Anti-poly

Narrower specificities have been obtained with antibodies to some unusual helical structures. Poly(dG)-poly(dC) induces antibodies specific for the immunogen and unreactive with other deoxyribonucleotide polymers, such as poly(dAT) or native DNA. Double-helical polyribonucleotides with modified furanoses, such as poly(A)-poly(2 -0-methylU), induce antibodies that react with a number of polymers bearing 2 -furanose substitutions (such as methyl or ethyl groups on either the purine or pyrimidine-containing strand). Poly(G)-poly(C) induced antibodies of narrow specificity in our studies, but Lacour and co-workers obtained anti-poly(G)-poly(C) that cross-reacted with several forms of viral RNA. ... 

Fig. 3. Counterimmunoelectrophoresis assay of anti-poly(A)-poly(U) antiserum. Serum (150 jrl) was placed in the trough, and 50 /ul containing 0.25 fig of antigen was placed in each well. The antigen preparations were, from left to right, poly(A) poly(A) + poly(U), 85 15 poly(A) + poly(U), 67 33 poly (A) + poly(U), 50 50 poly(A) + poly(U) 33 67 poly(A) + poly(U), 10 90 and poly(U). Electrophoresis was run at 300 V, giving 10 mA per slide, for 45 min. The gel was 0.8% agar in 50 mM Tris-HCl pH 8.

Pilz, I., Kratky, O., Licht, A., and Sela, M. (1975) Shape and volume of fragments Fab and F(ab )2 of anti-poly(D-alanyl) antibodies in the presence and absence of tetra-D-alanine as determined by small-angle x-ray scattering. Biochemistry 14,1326-1333. 

We thank Dr. Sugimura for the lOH anti-poly(ADP-ribose) antibody. We are gratefiil to Oidier Hentsch (IGBMC, lUkirch) and Jean-Christophe Laval (Leica Microsystems, France) for their help with the laser microbeam. This work was supported by funds from Centre Nadonal de la Recherche Scientifique, Association pour la Recherche Contre le Cancer, Electricity de France, Ligue Nationale Contre le Cancer and Commissariat k I Energie Atomique. 

The sera of patients with systemic lupus erythematosus contain antibodies to poly(ADP-ribose) (106, 163). Although the clinical and diagnostic implications are not clear, isolation and purification of the anti-poly(ADP-ribose) may provide a powerful research tool. 

Fig. lA-D. Immunofractionation of chromatin digested by micrococcal nuclease for various extents of time on an anti-poly(ADP-ribose) Sepharose column. HeLa nuclei from 10 cells were digested with micrococcal nuclease [50 units/10 nuclei at 37°C for 1 min (A), 5 min (B), 10 min (C), and 20 min (D)]. Soluble chromatin was subsequently prepared and immunofractionated on an anti-poly(ADP-ribose) Sepharose column. The columns were loaded and washed with phosphate buffered saline. Transmittance at 254 nm was continuously monitored. When no further UV-absorbing material was eluted, 6 M guanidinium hydrochloride was added to elute-bound material. The insets in each panel represent 1/50 of the DNA extracted from the pooled unbound U) and bound (B) peak fractions subjected to electrophoresis on 1.5% agarose. The gel was stained with ethidium bromide. (Taken from [10])... 

Wong M, Miwa M, Sugimura T, Smulson M (1983) Relationship between histone poly (adenosine diphosphate ribosylation) and histone HI phosphorylation using anti-poly (adenosine diphosphate ribose) antibody. Biochemistry 22 2384-2389... 

The specific activity of affinity-purified anti-poly(ADP-ribose) antibody was ten times greater than that of the 7-globulin fraction the specific activity was defined as the amount of protein required to give 1 A -os unit (antibody titer) under certain conditions. The poly(ADP-ribose) binding specificity of the rabbit antibody [8] was confirmed by both direct and competitive ELISA (data not shown). 

In our experiments using acetone-fixed HeLa cells as the substrate, the pattern of immunofluorescence of poly(ADP-ribose) was so-called homogeneous, irrespective of preincubation with NAD, with intense fluorescence after incubation with NAD. Thus, acetone-fixed HeLa cells after NAD incubation are of practical value for the screening of the presence of anti-poly(ADP-ribose) antibodies in the sera of patients with rheumatic diseases. The preparation of acetone-fixed HeLa cells is easy and the poly-(ADP-ribose) is distributed evenly in the cells particularly after incubation with NAD and behaves just like the immobilized poly(ADP-ribose) antigen as in the case of ELISA [10]. 

