ELSEVIER Role of Idiotype-Independent Anti-IgG Autoantibodies in Human Kidney Transplantation: Natural Anti-F(ab’)Z Antibodies Recognize an IgGl Hinge Region Epitope P. Terness, D. Navolan, I. Kohl, F. Siedler, L. Moroder, Ch. Dufter, M. Welschof, F. Schneider, D. Drugarin, and G. Opelz M ANY previous studies focused on the role of antiid- iotypic antibodies in graft rejection.’ The presence of circulating antiidiotypic antibodies at the time of trans- plantation was associated with reduced renal allograft re- jection in recipients with pretransplant antibodies to donor HLA antigens.* Anti-IgG antibodies whose isotype and specificity were not defined were also found to be associated with kidney graft survival.3 Another series of studies showed that kidney transplant recipients with high pretrans- plant IgG- or IgA-anti-F(ab’)2 autoantibody activity had a reduced graft rejection rate.4V5 In the current experiments we describe the reactivity of IgG- and IgA-anti-F(ab’)2 autoantibodies derived from sera of prospective kidney transplant recipients against a defined F(ab’)2 epitope. MATERIALS AND METHODS Patient Sera Sera were collected from kidney transplant recipients prior to transplantation. Synthetic Hinge Peptide A double-chain hinge peptide was synthesized as previously de- scribed.6-8 This peptide corresponds to the middle (core) and lower hinge region of IgGl (225-337/225’-237”). Anti-hinge Peptide Antibodies (ELISA) Microtiter plates were coated with 50 PLiwell of IgG-derived F(ab’)2 fragments (20 pg!mL) (Jackson Immunoresearch, Lab, West Grove, Penn) or double-chain hinge peptide (Mr = 2503) (18 pg/mL) and the remaining active groups were blocked with PBS + 5% human albumin. Test serum (50 ILL, dilution: 1:30 for IgG- anti-F(ab’)2, 1:15 for IgA-anti-F(ab’)2,1:16 for IgG-anti-hinge, 1:8 for &$-anti-hinge) was applied to precoated plates in triplicate. A reference serum with known anti-F(ab’)2 and anti-hinge activity was used as positive control. Neuropeptide Y (NPY) (Mr = 4271) (30 @mL) served as negative control. Alkaline-phosphatase con- H-Thr-Cys-Pro-Pro-Cys-Pro-Ala-Pro-GIu-Leu-Leu-Gly-Gly-OH I I H-Thr-Cys-Pro-Pro-Cys-Pro-Ala-Pro-GIu-Leu-Leu-Gly-Gly-OH Fig 1. Double-chain IgGl hinge region peptide (225-237/225’- 237’). 0041-l 315/97/$17.00 PII SO041 -1345(96)00614-8 1412 Table 1. Binding of IgG-anti-F(ab’)2 and IgA-anti-F(ab’)S Antibodies to Synthetic Double Chain IgGl Hinge Region Peptide Patients IgG-anti-hinge IgA-anti-hinge 1 1585 1098 2 2392 2823 3 1858 2489 4 2009 1418 5 1919 1343 6 1914 2322 7 1334 1693 8 1547 2331 9 2314 2004 10 1379 1078 mean % SD 1825 + 345 1860 2 588 Values are given as OD,,,nm X 1 03. In control experiments the hinge peptide was replaced with neuropeptide Y (IgG-anti-NPY = 196 -C122; IgA-anti-NPY = 196 2 126). jugated goat anti-human IgG(Fc) or IgA(Fc) antibody (Jackson Immunoresearch) was added. Each step was followed by extensive washing. After adding 250 pg PNP substrate, the optical density was measured at 405 nm every minute. The test was stopped at a defined extinction of the positive control. The binding activity to human albumin coated plates was subtracted from all values. Statistical Analysis Data are expressed as mean ? SD. Comparisons were performed by unpaired t test and Spearman rank correlation test corrected for ties. From the Department of Transplantation Immunology, Institute of Immunology, Heidelberg, Max Planck institute of Biochemis- try, Mattinsried, Germany; and Department of Physiology, Uni- versity of Medicine and Pharmacy, Timisoara, Romania. This work was supported by a grant from Forschungssch- werpunkt Transplantation Heidelberg of the Land Baden-Wtirt- temberg. Address reprint requests to Dr P. Terness, Department of Transplantation Immunology, Institute of Immunology, INF 305, University of Heidelberg, 69120 Heidelberg, Germany. 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 29, 1412-l 414 (1997)