io8 THE PRINCIPLES OF IMMUNOLOGY 



infants have been observed to precipitate the protein of cow milk. It 

 appears possible, then, that from absorption through the intestinal tract 

 early in life the protein may appear in the circulating- fluid in native 

 form and thus stimulate the formation of precipitins. These, of 

 course, are not normal precipitins in the sense indicated above for goat 

 serum but similarly are always precipitins of low titer and not 

 highly specific. 



Experimental Demonstration. For practical demonstration of the reaction 

 the serum proteins are the simplest to use. For immunization of animals the 

 intravenous route is by far the best, injecting 2.0 c.c. serum at five-day intervals 

 and bleeding ten days after the last dose. Three doses are usually sufficient, but 

 five doses frequently give a precipitin of very high titer. In order to get clear 

 serum it is necessary to fast the animal for twenty-four hours before bleeding, 

 thus eliminating fat from the serum. Rabbits are the animals usually selected 

 for this purpose because of their availability in the laboratory and because of the 

 relative ease of intravenous injection. Hektoen has shown, however, that the 

 domestic fowl is a prompt, reliable and liberal producer of precipitins, even more 

 so than the rabbit. A single intraperitoneal injection of 20 c.c. of defibrinated 

 blood or serum in most cases yields at the end of ten or twelve days a precipitating 

 serum of sufficient strength and specificity for practical purposes; but on ac- 

 count of an unwelcome tendency to give non-specific reaction, great care must 

 be exercised in all the tests with fowl antiserum, and it is necessary to use 

 salt solution 1.8 per cent, in strength. Man is also a good producer of precipi- 

 tins, as has been shown by investigation of human serum after the individual 

 has been given doses of horse serum. For performing the test, narrow tubes, 

 not more than 5 mm. in diameter are most suitable in order to save reagents 

 and get clear-cut results. Instead of diluting the antiserum, it is customary 

 here to dilute the antigenic serum. Nevertheless the titer thus obtained is 

 referred to the immune precipitin. Two methods are in use, the original 

 method of actual precipitation, and the Fornet ring test. In either case dilutions 

 of the antigenic serum are made i-io, i-ioo, 1-1,000, 1-10,000, 1-100,000, and 

 1-1,000,000, with provision for a salt solution control. After such a preliminary 

 test the serum may be more accurately titrated with intermediate dilutions. For 

 determining precipitation i.o c.c. of each dilution is run into tubes with a nipple 

 pipette, and to each is added o.i c.c. immune serum, the latter settling into the 

 dilutions, without shaking. Immediate observations are made and then the 

 mixtures incubated for one hour at 37 C., followed by subsequent observation, 

 and if desirable further observation after twenty-four hours in the ice chest. 

 The Fornet ring test is more clear-cut and is more commonly used. Here o.i c.c. 

 immune serum is placed in the tubes and the dilutions of antigen added with 

 nipple pipettes, so as to form a contact ring as in the Heller test for albuminuria. 

 A white ring gradually spreading both up and down indicates a positive re- 

 action. A good immune serum titrates 1-10,000 or more, although titers of 

 1-100,000 are obtainable. 



The production of bacterial precipitins is somewhat more difficult 

 and requires longer immunization, the precipitins appearing, as a rule, 

 somewhat later than the agglutinins. Zinsser recommends the use of 

 salt solution emulsions of agar cultures killed at 6o-7o C., rather 

 than extracts or filtrates of broth cultures. The intravenous route is 

 best unless the bacteria are extremely toxic, when the subcutaneous 

 or intraperitoneal method may serve. Intravenous injections should 

 be given four or five times at five- or six-day intervals, the animal 

 (rabbit) being bled eight or nine days after the last injection. Ex- 

 tracts of bacteria for similar purposes are obtained by growth for 

 three weeks to three months in slightly alkaline broth, filtration through 

 Berkefeld filters and injection of the filtrate. Salt solution suspen- 

 sions of agar cultures may be shaken in a machine for twenty-four 



