Immunochemistry. Pergamon Press 1965. Vol. 2, pp. 235-254. Printed in Great Britain IMMUNOCHEMICAL QUANTITATION OF ANTIGENS BY SINGLE RADIAL IMMUNODIFFUSION* G. MANCINI, A. O. CARBONARA and J. F. HEREMANS Research Department of Internal Pathology, Cliniques Universitaires St Pierre, Louvain, Belgium (Received 20 January 1965) Abstract--When an unknown amount of antigen is allowed to diffuse radially from a well in a uniformly thin layer of antibody-containing agar for a sufficient time to allow all antigen to combine, the final area reached by the precipitate is directly proportional to the amount of antigen employed, and inversely proportional to the concentration of antibody. It is also shown that the temperature at which the plates are incubated has no perceptible influence upon the results. By standardizing the technical conditions of the experiment it is possible to use this principle for the immunochemical determination of antigens. In the experimental albumin-antialbumin system here described, the lower limit of the method was found to correspond to 0'0025 tzg of antigen, and to an antigen concentration of 1.25 t~g per ml. The standard deviation of the antigen determinations was less than 2 per cent of the mean. INTRODUCTION AMONG the various immunochemical precipitin methods employing diffusion in in agar gels, the technique of single radial immunodiffusion has been the least studied. By definition the single-diffusion type of precipitin reaction is performed by incorporating one of the two partners of the reaction, usually the antibody, into the agar gel, at a uniform concentration, whereas the other reactant, usually the antigen, is introduced into a well from which it is allowed to diffuse into the gel where it will react with the 'internal reactant'. The system is termed 'linear' if such diffusion takes place in one dimension, as is the case in the technique of Oudin, C1)in which narrow glass tubes are employed. The term 'radial' immunodiffusion is applied to systems in which the gel is spread out on a surface, with diffusion taking place radially, starting from a circular well. Single radial immunodiffusion has apparently first been employed by Petrie(~) in his studies on the growth of bacterial colonies on gelified media containing specific antisera. He observed that such cultures became surrounded by one or more ring-shaped precipitates, which might eventually coalesce with similar precipitates formed around neighbouring cultures of the same strain of organisms. Ouchterlony~3) was the first to exploit single radial immunodiffusion for semi- quantitative purposes, in his studies on the toxin-producing ability of different strains of Corynebacterium diphtheriae. His method was based on the observation that there existed a relationship between the width of the precipitate and the amount of antigen produced by the cultures, and an inverse relationship between the size of the precipitate and the concentration of the antiserum employed. Un- fortunately these studies were not further pursued. Feinberg< 4), Hayward and * This work was supported by Grant No. 461 from the Fonds de la Recherche Scientifique M~dicale, Brussels, Belgium; and by P.H.S. Grant No. Am 04263-04 from the U.S. Department of Health, Education and Welfare. 235