title: Expression of an antibody-enzyme complex by the L-chain fusion method.
abstract: 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.
authors: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors: Chungyong
authors: C
authors: Han
authors: Masaki
authors: M
authors: Ihara
authors: Hiroshi
authors: H
authors: Ueda
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D058851:GPI-Linked Proteins / Q000235:genetics; D050439:Genes, Immunoglobulin Light Chain; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D006825:Hybridomas; D007075:Immunoglobulin M / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D008175:Lung Neoplasms / Q000235:genetics; D051379:Mice; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D058851:GPI-Linked Proteins; D007075:Immunoglobulin M; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental