title: Characterization of anti-thymoglobulin, anti-Atgam and anti-OKT3 IgG antibodies in human serum with an 11-min ELISA.
abstract: Patients treated with OKT3 may become resistant to OKT3 therapy following induction of anti-idiotypic antibodies. Antirabbit or antihorse antibodies following Thymoglobulin (rabbit antithymocyte globulin (ATG) or Atgam (horse ATG) therapy may not similarly inhibit drug efficacy due to an insufficient anti-idiotypic response against the multiple idiotypic specificities of the polyclonal anti-T cell preparations. However, no standardized assay had been developed to monitor antihorse and antirabbit antibodies. To address this issue, we developed three rapid (11-min) and standardized semiquantitative plate enzyme-linked immunoassays (ELISAs) to monitor human serum immunoglobulin-G (IgG) antibodies to Orthoclone OKT3 IgG2a, Atgam and Thymoglobulin. The format was identical for the three assays, with the exception of the immunoglobulin antigen coated on the ELISA plates. As OKT3 is a monoclonal antibody, OKT3 itself is used as the capture antigen. However, for antibody responses against the polyclonal antibody preparations Atgam and Thymoglobulin, it was found that horse IgG and rabbit IgG respectively were equivalent to Atgam and Thymoglobulin as capture antigens. Excellent correlation with a 3.5-h format was demonstrated (r values between 0.986 and 0.845). Specificity was demonstrated by inhibition experiments. Correlations between endpoint titres calculated from serial dilutions and 1:50 dilution OD values were 0.821, 0.983 and 0.937 respectively for the mouse, horse and rabbit assay. High titre sera were selected to assess their ability to block in vitro the binding of OKT3, Thymoglobulin and Atgam to T cells, using flow cytometry. None of the sera containing antihorse (n = 9) or antirabbit (n = 8) antibodies blocked T cell binding of Atgam or Thymoglobulin. In contrast, OKT3 binding was blocked by the four highest titre sera of the 13 anti-OKT3 sera tested. Consequently, prospective monitoring of treated patients using standardized 11-min assays may allow a better assessment of the effect of presensitization to OKT3, Atgam or Thymoglobulin.
authors.affiliation: SangStat Medical Corporation, Menlo Park, California 94025, USA.
authors.forename: J
authors.initials: J
authors.lastname: Regan
authors.forename: K
authors.initials: K
authors.lastname: Campbell
authors.forename: L
authors.initials: L
authors.lastname: van Smith
authors.forename: P
authors.initials: P
authors.lastname: Pouletty
authors.forename: T J
authors.initials: TJ
authors.lastname: Schroeder
authors.forename: R D
authors.initials: RD
authors.lastname: Guttmann
authors.forename: R
authors.initials: R
authors.lastname: Buelow
mesh_terms: D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000096:biosynthesis / Q000097:blood* / Q000737:chemistry; D019138:Antibodies, Blocking / Q000097:blood; D000918:Antibody Specificity; D000961:Antilymphocyte Serum / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006736:Horses; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000097:blood* / Q000737:chemistry; D019169:Jurkat Cells; D051379:Mice; D016853:Muromonab-CD3 / Q000276:immunology*; D011817:Rabbits; D011934:Reagent Strips; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000888:Antibodies, Anti-Idiotypic; D019138:Antibodies, Blocking; D000961:Antilymphocyte Serum; D007074:Immunoglobulin G; D016853:Muromonab-CD3; D011934:Reagent Strips