ABSTRACT
A modification of an immunoblotting procedure was developed to detect virus receptors: a nitrocellulose membrane containing electrotransferred cellular proteins separated by polyacrylamide gel electrophoresis in the presence of SDS is treated with the whole virus (viral proteins) and an antiserum against individual viral proteins, followed by the detection of the receptors after their incubation with peroxidase labeled anti-species antibodies by enhanced chemiluminescence. The method detects adenovirus receptors for the capsid proteins including 20- and 80-kD receptors for the fiber, 20- and 40-kD receptors for the penton, and 80- and 200-210-kD receptors for protein IIIa.
Subject(s)
Adenoviridae/metabolism , Blotting, Western/methods , Receptors, Virus/metabolism , Electrophoresis, Polyacrylamide Gel , Viral Proteins/metabolismABSTRACT
Using antigen-soaked nitrocellulose sheets stamped to Teraski plates, a microtechnique was devised for detecting bound biotinylated monoclonal antibodies which readily demonstrated non-overlapping epitopes. This procedure is simple, sensitive and rapid and is useful in screening hybridoma supernatants.
Subject(s)
Antibodies, Monoclonal , Antigens, Viral , Epitope Mapping/methods , Adenoviruses, Human/immunology , Animals , Antibodies, Viral , Avidin , Binding, Competitive , Biotin , Humans , Mice , Viral Proteins/immunology , Virology/methodsABSTRACT
Two descendants of the prototype strain AD71-Washington D. C. were obtained by independent passaging for at least 18 years in Kiev, and in Budapest (Ad h 1 kappa, and Ad h 1B, respectively). By restriction endonuclease mapping, the DNA was identical corresponding to the patterns of human adenovirus type 1. In spite of this, SDS-polyacrylamide gel electrophoresis revealed that the purified hexon of Ad h 1 kappa was of lower Mr than the subunit of Ad h 1B. In contrast to this, the native capsomer (hexon) of Ad h 1 kappa exhibited lower electrophoretic mobility in agarose gel electrophoresis than the native hexon of Ad h 1B. Oligopeptide mapping of the main hexon bands from SDS-polyacrylamide gels revealed the presence of unique spots among the chymotryptic oligopeptides of Ad h 1B, too. Thus, the differences in the sensitivity to proteolytic cleavage during purification seem to have a structural basis. Antigenic analysis of the native hexon capsomers was performed using polyclonal antihexon immunsera. Immunodiffusion, immunoelectrophoresis, and competitive RIA were used for comparison. The results indicate that native hexon capsomers of Ad h 1 kappa and Ad ha 1B possess antigenic differences within the type-specific regions, nevertheless, their genetic background could not be detected by the restriction endonucleases applied. It cannot be excluded that the differences were results of altered assembly of virions under different passage conditions.
Subject(s)
Adenoviruses, Human/analysis , Capsid Proteins , Capsid/analysis , Adenoviruses, Human/genetics , Adenoviruses, Human/growth & development , Antigens, Viral/analysis , Antigens, Viral/immunology , Capsid/genetics , Capsid/immunology , DNA, Viral/analysis , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Humans , Immunodiffusion , Immunoelectrophoresis , Peptide Mapping , RadioimmunoassayABSTRACT
The major internal protein of bovine leukemia virus (BLV p24) was isolated using ion exchange chromatography on phosphocellulose and gel filtration. The specificity of the BLV p24 isolated was checked by both the radioimmunoprecipitation (RIP) and the competitive radioimmunoassay (RIA). No cross reactivity between BLV p25 and the mammalian viruses of type C and D and the retrovirus isolated from human myeloma cells RPMI8226 [8, 17] was detected. The analysis of 429 leukemia-suspected bovine blood sera resulted in the detection of 165 (38.5%) positive and 264 (61.5%) negative blood sera. A correlation of the results of radioimmunoprecipitation reaction of major internal protein p24 and immunodiffusion test on the glycoprotein antigen of BLV was observed.
