368 IMMUNOGENETICS 



The cells are then centrifuged, washed once with 2 ml. 1/100 normal rabbit serum, and 

 then resuspended in 1 ml. of 1/100 normal rabbit serum. The saline at pH 6.4 is 

 prepared by adding 100 ml. of saline to 100 ml. of a buffer composed of 32.2 ml. of 

 0.15 M Na 2 HP0 4 and 67.7 ml. of 0.15 M KH 2 P0 4 . ThepH should be checked on a 

 pH meter and adjusted if necessary with either phosphate solution, 0.15 M. 



Many antigens work in this preparation, in mixtures as well as in pure solution. 

 Optimal amounts for sensitizing cells vary around 0.25 mg. of protein or other antigen 

 per ml. treated red-cell suspension. 



Compounds other than tannic acid are used as coupling agents. For formaldehyde 

 methods, see Ingraham 631 and Czimas. 235 The hemagglutination of antigen-coupled 

 red cells is a very sensitive technique, acceptably specific if proper controls are run. 



Precipitation system. — Classical precipitation systems have been described for single, 

 relatively pure, proteins in solution, in terms of reactions with their corresponding 

 antibodies. Typically, a constant amount of antiserum is placed in each of a series of 

 tubes, and increasing amounts of antigen are added to the tubes in sequence. The 

 classical precipitin curve rises to a maximum at some intermediate antigen quantity, 

 then decreases in antigen excess. Optimal relative concentrations are expressed in 

 various ways — in terms of the rapidity of appearance of a visible precipitate, in terms 

 of a maximum quantity of precipitate formed per mg. antibody nitrogen, and in terms 

 of an equivalence zone within which all of the detectable antigen and antibody are 

 included in the precipitate, none remaining in the supernate. These measures of 

 central tendency do not usually coincide; on either side of a rather broad central zone, 

 however, flocculation times increase, the quantity of precipitate decreases, and either 

 antigen or antibody begins to be detectable in the supernatant fluid after precipitation 

 is complete. 



Precipitin tests of this sort are subject to quantitative treatment through measure- 

 ment of the amount of precipitate by nitrogen determinations or other methods. A 

 chromatographic technique for the quantitative study of the precipitin reaction, 

 especially adaptable to very small amounts of serum, has recently been described by 

 Miquel et a/. 875 Absorptions can be conducted, in simple systems, by reacting the test 

 antigen with a given antiserum at relative concentrations within the equivalence zone, 

 removing the precipitate and using the partially absorbed supernatant as a further test 

 reagent. Somewhat different results are sometimes obtained if, instead, the antigen is 

 added in small increments, the precipitate being removed after each addition, until 

 precipitation no longer occurs. In the past, there has been a tendency on the part of 

 some geneticists familiar with only the elements of immunology to assume the properties 

 of a simple antigen-antibody system for complex mixtures of indeterminate numbers of 

 related and unrelated antigens, and correspondingly complex antisera. Unfortu- 

 nately, the system becomes very uncertain under such circumstances; two or more 

 systems, precipitating in a single tube, are not often at equivalence within the same 

 zone, so that antibodies to one may remain in the supernatant after the other has 

 passed into a region of antigen excess. Soluble complexes for the second system are 



