THE RELATION OF LIPOIDS TO IMMUNITY 525 



barium hydroxid, and finally 0.1 c.c. of the phenolphthalein strontium 

 chlorid mixture. 



It will be seen that beaker 4 the antigen control contains all the 

 constituents of the test-beakers 1 to 3 except serum. To make beaker 

 4 qualitatively as well as quantitatively equal to beakers 1 to 3, 0.1 c.c. 

 of the dilute serum (the average of the dilutions of serum which are used 

 in the test) is now added to beaker 4, the reaction having already taken 

 place. 



The addition of the sulphuric acid and barium hydroxid requires 

 great care. Since the reaction depends on small differences in acidity 

 or alkalinity, it is obvious that slight errors will vitiate the results. For 

 the acid and the alkali separate pipets are used. After emptying the 

 pipet of its content of acid or alkali, the last traces adhering to the 

 inside of the pipet are removed by washing. These washings are later 

 added to the beakers to which they belong. In filling the pipets with 

 acid or alkali, the latter should first be drawn up into the pipet at least 

 once, and then emptied again before the pipet is finally filled for delivery 

 into the next test-beaker. Only by careful attention to these points 

 in the technic can reliable results be obtained. 



Reading the Results. If beakers 1 to 3 contained the antiserum to the 

 antigen used, a positive epiphanin reaction will be obtained, and if the 

 barium hydroxid and sulphuric acid were previously carefully adjusted 

 to each other, it will be found that beakers 1 to 3 will be lighter than the 

 antigen control, beaker 4. The presence of a specific antigen-antibody 

 combination has shifted the phenolphthalein end-point in the sense of 

 increased H-ion concentration. The exact differences in the alkalinity 

 between beakers 1 to 3 and beaker 4 can be quantitatively determined 

 by titration with JQQ suphuric acid, and the results expressed as a curve. 



If the antigen and antibody were not specific, the epiphanin reac- 

 tion will be negative. Beakers 1 to 3 will be more alkaline than the 

 antigen control, beaker 4, because, as previously pointed out, serums 

 alone or antigen with non-specific serums shift the phenolphthalein 

 end-point in the sense of increased OH-ion concentration. 



The results may be plotted as curves. The titration values in JQQ 

 sulphuric acid are placed on the ordinates, and the serum dilutions on 

 the abscissae. The positive values are plotted above the line and the 

 negative values below the line. No reaction is regarded as positive 

 unless it gives a titration value of at least 0.05 c.c. ^QQ sulphuric acid. 

 Values below 0.05 c.c. are easily within the limits of error. 



