366 IMMUNOGENETICS 



mouse antibody, is then added. The mixture is incubated at 37° C. for 30 minutes. 

 After incubation the tubes are kept at 4-6° C. until the ratio of viable to nonviable 

 cells is determined. The suspension may be stored at this temperature for as long as 

 six hours. Immediately before viability is determined, the cells are resuspended with 

 a capillary pipette and the reaction mixture is allowed to come to room temperature. 

 To one drop of the mixture is added one drop of 0.4 per cent eosin in Tyrode's solution 

 and in a hemocytometer stained nucleated cells are counted as nonviable. Non- 

 stained nucleated cells are counted as viable. About 100-200 cells are counted from 

 each tube ; the degree of cytotoxicity of the antiserum is expressed in terms of the 

 fraction of nonviable cells observed, in comparison with controls treated only with 

 normal serum and complement. Standardization of the procedure, particularly in 

 terms of the numbers of cells in the suspension, the various dilution factors, and the 

 number of units of hemolytic complement added, is essential for quantitatively repeat- 

 able results. (Dr. Winn comments on this subject in more detail at the end of this 

 chapter.) 



An interesting point has been made by Amos and Wakefield: 21 although mouse 

 complement is ineffective for cytotoxic action of murine antibody on murine cells in vitro, 

 similar cells are lysed in diffusion chambers in vivo without the necessity of added 

 complement. 



In some instances, the effects of antibodies on mobile cells provide a good index of 

 serologic reaction. Unfortunately, we have not yet encountered in mammals a 

 system as sensitive and productive in this regard as the antibody-immobilizing systems 

 of the Ciliates. Mammalian sperm-cell suspensions, for example, do not generally 

 appear to be sensitive to antibody immobilization. The ameboid motions of some of 

 the leucocytes, however, are affected by antibody, and this has provided a test system of 

 some utility. 



SOLUBLE ANTIGENS 



Molecules in free suspension or solution in the body fluids, or obtainable in 

 extracts, should provide a rich source of material for the immunogeneticist. Until 

 recently, however, techniques for working with the serology of soluble antigens could 

 cope only with extreme difficulty and uncertainty with complex antigenic preparations. 

 A rather high degree of purification by chemical or physical methods was a usual 

 prerequisite. A number of powerful methods are now available for working with 

 mixtures, and we can sample this active area only inadequately here. 



Inhibition systems. — Materials having a specificity related to one of the red-cell 

 antigens can easily be tested, taking advantage of the red-cell reaction as an indicator. 

 The general procedure is as follows: A determined amount of antiserum, say antwl, is 

 mixed with a measured amount of the solution under test, such as saliva or a saline 

 extract. After an interval, A cells are added to the mixture. If the test material had 

 A specificity, it will have combined with the anti-yl antibodies, and thus will inhibit 



