Abstract
Antigen Used for Immunization
AKR mouse-derived thymoma cell line (BW5147) stably expressing human IgM Fc receptor (FcμR) on the cell surface.
Method of Immunization
BALB/c mice were subcutaneously injected at a 3-day interval six times with FcμR-bearing BW5147 cells (107 cells) at axillary, inguinal, and lower leg regions. The first injection was performed with complete Freund's adjuvant, the second with incomplete Freund's adjuvant, and the remainder in saline. One day after the last injection, mice were sacrificed.
Parental Cell Line Used for Fusion
P3-X63-Ag8.653 plasmacytoma line, a non-Ig-producing variant of the HAT-sensitive P3-X63-Ag8 cell line.
Selection and Cloning Procedure
Hybridomas were selected based on their reactivity by flow cytometric analysis with FcμR+ BW5147 cells and other FcμR-bearing cells (i.e., PMA-activated 697 human pre-B cell line and chronic lymphocytic leukemia [CLL] cells), but not with control or Fcα/μR+ BW5147 cells and pIgR-bearing FT-29 cell line. Single cell-derived subclones were obtained by limiting dilution.
Heavy and Light Chains of Immunoglobulin
HM2 (γ3κ), HM3 (γ2bκ), HM6 (γ2bκ), HM7 (γ2bκ), HM10 (γ1κ), and HM14 (γ1κ)
HM7 VH and Vκ: Ighv14-3, Ighd1-2, and Ighj2 for the γ2b heavy chain and Igkv3-4 and Igkj for the κ light chain.
Specificity
1. SDS-PAGE analysis of precipitated materials from membrane lysates of FcμR-positive and -negative cells with these HM MAbs.
2. Sequential immunoprecipitation analysis from membrane lysates of FcμR+ cells with these HM MAbs and IgM ligands.
3. Flow cytometric analysis of BW5147 cells stably transfected with human FcμR cDNA or control vector and of other different cells (PMA-activated 697 pre-B cell line, CLL cells, Fcα/μR+ BW5147 cells, pIgR+ FT-29 cells).
4. Flow cytometric analysis of BW5147 cells expressing recombinant human/mouse FcμR proteins for epitope mapping.
5. HM7 and HM10 MAbs recognize an epitope near the FcμR ligand-binding site, whereas the other four MAbs recognize an epitope in the stalk region.
Specific Antigen Identified
Human IgM Fc receptor (FcμR), which was initially designated incorrectly as an apoptosis inhibitor, Toso, or Fas apoptosis inhibitory molecule-3 (FAIM3).