Normal values of anti-poly(ADP-ribose), anti-dsDNA, and anti-ssDNA titers were less than 0.03 A405U... 

Thus, acetone-fixed HeLa cells that are incubated with NAD could be of practical use as substrates for screening for the presence of anti-poly(ADP-ribose) antibodies by the indirect immunofluorescent antibody technique. 

Kanai Y, Miwa M, Matsushima T, Sugimura T (1974) Studies on anti-poly(adenosine diphosphate ribose) antibody. Biochem Biophys Res Commun 59 300-306... 

Fig. 1. Immunoprecipitation of poly(ADP-ribosyl)ated cellular protein. (A) Nuclear proteins of human T-lymphoblastoid line CEM cells were poly(ADP-ribosyl)ated with [32p]NAD according to the method described before (4). Then, cells were lysed in NET-2 buffer ( 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 5 mM EDTA and 0.5 mM PMSF). Solubilized proteins were reacted for 2 hr with human serum IgG, either from control (C) or from patients (anti-poly(ADP-ribose) polymerase), which had been immobilized onto protein-A Sepharose CL-4B beads. After centrifugation and washing of the beads with NET-2 buffer, antibody-bound protein was solubilized in Laemmli s sample buffer and boiled for 2 min. Immunoprecipitated proteins were fractionated on a 12.5% polyacrylamide gel containing 0.1% SDS, and autoradiographed. (B) HeLa cell proteins were labelled with [ sjmetjiionine overnight, and solubilized in NET-2 buffer. Immunoprecipitation was processed as described above.

The patients with anti-poly(ADP-ribose) polymerase autoantibodies complained of persistent neuromuscular pain, principally affecting the lower extremities. One patient had Sjogren s syndrome the others did not have... 

Fig. 4. Cellular content of poly(ADP-ribose) polymerase by two-color fluorescent measurement. Human CEM T lymphoblastoid cells were fixed with 45% ethanol for 30 min and stained with (A) FTTC-labelled goat anti-human IgG, (B) FITC-labelled anti-poly(ADP-ribose) polymerase IgG, (C) normal human IgG followed by FTTC-labelled goat anti-human IgG or (D) affinity purified anti-poly(ADP-ribose) polymerase IgG followed by FTTC-labelled goat anti-human IgG (10 pg/ml in each experiment). Finally, cells were stained with propidium iodide (10 x g/ml) and two-color fluorescence was analyzed in a flow cytometer (11). Two-color contour plots shows DNA content (red fluorescence) on the X-axis, and HTC-antibody binding (green fluorescence) on the Y-axis. 

Fig. 2.4. Inhibition by alanine peptides of the precipitates obtained by reacting (A) IgG obtained from anti-poly-L-alanyl-human serum albumin with poly-L-alanyl-rabbit serum albumin (B) IgG obtained from anti-poly-o-alanyl-HSA with poly-D-alanyl-RSA. L, L-alanine D, D-alanine L2 is the polymer containing two L-alanine groups, etc. Reprinted by permission from Schechter et at. (22).

A change in conformation upon interaction with a hapten is indicated by the study of Pilz et al. (52a), who investigated the interaction of rabbit anti-poly-D-alanyl antibodies with tetra-D-alanine by the method of small-angle X-ray scattering. The interaction was associated with a significant contraction in volume (3-10%) and a small decrease in the radius of gyration of the antibody. The volume contraction was attributed to a conformational change. 

In semidilute solution and upon addition of salt (NaCl) an anti-poly-electrolyte effect was observed. That means that the viscosity ifacreased with the ionic strength and was several orders of magnitude higher than that measured in pure water [8-9]. 

Specificity of Anti-Poly I. Poly C Antibodies Induced in the Rabbit... 

Fig. 1. Precipitin reac on of anti-poly I poly C serum X 929 (0.5 ml of a 1 10 dilution)...

In order to define the specificity of the antibodies and to attempt to identify the antigenic determinants (or groups of antigenic determinants) Nahon-Merlin et al. (1973 a) studied the cross-reactions of anti-poly I poly C antibodies with different polynucleotides and polynucleotide complexes by direct precipitation and by specific absorption of antibodies. The rabbits respond to immunization with poly I poly C — MBSA, with a production of antibodies which varies according to the rabbit the sera contain from 700 to 1000 [i.g/ml of antibodies. 

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