Radioimmunoassay for infectious primate retrovirus reverse transcriptase: characterization, comparison with conventional immunologic assays and applicability to cellular extracts.
SSV reverse transcriptase (RT) was purified to homogeneity and used in a radioimmunoassay. Following iodination, the homogeneity of the protein and its identity with RT were confirmed by several criteria: (1) its molecular weight on an SDS-polyacrylamide gel; (2) its precipitation by anti-SSV RT but not by antisera to other SSV proteins; (3) its cross-reactivity in RIA with antisera to other retroviral polymerases; (4) its competition in RIA by active homogeneous SSV RT but not by other purified SSV proteins; and (5) its competition in RIA by only those fractions from a poly(U)-Sepharose column possessing SSV RT activity. Competition of the labelled probe with disrupted retroviruses of the infectious primate group showed that, while a homologous RIA detected only type-specific enzyme determinants, it did not distinguish the various woolly-gibbon retroviral DNA polymerases. A more broadly reactive heterologous assay utilizing an antiserum to R-MuLV RT detected group- but not interspecies-specific enzyme determinants. A comparison of immunologic assays for RT showed that: (1) highly purified RT is not essential for reliable results in enzyme neutralization or enzyme binding assays; (2) the greater sensitivity of enzyme binding compared to enzyme neutralization assays is a function of the antibody, not of the antigen. Competition RIAs using extracts of virus-infected cells showed that infectious primate retrovirus RT could be measured in a crude system and that cellular DNA polymerases alpha, beta and gamma did not compete with the labelled probe.
Robert-Guroff
M
M
Laboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Md. 20205, USA.
Kalyanaraman
V S
VS
Sarngadharan
M G
MG
D000818:Animals; D001859:Bone Neoplasms; D002144:Callithrix; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D016093:Mason-Pfizer monkey virus / Q000235:genetics / Q000302:isolation & purification*; D012516:Osteosarcoma; D012194:RNA-Directed DNA Polymerase; D011863:Radioimmunoassay; D012208:Rhabdomyosarcoma; D014407:Tumor Cells, Cultured
D012194:RNA-Directed DNA Polymerase