Microwell array chip for detecting antigen-specific lymphocytes, method of detecting and method of manufacturing antigen-specific lymphocytes, and method of cloning antigen-specific lymphocyte antigen receptor genes

a microwell array and lymphocyte technology, applied in the field of microwell array chips, can solve the problems of difficult to set the separation conditions of the device, inability to identify individual antigen-specific lymphocytes present in the lymphocytepopulation, and expensive and complex devices

Inactive Publication Date: 2006-06-22
VIVALIS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0046] (2) The antigen-specific lymphocyte is

Problems solved by technology

However, they are incapable of identifying individual antigen-specific lymphocytes present in the lymphocytepopulation.
However, the above-cited method requires an expensive and complex device known as a cell sorter for separating out individual lymphocytes, and presents the following problems as well:
(1) It is difficult to set the separating conditions of the device, requiring skills for operating device to separate out cells;
(3) cell separation is inefficient;
(5) although antigen binding can be determined, it is difficult to analyze the reaction of the lymphocyte that has bound the antigen.
However, it is not possible to analyze the reaction of the lymphocyte in binding the antigen (the metabolic or physiological reaction of the cell, such as intracellular signal transduction, RNA synthesis, or protein synthesis).
Further, the antigen-specific lymphocyte cannot be detected when the frequency of the antige

Method used

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  • Microwell array chip for detecting antigen-specific lymphocytes, method of detecting and method of manufacturing antigen-specific lymphocytes, and method of cloning antigen-specific lymphocyte antigen receptor genes
  • Microwell array chip for detecting antigen-specific lymphocytes, method of detecting and method of manufacturing antigen-specific lymphocytes, and method of cloning antigen-specific lymphocyte antigen receptor genes
  • Microwell array chip for detecting antigen-specific lymphocytes, method of detecting and method of manufacturing antigen-specific lymphocytes, and method of cloning antigen-specific lymphocyte antigen receptor genes

Examples

Experimental program
Comparison scheme
Effect test

embodiment 1

1. Separation of B Lymphocytes

[0178] Employing Ficoll-Paque (Pharmacia, Uppsala, Sweden), the lymphocyte fraction was separated from peripheral blood. The B lymphocyte fraction was then further separated and purified from the lymphocyte fraction using an AutoMACS (Miltenyi Biotec, Bergisch Gladbach, Germany)

2. Introduction of Fluo3 into Cells (see FIG. 1)

[0179] 2×106 cells of B lymphocyte were suspended in RPMI 1640 / 10 percent FCS solution containing 2 micromoles of Fluo3 / AM (Dojin, Kumamoto) and incubated for 30 min at room temperature. The cells were washed with RPMI 1640 / 10 percent FCS to remove the Fluo3 / AM that had not been incorporated into the cells. Subsequently, the cells were suspended in RPMI 1640 / 10 percent FCS solution.

3. Microwell Array Chip (see FIG. 2)

[0180] The microwell array chip was made of poly(dimethylsiloxane) (PDMS) and had microwells of 10 micrometers in diameter and 32 micrometers in depth arranged horizontally and vertically at a spacing of 30 micr...

embodiment 2

[0187] The efficiency of introduction of cells into the microwells was examined by fluorescence microscopy or microarray scanning.

[0188] Mouse lymphocytes were fluorescently labeled with CellTracker Orange (Molecular Probe Corp.).

[0189] The mouse lymphocytes were obtained as follows.

[0190] Spleens were extracted from mice and transferred to plastic Petri dishes containing PBS. The spleens were sandwiched between two pieces of mesh and crushed to remove lymphocytes. The lymphocytes that were removed were suspended in RPMI 1640 / 10 percent FCS solution and the number of lymphocytes was counted.

[0191] The fluorescent labeling of the murine lymphocytes was conducted as follows.

[0192] 2×106 cells of B lymphocytes were suspended in loading buffer (20 mM HEPES, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 1 mg / mL glucose, 1 mg / mL BSA) containing 1 micromole of CellTracker Orange (Molecular Probes, USA), 0.02 percent of Pluronic F-127 (Molecular Probes, USA) and incubated ...

embodiment 3

[Detection of Antigen-Specific B Lymphocytes by Microarray Scanner]

[0199] A healthy volunteer was inoculated with hepatitis B virus vaccine and B lymphocytes were prepared from peripheral blood on days 4 and 6 by the usual methods. Human B lymphocytes were suspended in buffer (loading buffer (20 mM HEPES, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl22H2O, 1 mM MgCl2, 1 mg / mL glucose, 1 mg / mL BSA)) containing 4 micromoles of the calcium fluorescence indicator Fluo-4 / AM (Molecular Probes Corp.), 1 micromole of CellTracker Orange (Molecular Probes Corp.), and 0.02 percent of Pluronic F-127 (Molecular Probes Corp.) to introduce Fluo-4 and CellTracker Orange into the cytoplasm. B lymphocytes loaded with Fluo-4 and CellTracker Orange were applied by the same method as in Embodiment 2 onto a microwell array chip identical to that in Embodiment 2.

[0200] Subsequently, the B lymphocytes loaded with Fluo-4 and CellTracker Orange that had been seeded in the microwell array chip were stimulate...

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Abstract

A microwell array chip that has multiple microwells and is employed to contain a single lymphocyte specimen in each microwell and detect antigen-specific lymphocytes in single units; wherein the microwell array chip is of a shape and of dimensions where only one lymphocyte is contained in each microwell. A method of detecting antigen-specific lymphocytes comprising the steps of adding antigen to each microwell in the above microwell array chip, stimulating the lymphocyte specimen, and detecting lymphocyte specimens reacting with the antigen.

Description

TECHNICAL FIELD [0001] The present invention relates to a microwell array chip employed in detecting antigen-specific lymphocytes, a method of detecting antigen-specific lymphocytes, and a method of manufacturing antigen-specific lymphocytes. The present invention further relates to a method of cloning antigen-specific lymphocyte antigen receptor genes. TECHNICAL BACKGROUND [0002] In the past, antigen-specific lymphocytes have been detected by placing about 200,000 individual lymphocytes per well in a 96-well plate such as that shown in FIG. 3 and culturing them for from three days to a week (“Lymphocyte Function Detection Methods”, ed. by Junichi Yano, Michio Fujiwara, Chugai Igaku Corp. (1994) (Nonpatent Reference 1) and “Methods of Conducting Immunological Experiments I, II”, ed. by Shunsuke Ishida, Susumu Konda, Morosuke Moto, and Toshiyuki Hamaoka, Nankodo (1995) (Nonpatent Reference 2)). [0003] These detection methods detect antigen-specific lymphocytes by: [0004] 1. Cell prol...

Claims

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Application Information

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IPC IPC(8): G01N33/567C12M1/34G01N33/53C07K16/08C40B60/14G01N33/543G01N33/569
CPCB01J2219/00317B01J2219/00743C07K16/082C07K2317/21C40B60/14G01N33/5302G01N33/54366G01N33/56972
Inventor MURAGUCHI, ATSUSHIKISHI, HIROYUKITAMIYA, EIICHISUZUKI, MASAYASU
Owner VIVALIS SA
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