshly prepared NaIO to it 4 Aqueous solution, after mixing, place in the refrigerator to avoid light and stir. The reaction solution was taken out and put into a dialysis bag, and t...

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Abstract

The invention discloses an enzyme-linked immunoimmunoassay kit and a method for preparing and utilizing it for pepsin detection. The kit includes: a box body; Pepsin monoclonal antibody, the enzyme standard plate is accommodated in the box body; the reagent bottle is accommodated in the box body, and the reagent bottle is respectively equipped with working solution, standard solution, buffer solution, washing solution and chromogenic solution , wherein the working solution contains enzyme-labeled pepsin antibody. The marker of the enzyme-labeled pepsin antibody is horseradish peroxidase, which is labeled by the sodium periodate method. The ELISA kit of the invention can accurately detect pepsin by using the chemiluminescent ELISA technology, so as to achieve the purpose of non-invasive diagnosis of throat reflux disease.

Description

technical field [0001] The present invention relates to the technical field of clinical diagnosis, more specifically, the present invention relates to a chemiluminescent enzyme-linked immunosorbent assay kit for detecting pepsin, which can detect the content of pepsin and perform non-invasive diagnosis on laryngopharyngeal reflux disease . Background technique [0002] Laryngopharyngeal reflux disease refers to the general term for a series of symptoms and signs caused by the reflux of gastric contents to the position above the upper esophageal sphincter. Symptoms such as chronic long-term cough, dyspnea, and laryngospasm, as well as mucosal hyperplasia and hypertrophy in the posterior commissure of the vocal cords, diffuse congestion and edema of the vocal cords, and in severe cases, laryngeal signs such as granuloma, disappearance of the laryngeal chamber, and subglottic stenosis. [0003] Laryngopharyngeal reflux disease is the source of some diseases in otolaryngology h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/76G01N33/573G01N33/577
Inventor 李进让吴立峰郭春雨
Owner 中国人民解放军总医院第六医学中心
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