OPTIMIZATION AND COMPARISON OF THE MTT ASSAY AND THE H-3-TDR ASSAY FOR THE DETECTION OF IL-2 IN HELPER T-CELL PRECURSOR ASSAYS

The helper T cell precursor (HTLp) assay is of value for predicting gr aft-versus-host disease after allogeneic bone marrow transplantation. The assay requires reliable detection of the amount of interleukin 2 ( IL-2) produced by one cell. To optimize the IL-2 sensitivity of our HT Lp assay we tested an IL-2 dependent cell line, CTLL-2, with two diffe rent measurement methods: a colorimetric assay with tetrazolium (MTT) and an isotope incorporation assay with H-3-thymidine (H-3-TdR). The t est conditions examined encompassed: time without IL-2, preincubation time in IL-2, CTLL-2 cell concentration and different human sera. Due to the different measurement procedures, the volumes of the IL-2 dilut ions were 75 mu l in assays with MTT and 150 mu l in assays with H-3-T dR. We found that it was the amount of IL-2, not the concentration, th at limited the growth of CTLL-2 cells. Ln the most optimal setting the MTT assay could detect 0.6 pg IL-2/well, corresponding to 8 pg/ml. Fo r the H-3-TdR assay the sensitivity was 0.6 pg/well, corresponding to 4 pg/ml. Because of the possibility of IL-2 detection in the whole cul ture volume (150 mu l), we found that the H-3-TdR assay was superior t o the MTT assay with a 10-fold better sensitivity in different human s era. (C) 1998 Elsevier Science B.V. All rights reserved.