METHODS IN MAMMALIAN IMMUNOGENETICS 859 



Dextran-diluted test reagent, the cells are allowed to settle for a period of an hour or 

 longer, and most of the supernatant fluid is then pipetted off with a capillary pipette. 

 The sedimcnted cells and a small amount of the remaining fluid are then streaked 

 across a microscope slide, and the slide is rocked back and forth manually five or six 

 times. In negative tests the cells may at first appear to be aggregated, but they spread 

 smoothly during the rocking procedure. In positive tests they remain conspicuously 

 clumped. The technique is very sensitive to handling; negative tests may be made to 

 appear positive if they are insufficiently rocked, or positive tests may be made to appear 

 negative if the fragile aggregates are broken up during the pipetting procedure or 

 through overmanipulation. With experience, however, this test becomes entirely 

 reliable and is stil! the technique of choice for routine typing and testing of red cell 

 antigens in the mouse, f Two other techniques for red-cell typing in the mouse, 

 agglutination in saline medium after enzyme treatment of the red cells and hemolysis, 

 will be mentioned below. 



Treatment of red-cell preparations with particular enzymes sometimes renders 

 them subject to agglutination by antibody that does not cause the reliable agglutina- 

 tion of untreated cells. The technique for trypsin treatment recommended by Morton 

 and Pickles 900 is essentially as follows: A stock solution of 0.1 gram of crystalline 

 Armour trypsin is prepared in 10 ml. of N/20 HC1; this can be kept for several months 

 at 4° C. One part of this stock solution is diluted with nine parts of M/10 phosphate 

 buffer (/>H 7.7) on the day it is to be used. Four volumes of the diluted trypsin are 

 added to one volume of well-washed packed cells; the mixture is held at 37° G. for \ to 

 1 hour. After a single additional washing the cells are made up to 5 per cent concentra- 

 tion in saline. In Rh testing, the test sera as well as the suspensions of cells are equili- 

 brated to 37° C, and the test is incubated at that temperature in order to avoid false 

 positive reactions caused by cold agglutinins. In applying enzymatic treatments to 

 other mammalian red cells, adjustments frequently have to be made for the particular 

 system under test, in order to avoid false positive reactions. 



Numerous other enzymes have also been used to treat red cells; in addition to 

 trypsin, papain and ficin are most frequently used. 1388 A rapid and simplified enzyme 

 test has come into common use in human red-cell typing. Low's technique 806 is as 

 follows: a papain solution is made up by grinding 2 g. of papain (papayotin Merck 

 1 : 350) in a mortar with 100 ml. of M/15 phosphate buffer, />H 5.4. This is filtered, 

 and 10 ml. of 0.5 M cystein is added to activate the enzyme. The solution is diluted 

 to 200 ml. with the buffer, incubated for 1 hour at 37° C, and stored at -20° G. It 

 retains its activity over long periods under these circumstances. Three parts of this 

 enzyme solution are mixed with one part of the test serum. Then equal parts of the 

 serum-enzyme mixture and a 3 per cent red-cell suspension are mixed in a test tube 

 and incubated for 2 hours at 37° C. before being read for agglutination. Hekker et 



fSee, however, Stimpfling, J. H., Transplant. Bull. 27:109, 1961 for a technique using 

 polyvinylpyrrolidone. 



