METHODS IN MAMMALIAN IMMUNOGENETICS 357 



factors may sometimes be involved in the fixation of antibody in absorption, part of the 

 logic upon which absorption analyses are based becomes doubtful. Most immuno- 

 geneticists generally choose to believe that absorption tests are the last compelling 

 resort for ascertaining the specificity of questionable test cells ; it is now clear, however, 

 that even this resort is not unfailingly reliable. Nevertheless, in addition to their 

 utility in providing simpler test fluids as reagents, antibody-absorption procedures are 

 a useful and often irreplaceable source of detailed information about cell and antibody 

 specificities. If one were to give a value estimate, he would probably conclude that 

 more misleading conclusions have entered the literature through failure to conduct 

 adequate absorption analyses than through the overzealous application of this 

 technique. 



When one is working with so small a mammal as the mouse, and particularly in 

 segregating generations when recourse cannot be had to pooled bloods from numerous 

 representatives of an homogeneous group, shortage of absorbing cells and of antisera 

 may become a limiting consideration. Under these conditions, in vivo absorptions, as 

 conducted by Amos, 19 may prove useful. For example, when 0.1 ml. of an antiserum 

 produced in BALB/c mice against the C57 black leukosis EL4 was injected intravenously 

 into C57 black mice and the recipient animals were bled at intervals, the titer of the 

 serum of the recipient animal against ^4-cells had fallen below detectable levels after 

 30 minutes. Injected into the BALB/c mice, the same amount of serum remained at a 

 titer of 1 /256. The passively administered antibodies persisted in the serum of the 

 nonreactive mouse with a half-life of about two days or longer. Intraperitoneal 

 rather than intravenous injections are now commonly used for this technique. Given 

 an antiserum that can withstand the dilution factor observed (about 1 /20) , this tech- 

 nique therefore provides a convenient method of absorption analyses in murine test 

 systems. 



Red-cell test systems. — A variety of test systems have been elaborated for immuno- 

 genetic studies of red cells. These have been described in detail in research papers and 

 books on technique in the literature, 241 ' 1290 and only a brief survey will be undertaken 

 here. 



1. Saline agglutination. Tests for saline agglutination of red cells are commonly 

 performed in small test tubes (10 x 78 mm.), into which one or two drops of the 

 diluted test reagent are dropped from a 1-ml. pipette equipped with a 1-ml. rubber 

 bulb. A drop of a suspension of the washed test cells is then added in the same manner 

 — usually, about a 2 per cent suspension. When a number of samples of cells are to 

 be tested against a number of reagents, usually the reagents are arranged in rows in a 

 series of racks, and the test cells are added in columns, to provide a complete test 

 pattern. For some reagents, it may be necessary to allow the test to stand for an hour 

 or two or more after shaking the cells into smooth suspension, allowing the cells to 

 settle by gravity and then reading for agglutination either macroscopically or micro- 

 scopically or both. Frequently, however, the test can be read only five minutes or so 

 after the cells have been added, sedimenting the cells by brief centrifugation. The 



