Selection of phage-displayed anti-guinea pig C5 or C5a antibodies and their application in xenotransplantation

Xenogeneic liver transplantation in the discordant guinea pig (gp) to rat m odel results in hyperacute rejection within a few minutes, which is due to activation of the complement system. Currently no antibodies against gp com plement factors are available, which allow activation of the gp complement system in serum or complement deposition in tissue to be detected. To close this gap, we started developing single chain Fvs (scFvs) against gpC5 and gpC5a. We generated a combinatorial library of scFv antibodies comprising the Vari able heavy and light chain repertoire from mice immunized with gpC5. Out of this library we selected several antibodies against gpC5 and C5a after fou r and six rounds of biopanning, respectively. Selected gpC5-specific scFvs were purified by metal affinity chromatography followed by size exclusion c hromatography or by affinity chromatography using Protein L. Purified scFvs were able to inhibit gp complement system in a hemolytic assay and to dete ct gpC5 deposition in tissue. A surface plasmon resonance based assay on BI Acore was established, with which the C5 concentration in gp serum was dete rmined to 240 mu g/ml. As at least 0.04% of the normal gpC5 concentration c an be detected, the test provides a powerful tool to investigate the develo pment and the consequence of a hybrid complement system after liver xenotra nsplantation from gp to rat. (C) 2000 Elsevier Science Ltd. All rights rese rved.