METHODS IN MAMMALIAN IMMUNOGENETICS 367 



them from reacting with the A test cells. If, on the other hand, the test material 

 lacked A specificity, the antibodies will not be inhibited and will therefore promote the 

 agglutination of the test cells. This is the kind of system used for the definition of the 

 blood-group substances in animal plasmas and other fluids. The techniques have been 

 discussed in detail recently by Boyd, 121 and will not be further elaborated here. 



Enzymatic inhibition. — Enzymes under genetic control are often excellent antigens 

 and the effects of antibodies on enzymatic preparations are sometimes useful in dis- 

 tinguishing genetic differences. This approach has been used more productively with 

 microorganisms than with mammalian material in the past, but there is no reason why 

 it should not be useful in mammalian genetics as well. An antiserum is prepared by 

 injecting an enzymatically active material into an animal, generally a rabbit. The 

 antibodies that are formed may precipitate the enzyme from solution; the enzymatic 

 activity provides a sensitive and convenient tag for the removal of the enzyme from 

 the supernatant by such precipitation. Precipitin tests will be discussed below; we 

 will only note here that the complications of complexity in mixtures are to a consider- 

 able degree avoided in enzyme serology, because of the easy identification of the 

 particular antigen, the enzyme, in the mixture by means of its activity. In many but 

 by no means all enzyme-anti-enzyme systems, the antibody may inactivate the enzyme 

 without precipitating it. Under such circumstances, tests for enzymatic activity in 

 mixtures can be made immediately after the addition of antiserum and substrate to an 

 enzyme preparation; the results are quickly and easily read if convenient measures of 

 enzymatic activity are at hand and if there is no interference by nonantibody serum 

 factors in the assay. 



Coupling antigens to red cells. — Erythrocytes to which soluble antigens have been 

 coupled are often endowed with the property of agglutinating or giving other visible 

 reactions with antibody to the test antigens. A common technique is the use of 

 "tanned" red cells; the procedure we use is based on a report by Stavitski, 1270 and is 

 derived from the original report by Boyden. 123 Sheep blood in Alsever's solution is 

 washed three times with saline, and 1 ml. of the packed cells is diluted with about 40 ml. 

 of/>H 7.2 buffered saline, so that 1 ml. of this diluted cell suspension plus 5 ml. of dis- 

 tilled water gives a reading of 400 with a no. 54 filter in the Klett colorimeter. The 

 buffered saline, pH 7.2, is prepared by diluting 100 ml. of a buffer containing 23.9 ml. 

 of 0.15 M KH 2 P0 4 and 76.0 ml. of 0.15 M Na 2 HP0 4 , with 100 ml. saline. A stock 

 solution of tannic acid (Merck or Mallinckrodt reagent grade) is diluted with saline, 

 1/100. A further dilution, to 1/20,000 of the acid, is prepared daily. 



One ml. of the cell suspension is incubated in a water bath at 37° C. for 10 minutes 

 with 1 ml. of the 1 /20,000 dilution of tannic acid. The cells are then centrifuged gently 

 and washed with 1 ml. of pH 7.2 buffered saline, resuspended in 1 ml. saline. The 

 treated cells cannot be kept more than 1 8 hours before use. 



The antigen is prepared by mixing 4 ml. of/?H 6.4 buffered saline with 1 ml. of the 

 antigen solution in saline and 1 ml. of the tannic-acid-treated suspension of cells, in 

 this order, and allowing the mixture to stand at room temperature for ten minutes. 



