Generation of a polyclonal rabbit anti-mouse tissue factor antibody by nucleic acid immunisation

Abstract

Tissue factor (TF) the cellular receptor and cofactor for factor VII, initiates coagulation and has also been implicated in several coagulation-independent functions, including inflammation, angiogenesis and tumour metastasis. Investigations of TF expression in mouse models of these processes has been limited by the availability of antibodies that specifically recognise mouse TF. We have generated a rabbit polyclonal antibody to mTF by DNA immunisation. This has yielded an antiserum that recognises native mTF in immunohistochemical and flow cytometric analyses. Furthermore, the antiserum is inhibitory in coagulation assays. This antiserum will be a valuable investigative tool in the analysis of mTF expression.

Protease‐activated receptor‐2 signalling by tissue factor on dendritic cells suppresses antigen‐specific CD4+ T‐cell priming

“…1 × 10 7 cells were injected into the tail vein of BALB/c mice. After 24 hr, the mice were given an intraperitoneal injection of either saline, 250 μg anti‐TF antibody, control rabbit immunoglobulin, 25 μg LPS ( E. coli serotype 0127:B8) (Sigma‐Aldrich) or LPS alone followed by 5 mg whole OVA. After 72 hr, lymph node (cervical, axillary, brachial and inguinal) cells were harvested for analysis of proliferation and IFN‐γ, IL‐4 and IL‐10 production by flow cytometry.…”

Postinjury vascular intimal hyperplasia in mice is completely inhibited by CD34+ bone marrow‐derived progenitor cells expressing membrane‐tethered anticoagulant fusion proteins

“…Vessels were embedded in OCT (VWR International, Dorset, UK) by freezing with CO 2 , sectioned and fixed in 100% methanol at −20 °C. Frozen sections were immersed in 1% BSA‐PBS and 10% goat serum (Sigma) for 30 min, then incubated overnight at 4 °C with one of the following antibodies (Ab): rabbit antihuman TFPI, sheep anti‐Hir (American Diagnostica Inc., Greenwich, CT, USA), mouse antihuman fibrin II β chain Ab (NYB‐T2G1, Accurate Chemical Scientific Corp.), rat antimouse CD41 (Pharmingen, San Diego, CA, USA), rat antimouse CD68 (Serotec, Oxford, UK), rabbit anti mouse TF [26] or mouse antihuman α ‐SMA (Sigma). Second layer staining was with a goat antirabbit IgG‐FITC, donkey antisheep IgG‐FITC, sheep antimouse IgG–FITC, goat antirat IgG‐FITC, rabbit antichicken IgG‐FITC, (all from Sigma), goat antirabbit IgG‐Texas red, horse antimouse IgG‐Texas red or goat antirat IgG‐Texas red (all from Vector Labs, Burlingame, CA, USA).…”

Tissue Factor and Thrombosis Models

“…Furthermore, a recently described mouse expressing WT levels of human TF 102 may be useful for testing an extensive library of TF-directed antibodies, 103 as will inhibitory antimouse TF antibodies. 104 Also, small and interfering RNA might be useful to study the temporal requirements of TF in animal models.…”