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Pepsin enzyme-linked immunoassay detection kit

A technology of enzyme-linked immunoassay and detection kit, applied in the field of pepsin enzyme-linked immunoassay detection kit, can solve few problems

Inactive Publication Date: 2012-08-01
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no research and application report on the establishment of a sandwich enzyme immunoassay for the detection of pepsin by using the same strain polyclonal antibody, and there are few related foreign literature reports

Method used

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Experimental program
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Embodiment 1

[0013] The preparation of embodiment 1 pepsin ELISA detection kit

[0014] 1. Materials

[0015] Three big-eared white rabbits for immunization, male, weighing 1.5 kg, were provided by the Animal Experiment Center of 301 Hospital. Human pepsin was purchased from Sigma, Freund's complete and incomplete adjuvants were self-made in our laboratory, trypsin, amylase, antitrypsin, and secretory IgA were all purchased from Sigma, normal human saliva and reflux esophagitis The samples of patients' bronchial secretions were all from the General Hospital of the Chinese People's Liberation Army.

[0016] 2. Preparation and purification of pepsin antibody

[0017] (1) Preparation of antiserum for the first immunization Mix 600ug of pepsin and 4ml of Freund's complete adjuvant fully, and inject into the skin at multiple points on the back of 3 rabbits respectively, and boost immunization every 4 weeks (300ug of immunogen and incomplete 4ml of adjuvant is fully mixed and injected in the ...

Embodiment 2

[0023] Embodiment 2 pepsin ELISA detection method

[0024] 1. Preparation of solid-phase antibody microtiter plate

[0025] Take the purified pepsin antibody, prepare 10ug / ml with 0.05M citrate buffer, add 100ul per well to 96-well ELISA plate, incubate at 37°C for 3h, wash the plate, add 3% In the Tris.HCl blocking solution of bovine serum albumin, stay overnight at 4°C, shake off the blocking solution, remove excess water in the hole with a freeze dryer, put it in a tin foil bag, vacuum seal it with a vacuum packaging machine, and store it at 4°C .

[0026] 2. Preparation of reagents and instruments

[0027] 7 bottles of freeze-dried standard products: each bottle was dissolved in 1 mL of experimental buffer and the concentrations were 8, 4, 2, 1, 0.5, 0.25, 0 ng / mL; horseradish peroxidase-labeled pepsin antibody working solution ( HRP-IgG) 1 bottle; Chromogenic agent A: 1 stick; Chromogenic reagent B: 1 bottle; Chromogenic working solution: Mix A and B at a ratio of 1:19...

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Abstract

The invention discloses a pepsin enzyme-linked immunoassay detection kit, belonging to the technical field of enzyme-linked immunodetection. The kit consists of a solid-phase enzyme labelled plate, a pepsin standard substance, a pepsin antibody marked by horse radish peroxidase, and auxiliary reagents. When the kit is used, the pepsin standard substance and pepsin to be detected in a sample are added into a solid phase antibody plate, wherein the pepsin standard substance and the pepsin to be detected have the same volume; after the pepsin standard substance and the pepsin to be detected are washed repeatedly, an enzyme labelled antibody is added into the pepsin standard substance and the pepsin to be detected; and after the mixture is washed repeatedly, a colour developing agent is added into the mixture, and the content of pepsin in the sample to be detected is judged according to colour shades of the mixture. The method is proved to be practical and reliable by detection on 62 samples of saliva of healthy people and 23 samples of saliva of patients suffering from reflux esophagitis.

Description

technical field [0001] The invention belongs to the technical field of ELISA detection, in particular to a pepsin ELISA detection kit. Background technique [0002] Gastroesophageal reflux disease (CERD) is a disease in which the reflux of gastric contents causes discomfort symptoms and (or) complications. It is a common disease of the digestive system. The incidence rate in Western countries is about 7%-27%. CERD in our country The incidence also shows an increasing trend, about 9.1%-22.5%, but the detection of CERD is quite troublesome at present. Therefore, we established a detection method of pepsin enzyme-linked immunoassay to assist clinical differential diagnosis. Pepsin (Pepsin) is an acidic protease transformed from pepsinogen released by gastric mucosal chief cells, with a molecular weight of 35Kd. However, in some cases when gastric reflux occurs, a small amount of pepsin in the gastric juice can destroy the normal structure of the original mucosa, damage the pr...

Claims

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Application Information

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IPC IPC(8): G01N33/573
Inventor 颜光涛薛辉
Owner GENERAL HOSPITAL OF PLA
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