title: Measurement of anti-CD154 monoclonal antibody in primate sera by competitive inhibition ELISA.
abstract: BACKGROUND
  Anti-human CD154 monoclonal antibody (mAb)-based regimens have been demonstrated to prevent T cell-dependent elicited antibody response in baboon recipients of pig hematopoietic progenitor cells, organs and islets. Monitoring of anti-CD154 mAb in serum is important to ensure maintenance of adequate levels and for adjusting dosage of the anti-CD154 mAb. We describe a method for measuring the level in primate sera.
  
  
  METHODS
  The anti-CD154 mAb level in primate serum was measured with a competitive inhibition enzyme linked immunosorbent assay in which the extent of inhibition of binding by anti-CD154 mAb conjugated to horseradish peroxidase (anti-CD154-HRP) to soluble CD154 was used to determine the serum level. Briefly, a 96-well maxisorb plate coated with soluble human CD154, and blocked with bovine serum albumin, was loaded with graded doses of anti-CD154 mAb or primate sera containing anti-CD154 mAb. Both were mixed with a known dosage of anti-CD154-HRP before loading. Bound anti-CD154-HRP was detected by color developed using 3,3',5,5' tetramethyl-benzidine as substrate. Absorbance was measured in a Synergy HT Multi-Detection Microplate Reader at a wavelength of 450 nm. Data analysis was carried out using BioTek's KC4 Data Analysis Software. The standard curve was generated from the wells loaded with the mixture of anti-CD154 mAb and anti-CD154-HRP.
  
  
  CONCLUSIONS
  The assay has been used successfully to measure anti-CD154 mAb levels in the serum of both baboons and monkeys.
authors.affiliation: The Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Hao-Chih
authors.initials: HC
authors.lastname: Tai
authors.forename: Nathalie
authors.initials: N
authors.lastname: Campanile
authors.forename: Mohamed
authors.initials: M
authors.lastname: Ezzelarab
authors.forename: David K C
authors.initials: DK
authors.lastname: Cooper
authors.forename: Carol
authors.initials: C
authors.lastname: Phelps
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001667:Binding, Competitive; D023201:CD40 Ligand / Q000097:blood* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000882:Haplorhini; D006801:Humans; D010215:Papio
chemical_list: D000911:Antibodies, Monoclonal; D023201:CD40 Ligand