Studies with FcgRI/III-deficient mice and Fab fragments have shown protection against human IgG-induced cell death (He et al., 2002).
To eliminate the possibility that the protection afforded by immunization of CD200KO mice with CpG and EMT6 cells reflected development of an immune response to CD200-dependent epitopes, as has been described for Stat6+ adenocarcinoma cells in Stat6KO mice [ 26], we confirmed that protection was observed in wild-type mice receiving Fab anti-CD200 Mab (Fig. 4).
In this study, we compared microSPECT/CT and microPET/CT for imaging s.c. human tumor xenografts expressing a wide range of HER2 density in athymic mice using trastuzumab Fab fragments modified with 1,4,7,10-tetraazacyclododecane N, N″, N'″-tetraaceticetic acid (DOTA) for complexing 111In or 64Cu.
For immunolabeling, sections were incubated with anti-Cx43 or anti-drebrin antibodies for 1 h followed by a 30 min incubation with 10 or 5 nm colloidal gold-labeled goat-anti-rabbit or goat-anti-mouse Fabs (British BioCell) or protein A-Gold as described by us earlier (Butkevich et al. 2004).
Patients' IgG autoantibodies are pathogenic in C5-deficient mice and F ab' 2, Fab, and scFv fragments of autoantibodies induce acantholysis by passive transfer in wild type mice showing that complement activation or other Fc-mediated effects are not required for pathogenicity [ 4, 21, 45, 55].
Briefly, after treatment with pepsin for 10 min for antigen retrieval, sections were incubated with anti-TNC antibody 4F10TT and subsequently with peroxidase-conjugated anti-mouse IgG Fab' (Medical and Biological Lab Co. Ltd., Nagoya, Japan).
Rit is a chimeric mouse/human monoclonal antibody, consisting of the human IgG1 and k constant Fc region, and a mouse variable Fab region specific against the B-cell antigen CD20.
For Pax7 immunostaining, fresh sections were fixed in 4% paraformaldehyde for 20 min, permeabilized with methanol (-20°C) for 6 min, and blocked first with a solution containing 4% bovine serum albumen (BSA; Jackson) in PBS and then with goat anti-mouse AffiniPure Fab fragment (1 100; Jackson) after antigen retrieval with 100 mmol/L sodium citrate.
Goat anti-mouse IgG (Fab)'2 Alexa 488, nuclear TOTO stain were obtained from Molecular Probes (Eugene, Oregon).
Anti-mouse IgG (Fab specific) and anti-rabbit IgG secondary antibodies conjugated to peroxidase were purchased from Sigma.
