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Sandwich ELISA (Enzyme Linked Immunosorbent Assay) kit for Hum j3 detection

A kit and sandwich technology, applied in the field of sandwich ELISA kits, can solve the problem of not establishing Humj3, and achieve the effect of good sensitivity

Inactive Publication Date: 2011-03-23
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, so far, there is no established method for the simple and efficient detection of Hum j3

Method used

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  • Sandwich ELISA (Enzyme Linked Immunosorbent Assay) kit for Hum j3 detection
  • Sandwich ELISA (Enzyme Linked Immunosorbent Assay) kit for Hum j3 detection
  • Sandwich ELISA (Enzyme Linked Immunosorbent Assay) kit for Hum j3 detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The establishment of embodiment 1 sandwich ELISA method

[0038] 1. Acquisition of the main allergenic protein Hum j3 from natural purification of Humulus japonicus

[0039] The main allergenic protein Hum j3 was purified from natural Humulus japonicus pollen by molecular gel chromatography, ion exchange chromatography and affinity chromatography.

[0040] 2. Obtaining the mouse hybridoma monoclonal antibody against Hum j3

[0041] After obtaining the natural purified main allergenic protein Hum j3 of Humulus japonicus, complete the small study with reference to "hybridoma technology and monoclonal antibody preparation" (Edited by Bardenian, "Technology and Application of Contemporary Immunology", Chapter 11, P351-364). Preparation and primary screening of mouse hybridoma monoclonal antibody. For the specific method, refer to the patent application CN 101392022.

[0042] Multiple monoclonal antibody cell lines were obtained through screening, and the identification r...

Embodiment 2

[0068] The formation of embodiment 2 sandwich ELISA kit

[0069] Based on the above experiments, this example provides a kit for the detection of Hum j3, the main allergenic protein of Humulus pollen, which consists of the following:

[0070] 1) ELISA plate coated with monoclonal antibody; (#87 monoclonal antibody, coating amount is 0.5 μg / well)

[0071] 2) Horseradish peroxidase-labeled antibody; (#66 monoclonal antibody)

[0072] 3) washing solution; (PBST)

[0073] 4) Substrate chromogenic solution:

[0074] Substrate buffer (pH 5.0):

[0075] Solution A: 0.1mol / L citric acid (2.1g C 6 h 8 o 7 ·H 2 O / 100ml), stored at 4°C;

[0076] Liquid B: 0.2mol / L dibasic sodium phosphate (7.163gNa 2 HPO 4 12H 2 O / 100ml), stored at room temperature;

[0077] Take 24.3ml of liquid A and 25.7ml of liquid B, mix, add 50ml of distilled water, a total of 100ml. Store at 4°C.

[0078] OPD-H 2 o 2 Chromogenic solution buffer: substrate buffer 10ml, OPD 4mg, 30% H 2 o 2 15 μl. ...

Embodiment 3

[0081] The application of embodiment 3 sandwich ELISA method in actual clinical practice

[0082] The content of Hum j3 in different product types of Humulus japonicus allergen infusion was determined by the constructed sandwich ELISA method, so as to determine the clinically effective dose.

[0083] Operate with reference to the condition of item 4 of embodiment 1. The standard curve was made from the dose-effect curve of 11 consecutive 2× gradient dilutions of the natural purified Humulus japonicus main allergen protein Hum j3 standard with an initial concentration of 0.05 μg / ml. The samples to be tested are 50% glycerin Humulus allergen infusion puncture solution provided by Macrolink Allergen Pharmaceutical Co., Ltd., the batch numbers are #20061213, #20061214, #20061215, #20070117. The detection was repeated three times for each batch number. The total protein content was determined by the Bradford method and completed with a protein content determination kit (Protein A...

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Abstract

The invention provides a sandwich ELISA (Enzyme Linked Immunosorbent Assay) kit for Hum j3 detection. A sandwich ELISA method is established by the following steps of: preparing main sensitization protein Hum j3 by purifying natural humulus scandens pollen; preparing and screening mouse hybridoma monoclonal antibodies; selecting two kinds of monoclonal antibodies for the sandwich ELISA method; determining the experimental conditions of the sandwich ELISA method; and determining the methodology indexes of sensitivity, specificity, linear range, stability and the like of the sandwich ELISA method, and so on. The application of the method in the kit of the invention comprises a capture antibody and a detection antibody, wherein the capture antibody is coated in an ELISA plate, the detection antibody is an enzyme labeled antibody, and the capture antibody and the detection antibody are respectively specific to different antigenic determinants of Hum j3. The sandwich ELISA kit has an important application value in clinic practice, pharmacy industry production of allergens, quality control and new drug research and development and Hum j3 content monitoring and forecasting in climate and the environment.

Description

technical field [0001] The invention relates to a sandwich ELISA kit for detecting the content of Hum j3 based on mouse hybridoma monoclonal antibody and natural humulus pollen-purified main allergenic protein Hum j3, which is used in clinical practice, allergen pharmaceutical industry production, quality It has important practical application value in the control and development of new drugs, the monitoring and forecasting of Hum j3 content in climate and environment. Background technique [0002] Humμlus japonicus or Humμlus scandens is an annual twining herb of the Rosaceae Cannabis family. It is native to my country and widely distributed in the Far East. It is distributed in all provinces and regions except Xinjiang and Qinghai. East Asian countries (Korea, Japan, Russia, etc.) , Vietnam) and Europe, the west coast of the United States are also distributed. In the first national airborne allergenic pollen survey led by Peking Union Medical College Hospital in the 1980s,...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/577G01N33/543
Inventor 尹佳周俊雄程璇孙劲旅
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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