PHOTOMETRIC MICRODETERMINATION OF HUMAN GAMMA GLOBULIN. II. COMPARISON OF QUANTITATIVE FLOCCULATION-NINHYDRIN METHOD WITH ELECTROPHORETIC METHOD 1

Abstract

The application to human sera of a previously described photometric flocculation-ninhydrin method for the quantitative microdetn. of gamma globulin is described. The principle of the method is based on precipitation of gamma globulin quantitatively, with a minimum contamination from the other globulin fractions, by means of 20% Na sulfite prior to the appln. of the flocculation reaction to the dialyzed globulin clot. Results obtained with this procedure are called the globulin clot-gamma globulin values. When the flocculation-ninhydrin method is applied directly to diluted whole sera, the values obtained are usually about 50% higher than the corresponding globulin clot values and are called total serum-gamma globulin values. 50 sera from both normal and disease cases were run comparatively with the 2 procedures described above and with the Tiselius electrophoresis apparatus. From these data it was detd. that the globulin clot-gamma globulin value corresponds to the electrophoretic gamma globulin value. Also that the total serum-gamma globulin value corresponds to gamma globulin plus another fraction which is believed to be composed of beta2 globulins and beta lipoproteins. 32 normal sera and 25 tuberculosis sera were run with the photometric flocculation-ninhydrin method. The gamma globulin values obtained for each series checked closely with the electrophoretic data reported in the literature by other investigators for a similar series of cases. The total serum/globulin clot ratio was found to be relatively constant for normal sera, i.e., 1.55 [plus or minus] 0.12, and to vary markedly in disease cases. It is not affected by changes in the alpha globulins, e.g., reticulocell sarcoma. It is markedly elevated by increases in beta lipoprotein, especially when accompanied by decreases in the gamma globulin, e.g., lipoid nephrosis. It decreases with increases in the gamma globulin, e.g., multiple myeloma and liver disease, but increases with decreasing gamma globulin e.g., baby sera. The many advantages of the photometric flocculation-ninhydrin method over the laborious single detn. electrophoretic method for clinical work are discussed particularly with respect to the equipment needed and volume of work performed. A statistical analysis of the data obtained with the two procedures shows that the photometric flocculation-ninhydrin method has about half of the variation of the electrophoretic method in spite of the much smaller sample, i.e., 0.01 to 0.02 ml. of serum used. The method is reproducible to about a [plus or minus] 5% error and recovers gamma globulin from sera with an avg. value of about + 105% of the amt. of protein added.