ABSTRACT
A method of detection of BLV proviral DNA in the peripheral blood leukocytes of cattle is reported. Leukocytes were used without preliminary cultivation. Cell DNA was dot-hybridized with P-labeled plasmid containing a fragment of BLV proviral DNA. The sera samples taken from the cattle under study were also tested using immunodiffusion assay (ID). The results of comparison showed that dot-hybridization assay is a more sensitive diagnostic test than ID, because it detects BLV infection in apparently normal cattle, which was seronegative in ID.
Subject(s)
Enzootic Bovine Leukosis/diagnosis , Leukemia Virus, Bovine/isolation & purification , Leukocytes/virology , Proviruses/isolation & purification , Animals , Antibodies, Viral/blood , Cattle , DNA Probes , DNA, Viral/analysis , Enzootic Bovine Leukosis/blood , Enzootic Bovine Leukosis/virology , Immunoblotting/methods , Immunodiffusion , Leukemia Virus, Bovine/genetics , Nucleic Acid Hybridization , Proviruses/genetics , Sensitivity and SpecificityABSTRACT
The coprecipitation test under conditions of rocket immunoelectrophoresis was used for the analysis of human normal serum antigens associated with HBsAg structures. Antigenic determinants of human serum albumin were detected in the HBsAg composition. In the multicomponent test system, 125I-HBsAg coprecipitated with some more distinct antigenic systems in addition to albumin. Possible mechanisms of formation of classes of HBsAg structures differing in host antigens are discussed.
Subject(s)
Epitopes/analysis , Hepatitis B Surface Antigens/analysis , Serum Albumin/immunology , Antigens/analysis , Carrier State/immunology , Hepatitis B/immunology , Humans , Immunoelectrophoresis/methods , Precipitin Tests/methods , Protein BindingABSTRACT
In competitive radioimmunoassay of purified HBsAg preparations less than 1% of normal human serum antigens was found in weight equivalents. In radioimmune precipitation 125I-HBsAg was precipitated by antiserum to normal human serum proteins. The use of successive precipitation of the label with virus-specific and polyprecipitating antisera established the association of the detected antigenic determinants of the host with some portion (52%) of HBsAg structures.
Subject(s)
Hepatitis B Surface Antigens/analysis , Animals , Epitopes/analysis , Glycoproteins/analysis , Guinea Pigs , Hepatitis B Surface Antigens/isolation & purification , Humans , Immune Sera/analysis , Immunization , Precipitin Tests/methods , Rabbits , Radioimmunoassay/methodsABSTRACT
A comparative study of the antigenic and immunogenic properties of hepatitis B virus surface antigen (HBsAg) and a glycopeptide containing protective antigenic determinant "a" (aAg) isolated from purified HBsAg structures was carried out. In contrast to HBsAg structures, aAg preparations contained no antigens of normal human serum. Anti-aAg antisera unlike immune sera against HBsAg contained no antibody against normal serum proteins. The aAg preparations were at least as immunogenic as HBsAg for laboratory animals. Inoculation of aAg-immunized mice with 125I-HBsAg resulted in no accumulation of the label in the kidneys whereas it was demonstrated in the animals immunized with HBsAg. The prospects of using minimal antigenic structures carrying protective antigenic determinants as split-HBV vaccines are discussed.
Subject(s)
Hepatitis B virus/immunology , Hepatitis B/prevention & control , Viral Vaccines/isolation & purification , Animals , Dose-Response Relationship, Immunologic , Drug Evaluation, Preclinical , Epitopes/immunology , Glycoproteins/isolation & purification , Guinea Pigs , Hepatitis B Surface Antigens/immunology , Hepatitis B Surface Antigens/isolation & purification , Humans , Immune Sera/isolation & purification , Immunization , Mice , RabbitsABSTRACT
A method for obtaining a subunit inactivated vaccine preparation from the 22-nm particles of HBsAg is proposed. For inactivation of the residual infectious hepatitis B virus (HBV) the preparations were successively treated with 1% sodium dodecyl sulfate (SDS) and nucleases. In addition, thermal denaturation and ultraviolet irradiation of HBV DNA were used. As a control the biologic activity of a reference virus (SV40) was tested after the same treatment. The effectiveness of DNA inactivation was monitored by adding 3H-thymidine labeled reference virus to the vaccine preparations. The purified and inactivated HBsAg was adsorbed on Al2O3. Antigenicity was calculated on the basis of the determination of antibody in guinea pigs immunized with various doses of the vaccine, and the release of 125I- HBsAg from blood and kidneys in immunized and control mice was analyzed. Possible methods of inactivation and control of HBV vaccine is discussed.
Subject(s)
Hepatitis B Surface Antigens/immunology , Hepatitis B virus/immunology , Viral Vaccines/immunology , Animals , Deoxyribonucleases/pharmacology , Guinea Pigs , Hepatitis B Antibodies/biosynthesis , Hepatitis B Surface Antigens/isolation & purification , Hepatitis B virus/drug effects , Hepatitis B virus/radiation effects , Hot Temperature , Humans , Mice , Nucleic Acid Denaturation , Ribonucleases/pharmacology , Sodium Dodecyl Sulfate/pharmacology , Ultraviolet Rays , Vaccines, Attenuated/immunologySubject(s)
Hepatitis Viruses , Orthomyxoviridae , Chemistry , Congresses as Topic , Molecular Biology , MoscowABSTRACT
An original method for purification of HBsAg from the blood plasma of donors is described. Preparations have been obtained containing structures 22 nm in size the proteins of which form 9 bands in polyacrylamide gel electrophoresis and containing no detectable amounts of admixtures accompanying HBsAg. The purified HBsAg meets the requirements for test-antigens for radioimmuniassay and immune autoradiography. The reported method may be used for preparation of HBsAg under production conditions.
Subject(s)
Hepatitis B Surface Antigens/isolation & purification , Hepatitis B/blood , Centrifugation, Isopycnic , Cesium , Chlorides , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Humans , Immunoelectrophoresis , Methods , Radioimmunoassay , SucroseABSTRACT
A modification of radioimmunoassay (RIA) for detection of HBsAg is described. The schedule for purification and the method of iodinization of purified HBsAg are presented. The sensitivity of RIA and other immunological methods for detection of HBsAg was analysed comparatively. RIA in the modification described detected HBsAg in a concentration of 50 mg/ml.