A dot-blot method for screening polyclonal and monoclonal antisera to poly(ADP-ribose).
ADP-ribosylation reactions play a key role at several points in cellular regulation and repair of DNA damage. The use of polyclonal or monoclonal antisera to poly(ADP-ribose) as probes to localize the site(s) of action of the polymer offers a promising tool for these studies. We report here a simple, sensitive method for detection and titration of these antisera to poly(ADP-ribose) using nitrocellulose membrane (NC) as a support for a dot-blot analysis. We take advantage of the fact that a highly labeled poly(ADP-ribose) preparation can be obtained by incubation of a 0.3 M KCl extract prepared from calf thymus nuclei with 32P-NAD. Such a preparation of labeled antigen is used as a reagent to detect the positive antibody spots on the NC with negligible background. Subsequent titration of the antisera and their semi-quantitative evaluation are also feasible using the dot-blot method. The sensitivity of the assay is only limited by the specific activity that can be achieved for the labeled polymer prepared as the antigen probe. The advantage of this method is that it eliminates the need to prepare pure, highly radiolabeled polymer as well as the fact that several samples can be handled on the membrane simultaneously. We demonstrate application of this technique for screening sera from patients with systemic lupus erythematosus (SLE) for anti-poly(ADP-ribose) antibodies. Further, we also extend the use of these sera for immunoquantitation of ADP-ribosylated proteins in six human tumor cells in tissue culture.
Prasad
S
S
Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC.
Thraves
P
P
Kanai
Y
Y
Smulson
M
M
Dritschilo
A
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002417:Cattle; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D008180:Lupus Erythematosus, Systemic / Q000175:diagnosis / Q000276:immunology; D009687:Nuclear Proteins / Q000276:immunology*; D009702:Nucleoside Diphosphate Sugars / Q000276:immunology*; D011064:Poly Adenosine Diphosphate Ribose / Q000276:immunology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D013950:Thymus Gland / Q000276:immunology
D000911:Antibodies, Monoclonal; D009687:Nuclear Proteins; D009702:Nucleoside Diphosphate Sugars; D011064:Poly Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases