ABSTRACT
A new mathematical model for antigen-antibody interactions has been developed. The new model is based on the assumption that the formation of complexes between a bivalent antibody and a multivalent antigen is determined thermodynamically by the concentrations and valences of antigen as well as antibody, together with one association constant which is common to all mutual interactions. Formulae have been derived for calculation of the distributions of compositionally different antigen-antibody complexes either from knowledge of equilibrium concentrations of free antigen and antibody, or from knowledge of total amounts of antigen and antibody in the system. A computer program for these calculation is described. The model is found to yield precise predictions of the formation of soluble immune complexes, as studied by zonal centrifugation. It is found through use of the model that 'complex formation' as such differs in binding characteristics from adsorption, especially for high concentrations of antigens and antibodies. 'Complex formation' implies that association constants estimated through a Sips plot method will vary with antibody concentration, and that certain curvatures of the lines in a Sips plot reflect inherent properties of complex-forming systems.
Subject(s)
Antigen-Antibody Complex , Models, Biological , MathematicsABSTRACT
The zonal ultracentrifuge was used for separation of Treponema pallidum from large volumes of rabbit testicular syphiloma extracts by continuous-flow centrifugation in a cesium chloride density gradient. The gradient was linear with radius from a density of 1.05 to 1.36 g/ml. Operating speeds were 15,000 rev/min for the continuous-flow phase and 25,000 rev/min for a 30-min banding period. A total of 9 x 10(9) (24.3%) treponemes were recovered from the original extract. Of the treponemes recovered, 88% formed a band at a density of 1.170 to 1.211 g/ml. Within the limits of present methods of assay, these fractions were relatively free from testicular particulates and protein when compared with treponemes recovered after differential centrifugation. Observations of the isolated fractions by dark-field and electron microscopy indicated a lack of gross morphological damage to T. pallidum. Their antigenic characteristics were also retained, as evidenced by their ability to react with syphilitic sera in the indirect fluorescent-antibody procedure.
Subject(s)
Centrifugation, Zonal , Treponema pallidum/isolation & purification , Animals , Centrifugation, Density Gradient , Cesium , Chlorides , Evaluation Studies as Topic , Fluorescent Antibody Technique , Goats/immunology , Immune Sera , Immunodiffusion , Male , Microscopy, Electron , Rabbits , Syphilis/microbiology , Testis/microbiology , Tissue Extracts , Treponema pallidum/cytology , Treponema pallidum/enzymology , UltracentrifugationABSTRACT
New zonal centrifuges can conveniently process as much as five orders of magnitude (10(5)) greater sample volumes than conventional swinging-bucket rotors. The continuous-sample-flow-with-banding versions may be used in series with ancillary purification procedures. Here we have studied the combined process: absorption and elution of influenza virus with barium sulfate followed by concentration and isopycnic banding of the virus in a buffered sucrose gradient. Kilogram quantities of impurity have been rapidly separated from grams of purified virus, which have been conveniently concentrated several hundred-fold by the purification process. Experimental vaccines made by these procedures are being evaluated.
Subject(s)
Centrifugation, Density Gradient , Influenza Vaccines , Orthomyxoviridae/isolation & purification , Animals , Barium Sulfate , Chick Embryo , Culture Techniques , Hemagglutination, Viral , Influenza Vaccines/isolation & purification , Methods , Mice , Orthomyxoviridae/immunology , Rabbits , Virus CultivationSubject(s)
Centrifugation, Zonal/instrumentation , Animals , Blood Proteins , Methods , Pollen , Rats , Ribosomes , Spleen/cytologyABSTRACT
A new zonal centrifuge rotor (B-IX) which combines continuous sample flow centrifugation with isopycnic banding has been used to isolate and concentrate respiratory syncytial virus from liter volumes of culture fluid. This isolation technique utilizes a sucrose density gradient to trap and isopycnically band the virus particles, and permits recovery of the particles from the rotor in an unaggregated condition.
