Cytotoxic T lymphocyte activation assay

• US 5,180,662 A
• Filed: 11/08/1990
• Issued: 01/19/1993
• Est. Priority Date: 01/05/1988
• Status: Expired due to Term

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Sitkovsky, Michail V.
• Primary Examiner(s): SAUNDERS, DAVID A
• Application Number:
  US07/610,332
• Time in Patent Office: 803 Days
• Field of Search: 435/5, 435/7.23, 435/7.29, 435/19, 435/7.24, 435/19, 435/34, 435/29, 436/506, 436/519, 436/530, 436/548, 436/63, 436/811, 436/815, 436/503, 436/518, 436/548, 436/827
• US Class Current: 435/7.24
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: Y10S 436/827 Lectins

Abstract

A method is provided for the quantitative study of cytotoxic T-lymphocyte activation by measuring secreted granule-associated BLT esterase activity after incubating the cytotoxic T-lymphocytes with activating stimuli. This method can be used to screen inhibiting (drugs) or activating agents (monoclonal antibodies) (at selected sub-optimal levels of activation of cytotoxic T-lymphocytes.

Claims

1. A method for directly assaying cytotoxic T lymphocyte activation comprising the steps of:

1) incubating cytotoxic T lymphocytes with one or more lymphocyte activating stimuli selected from the group consisting of immobilized anti-TcR monoclonal antibody, an immunocomplex of anti-TcR monoclonal antibody and immobilized rabbit anti-mouse antibody, a mixture of beta-phorbol-12-myristate-13 acetate and ionophore A23187, and immobilized concanavalin A, said activating stimuli inducing the exocytosis of cytotoxic T lymphocyte granules and resulting in the secretion of BLT-esterase; and

2) quantitatively or qualitatively measuring the secretion of said BLT-esterase resulting from the activation of cytotoxic T lymphocyte by the lymphocyte activating stimuli.

2. The method of claim 1 wherein a supplemental, lymphocyte activation-enhancing protein containing composition is added at step 1), said protein containing composition being selected from the group consisting of bovine serum albumin (BSA) and fetal calf serum (FCS).

3. The method of claim 1, wherein said immobilized anti-TcR monoclonal antibody consists of a support-immobilized first monoclonal antibody (mAb), said first mAb being immobilized directly or indirectly via a second or additional mAb to said support.

4. The method of claim 3, wherein said immobilization occurs on a solid support comprising a multi-well plate.

5. The method of claim 3, wherein the surface of said support whereon said mAb is immobilized consists essentially of plastic.

6. The method of claim 1, wherein the secretion of said BLT-esterase is measured as a function of enzymatic activity.

7. The method of claim 6, wherein said enzymatic activity is caused by a reaction of N-alpha-benzyloxy-carbonyl-L-lysine thiobenzyl ester with (BLT) esterase activity.

8. The method of claim 7, wherein said enzymatic activity is measured by:

1) mixing said secreted BLT esterase with BLT and 5,5a'"'"'-dithio-bis-(2-nitrobenzoic acid) (DTNB);

2) incubating the mixture of step

     1); and

3) measuring the absorbance of said incubated mixture at 412 nm by spectrophotometric analysis.

9. The method of claim 8, wherein phenylmethanesulfonyl fluoride (PMSF) is added to the mixture of step 2) prior to spectrophotometric analysis.

10. A method for screening monoclonal antibodes or pharmacological agents for cytotoxic T lymphocyte activation comprising the steps of:

1) incubating cytotoxic T lymphocytes with one or more monoclonal antibodies or pharmacological agents;

2) quantitatively or qualitatively measuring the exocytosis of cytotoxic T lymphocyte granules by the resulting secretion of BLT-esterase, said secretion, if any, being induced by cytotoxic T lymphocyte activating monoclonal antibodies or pharmacological agents.

11. A method for screening monoclonal antibodies or pharmacological agents for use in inhibiting activation of cytotoxic T lymphocytes comprising the steps of:

1) incubating cytotoxic T lymphocytes with;

a) one or more monoclonal antibodies or pharmacological agents, and

b) one or more lymphocyte activating stimuli selected from the group consisting of immobilized anti-TcR monoclonal antibody, an immunocomplex of anti-TcR monoclonal antibody and immobilized rabbit anti-mouse antibody, a mixture of beta-phorbol-12-myristate-13 acetate and ionophore A23187, and immobilized concanavalin A, said activating stimuli inducing the exocytosis of cytotoxic T lymphocyte granules resulting in the secretion of BLT-esterase; and

2) quantitatively or qualitatively measuring the exocytosis of cytotoxic T lymphocyte granules by the resulting secretion, if any, of BLT-esterase.