In this report, we describe a novel method for directly preparing enzyme-labeled antibodies harvested from IgM-producing hybridoma cells. We constructed expression vectors for antibody light (L) chain-enzyme fusion proteins by linking either the genes for the murine lambda L chain or its constant region (C-L) with one of two proteins, either the secreted placental alkaline phosphatase or Gaussia luciferase (Gluc). When the vectors were transfected into anti-NP (4-hydroxy-3-nitrophacetyl) IgM-producing myeloma cells, secretion of the IgM-enzyme complex from the gene-transfected cells was confirmed by a direct enzyme-linked immunosorbent assay with an immobilized antigen. Furthermore, when human hybridoma HF10B4, a cell line that produces anti-human lung cancer IgM, was transfected with the vector containing L-Gluc, a significantly stronger signal was obtained for the human lung carcinoma SBC-1 cells than for cervical HeLa cells. Because successful production of an active IgM-enzyme complex containing a heterologous L chain-enzyme fusion was observed, the L-chain fusion method will be a generally applicable method for preparing various IgM-enzyme complexes. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.