ABSTRACT
A method for simultaneous qualitative and quantitative characterization of virus envelope proteins and virus-neutralizing antibodies is described. The quantitative estimate is based solely on the precipitation of the antibody/antigen (Ab/Ag) complexes at equivalence under equilibrium conditions. The qualitative analysis is performed by determining the diffusion coefficients, i.e., molecular masses of antigen and antibodies. Isolation, purification, labelling of antigen and antibodies and use of any standards are not required. The analyses are carried out directly in crude biological fluids in which antigen and antibodies naturally occur. The results obtained are independent on the avidity of the Ab/Ag system to be analysed. The method was tested by using antigenic subunits of Newcastle disease virus (NDV) and four chicken anti-NDV immune sera of different avidities.
Subject(s)
Antibodies, Viral/analysis , Antigen-Antibody Complex/analysis , Antigens, Viral/analysis , Newcastle disease virus/immunology , Viral Envelope Proteins/analysis , Animals , Antibodies, Viral/blood , Antibodies, Viral/isolation & purification , Antigen-Antibody Complex/blood , Antigen-Antibody Complex/isolation & purification , Antigens, Viral/blood , Antigens, Viral/isolation & purification , Chick Embryo , Chickens , Electrophoresis, Polyacrylamide Gel/methods , Immunodiffusion/methods , Immunoglobulin G/blood , Molecular Weight , Neutralization Tests , Newcastle disease virus/metabolism , Viral Envelope Proteins/blood , Viral Envelope Proteins/isolation & purificationABSTRACT
The two-dimensional double immunodiffusion, the so-called 'two-cross' method, is introduced for determination of the size of antigenic subunits released after solubilization of enveloped viruses with non-ionic detergents. The method enables the determination of diffusion coefficients and calculation of relative molecular masses of immunoreacting components without using any standard and without prior isolation, purification or labelling of material to be analysed. The molecular masses of surface antigenic fragments of the Newcastle disease virus (NDV) disrupted by non-ionic detergent Triton X-100 were determined. Two antigenic fragments, the larger having a molecular mass between 350 and 187 kDa and the smaller from 140 to 86 kDa, were released by the action of 0.1-1.0% detergent at 20 degrees C. One fragment, whose molecular mass varied from 210 to 187 kDa, was obtained at 37 degrees C after treatment of NDV with 0.2-1.0% detergent, respectively The detergent disruption of purified NDV is studied for comparison. No difference was found whether purified or NDV in allantoic fluid was subjected to the same detergent extraction.
Subject(s)
Antigens, Viral/chemistry , Detergents/pharmacology , Immunodiffusion/methods , Newcastle disease virus/drug effects , Allantois/microbiology , Animals , Antigens, Viral/drug effects , Chick Embryo , Chickens , Newcastle disease virus/immunology , Newcastle disease virus/ultrastructure , Octoxynol/pharmacology , Particle Size , Peptide Fragments/chemistry , SolubilityABSTRACT
An immune complex (IC), composed of antigenic subunits of the Newcastle disease virus (NDV) and specific polyclonal allogeneic antibodies, was used to protect chickens against NDV. Antibodies in the IC were chicken immunoglobulin G. The antibody:antigen ratio in IC was 2.03. The IC was prepared at equivalence by direct mixing of NDV-infected allantoic fluid, treated with Triton X-100, and chicken anti-NDV serum. In order to bind NDV antigenic subunits to specific antibodies, previous isolation and purification of antigen is not required. Chickens were immunized with 1 mg IC, containing 0.3788 mg of viral antigens. The IC, prepared in the form of an oil-emulsion, was administered intramuscularly. The IC generated high levels of anti-NDV antibodies and successfully protected chickens against live virus challenge. Therefore, the IC could be recommended as a safe and environmentally convenient vaccine.
