Radioimmunoassay for Avian C-Type Virus Group-Specific Antigen: Detection in Normal and Virus-Transformed Cells

Abstract

MATERIALS AND METHODS Media. Cells were grown in Dulbecco's modification of Eagle medium supplemented with 10% calf serum (Colorado Serum Co.) in 50-mm petri dishes (Falcon Plastics, Los Angeles). Cells and viruses. The control cell lines used included BALB/3T3 (3) and normal rat kidney (NRK) cells (8). The Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) subgroup D (5) was used to transform BALB/3T3 and NRK. Two weeks after infection with this avian tumor virus, focal areas of transformed cells were visible; these were selected by means of cloning cylinders. The resulting mixed cultures of normal and transformed cells were cloned in microtiter plates (34). The SR-RSV-transformed lines obtained were designated SR-BALB and SR-NRK. A B77 avian sarcoma virus (36) transformant of BALB/3T3, B77-BALB, was provided by P. Vogt. This line was also cloned prior to use. The Kirsten (Ki-) and Rauscher (R-) strains of murine leukemia virus (MuLV) were propagated as previously described (34). The Carr-Zilber strain of RSV and Rousassociated viruses, RAV-1 and RAV-2, were obtained from the Resources and Logistics Segment, National Cancer Institute (NCI). Purification of avian gs antigen. Chicken serum containing high-titered avian myeloblastosis virus (AMV) was provided by J. W. Beard, Duke University, through the Resources and Logistics Segment, NCI. After clarification by centrifugation at 10,000 rpm for 15 min, the virus was pelleted at 30,000 rpm for 90 min, banded in a 15 to 60% sucrose gradient, and then repelleted. After resuspension and dialysis overnight against 0.01 M Tris (pH 7.8)-0.1 M 893

Influence of 5‐bromodeoxyuridine on the expression of avian GS antigen in mammalian rous sarcoma virus‐transformed cells

“…Our results also confirm recent data obtained by Stephenson et al (1973), who, using the radioimmunoassay, observed a significant increase in the level of gs antigen in rat cells transformed by RSV after treatment with IUdR.…”

Relationship of Friend murine leukemia virus production to growth and hemoglobin synthesis in cultured erythroleukemia cells

“…P (30), the major internal polypeptide of the virion, was generally measured by the microtiter complement fixation method (26). P(30) and P(12) were also assayed by a modification of the competition radioimmunoassay of Stephenson et al (35). Growing and stationary F4-6/K cells were collected by centrifugation and disrupted with PE buffer (10 mM potassium phosphate, pH 7.8; 10 mM EDTA; 0.6% bovine serum albumin) plus 0.5% Triton X-100 in a glass homogenizer with Teflon pestle.…”

Radioimmunoassay for the Major Structural Protein of Hamster Type C Viruses

“…In contrast, a hamster type C virus with no history of contact with other species showed no cross-hybridiza-tion with mouse or rat viruses. Sensitive radioimmunoassays (RIA) demonstrate gs antigens in normal mouse and chicken cells which were negative in complement-fixation (CF) or gel diffusion assays (4,26,34,36,37,41). Additionally, RIA has proven useful for demonstration of type-specific differences on MuLV p30 (35,39), and other viral proteins including gp 70 (38) and p12 (36,40).…”