360 IMMUNOGENETICS 



a/. 543 add a drop of a papain solution directly to a drop of test antiserum on a slide and 

 then add a drop of the red-cell suspension. This is reported to give an accurate and 

 rapid test system. These and other test systems are ably described by Race and 

 Sanger. 1036 



Another method of setting up and reading tests is to examine the pattern of the 

 sedimented cells after they have settled out by gravity. This may be done in ordinary 

 round-bottomed test tubes, or through the use of plates containing round- or sloping- 

 bottomed depressions. Antibody-sensitized cells often behave in a nonspecifically 

 sticky fashion so that they do not sediment to the lowest point of the depression but 

 remain attached to the sides and bottom of the tube or depression. Frequently, 

 agglutination is easily read under low-power magnification or even macroscopically. 

 In such receptacles, and in cases in which the agglutinates are very fragile, positive 

 reactions can often be identified in the undisturbed plates much more easily than they 

 can be read by ordinary tube and microscopic methods of examination. Hildemann 

 (personal communication) reports good results through the application of such a 

 system to murine red-cell typing. 



3. Hemolysis. Agglutinating tests are sometimes convertible to hemolytic ones 

 through the addition of complement to the system. The most common source of 

 complement is fresh normal guinea-pig serum, a drop of which, usually at a dilution 

 of 1/8, added to the saline tube-agglutinating system described in the preceding 

 section, may cause sensitized red cells to lyse. Another common source of complement 

 is fresh normal rabbit serum, which is generally used at a somewhat lower dilution, 

 usually 1/2. The optimal source of complement in any new system is unpredictable; 

 for example, certain antibody reagents will lyse positive cattle red cells far better with 

 rabbit complement than with guinea pig, whereas others work much better with guinea 

 pig complement. Rabbit complement is best for testing mouse cells with murine 

 antibody. 574 



Rabbit, guinea-pig, or other normal sera sometimes contain antibodies against the 

 test cells and may cause the cells to lyse without the addition of reagent antibody. In 

 such a circumstance it is often advisable to screen individual rabbits or guinea pigs in 

 advance and to select as complement sources those whose sera lack normal antibody 

 against the red cells to be tested. Alternatively, the normal antibody can be removed 

 from a serum by a quick absorption at 0° C. ; we generally use a volume of packed red 

 cells for this absorption about equal to the volume of undiluted normal serum to be 

 absorbed, and conduct a 5-minute absorption with prechilled cells and serum and with 

 the tubes in an ice bath, followed by centrifugation in a refrigerated centrifuge. The 

 antibody is removed by the absorbing cells, but the complement activity is little 

 affected. Complement is a relatively labile material; it should never be permitted to 

 sit out at room temperature for any extended interval before its use in the tests. It is 

 inactivated by heating at 56° C. for 20 minutes. 



Hemolytic systems can often be rendered more sensitive by the use of an isotonic 

 buffer containing magnesium and calcium for the diluting and suspending medium. 



