The ratios of hapten and bovine serum albumin (BSA) in an antigen conjugate were determined by matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Hybridomas secreting monoclonal antibodies against 2,4-dichlorophenoxyacetic acid (2,4-D) were produced by fusing 2,4-D-BSA conjugate-immunized splenocytes with a HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A substantial cross-reaction was observed for 2,4-dichlorophenol (2,4-DP) when compared with that observed for 2,4-D. The full measurement range for this assay is 0.2-3 g ml À1 for 2,4-DP. On the other hand, the range for 2,4-D is between 1 and 20 g ml À1 .Abbreviations: ABTS -2,2 0 -azino-bis(3-etylbenzothiazoline-6-sulfonic acid) diammonium salt; BSAbovine serum albumin; 2,4-D -2,4-dichlorophenoxyacetic acid; 2,4-DP -2,4-dichlorophenol; ELISAenzyme-linked immunosorbent assay; MAb -monoclonal antibody; MALDI -matrix-assisted laser desorption/ionization; PBS -phosphate-buffered saline; TPBS -PBS containing 0.05% of Tween 20; SPBS -PBS containing 5% of skim milk