198 



THE PRINCIPLES OF IMMUNOLOGY 



TlTRATION OF ANTIGEN FOR FIXING PROPERTY. 



Other Reagents for the Test. The methods of obtaining guinea-pig blood, 

 the hemolytic amboceptor and sheep blood have been described (see pages 

 117 and 127). Various methods are in vogue for obtaining human blood. In adults 

 the simplest satisfactory method is to obtain the blood by puncture of one of 

 the large veins in the cubital fossa anterior to the elbow-joint. The needle should 

 have a calibre of about I. m.m. and although sharp should not have an elongated 

 point. The fossa is cleansed with soap and water followed by alcohol. A tourni- 

 quet is applied at the middle of the upper arm and the patient instructed to 

 " make a fist " several times until the veins stand out prominently. The sterile 

 needle is inserted and the blood collected in amounts of 5 to 10 c.c. in a 15 c.c. 

 centrifuge tube. The tourniquet is released before the needle is withdrawn 

 and the wound sealed with collodion. The blood is allowed to clot, the clot 

 separated from the side of the tube by means of a sterile needle, and allowed 

 to contract for several hours in the refrigerator. The tube is then centrifuged 

 and the serum pipetted into another tube so as to avoid hemolysis. The serum 

 is inactivated at 56 to 60 C. for one-half hour before testing, unless the test 

 is to be made by a modification which employs human complement. Methods 

 have been suggested in which the amount of blood obtained by puncture of finger 

 tip or ear lobe provides sufficient blood. In infants or obese adults blood may be 

 obtained by the use of a scarifier and cupping. Bleeding from the longitudinal 

 sinus, from the great toe and from the heel are also practised in infants. 



The Test. With the reagents at hand the test is set up with one or more 

 antigens. In many laboratories different types of antigen are employed, as for 

 example an acetone insoluble antigen, a cholesterolized alcoholic extract of 

 heart muscle and a non-cholesterolized alcoholic extract of heart muscle. Others 

 are employed as the operator sees fit. Antigens .may deteriorate, so that it is 

 wise to have several on hand and under observation in the test. The protocol 

 shows 2 antigens of the same strength. All the elements in the test are to be 

 controlled to prove that they are not antilytic and to show that the hemolytic 

 system operates properly. In addition it is essential to have controls with a 

 known positive and a known negative serum. The antigen, complement and 

 hemolysins are diluted so that the proper quantity of each is contained in i.o c.c. 

 It appears to be desirable to add the human serum without dilution. This is 

 done with a i.o c.c. pipette graduated in hundredths of a cubic centimeter. 

 The dotted lines in the body of the protocol indicate that salt solution is to be 

 substituted in quantities of i.o c.c. 



