Two Widely Used Anti-DRm Monoclonal Antibodies Bind to an Intracellular C-Terminal Epitope Ulrike Grtineberg, Tina Rich, Corinne Roucard, S. Marieke van Ham, Dominique Charron, and John Trowsdale ABSTRACT: In this report we show that two widely- used monoclonal antibodies, TAL-lB5 and DA6.147, which react with the HLA-DRa chain on immunoblots, recognize the C-terminal intracellular tail of this HLA- DR subunit. We demonstrate that both MoAbs react with a synthetic peptide representing the intracellular C- ABBREVIATIONS AA amino acid ABTS 2.2’-Azino-di-(3-ethylbenzthiazolinsulfonate (6)) ECL enhanced chemiluminescence ELISA enzyme-linked immunosorbent assay FACS fluorescence activated cell sorting INTRODUCTION Major histocompatibility complex (MHC) class II mol- ecules are heterodimeric cell surface molecules that bind peptides and present them to CD4-positive T cells. Hu- man MHC class II molecules comprise three subclasses: HLA-DR, DP, and DQ, which are highly polymorphic. The DR subset in particular has been studied extensively and the biochemistry, peptide binding characteristics and mode of interaction with the T cell receptor of HLA- DR molecules are known in great detail [I, 2). A variety of MoAbs react with the fully folded DR molecule and recognize both polymorphic and mono- morphic determinants (e.g. L243 {?I, Dl-12 141, and From the Human lmmunogenetzrs Group. lmpeper& Cancer Research Fund (U. G., C. R., S. M. $1. H., J. T. ), London. United Kuzgdom: and the Labora- toire d’lmmunogei26tiqw lnstitut des Cord&w (7. R., D.C.). Paris, France. Address reprint requesrs to Dr. John Trousdale, Human Immunogenetics Group. lmperzal Cancer Research Fund. 44 Lincoln’s Inn Fields. London WCZA 3PX. United Kzngdom. Received Ortohev 23, 1996: accepted December 5, 1996. terminal tail of the DRa chain and that mutant DR molecules lacking this part of the (Y chain lose reactivity with TAL-lB5 and DA6.147, both in Western blot analysis and in intracellular FACS staining. Human Immu- nology 53. 3438 (1997). 0 American Society for His- tocompatibility and Immunogenetics, 1997. HLA human leukocyte antigen HPLC high performance liquid chromatography HRP horse-radish peroxidase Ii invariant chain MoAb monoclonal antibody MHC major histocompatibility complex 16.23 IS]). However, only very few MoAbs are suitable for Western blotting of DR molecules although this is a convenient method to analyze expression of class II mol- ecules. Two antibodies that react very strongly on West- ern blots with HLA-DR are DA6.147 {6} and TAL-lB5 171. Both MoAbs reveal bands for the DRar chain and the DR c$ heterodimer indicating that the epitopes for both antibodies are on the OL chain. Besides immuno- blotting, both MoAbs are useful reagents for immuno- precipitations of MHC class II proteins but do not stain MHC class II-positive cells in FACS analysis unless the cells have been permeabilized. TAL-lB5 and DA6.147 have been widely used for immunoblotting, immunohis- tochemistry, immunoprecipitations, and tumor analysis [S-13}. Despite their extensive use, however, the exact epitope for neither TAL-lB5 nor DA6.147 has been identified so far. Here we show that both DA6.147 and TAL-lB5 react with the intracellular tail of the DRa chain using ELISA, Western blot, and FACS analysis of Human Immunologp 53, 34-38 (1997) 0 Amencan Society for Hlstocompanbility and Immunogenetics, 1997 019%8859/97/$17.00 PII SO1 98-8859(97)00025-6