362 IMMUNOGENETICS 



The principle of the test is as follows: Antibody globulin combines firmly with its 

 corresponding antigenic sites on cell surfaces and remains attached during washing of 

 the positive cells. Unbound globulin will be removed from the system during the 

 washing. The washed cells, having the antibody on their surface if they are positive, 

 are then exposed to antibody to the globulin itself. Reaction between the cell-bound 

 globulin and the antiglobulin results in the aggregation of positive test cells. 



To illustrate the technique, our procedure (entirely derivative from the experiences 

 of others with other systems) for the detection of individual differences among rhesus 

 monkeys may be used as an example. The tests are set up in small agglutination tubes, 

 as for saline agglutination. The test fluids are rabbit-anti-rhesus red-cell antisera 

 fractionated by antibody absorption to leave reagents that react with the red cells of 

 some monkeys and not others. Two drops of the reagent diluted in buffered saline 

 (130 g. NaCl, 12.3 g. Na 2 HP0 4 , 3.6 g. KH 2 P0 4 , 17 liters distilled water, pU 7.08) are 

 first placed in the appropriate tubes. One drop of a 2 per cent suspension of washed 

 test red cells is then added to each proper tube. The tubes are shaken and allowed to 

 stand at room temperature for at least one-half hour, after which they are shaken, 

 filled with buffered saline, and centrifuged ; the supernatant is poured off and the cells are 

 washed again with saline. To the pellet of cells remaining after pouring off the last 

 supernatant, one drop of a 1/10 dilution of goat-anti-rabbit-globulin serum, which has 

 been absorbed with rhesus red cells in order to remove anti-rhesus red-cell antibody, 

 is added. The procedure for preparing this anti-rabbit-globulin serum will be des- 

 cribed below. The test is shaken again, allowed to stand for another hour, then 

 centrifuged briefly and read for agglutination as the pellet of centrifuged cells is shaken 

 back into suspension. With this test system, antibody reagents that produce little or 

 no reliable agglutination of positive test cells in saline become very sharply discrimi- 

 nating. 



The type of antiglobulin serum used in this kind of test depends, of course, on the 

 source of the cell-sensitizing antibody. In the rhesus test described above, since the 

 test antibody was rabbit, the Coombs antiserum was anti-rabbit globulin, in this case 

 prepared in a goat. For test systems in which the cell-sensitizing antibody is other 

 than rabbit, it is commonly the rabbit that is used as the source of the Coombs reagent. 

 For example, sheep cells and rat-anti-sheep red-cell antibody can be used in a Coombs 

 system if rabbit-anti-rat globulin is used as the developing reagent. Similarly, in 

 human tests, where the Coombs system has become a very important test procedure, it 

 is rabbit-anti-human globulin that is generally used as a Coombs reagent. 



In preparing the Coombs reagent, first a predominantly globulin fraction is salt- 

 precipitated from an immune serum — for example, to prepare an anti-rabbit-globulin 

 serum in a goat, we first take a rabbit antiserum to bovine serum albumin which we 

 know to contain a good deal of antibody and precipitate this antibody by the addition, 

 at room temperature, of an equal volume of saturated ammonium sulfate solution to a 

 1/2 dilution of the serum in saline. After adjustment to pH 7.8 with 10 M NaOH, 

 the precipitated globulin is centrifuged out and redissolved in saline. For this purpose, 



