Differing Methodology and Equations Used in Quantitating Immunoglobulins by Radial Immunodiffusion—A Comparative Evaluation of Reported and Commercial Techniques

Abstract

Differing methods of plotting have been used to describe the results of radial immunodiffusion (RID). Two, logarithm of antigen concentration (log c) vs. precipitin-ring diameter (d) (Fahey) and log c vs. area (A or d²) (Mancini), are linear while rings are enlarging. Another, c vs. A (or c vs. d²), becomes linear when enlargement ceases at equivalence (Mancini). Because of methodological inaccuracy, I could not experimentally determine whether log c = d or log c = d² is appropriate; either one or both semilogarithmic graphs may be linear as precipitin circles enlarge. I could confirm the c = d² linearity at equivalence. Because smaller circles cease growth first, intermediate readings produce plots that are partially curved and partially straight. Commercial RID plates produce linear and curved plots of log c vs. d and of c vs. d². They often create too small a diameter range for great accuracy. I conclude that semilogarithmic plots are of questionable value. For greatest accuracy plates should be designed for measurement at equivalence. At this time, a straight-line graph of c = d² will include a maximal diameter range.