Studies on defined immunofluorescence in clinical immunopathology. III. Past progress, some new methods, and prospects for the future.

Experience with chessboard titrations over the past 9 years has shown that essentially all indirect IF staining systems that employ anti-IgG conjugates can be defined with the aid of assays for F/P ratios and/or antibody concentrations. Some improvements in the radial diffusion assays for antibody protein have been effected by refinements in methodology. Studies with a form of the Dass system showed that over a 16-fold range of light intensities, relatively little change occurred in "titers" of IgG (comparably to titers of patient sera) or in PEP conjugates as seen in DASS chessborad titrations. An analysis of 233 PEP values included in 12 literature reports on indirect IF tests indicate that 88% fall within one doubling dilution of 1/16 unit/ml or about 12.5 mug labeled Ab/ml. The DASS reactions also are within this range. Factors responsible for variation beyond these limits in indirect IF tests were analyzed. The most important factor appears to be experimental errors. Other factors include low titers of primary antibodies, some aberrant conjugates, and a recent shift toward greater sensitivity of chessboard titrations. The latter appears to be due primarily to modes of reading and should be controlled by the constant use of reference sera. It now seems possibe that future IF studies may be defined with the aid of this DASS system. To achieve this goal, it will be necessary to not only elaborate microanalytic techniques of the type that have been considered at this Conference but also to generate macroanalytic methods that can be used in parallel with them as a basis for interpreting microscopic observations both in the DASS system and in IF studies of sera and tissues.