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 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 method of cloning an antigen-specific antigen receptor gene, comprising the steps of:
(a) providing a microwell array having multiple microwells which contain lymphocytes, wherein said microwells are of a shape and of dimensions where only one lymphocyte can be contained in each microwell;
(b) supplying antigen to said microwell array;
(c) detecting said microwells containing lymphocytes that have been stimulated and are reacting with said antigen;
(d) recovering a single lymphocyte that is an antigen-specific lymphocyte reacting with said antigen from one of said detected microwells; and
(e) cloning at least an antigen-specific antigen receptor gene from said selected single antigen-specific lymphocyte, wherein said antigen-specific lymphocyte is a B lymphocyte.
2. The method of claim 1 , wherein a glass cover is placed on the top of the microwells into which cells have been loaded to prevent the microwells from drying out.
3. The method of claim 1 , wherein step (b) comprises supplying an antigen solution in a manner that covers the surface of the microwell array.
4. The method of claim 1 , wherein step (b) comprises supplying an antigen solution to each well.
5. The method of claim 1 , wherein antigen-specific lymphocytes present in said microwells of said microwell array occur at a frequency of 0.1 percent or less among the lymphocytes present in said array.
6. The method of claim 1 , wherein said cloning comprises the steps of breaking down said antigen-specific lymphocyte using a cytolytic agent and amplifying said antigen-specific antigen receptor gene by RT-PCR.
7. The method of claim 6 , wherein said RT-PCR is conducted by preparing cDNA with reverse transcriptase and carrying out PCR twice with primer mixes for the antigen-specific antigen receptor gene.
8. The method of claim 1 , wherein said antigen-specific antigen receptor gene is an immunoglobulin gene.
9. The method of claim 6 , wherein said antigen-specific antigen receptor gene amplification is conducted directly in the microwell containing the detected antigen-specific lymphocyte.
10. The method of claim 2 , wherein a buffer solution fills the space between the glass cover and the microwells.
11. The method of claim 2 , wherein the detection of the lymphocytes that have been reacted with the antigen is conducted with the glass cover in place.