METHODS IN MAMMALIAN IMMUNOGENETICS 351 



particular individuals. In such cases no injections are necessary, although injections 

 may increase the titer or the reactivity or change somewhat the character of the anti- 

 body normally present. Materials that will aggregate the red cells of mammals, with 

 some degree of specificity, are not restricted to mammalian sera ; lower vertebrates and 

 even many invertebrates have such hemagglutinating materials in their plasma. Even 

 some plant-seed extracts provide useful reagents for distinguishing individual differences 

 within mammalian species; practically all of the useful extracts of plant seed are from 

 legumes. 101 The plant-seed extracts have been used mainly for studies of human red 

 cells, and to a lesser degree those of birds; they have as yet found relatively little 

 application in the immunogenetics of mammals other than man. The preparation of 

 the plant-seed extracts is generally simple; the pulverized seed is extracted with saline, 

 and the extract clarified by filtration or centrifugation before it is used as a test fluid on 

 red-cell suspensions. 



The classical and best-studied example of normal antibodies that make individual 

 distinctions within a species is, of course, concerned with the ABO blood groups of man. 

 Similar normal antibodies are useful in studies of cattle and sheep and occur in a 

 number of other species as well. In rats, normal antibodies reacting with the cells of 

 other individuals are rarely encountered; there is, however, at least one simply in- 

 herited difference displayed by normal antibodies in the plasma of certain rather rare 

 rats, which agglutinate in saline the red cells of almost all other rats. 147, 980 Normal 

 antibodies for murine cells also occur in certain sera of the mouse; they are, however, 

 irregular and weak. Normal antibodies for the red cells of other species are very 

 common. 



The techniques used to obtain test sera, either normal or immune, vary with the 

 kind of animal being used. In such animals as cattle or sheep, blood can be collected 

 in quantity by needle puncture of a large vein at the side of the neck. In rabbits, 

 small quantities of blood are conveniently obtained by nicking the marginal ear vein 

 with a razor blade, and permitting the blood to drop into dry test tubes. Warming 

 the ear or rubbing a drop of xylol on its tip distends the vein and promotes more rapid 

 bleeding. It is useful also to rub a light film of vaseline on the ear at the site of the 

 incision to reduce blood clotting during bleeding. Bleeding can be stopped by pressing 

 a bit of dry absorbent cotton over the incision and compressing the vein with the fingers 

 distal to the incision for a minute or two. Larger quantities of blood are obtained 

 from the rabbit by cardiac puncture, a procedure that in experienced hands is of little 

 danger to the animal. The rabbit is tied down on a board, without anesthesia; 

 anesthesia is a greater threat to the survival of the rabbit than is the operation itself. 

 Bleeding is generally done with an 18-gauge 1-^-inch needle on a 50-ml. syringe. 

 Quantities of 50 or 60 ml. can easily be taken at a time without killing the rabbit; he 

 may be bled in this manner on at least two successive days. Several different ap- 

 proaches to cardiac puncture are in use ; in one, the V formed by the lowest attached 

 ribs and sternum provides the point of orientation. The needle, on the syringe, is then 

 inserted through the second costal space, close to the sternum on the animal's left side, 



