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Abstract: Dispersions of various concentrations (15–35%) were prepared in silicone oils of vastly different viscosities (40, 1000, 10,000, and 30,000 mPa s) and compressed to high values of stress while under an electric field of 2 kV/mm. A purpose of this study was to observe the effect of compression and E field simultaneously on these dispersions and assess predictions of most common and relevant theories. As shown, static stresses of over 1000 kPa could readily be obtained although the data presented here were held below 300 kPa to protect the load cell and equipment. The results are compared to and discussed in terms of a power law fit for stress vs gap since most theories predict such a dependence. The PL exponents fall around 3 ranges: (−2), (−3), and much less than (−3). The PL coefficients however reflect in systematic way the viscosities of the dispersing oils. The compressive stress vs strain behavior is studied with regard to particle concentration and dispersing oil viscosity. [Copyright &y& Elsevier]

6A6 is a murine monoclonal antibody raised against the humanized anti-tissue factor antibody D3H44. 6A6 is able to completely neutralize the anticoagulant activity of D3H44 in tissue factor-dependent functional assays, such as endotoxin-induced whole blood clotting, prothrombin time, as well as factor X and factor IX activation. ELISA-type assays further showed that 6A6 binds to an epitope with critical determinants on the VL domain of D3H44. The possibility that the anti-idiotypic 6A6 might carry an “internal image” of the original antigen (tissue factor) was examined using the X-ray structure of the 6A6-Fab/D3H44-Fab complex determined at 2.5 A˚ resolution. We find that 6A6 structurally mimics tissue factor only so far as it combines with the antigen recognition surface of D3H44. While 6A6 contacts both VL and VH domains of D3H44, as does tissue factor, there is more contact with the D3H44 VL domain and less with the D3H44 VH domain relative to the tissue factor contacts on D3H44. Additionally, there is an almost total lack of correspondence between 6A6 and tissue factor at the level of amino acid side-chain functional groups. Despite the fact that both tissue factor and 6A6 are composed largely of β-sheets, they present fundamentally different elements of secondary structure to D3H44; tissue factor presents β-sheets edge-on, while 6A6 uses mostly loops. Finally, the finding that 6A6 competes with tissue factor for D3H44 binding raises the possibility of using 6A6 as an antidote for D3H44 anticoagulant therapy. To this end, we constructed a chimeric murine/human 6A6-Fab, which effectively neutralized D3H44 and fully restored tissue factor function in enzymatic assays. [Copyright &y& Elsevier]

Abstract: Short interfering RNA (siRNA) has therapeutic potential. However, efficient delivery is a formidable task. To facilitate delivery of siRNA into cells, we covalently conjugated siRNA to antibodies that bind to cell surface proteins and internalize. Understanding how these antibody–siRNA conjugates function in vivo requires pharmacokinetic analysis. Thus, we developed a simple real-time antigen capture reverse transcription–polymerase chain reaction (RT–PCR) assay to detect intact antibody–siRNA conjugates. Biotinylated antigen bound to streptavidin-coated PCR tubes was used to capture antibody–siRNA conjugate. The captured antibody–siRNA conjugate was then reverse-transcribed in the same tube, avoiding a sample transfer step. This reproducible assay had a wide standard curve range of 0.029 to 480ng/ml and could detect as low as 0.58ng/ml antibody–siRNA conjugates in mouse serum. The presence of unconjugated antibody that could be generated from siRNA degradation in vivo did not affect the assay as long as the total antibody concentration in the antigen capture step did not exceed 480ng/ml. Using this assay, we observed a more rapid decrease in serum antibody–siRNA conjugate concentrations than the total antibody concentrations in mice dosed with antibody–siRNA conjugates, suggesting loss of siRNA from the antibody. This assay is useful for optimizing antibody–siRNA and likely aptamer–siRNA conjugates to improve pharmacokinetics and aid siRNA delivery. [Copyright &y& Elsevier]