Within the field of immunology, ultracentrifugation has been used extensively for studies of immune complexes and immune complex formation. Sedimentation velocity studies in analytical centrifuges, and rate zonal centrifugation in preparative swing-out or zonal rotors have revealed size distributions of immune complexes formed in vivo and in vitro, and have allowed measurements of association constants of complexes formed at various antigen:antibody ratios. Density gradient centrifugation has been used both for detection and for preparative isolation of immune complexes from biological fluids.
Centrifugation studies of immune complex formation in vitro have revealed that the outcome of antigen-antibody interactions is a mixture of com-positionally different complexes, and not a single reactant as is otherwise typical of macromolecular reactions. The smallest complexes are formed in the greatest amounts, and at a given antigen:antibody ratio, immune complex formation seems to be self-limiting with respect to the maximal size of the complexes. Association constants for immune complexes have been shown to depend on 1) antigen valence, 2) antibody affinity and 3) the concentration of antibody combined with 4) the degree of antibody excess. The association constants observed in antibody excess are higher than those recorded in antigen excess for the same set of antigen and antibody. These observations demonstrate that the interactions between antigens and antibodies are a unique set of biological reactions that are different from ordinary ligand binding reactions.
Rate zonal centrifugation in sucrose gradients has for many years been the 'gold standard' among procedures devised for the detection of immune complexes in biological fluids. After centrifugation, immune complexes are quantitated as immunoglobulins with increased sedimentation rates. The procedure is inconvenient for screening large numbers of samples, but has been used to confirm the presence of immune complexes in patients with a wide range of neoplastic and chronic inflammatory disorders.
Animal studies have shown that several physico-chemical properties of immune complexes are important for the development of disease. When complexes are formed with positively charged antigen or antibody, or with antibodies of low affinity or avidity, there is increased risk for the development of glomerulonephritis. In addition, the size of the complex is important, but the critical size, as measured by rate zonal centrifugation, may vary between individual antigen-antibody complexes.
For analysis of the components present in immune complexes, preparative studies have been accomplished using rate zonal centrifugation in sucrose or in combined sucrose-polyethylene glycol gradients. DNA-antibody complexes were isolated by the former procedure from the serum of patients with systemic lupus erythematosus. The sedimentation coefficients of immune complexes varies, however, and rate zonal centrifugation is therefore not an ideal method for preparative work because the complexes will be distributed over several gradient fractions. In contrast, after isopyknic centrifugation, immune complexes can be recovered as sharp bands in the gradients. Analysis of immune complexes isolated by isopyknic centrifugation in sucrose gradients has indicated that antigens related to those present in a retrovirus-like particle may participate in immune complex formation in patients with psoriasis and seronegative arthritis.
Recent developments within the field of ultracen-trifugation include air-cooled, benchtop centrifuges and a novel analytical centrifuge. The benchtop centrifuges are ideal for rapid, preparative work in small volumes and have also been used for analytical studies. Compared to the previous generation of analytical ultracentrifuges, the new centrifuge permits analytical centrifugation to be carried out with considerably increased sensitivity. Although ultracentri-fugation has to some extent been replaced by other techniques, it still has great potential, both in analytical and preparative work, and remains an invaluable tool for the immunologist.
See also: Affinity; Immune complexes; Protein separation techniques; Valency of antigens and antibodies.
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