Figure 1.
The inhibition of murine monoclonal anti-DNA binding to DNA by NABPs.
The ability of polymers PAMAM, HDMBr, and CDP to inhibit binding of a murine monoclonal anti-DNA antibody QB1 to DNA was tested by ELISA. Binding of QB1 at 35 ng/ml final concentration was assayed by ELISA in the presence of PAMAM, HDMBr, or CDP at final concentrations from 10 ng/ml to 1,250 ng/ml. Control wells had buffer alone. Antibody was measured as described in Experimental Procedures. The OD450 values of 3 wells of each polymer concentration or of 6 wells without polymer were averaged. Each point shown is 100% x average OD450 with polymer/average OD450 without polymer. Circles show data for PAMAM; squares show data for HDMBr; triangles show data for CDP.
Figure 2.
The inhibition of SLE antibodies to DNA by NABPs.
The ability of polymers PAMAM, HDMBr, and CDP to inhibit DNA binding by anti-DNA antibodies in three SLE patient plasmas to DNA was tested by ELISA. Binding by antibodies in Plasma 1 (final dilution 1/1,000), Plasma 2 (final dilution 1/2,800), and Plasma 3 (final dilution 1/3,000) was assayed in the presence of PAMAM, HDMBr, or CDP at final concentrations ranging from 300 ng/ml to 10,000 ng/ml or with dilution buffer alone. Antibody levels were determined by ELISA as described in Materials and Methods. The OD450 values of 2 wells for each condition were averaged. Each point shown is 100% x average OD450 with polymer/average OD450 without polymer. Circles show data for Plasma 1; squares show data for Plasma 2; triangles show data for Plasma 3.
Figure 3.
The effects of NABPs on the binding of SLE antibodies to the Sm antigen and tetanus toxoid.
The specificity of inhibition of SLE antibody binding to dsDNA by NABPs was investigated by examining the effects of the polymers on SLE antibody binding to the Sm antigen and to tetanus toxoid in ELISA assays. (A) The effects of polymers PAMAM, HDMBr, and CDP on antibody binding to the Sm antigen were tested by ELISA using the three lupus plasmas and a human polyclonal anti-Sm antibody preparation (anti-Sm Ag) from USB. Plasmas were tested at 1/200 final dilution in the presence of inhibitors at concentrations from 80 ng/ml to 10,000 ng/ml of each polymer or with no polymer. The OD450 values of 2 wells for each condition were averaged. Each point shown is 100% x average OD450 with polymer/average OD450 without polymer. Circles show data for Plasma 1; squares show data for Plasma 2; triangles show data for Plasma 3; and open diamonds show data for the USB anti-Sm antibody. (B) The effects of polymers PAMAM, HDMBr, and CDP on antibody binding to tetanus toxoid were tested by ELISA using the three lupus plasmas. Plasma 1 (final dilution 1/720), Plasma 2 (final dilution 1/6,400), or Plasma 3 (final dilution 1/14,400) was incubated in wells coated with tetanus toxoid in the presence of PAMAM, HDMBr, or CDP at concentrations of 1,250 to 10,000 ng/ml or with no polymer. Antibody binding was determined as described in Materials and Methods. The OD450 values of 2 wells for each condition were averaged. Each point shown is 100% x average OD450 with polymer/average OD450 without polymer. Circles show data for Plasma 1; squares show data for Plasma 2; triangles show data for Plasma 3.
Figure 4.
The effects of NABPs on the binding of SLE antibodies to biotinylated DNA.
Native CT DNA was biotinylated and then captured in ELISA plate wells coated with streptavidin. The binding of Plasma 1 (final dilution 1/3,500), Plasma 2 (final dilution 1/8,000), and Plasma 3 (final dilution 1/4,500) was measured by ELISA in the presence of PAMAM, HDMBr, or CDP at final concentrations from 80 ng/ml to 10,000 ng/ml or with dilution buffer alone. The OD450 values of 2 wells for each condition were averaged. Each point shown is 100% x average OD450 with polymer/average OD450 without polymer. Circles show data for Plasma 1; squares show data for Plasma 2; triangles show data for Plasma 3.
Figure 5.
The effects of NABPs on the pre-formed complexes.
In this assay, 50 µl/well of Plasma 1 diluted 1/1,700, Plasma 2 diluted 1/3,950, and Plasma 3 diluted 1/2,250 were incubated in wells of microtiter plates with biotinylated DNA bound to streptavidin. After 1 hour to allow the formation of immune complexes, 50 µl of dilutions of PAMAM, HDMBr, or CDP or 50 µl of dilution buffer alone were then added to produce concentrations of the polymers of 10,000 ng/ml, 2,500 ng/ml, 620 ng/ml, 160 ng/ml or 0 ng/ml. Antibody binding was then determined by ELISA. The OD450 values of 2 wells for each condition were averaged. Each point shown is 100% x average OD450 with polymer/average OD450 without polymer. Circles show data for Plasma 1; squares show data for Plasma 2; triangles show data for Plasma 3.