450 IMMUNOLOGY 



Ratio of Components. — The ratio of concentrations between in- 

 gredients is of great importance. To appreciate this it would 

 seem best to discuss separately the ratio between the various in- 

 gredients. It must be remembered, however, that they are 

 definitely interdependent. 



Concentration of Lipoids. — Tlie sensitivity of the reaction i.s 

 dependent upon the number of milligrams of lipoids per cubic 

 centimeter of extract. An optimum range is determined by 

 empirical titration, using strongly positive, weakly positive, and 

 negative sera. The preceding statement must be qualified, since 

 it holds only in general for the lipoids wdthin limited concentra- 

 tions. The lipoidal solids obtained by the extraction methods 

 used are crude mixtures of antigenic and nonantigenic lipoids, 

 while the activity of the extract is due only to the antigenic 

 lipoids. Until new methods of separation are devised, an exact 

 quantitative ratio cannot be reached. 



(1) Fraction of total lipoids extracted from heart muscle 



Cholesterol-Lipoid Ratio. — From the viewpoint of sensitivity 

 of a newl}^ prepared tissue extract, the cholesterol-lipoid ratio 

 is of still greater importance. As the percentage of cholesterol 

 in ratio to a given concentration of extractives increases, the 

 precipitation value of this complex by human sera, especially 

 syphilitic sera, increases to a maximum. Opinions differ con- 

 cerning the cholesterol-lipoid ratio. Eagle (1931) believes the 

 sensitivity of the antigen can be increased u]) to the cholesterol 

 saturation point and recommends 0.8 per cent cholesterol and 

 0.6 per cent sitosterol in antigens. Levine (1932, 1933), on the 

 other hand, believes these statements unwarranted. He contends 

 that the cholesterol concentration required for maximum sensi- 

 tivity depends upon the lipoid concentration of the individual 

 antigen. According to the Levine theory the sensitizing activity 

 increases as the cholesterol concentration increases and after 

 reaching a maximum (optimum lipoid-cholesterol ratio), addi- 

 tional cholesterol will reduce the sensitivity of the antigen, fol- 

 lowing the so-called ''colloidal type curve." It has been the 

 experience in this laboratory that excessive cholesterol concentra- 

 tions reduce the specificity of the test. Until more data are ob- 

 tained, an intermediate view between the two extremes appears 

 reasonable. 



