SEROLOGICAL METHODS 201 



vials with self-sealing rubber caps. The vials of antigen are kept under 

 refrigeration and should be warmed to 37°C immediately before use. 

 Before filling a syringe for immunization, the rubber cap is swabbed with 

 70 per cent ethanol. 



To fill a syringe, the plunger is drawn back so that an amount of air 

 equivalent to the volume desired can be forced into the vial. While the 

 bottle is held inverted, the needle is inserted through the cap, the plunger 

 depressed and then withdrawn, pulling fluid from the vial. The plunger 

 is manipulated back and forth until all air bubbles have been worked out, 

 and then the desired volume is withdrawn. 



Adjuvants. Antibody formation may be enhanced by the addition of 

 various substances to the antigen. These substances, called adjuvants, 

 include alum, tapioca, certain bacteria, and paraffin oil. 



The water-oil emulsion adjuvants (Freund, 1947) may be used with 

 either particulate or soluble antigens. Water and paraffin oil are emulsi- 

 fied by the addition of lanolin or a derivative of lanolin. The addition of 

 killed Mycohacterium tuberculosis or M. hutyricum also contributes to the 

 adjuvant action. While various ratios of these ingredients have been 

 tried in different laboratories, the following proportions are recom- 

 mended: 1 part of saline solution containing the antigen, 1 part of 

 anhydrous lanolin, U.S. P., 1-2 parts of paraffin oil and 0.5 mg of killed 

 dry tubercle bacilli per milliliter of the emulsion. The tubercle bacilli 

 should be ground and incorporated into the paraffin oil before the other 

 ingredients are added. Emulsification can be hastened by alternately 

 dramng the mixture into a large syringe and expelling it. Adjuvants of 

 this type are injected intramuscularly or subcutaneously. 



When soluble protein antigens are used, adjuvant action may be 

 secured by precipitating the protein with alum. The protein solution is 

 made up at a concentration of 1-2 mg per ml, and 5 ml of sterile 1 per cent 

 alum solution is added to each 100 ml of protein solution. The mixture 

 is adjusted to pH 7.0 with NaOH. 



Immunization, Because of its convenient size and superior antibody 

 production, the rabbit is employed more frequently than other laboratory 

 animals for immunization. Antigen may be introduced intravenously, 

 intraperitoneally, or subcutaneously. The following procedures are 

 essentially the same as those recommended for inoculation in Chap. X: 



Intravenous injections are made into the marginal ear vein of the rabbit. A state of 

 hyperemia is induced by rubbing the ear with cotton soaked in xylene until the veins 

 become prominent. ^ The xylene is then washed off with 70 per cent ethanol, and the 

 antigen introduced through a 1-, 5-, or 10-ml syringe and a 25- or 26-gauge needle. 

 The needle is held with the beveled edge upward and inserted into the vein. It is 



^ It may not be necessary to use xylene if suflEicient hyperemia can be induced by 

 rubbing the ear. 



