Improved methodology for quantitative determination of serum and milk proteins by single radial immunodiffusion

Single radial immunodiffusion is a simple inexpensive method for quantitating specific proteins in a heterogenous mixture. However, it has been criticized for its lack of accuracy and repeatability; coefficients of variation of 25% have been reported by some workers. We have adopted several refinements to the single radial immunodiffusion techniques which have appeared in the literature and have made several innovations of our own. Also, as an integral part of the technique, we have developed a computer program that determines the best-fitting curve for a given set of standards, applies this curve to the raw ring-diameters, and corrects the data for plate variation and sample dilution. This program has reduced errors greatly in calculating protein concentration since all data handling beyond the recording of the precipitant ring diameter is by computer. It also allows for the storage, retrieval, and analysis of large volumes of data on many different proteins simultaneously. The overall result of this system has been to decrease the coefficients of variation to below 5% for all the milk and serum proteins tested.