flat bottomed elisa strip plates  (Greiner Bio)

Journal: Chemical research in toxicology

Article Title: Detection of acrolein-derived cyclic DNA adducts in human cells by monoclonal antibodies

doi: 10.1021/tx3004104

Figure Lengend Snippet: (A) Different amounts of dG, dA, dC and T were used to determine their effects on the binding to mAbs to immunogens. Of all the mAbs examined, A3-mAb4 did not bind to normal nucleosides, as did A3-mAb1 (not shown). (B) All other mAbs, represented by A3-mAb3, showed cross-reactivity to dG at the highest concentration. (C) The A3-mAb4 and A3-mAb1 were further studied in a competitive ELISA assay coated with Acr-Guo3-conjugated BSA for their reactivity towards Acr-dG1/2, Acr-dG3, Acr-Guo1/2, Acr-Guo3, Cro-dG, HNE-dG, 8-oxo-dG, edA. No stereospecificity was observed; A3-mAb4 bound equally well to Acr-dG3 and Acr-dG1/2 as to Acr-Guo1/2 and Acr-Guo3. A3-mAb4 displayed significant cross-reactivity towards Cro-dG, but less for HNE-dG. It did not at all recognize 8-oxo-dG and showed minimal reactivity towards edA only at the highest concentration. (D) Acr modified CTDNA was coated on plates and Acr-dG3 and ring-opened form of AcrdG3 (Acr-dG3 RO) were added in competitive ELISA. A3-mAb4 showed binding to Acr-dG3 and no reactivity towards ring-opened form of Acr-dG3. The coated antigens were different in (C) and (D), so the competitive binding curves for Acr-dG3 were different. Data were obtained from duplicate experiments. (E) Slot-blot assay further demonstrated the specificity of these antibodies towards Acr modified plasmid DNA with no reactivity towards BPDE, H2O2, MDA modified DNA. The left panel of blot image shows the binding of antibodies to the modified DNA samples and binding only occurred with Acr-Modified DNA in a dose-dependent manner, the middle panel shows the DNA loading markers and the right panel is the information for the corresponding modified DNA samples.

Article Snippet: Characterization of monoclonal antibodies with ELISA The ELISA plate wells (Immulon 4 HBX flat bottom 1×12 strips from Thermo, part number 6404) were coated with 100 μl of 1 μg/ml antigens of Acr-Guo 1/2- or Acr-Guo 3-cojugated BSA in PBS at 37°C for 2 h and washed twice with PBS containing 0.1% (v/v) Tween 20 (PBST).

Techniques: Binding Assay, Concentration Assay, Competitive ELISA, Modification, Slot Blot Assay, Plasmid Preparation

Journal: Chemical research in toxicology

Article Title: Detection of acrolein-derived cyclic DNA adducts in human cells by monoclonal antibodies

doi: 10.1021/tx3004104

Figure Lengend Snippet: (A) In the FACS assay, the left panel shows that the average fluorescence intensity (F mean) indicates the antibodies bound in a concentration-dependent manner to the cellular DNA containing Acr-dG. The right panel shows the original histograms with X-axis as the fluorescence (F) and Y axis as the number of cells (N). The p values from t-test for 0 vs 50, 50 vs 100, 100 vs 150 and 150 vs 200 μM comparisons are 0.4, 0.14, 0.14 and 0.08, respectively. (B) A highly sensitive ELISA assay shows the Acr-dG levels in DNA extracted from HT29 cells. Only one μg DNA from each sample was used, and the chemiluminescence signal (CL), representing the binding of antibodies to DNA, showed that the Acr-dG levels increased in a concentration-dependent manner. The corresponding p values as (A) are 0.26, 0.3, 0.3 and 0.02, respectively. (C) To validate this, Acr-dG levels in the same DNA samples were quantified with by LC-MS/MS-MRM. The corresponding p values as (A) are 0.2, 0.3, 0.08 and 0.06, respectively. All results were obtained from duplicate experiments. (D) Quantification of Acr-dG by a competitive ELISA assay. To the ELISA plates were coated with different levels of modified CTDNA, different amount of Acr-dG standards were added together with the anti-Acr-dG antibodies. The approximate detection ranges that can be used to quantify the Acr-dG adducts are: 100pmol to more than 1000pmol for the 20% Acr-CTDNA coated plates, 10pmol to 1000pmol for the 2% Acr-CTDNA coated plates and <100fmol to 100pmol for and the un-modified CTDNA coated plates.

Article Snippet: Characterization of monoclonal antibodies with ELISA The ELISA plate wells (Immulon 4 HBX flat bottom 1×12 strips from Thermo, part number 6404) were coated with 100 μl of 1 μg/ml antigens of Acr-Guo 1/2- or Acr-Guo 3-cojugated BSA in PBS at 37°C for 2 h and washed twice with PBS containing 0.1% (v/v) Tween 20 (PBST).

Techniques: Fluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Competitive ELISA, Modification