Antibody-based method for isolating Th1 and Th2 helper lymphocytes from human peripheral blood
A negative isolation method for separately isolating preparations of Th1 and Th2 helper lymphocytes from peripheral blood mononuclear cells involving the use of novel combinations of monoclonal antibodies to separately sequester specific Th1 and Th2 lymphocytes and contaminating leukocytes and erythrocytes, adding a magnetic colloid to the cells, and using a magnetic column for fractionation of Th1 and Th2 cells. Imbalances in the relative numbers of Th1 and Th2 lymphocytes can be used in the diagnosis and prognosis of human diseases.
1. A method for isolating Th2 lymphocytes from a mixture of human peripheral blood mononuclear cells (PBMCs), comprising the steps of:
(i) contacting said mixture with anti-Th1 monoclonal antibodies comprising anti-CD29-biotin and anti-CXCR3-biotin clones,
(ii) contacting said mixture with antibodies that bind to cell surface antigens of non-Th2 leucocytes, wherein the cell surface antigens of the non-Th2 leucocytes comprises CD8, CD14, CD16, CD19, and CD56;
(iii) contacting said mixture with an antibody to an erythrocyte cell surface antigen;
(iv) contacting the mixture in step in with a magnetic colloid that binds to the antibodies in steps (i) to (iii) to magnetically label the Th1 lymphocytes, non-Th2 leukocytes, and erythrocytes; and
(v) separating the magnetically labeled cells from the Th2 lymphocytes with a magnet to obtain isolated Th2 lymphocytes.
2. The method of claim 1 , wherein said antibody directed to said erythrocyte antigen is an anti-glycophorin A (CD235a) antibody.
3. The method of claim 1 , wherein said Th2 lymphocytes collected from step (v) are at least 84% pure.