Monoclonal antibodies to poly(adenosine diphosphate ribose) recognize different structures

Abstract

Two hybridomas producing monoclonal antibodies to poly(adenosine diphosphate ribose) [poly(ADP-Rib)] were established. One antibody, 10H (IgG3, kappa), bound to most of the poly(ADP-Rib) preparation, which consisted of molecules of various sizes of more than 20 ADP-Rib residues. The binding of this antibody was inhibited by not only poly-(ADP-Rib) but also a monomer unit of poly(ADP-Rib), Ado(P)-Rib-P. The sites protected by antibody 10H were isolated and analyzed by hydrolysis with alkaline phosphomonoesterase and then snake venom phosphodiesterase. The sites contained the same amounts of monomer units and branched portions [Ado(P)-Rib(P)-Rib-P] as the original poly(ADP-Rib) molecules but a lower average number of branched portions per molecule than in the original molecules. The other antibody, 16B (IgM, lambda), reacted with only 50% of the radioactive poly(ADP-Rib), and its binding was not inhibited by a monomer unit. This antibody protected 25% of all the poly(ADP-Rib) molecules from hydrolysis by snake venom phosphodiesterase. The protected sites contained twice as many branched portions per molecule as the original poly(ADP-Rib) molecules. These results show that the two monoclonal antibodies recognize different structures of poly-(ADP-Rib); 10H antibody recognizes the linear structure with ribose-ribose linkages, and 16B antibody may recognize specific structures, including the branched portions of poly-(ADP-Rib).

Functional Interaction between Poly(ADP-Ribose) Polymerase 2 (PARP-2) and TRF2: PARP Activity Negatively Regulates TRF2

“…Cells were washed with ice-cold phosphate-buffered saline (PBS), fixed for 10 min with ice-cold methanol-acetone (1:1), followed by washing three times with ice-cold PBS-0.1% Triton-0.1% bovine serum albumin (BSA). Cells were then incubated overnight at 4°C with either rabbit polyclonal anti-PML antibody (1:100; H-238; Santa Cruz Biotechnology), goat polyclonal anti-TRF2 antibody (1:100; N-20; Santa Cruz Biotechnology), or mouse monoclonal antipolymer antibody (1:100; H10 [36]). After three washes with ice-cold PBS-0.1% Triton-0.1% BSA, cells were incubated for 1 h at 4°C with the appropriate conjugated secondary antibodies: an Alexa Fluor 568 goat anti-rabbit immunoglobulin G (1:1,500; Molecular Probes), an Alexa Fluor 594 donkey anti-goat immunoglobulin G (1:1,500; Molecular Probes), or a fluorescein isothiocyanate-conjugated sheep anti-mouse antiserum (1:400; Sigma).…”

Tumorigenic conversion of immortal human skin keratinocytes (HaCaT) by elevated temperature

“…Immunostaining procedure was performed as described previously (BuÈ rkle et al, 1993) using a monoclonal antibody (10 H) against poly(ADP-ribose) at a concentration of about 5 mg/ml in PBS/1% BSA. The speci®city of the antibody was determined as described (Kawamitsu et al, 1984). The second antibody (goat anti-mouse) was labeled with FITC (Dianova, Hamburg, Germany) and used at a dilution of 1 : 50.…”

Overexpression of dominant negative PARP interferes with tumor formation of HeLa cells in nude mice: Evidence for increased tumor cell apoptosis in vivo

“…Mouse monoclonal antibody 10H (Kawamitsu et al, 1983) against poly(ADP-ribose) was kindly provided by M Miwa and T Sugimura, Tokyo, Japan. Monoclonal antibody 6A7 against Bax was from Pharmingen, Hamburg, Germany.…”