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Monoclonal antibody, hybridoma, immunoassay method and diagnosis kit

A monoclonal antibody, diagnostic kit technology, applied in the direction of anti-enzyme immunoglobulin, anti-bacterial immunoglobulin, immunoglobulin, etc., can solve the diagnosis of antibody titer and specificity changes, poor specificity, polyclonal antibody Tests are difficult to control quality and other problems

Inactive Publication Date: 2002-12-11
WAKAMOTO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, polyclonal antibodies are often cross-reactive and poor in specificity, and also have the disadvantage that antibody titers and specificities can vary from batch to batch of antisera
Therefore, the problem is that diagnostic tests using polyclonal antibodies are particularly difficult to control in quality

Method used

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  • Monoclonal antibody, hybridoma, immunoassay method and diagnosis kit
  • Monoclonal antibody, hybridoma, immunoassay method and diagnosis kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] [Monoclonal antibody preparation]

[0113] (1) Preparation of Helicobacter pylori coccal cell suspension (immunogen)

[0114] Helicobacter pylori (ATCC 43504) was streak cultured step by step on a brain-heart drip agar medium (product of Difco) supplemented with 5% defibrinated horse blood. Plates were incubated at 37°C for 3-4 days in a microaerobic environment, followed by 7 days at 37°C in anaerobic environment, resulting in spherical cells. Each colony was scraped off with a platinum ring or the like, and suspended in phosphate-buffered saline (PBS). Centrifuge at 10,000xg at 4°C for 10 minutes to collect Helicobacter pylori, suspend in 0.5% formaldehyde, and keep the suspension at 4°C for 4 days to inactivate it. Then, the cells were washed by resuspension in PBS, followed by centrifugation at 10,000 xg for 10 minutes at 4°C, and then resuspension in PBS in triplicate.

[0115] (2) Preparation of Helicobacter pylori spherical cell breakdown product (immunogen) ...

Embodiment 2

[0122] [Using sandwich ELISA to select monoclonal antibodies that specifically recognize Helicobacter pylori in gastrointestinal excreta]

[0123] (1) Preparation of monoclonal antibody immobilized plate

[0124] The ascitic fluid of 32 clones was diluted twice with PBS, 1 ml each, 2 ml of saturated ammonium sulfate was added dropwise, and the mixture was left at 4°C for 4 hours. The mixture was then centrifuged at 3000 rpm for 20 minutes, the pellet was suspended in 2 ml of PBS and dialyzed. These monoclonal antibodies were immobilized in 96-well ELISA plates in the following manner. That is, each monoclonal antibody was diluted to 5 µg / ml, and 0.2 ml of the dilution was added to each well of a 96-well plate. After overnight at 4°C, the plate was washed with PBS, and then 0.25 ml of 1% skim milk-PBS was added to each well, and the plate was allowed to stand at 4°C for 1 hour for blocking. Then, the plate was washed with the above washing solution.

[0125] (2) Preparation...

Embodiment 3

[0132] [Comparison of reactivity among strains and reactivity with other types of bacteria]

[0133] (1) Preparation of Helicobacter pylori cell suspension

[0134] Various Helicobacter pylori strains (ATCC 43504; Tokai University Hospital clinical isolate No. 130 and 112; Hyogo Medical College clinical isolate Nos. 526, 4484, 5017, 5025, 5049, 5142, 5287, 5308, 5314 and 5330). Plates were cultured at 37°C in a microaerobic environment for 3-4 days to obtain spiral cell colonies, or cultured at 37°C in anaerobic environment for 7 days, resulting in spherical cells. Each colony was scraped off with a platinum ring or the like, and suspended in PBS. Cells were collected by centrifugation at 10,000xg at 4°C for 10 minutes, suspended in 0.5% formaldehyde, and kept at 4°C for 4 days to inactivate. Then, the cells were washed by resuspension in PBS, followed by centrifugation at 10,000 xg for 10 minutes at 4°C, and then resuspension in PBS in triplicate. Helicobacter pylori heli...

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Abstract

The present invention provides a diagnostic method by which Helicobacter pylori infection can be diagnosed economically without causing any pain to the patient and without resorting to any special devices. Due to the use of one antibody in this method, a high degree of specificity can be achieved without showing any cross-reactivity resulting in batch-to-batch variation, thereby facilitating quality control. Furthermore, high sensitivity can be achieved even when using simple monoclonal antibodies. The invention also provides a monoclonal antibody with Helicobacter pylori catalase as antigen.

Description

technical field [0001] The present invention relates to a monoclonal antibody recognizing Helicobacter pylori catalase as an antigen, a hybridoma producing said monoclonal antibody, an immunoassay and a diagnostic kit. Background technique [0002] Helicobacter pylori is a bacterium found in the human stomach lining. The prevalence of H. pylori infection is closely related to social and economic factors and is higher in developing countries and lower in developed countries. However, among developed countries, the infection rate in Japan is particularly high, and it has even been reported that 80% of people aged 40 to over 40 have been infected. In recent years, it has been revealed that Helicobacter pylori can cause various gastric and duodenal diseases, such as gastric ulcer, duodenal ulcer, chronic gastritis and more serious gastric cancer. [0003] Due to the proven possibility of reducing the likelihood of being afflicted by these diseases by eradicating H. pylori, the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12P21/08G01N33/569
CPCC07K16/121G01N33/56922G01N2333/908C07K16/40
Inventor 若杉昌彦持田立子平田晴久
Owner WAKAMOTO PHARMA
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