Mouse Anti-dsDNA Recombinant Antibody (clone 2C10)

A, antibody 2C10 was preincubated with various double- or single-stranded oligonucleotides, and the mixture was added to microtiter plate wells coated with native calf DNA. ●, 5'-CGCGCATATATATATGCGCG-3' and 3'-GCGCGTATATATATACGCGC-5'; ▲, 5'-CGCGCATAGATCTATGCGCG-3' and 3'-GCGCGTATCTAGATACGCGC-5'; ○, 5'-CGCGCAGAGAGAGAGGCGCG-3'; ∆, 5'-CGCGCCTCTCTCTCTGCGCG-3'; ■, 5'-CGCGCAGAGAGAGAGGCGCG-3' and 3'-GCGCGTCTCTCTCTCCGCGC-5'. B, 2C10 was preincubated with polynucleotides, and the mixture was added to microtiter plate wells coated with native DNA. +, poly(dA-dT); ∆, poly(dG-dC); ○, poly(dG-dmC).

Kubota, T., Watanabe, N., Kanai, Y., & Stollar, B. D. (1996). Enhancement of oxidative cleavage of DNA by the binding sites of two anti-double-stranded DNA antibodies. Journal of Biological Chemistry, 271(11), 6555-6561.

DNA was incubated with anti-dsDNA antibody 2C10 (A) or H241 (B) or with a control myeloma protein, MPC11, and loaded on a 10% polyacrylamide gel with a 4% stacking gel. Molar ratios of DNA to antibody are indicated at the top of each lane.

Kubota, T., Watanabe, N., Kanai, Y., & Stollar, B. D. (1996). Enhancement of oxidative cleavage of DNA by the binding sites of two anti-double-stranded DNA antibodies. Journal of Biological Chemistry, 271(11), 6555-6561.

Antibodies 2C10 (lanes 2–5) and H241 (lanes 7–10) were incubated with the 274-bp dsDNA probe alone at DNA/antibody ratios of 1:40 (lanes 2 and 7) and 1:80 (lanes 4 and 9) or with a DNA/distamycin A mixture at DNA/antibody ratios of 1:40 (lanes 3 and 8) and 1:80 (lanes 5 and 10). The mixtures were then loaded on a 10% polyacrylamide gel with a 4% stacking gel. Lanes 1 and 6 are the DNA without antibodies.

Kubota, T., Watanabe, N., Kanai, Y., & Stollar, B. D. (1996). Enhancement of oxidative cleavage of DNA by the binding sites of two anti-double-stranded DNA antibodies. Journal of Biological Chemistry, 271(11), 6555-6561.

The 274-bp dsDNA probe was incubated with anti-dsDNA antibody 2C10 (A) or H241 (B) or with a control myeloma protein, MPC11, for 30 min in PBS. Then a 100 mM concentration of the divalent metal ion indicated at the top of each lane was added. Four min later, an excess of Na₄EDTA and thiourea was added to quench the reaction. In C, the dsDNA probe was incubated with H241 for 30 min, and the complexes were then incubated further with no additions (lane 1) or with 1 mM sodium ascorbate plus 9 mM H₂O₂ (lane 2); ascorbate alone (lane 3); H₂O₂ alone (lane 4); or ascorbate plus H₂O₂ plus 10 mM EDTA-chelated Fe(II) (lane 5), Mg(II) (lane 6), Zn(II) (lane 7), or Ca(II) (lane 8). Four min later, the reaction was quenched by EDTA and thiourea. In D, the dsDNA probe was incubated with MPC11 or 2C10 for 30 min and then for 4 min with no additions (lane 1) or with ascorbate and H₂O₂ (lanes 2 and 4) or ascorbate plus H₂O₂ plus EDTAchelated Fe(II) (lanes 3 and 5).

Kubota, T., Watanabe, N., Kanai, Y., & Stollar, B. D. (1996). Enhancement of oxidative cleavage of DNA by the binding sites of two anti-double-stranded DNA antibodies. Journal of Biological Chemistry, 271(11), 6555-6561.

The DNA probe was cleaved by Udenfriend's system with anti-dsDNA antibody 2C10 (A) or H241 (B) or with a control myeloma protein, MPC11. The standard reaction mixture (1×) contained 10 μM Fe(II), 20 μM Na4EDTA, 9 mM H₂O₂, and 1 mM sodium ascorbate (A, lanes 2 and 6; and B, lane 4). These concentrations were 0.25 × (B, lane 2), 0.5 × (B, lane 3), 2 × (A, lane 3 and 7), and 4 × (A, lanes 4 and 8) the standard. Lanes 1 and 5 in A and lane 1 in B are uncleaved controls. Shown in C is the densitometric estimation of the amounts of the starting DNA incubated with the hydroxyl radicalgenerating mixture and H241 (∆), 2C10 (●), or MPC11 (○).

Kubota, T., Watanabe, N., Kanai, Y., & Stollar, B. D. (1996). Enhancement of oxidative cleavage of DNA by the binding sites of two anti-double-stranded DNA antibodies. Journal of Biological Chemistry, 271(11), 6555-6561.

A, the DNA probe was incubated with anti-dsDNA antibody 2C10 or H241 or with a control myeloma protein, MPC11, and then cleaved by the reaction with the standard concentration of Udenfriend's system reagents for continuous hy_x0002_droxyl radical generation along with oxidation of Fe(EDTA)² ⁻. The reaction time was varied from 0 to 480 s as indicated. B, shown is the densitometric estimation of the amounts of the starting DNA in reactions with H241 (∆), 2C10 (●), and MPC11 (○).

Kubota, T., Watanabe, N., Kanai, Y., & Stollar, B. D. (1996). Enhancement of oxidative cleavage of DNA by the binding sites of two anti-double-stranded DNA antibodies. Journal of Biological Chemistry, 271(11), 6555-6561.

Oligonucleotide K5 (5'-AAAATATATATTT-3') or K6 (5'-GGGGCGCGCGCCC-3') was added to the DNA cleavage reaction using Udenfriend's system in the presence of anti-dsDNA antibodies 2C10 and H241 or a control myeloma protein, MPC11. Based on the densitometric estimation of the amounts of the starting materials, percent inhibition was calculated as follows: inhibition = (1 - ((cleavage with anti-DNA and inhibitor) - (cleavage with MPC11))/((cleavage with anti-DNA) - (cleavage with MPC11))) × 100.

Kubota, T., Watanabe, N., Kanai, Y., & Stollar, B. D. (1996). Enhancement of oxidative cleavage of DNA by the binding sites of two anti-double-stranded DNA antibodies. Journal of Biological Chemistry, 271(11), 6555-6561.

A, the DNA probe was cleaved by hydroxyl radical generated by reduction of H₂O₂ in Udenfriend's system in the presence of anti-DNA antibodies 2C10 and H241 or a control myeloma protein, MPC11. G, Maxam-Gilbert's G-reaction; G-control, Maxam-Gilbert's G-reaction without alkaline cleavage. B, densitometric analysis corresponds to the position of the oligonucleotide insert. The ordinate represents the relative sensitivity to the cleavage calculated as follows: relative cleavage sensitivity = (density of cleaved ladder in the presence of anti-DNA)/(density of cleaved ladder in the presence of MPC11). Hatched columns, H241; closed columns, 2C10.

Kubota, T., Watanabe, N., Kanai, Y., & Stollar, B. D. (1996). Enhancement of oxidative cleavage of DNA by the binding sites of two anti-double-stranded DNA antibodies. Journal of Biological Chemistry, 271(11), 6555-6561.