354- IMMUNOGENETICS 



have sufficient "adjuvant" activity of their own. In some systems, such as the auto- 

 sensitization of mice to red cells of their own type, Freund's adjuvant may play an 

 irreplaceable role. 



Avoid young animals, or animals in poor physical condition, for the preparation 

 of antisera. We commonly use a market strain of New Zealand white rabbits and 

 require that each animal weigh at least 5 lb. before injections are begun. Mice 

 achieve sufficient serologic maturity after they are about 6 weeks of age, but they are 

 commonly used at 12 weeks. 



Mice are commonly immunized by way of intraperitoneal or subcutaneous injec- 

 tions, although intravenous injections into the lateral tail veins are not difficult. Rats 

 are somewhat more difficult to inject intravenously than are mice; intravenous injec- 

 tions of rats are possible, but intracardiac injections or injections by cannulating a 

 femoral or carotid vein are often easier. 237 Mice do not produce good antisera to 

 murine red cells in response to the injection of blood; instead, macerated tissues 

 (particularly spleen) or tumor transplants are commonly used to sensitize them. We 

 have evidence that better hemolytic antisera are likely to be produced in mice if they 

 first reject a skin transplant from the donor line; shortly after the rejection of the 

 sensitizing transplant we give the first of two intraperitoneal injections of splenic cell 

 suspensions (1 donor spleen per 3 recipients) at weekly intervals, then bleed the reci- 

 pients for test serum about ten days after the second injection. 



Preparation of antibody reagents. — Under the simplest and most fortunate circum- 

 stances an antiserum, either normal or immune, may contain an antibody population 

 recognizing only a single difference among the individuals tested — those having the 

 specificity recognized by the antibodies, and those lacking it. Serologically, this simple 

 situation is revealed when absorptions are conducted with the red cells of a panel of test 

 individuals. Packed, washed, red cells of each test individual are mixed with a saline 

 dilution of the test serum and thrown down in a centrifuge. The supernatant, absorbed 

 serum is then used as a test reagent; in the simplest situation all positive cells will 

 absorb the antibody for all other reactive cells, whereas negative cells have no effect on 

 the antibody population. Genetically, this simple serologic situation is almost always 

 found to reflect a single genetic alternative; positive individuals are found to be either 

 homozygous or heterozygous for an allele producing the test specificity, and individuals 

 lacking it are homozygous for an allele of the gene so defined. 



Much more commonly, an antiserum will be found to contain two or more anti- 

 body populations of discrete specificity. Serologically, this situation is revealed by 

 absorption analyses; not all reactive cells will remove the antibody for all others. 

 Table 60 shows an absorption analysis leading to the recognition of two specificities, 

 designated A and B, by means of anti-^4 and anti-Z? antibody populations in a particular 

 antiserum. An analysis like that illustrated in table 60 should be followed by further 

 absorptions on the reagents postulated from this set of absorptions to be of single 

 specificities; the test fluid remaining after absorption with cell no. 2, for example, 

 should now be further absorbed with each of the reactive cells separately, and it should 



