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.
Results and conclusions: The assay has been used successfully to measure anti-CD154 mAb levels in the serum of both baboons and monkeys.