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

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.

1 . 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.  
     
     
         2 . The microwell array chip of  claim 1 , wherein the microwell is of cylindrical, rectangular parallelepiped, inverse conical, or inverse pyramid shape, or some combination of two or more thereof.  
     
     
         3 . The microwell array chip of  claim 1 , wherein the diameter of the maximum circle inscribable in a planar configuration of the microwells falls within a range of from one to two times the diameter of the lymphocytes that are to be contained in the microwells, and the depth of the microwells falls within a range of from one to two times the diameter of the lymphocytes to be contained in the microwells.  
     
     
         4 . A microwell array chip that is employed to detect antigen-specific lymphocytes and has multiple microwells each containing a single lymphocyte specimen.  
     
     
         5 . The microwell array chip of  claim 4 , wherein the microwell has a diameter of from 5 to 100 micrometers and a depth of from 5 to 100 micrometers.  
     
     
         6 . The microwell array chip of  claim 4 , wherein the lymphocyte specimen is contained in the microwell together with a culture medium.  
     
     
         7 . The microwell array chip of  claim 4 , wherein the lymphocyte specimen is derived from blood.  
     
     
         8 . The microwell array chip of  claim 4 , wherein the lymphocyte specimen is a B lymphocyte or T lymphocyte.  
     
     
         9 . A method of detecting antigen-specific lymphocytes comprising the steps of adding antigen to each microwell in the microwell array chip according to  claim 4 , stimulating the lymphocyte specimen, and detecting lymphocyte specimens reacting with the antigen.  
     
     
         10 . The method of  claim 9 , wherein the detection of cells reacting with antigen is conducted by using Ca ion dependent fluorescent dye.  
     
     
         11 . The method of  claim 9 , wherein the detection of cells reacting with antigen is conducted by employing, as a marker, an activated marker protein expressed on the surface of the activated lymphocyte specimen that has been stimulated with antigen.  
     
     
         12 . The method of  claim 9 , wherein the detection of cells reacting with antigen is conducted by employing, as an indicator, the degree of polarization of fluorescence emitted by a fluorescent substance in the lymphocyte specimen.  
     
     
         13 . The method of  claim 9 , wherein the detection of cells reacting with antigen is conducted by employing, as an indicator, the proliferation of the lymphocyte specimen or the production of antibody.  
     
     
         14 . The method of  claim 9 , wherein the antigen is a protein, peptide, DNA, RNA, lipid, sugar chain, or organic macromolecular compound.  
     
     
         15 . The method of  claim 9 , wherein the antigen is a bacterium, virus, autoantigen, tumor antigen, or allergen.  
     
     
         16 . A method of manufacturing antigen-specific lymphocytes comprising the step of recovering from microwells lymphocyte specimens reacting with antigen that have been detected by the method of  claim 9 .  
     
     
         17 . A method in which a single lymphocyte reacting specifically with a certain antigen (referred to hereinafter as an antigen-specific lymphocyte) is selected and an antigen-specific antigen receptor gene is cloned from the single antigen-specific lymphocyte.  
     
     
         18 . The method of  claim 17 , wherein the selection of the antigen-specific single lymphocyte is conducted by adding antigen to each microwell in an antigen-specific lymphocyte detection-use microwell array chip having multiple microwells each containing a single lymphocyte specimen, detecting which lymphocytes have reacted with the antigen, and removing the antigen-specific lymphocytes that have been detected from the microwells.  
     
     
         19 . The method of  claim 17 , wherein the antigen-specific lymphocyte is present in a frequency of 0.1 percent or less.  
     
     
         20 . The method of  claim 17 , wherein the antigen-specific lymphocyte is broken down using a cytolytic agent and the antigen-specific antigen receptor gene is amplified by RT-PCR.  
     
     
         21 . The method of  claim 20 , wherein the RT-PCR is conducted by preparing cDNA with reverse transcriptase and carrying out PCR twice with primer mixes for antigen receptor gene.  
     
     
         22 . The method of  claim 17 , wherein the antigen-specific lymphocyte is a B lymphocyte or T lymphocyte.  
     
     
         23 . The method of  claim 22 , wherein the antigen-specific antigen receptor gene is an immunoglobulin gene when the antigen-specific lymphocyte is a B lymphocyte, and a T-cell receptor gene when the antigen-specific lymphocyte is a T lymphocyte.  
     
     
         24 . The method of  claim 17 , wherein the antigen-specific lymphocyte is a B lymphocyte and an antigen-specific immunoglobulin gene is cloned.  
     
     
         25 . The method of  claim 17 , wherein the antigen-specific lymphocyte is a T lymphocyte and an antigen-specific T-cell receptor gene is cloned.  
     
     
         26 . The method of  claim 18 , wherein gene amplification is conducted in the microwell without removing from the microwell the antigen-specific lymphocyte that has been detected.  
     
     
         27 . A method of manufacturing monoclonal antibody using the antigen-specific immunoglobulin gene that has been cloned by a method of  claim 24 .  
     
     
         28 . A method of manufacturing material for gene therapy employing the antigen-specific T-cell receptor gene that has been cloned by a method of  claim 25.