Identification of woodchuck class I MHC antigens using monoclonal antibodies

Abstract

Two class I major histocompatibility complex (MHC) proteins with molecular masses of 43- and 39-kDa were identified in the cell surface membranes of normal woodchucks using a newly developed antiwoodchuck class I monoclonal antibody (mAb) B1b.B9 and immunoblotting. B1b.B9 was generated by immunizing mice with viable woodchuck peripheral blood mononuclear cells and was selected for anti-class I MHC reactivity using a cellular enzyme-linked immunoassay, indirect immunofluorescence on tissue sections and flow cytofluorimetry. The distribution pattern of class I MHC antigen on woodchuck lymphoid cells was found to be similar to that reported in other species. Also, the antigen expression on normal woodchuck hepatocytes was comparable to that observed on normal human liver parenchymal cells; thus, the antigen was not detected on hepatocytes by staining of liver tissue sections, but was found by indirect immunofluorescence staining of isolated liver cells. Western blot analysis of the plasma membranes from normal woodchuck hepatocytes revealed the presence of a single species of class I MHC heavy chain protein with a molecular mass of 43-kDa, whereas splenocyte plasma membranes showed intense expression of a 43-kDa species, as well as the presence of a 39-kDa protein. The 39- and 43-kDa proteins were extracted with Triton X-114 to the hydrophobic protein phase, suggesting that they both contain a hydrophobic transmembrane domain. The data obtained indicate that the B1b.B9 identifies a nonpolymorphic epitope of woodchuck class I MHC heavy chains, providing an important reagent for the study of the pathogenesis of hepatitis B virus infection in a woodchuck model.

Inhibition by Woodchuck Hepatitis Virus of Class I Major Histocompatibility Complex Presentation on Hepatocytes Is Mediated by Virus Envelope Pre-S2 Protein and Can Be Reversed by Treatment with Gamma Interferon

“…To evaluate the presentation of class I MHC antigen on the transfected and control WCM-260 hepatocytes, the unfixed cells were incubated for 45 min on ice with mouse monoclonal antibody (MAb) against woodchuck class I MHC heavy chain (B1b.B9), previously prepared in this laboratory (21) or with phosphatebuffered saline (PBS), pH 7.6, a negative control. After a washing with PBS, cells were exposed to FITC-conjugated goat anti-mouse IgG (heavy plus light chain) antibodies (Jackson ImmunoResearch Laboratories Inc.) for 45 min, followed by washing with PBS.…”

Occult Lifelong Persistence of Infectious Hepadnavirus and Residual Liver Inflammation in Woodchucks Convalescent From Acute Viral Hepatitis

“…Peripheral blood mononuclear cells (PBMC) were isolated typically at monthly or bimonthly intervals from ethylenediaminetetraacetic acid-treated blood using Histopaque 1119 (Sigma Chemical Co., St. Louis, MO) density gradients. 22 In some instances, isolated viable PBMC were subjected to DNAase and trypsin digestion as described below. Liver biopsies were performed by surgical laparotomy under inhalant isofluorane anesthesia.…”

Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis

“…Beginning at 2 months of age and continuing up to 42 months after birth, blood was collected monthly for isolation of serum or plasma and PBMCs. PBMCs were harvested on Histopaque-1119 (Sigma Chemical Co., St. Louis, Missouri, USA) gradients as described previously (20). In offspring observed for longer than 6 months, liver biopsies were done annually by laparotomy.…”