Pepsase chemiluminescent immunoassay kit and preparation method thereof

A technology of protease chemistry and detection kits, applied in the fields of chemiluminescence/bioluminescence, analysis through chemical reaction of materials, measuring devices, etc.

Inactive Publication Date: 2011-07-20
GENERAL HOSPITAL OF THE SECOND ARTILLERY OF CHINESE PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, when using the immunoassay method for specific detection, it is necessary to maintain the immunological activity of pepsin and avoid its ability to decompose protein by its enzymatic activity. This is also one of the difficulties in the development of immunoassay kits, and chemiluminescence immunoassay As a new analytical technique, its application to pepsin detection has not been reported

Method used

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  • Pepsase chemiluminescent immunoassay kit and preparation method thereof

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Embodiment 1

[0049] Example 1 Application of horseradish peroxidase system to prepare pepsin chemiluminescence immunoassay kit of the present invention

[0050] 1. Preparation of horseradish peroxidase-labeled antibody

[0051] Dissolve 5 mg of HRP in 0.5 mL of distilled water, add 0.5 mL of sodium periodate (50 mmol / L) and stir at room temperature for 30 min, dialyze through 10 mM sodium acetate buffer with a pH value of 4.4, add 5 mg of pepsin antibody, stir for 2 h, and finally use 200 mM NaBH 4 For reduction, after dialysis with 0.02M PBS buffer, add an equal volume of glycerol, and store below -20°C.

[0052] 2. Enzyme-labeled antibody diluent

[0053] NaH 2 PO 4 2H 2 O 0.2g

[0054] Na 2 HPO 4 12H 2 O 2.9g

[0055] NaCl 8.766g

[0056] Enzyme Stabilizer 1g

[0057] BSA 10g

[0058] Proclin 1mL

[0059] Double distilled water 1000mL

[0060] 3. Enzyme-labeled antibody concentration selection

[0061] It has been proved by experiments that the working concentration rang...

Embodiment 2

[0110] Example 2 Application of alkaline phosphatase system to prepare pepsin chemiluminescence immunoassay kit of the present invention

[0111] 1. Preparation of alkaline phosphatase-labeled antibody

[0112] Since this kit uses alkaline phosphoric acid, glutaraldehyde cross-linking is generally used to prepare enzyme markers. In this cross-linking method, a two-step method is adopted, that is, the enzyme is first reacted with glutaraldehyde, and the excess glutaraldehyde is removed after dialysis, and then the enzyme marker is formed by reacting with the antibody. Since the enzyme and antibody in the enzyme-labeled antibody are biologically active substances and are easily inactivated, an equal volume of glycerol should be added to the conjugate solution and stored in a low-temperature refrigerator (-20°C).

[0113] 2. Chemiluminescence substrate

[0114] The preparation method of the chemiluminescence substrate liquid of alkaline phosphatase (ALP) used in the present inv...

Embodiment 3

[0130] Example 3 Application of biotin streptavidin system to prepare pepsin chemiluminescent immunoassay kit of the present invention

[0131] 1. Preparation of biotin-labeled antibody

[0132] The biotin-labeled pepsin antibody was conjugated with biotin succinimide ester under slightly alkaline conditions, fully dialyzed against PBS, the biotin-labeled product was diluted with newborn calf serum, added to Proclin300, and stored below -20°C .

[0133] 2. Preparation of horseradish peroxidase-labeled streptavidin

[0134] Dissolve 5 mg of HRP in 0.5 mL of distilled water, add 0.5 mL of sodium periodate (50 mmol / L) and stir at room temperature for 30 min, dialyze through 10 mM sodium acetate buffer with a pH value of 4.4, add 2 mg of pepsin antibody, stir for 2 h, and finally use 200 mM NaBH 4 For reduction, after dialysis with 0.02M PBS buffer, add an equal volume of glycerol, and store below -20°C.

[0135] The configuration method of other reagents and components is con...

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Abstract

The invention relates to the field of immunological detection, in particular to a pepsase chemiluminescent immunoassay kit and a method thereof. The pepsase chemiluminescent immunoassay kit provided by the invention comprises: 1) a pepsase antigen calibrator; 2) sample collecting solution at a pH value of 2 to 3; 3) sample diluents at a pH value of 6 to 8; 4) a microporous plate coated by a pepsase antibody; 5) a pepsase antibody marker; 6) a chemical luminescence substrate liquid; and 7) concentrated cleaning solution. The kit provided by the invention can detect the pepsase content in gastric juice, esophageal content, throat secretion and tracheal bronchus secretion, judge if gastroesophageal reflux happens according to the result of the pepsase detection and judge the treatment effectof stomach diseases and change of state of illness according to the detected pepsase content. The kit has the advantages of noninvasiveness, simplicity, convenience, quickness, sensitivity, stabilityand the like.

Description

technical field [0001] The invention relates to the field of immunoassay, in particular to a pepsin chemiluminescent immunoassay kit and a method thereof. Background technique [0002] Pepsin (pepsin) belongs to aspartate proteolytic enzymes and is a gastric digestive proteolytic enzyme with a relative molecular weight of 31-36kd. Its precursor is pepsinogen, which is synthesized in the gastric mucosa of adult vertebrates. Secreted by gastric principal cells, converted to pepsin under the acidic conditions of gastric juice. Its function is to break down the proteins in food into small peptide fragments. [0003] Gastroesophageal reflux disease (GERD) refers to a disease in which gastrointestinal contents reflux into the esophagus, causing acid reflux, heartburn, chest pain and other gastrointestinal symptoms, which seriously affect the quality of life and can lead to precancerous lesions of the esophagus. It is a very common esophageal disease that affects the quality of l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/573G01N21/76
Inventor 陈秀柳静
Owner GENERAL HOSPITAL OF THE SECOND ARTILLERY OF CHINESE PLA
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