title: A novel tool for B-cell tolerance research: characterization of mouse alloantibody development using a simple and reliable cellular ELISA technique.
abstract: In animal-based transplantation research, the measurement of anti-donor antibodies in transplant recipients is limited by lack of an appropriate technique. We have developed a novel immunoassay capable of quantifying antibody bound to cell-surface major histo- compatability complex (MHC) and non-MHC antigens, using splenocytes from wild-type and MHC-deficient mice as antigen-bearing target cells. We utilized our "cellular ELISA" (CELISA) technique to study the development of tolerance versus immunity in the B-cell compartment in response to neonatal exposure to allogeneic fetal liver cells (FLC). This neonatal tolerance protocol typically induces permanent acceptance of donor-type and third-party cardiac allografts, but rejection of both donor-type and third-party skin grafts occurs. C3H/He (C3H; H-2(k)) mice were injected as neonates with BALB/c (BALB; H-2(d)) FLC and transplanted as adults with C57BL/6 (B6; H-2(b)) cardiac grafts. Despite long-term acceptance of third-party B6 cardiac grafts, serum contained increased anti-B6 IgG and IgM levels as measured by CELISA; IgM production was elevated by 2 weeks posttransplant and remained stable, while IgG production increased rapidly between 2 and 5 weeks posttransplant. In another experimental setting, CELISA assays were able to detect that neonatal injection of C3H mice with FLC from wild-type B6 mice or from MHC class II-deficient or class I/II-deficient (B6 background) mice (CI(+)CII(+), CI(+)CII(-), CI(-)CII(-), respectively) prevented sensitization to B6 antigens by subsequent skin transplants but did not induce graft acceptance, whereas FLC from MHC class I-deficient-only (CI(-)CII(+)) did not prevent B6 sensitization. The CELISA technique is a simple and sensitive means for quantifying alloantibodies in mice and will assist in further delineating the role of the B-cell compartment in neonatally induced cardiac allograft acceptance.
authors.affiliation: Division of Cardiology, Department of Paediatrics, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada.
authors.forename: X
authors.initials: X
authors.lastname: Fan
authors.forename: K
authors.initials: K
authors.lastname: Tyerman
authors.forename: A
authors.initials: A
authors.lastname: Ang
authors.forename: K
authors.initials: K
authors.lastname: Koo
authors.forename: K
authors.initials: K
authors.lastname: Parameswaran
authors.forename: K
authors.initials: K
authors.lastname: Tao
authors.forename: L
authors.initials: L
authors.lastname: Mai
authors.forename: H
authors.initials: H
authors.lastname: Lang
authors.forename: L J
authors.initials: LJ
authors.lastname: West
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006338:Heart Neoplasms / Q000276:immunology*; D015395:Histocompatibility Antigens Class I / Q000235:genetics; D000949:Histocompatibility Antigens Class II / Q000235:genetics; D007108:Immune Tolerance; D007518:Isoantibodies / Q000276:immunology*; D008285:Major Histocompatibility Complex; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016038:Skin Transplantation / Q000276:immunology*; D013154:Spleen / Q000276:immunology
chemical_list: D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D007518:Isoantibodies