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Characterization of four new monoclonal antibodies that recognize mouse natural killer activation receptors
1Department of Medical Biophysics, Ontario Cancer Institute, University of Toronto, Canada.
With the aim of identifying natural killer (NK) activation receptors, we immunized BALB/c mice with (BALB/cxB6)F1 NK LAK cells and made B-cell hybridomas. These were screened for monoclonal antibody (MAb) reacting with an NK activation receptor by using an antibody-induced redirected lysis (AIRL) assay against FcR-bearing P815 targets. Four hybridomas, clones 1C10, 1F10, 2D10 and 4G4, were selected for further characterization. Protein G-purified MAbs from these clones activated both resting and IL-2 activated B6 or F1 NK cells in the AIRL assay. 1F10 MAb, but not the other three MAbs, could compete for the binding of anti-NK1.1 (PK136) MAb to F1 NK cells. The four MAbs were screened for their ability to bind to or activate NK cells from the mouse strains SJL/J, DBA/2, 129/J, C3H/J, and BALB.K. None showed activity except IC10, which could bind to and activate SJL/J NK cells. When members of the NKR-P1 family from both B6 mice (A, B, and C genes expressed) and SJL mice (only A and B genes expressed) were expressed in Jurkat cells and tested for their antibody reactivity, PK136 MAb was found to recognize B6 NKR-P1C and SJL/J NKR-P1B; IC10 MAb was found to recognize NKR-P1-A, -B and -C from B6, but not NKR-P1A or -B from SJL/J; and 1F10 MAb was found to react only with B6 NKR-P1C.
With the aim of identifying natural killer (NK) activation receptors, we immunized BALB/c mice with (BALB/cxB6)F1 NK LAK cells and made B-cell hybridomas. These were screened for monoclonal antibody (MAb) reacting with an NK activation receptor by using an antibody-induced redirected lysis (AIRL) assay against FcR-bearing P815 targets. Four hybridomas, clones 1C10, 1F10, 2D10 and 4G4, were selected for further characterization. Protein G-purified MAbs from these clones activated both resting and IL-2 activated B6 or F1 NK cells in the AIRL assay. 1F10 MAb, but not the other three MAbs, could compete for the binding of anti-NK1.1 (PK136) MAb to F1 NK cells. The four MAbs were screened for their ability to bind to or activate NK cells from the mouse strains SJL/J, DBA/2, 129/J, C3H/J, and BALB.K. None showed activity except IC10, which could bind to and activate SJL/J NK cells. When members of the NKR-P1 family from both B6 mice (A, B, and C genes expressed) and SJL mice (only A and B genes expressed) were expressed in Jurkat cells and tested for their antibody reactivity, PK136 MAb was found to recognize B6 NKR-P1C and SJL/J NKR-P1B; IC10 MAb was found to recognize NKR-P1-A, -B and -C from B6, but not NKR-P1A or -B from SJL/J; and 1F10 MAb was found to react only with B6 NKR-P1C.