METHODS IN MAMMALIAN IMMUNOGENETICS 375 



Dr. Winn: Dr. Owen's paper is a very impressive manuscript in which he has 

 skillfully interwoven the complex terminology and methodology of immunology with 

 what I consider the equally complex terminology and conceptions of genetics. 



As most of you know, the methodology of immunogenetics would have been a 

 great deal simpler to discuss about ten to fifteen years ago. At that time it consisted 

 largely of the study of the interactions of serum antibodies with intact red blood cells. 

 Now the very rapid growth and development of the field of tissue transplantation has 

 changed all this, and we find ourselves studying a very large variety of reactions in- 

 volving not only serum antibodies but cellular or cell-bound antibodies. For the most 

 part the work that has been done with the cell-bound or cellular antibodies does not 

 really lend itself to any particular experiments that I know of in immunogenetics. 

 Generally, this work has been directed to some understanding of the mechanism of 

 graft rejection or the relationship, metabolic or otherwise, between soluble and cell- 

 bound antibodies. 



Our interest in the cytotoxic technique came about because of the limitations of 

 the red-cell agglutination test. In the mouse one finds that the red-cell agglutination 

 technique devised by Gorer and colleagues makes a really fine tool for the study of 

 antigens controlled by the genes at the H-2 locus, but there are some fourteen or 

 fifteen or maybe twice that number of loci which control the acceptance or rejection of 

 grafts. We had hoped that by studying the effects of antibodies on white cells we 

 might be able to analyze the antigens controlled by the genes at these other loci. The 

 technique is relatively simple. One mixes antiserum with white cells from either 

 lymph nodes or spleen (for some reason we do not completely understand, thymus is 

 not suitable). To this mixture is added measured amounts of complement, and after 

 a suitable period of incubation some vital dye to determine how many of the cells are 

 dead. 



Gorer has done some work on this and Schrek has also. For the most part they 

 have added enormous quantities of complement to mixtures of undiluted or only 

 slightly diluted serum and cells. We wanted to use the technique in a more quantita- 

 tive fashion, and we decided to standardize the requirement for complement and anti- 

 bodies. The longer we worked at standardizing the technique, the more difficult we 

 made it, and we soon reached the point where in one day we could analyze only a 

 single serum. (With the red-cell agglutination technique I estimate something over a 

 hundred serums could be analyzed in one day.) 



It occurred to us that one of the problems we had was the fact that this system 

 required an enormous amount of complement in terms of the amounts that are 

 normally used to lyse red blood cells, described by Dr. Owen. This suggested that 

 instead of going through this very laborious procedure of incubating the cells and 

 obtaining differential counts for each tube, we might actually add a very large excess of 

 complement and measure the amount that was used up. This has worked out very 

 nicely and we are now using a test which is patterned after that used by Osier and his 

 colleagues for the study of soluble proteins and carbohydrates. 



