Nonradioactive alternative to clinical mixed lymphocyte reaction.
The MLR is used clinically as a functional assay for genetic HLA disparity. Traditionally this test relies on [3H]thymidine incorporation to detect T-cell proliferation as an indicator of alloantigenicity. Environmental and administrative concerns regarding radioisotope use and disposal have encouraged the development of sensitive, nonradioactive assay for T-cell alloactivation. We describe a nonradioactive alternative to the clinical MLR which uses an IL-2-dependent cell line, CTLL20.3, and MTT reduction to detect IL-2 accumulation in MLC SNs as an index of T-cell alloactivation. We first determined (a) the optimal number of CTLL20.3 cells for the assay, (b) the optimal time of SN analysis for IL-2, and (c) additional manipulations that significantly increase the assay sensitivity for IL-2. Using this assay system with patient lymphocyte combinations, we demonstrated that the CTLL20.3-MTT assays correlate well with the [3H]thymidine assays of T-cell proliferation for the detection of MHC incompatibility. Indeed, the CTLL20.3-MTT assay may be slightly more sensitive than the traditional clinical MLR.
VanBuskirk
A M
AM
Department of Surgery, Ohio State University College of Medicine, Columbus 43210, USA.
Adams
P W
PW
Orosz
C G
CG
D002478:Cells, Cultured; D003124:Colorimetry; D004396:Coloring Agents; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis*; D008213:Lymphocyte Activation; D007959:Lymphocyte Culture Test, Mixed; D008297:Male; D013053:Spectrophotometry; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D013778:Tetrazolium Salts; D013844:Thiazoles
D004396:Coloring Agents; D007376:Interleukin-2; D013778:Tetrazolium Salts; D013844:Thiazoles; C022616:thiazolyl blue