Production of anti-(ADP-ribose) antibodies with the aid of a dinucleotide-pyrophosphatase-resistant hapten and their application for the detection of mono(ADP-ribosyl)ated polypeptides.
Previous attempts to produce anti-(ADP-ribose) antibodies by immunization of rabbits with ADP-ribose conjugated to serum albumin had resulted in the production of 5'AMP-specific antibodies [Bredehorst et al. (1978) Eur. J. Biochem. 82, 105-113]. To obtain true anti-(ADP-ribose) antibodies an antigen was constructed that was resistant to enzymic degradation at the pyrophosphate group. The enzymically active beta-methylene derivative of NAD (NAD[CH2]) was synthesized from ADP containing a methylene bridge (CH2) instead of an oxygen in the diphosphate group. NAD[CH2] was converted to its N6-[(2-carboxyethyl)thiomethyl] derivative and hydrolyzed to the corresponding ADP[CH2]-ribose derivative which was then coupled to bovine serum albumin. The antibodies obtained with this antigen were specific for free or protein-bound ADP-ribose groups, except for a cross-reaction with FAD, AMP, ADP, ATP or poly(ADP-ribose) interfered with [3H]ADP-ribose tracer binding only at higher concentrations. No interference was observed with poly(A), RNA and DNA at 6000-fold excess. The antibodies were purified on a novel type of affinity matrix. This was formed from NAD and guanidinobutyrate by a cholera-toxin-catalyzed reaction and the product, ADP-ribosyl guanidinobutyrate, was bound to Affi Gel by carbodiimide-aided condensation. The purified antibodies allowed the detection of ADP-ribose conjugated to polypeptides in amounts lower than 1 pmol as demonstrated by immunoblotting of [14C]ADP-ribosylated elongation factor 2. They also could be used to observe in situ, by indirect immunofluorescence, the increased mono(ADP-ribosyl)ation of nuclear proteins in dimethyl-sulfate-treated cells, and to show that histone H2B was the principal histone acceptor of single ADP-ribose groups in alkylated 3T3 cells.
D000246:Adenosine Diphosphate Ribose / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D000917:Antibody Formation; D000918:Antibody Specificity; D003101:Collodion; D005455:Fluorescent Antibody Technique; D006241:Haptens; D007120:Immunochemistry; D007074:Immunoglobulin G / Q000032:analysis; D009702:Nucleoside Diphosphate Sugars / Q000276:immunology*; D010455:Peptides / Q000032:analysis*; D011755:Pyrophosphatases; D011817:Rabbits
D006241:Haptens; D007074:Immunoglobulin G; D009702:Nucleoside Diphosphate Sugars; D010455:Peptides; D000246:Adenosine Diphosphate Ribose; D003101:Collodion; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase