850 THE KAHN REACTION 



into a precipitation test hastened the formation of precipitates and dilution delayed 

 their formation. Thus, if the same amount, such as o.i cc, of antigen suspension- 

 serum mixture is pipetted into a series of tubes and increasing amounts of saline, 

 let us say, in 0.2-cc. quantities, are added to each tube, the delay in precipitation is 

 directly proportional to the amount of saline added, and when i cc. is added, pre- 

 cipitation may be inhibited. This observation suggested that for a precipitation test 

 in s}'philis the serum should be used in an undiluted form, the antigen employed 

 should have a high concentration of lipoids, and a minimum amount of saline 

 should be used for preparing the antigen suspension. Subsequent studies indicated 

 that excessive concentration of antigen also delayed or inhibited precipitation. This 

 necessitated the preparation of an antigen of optimum concentration — to conform 

 with the desired degree of sensitivity with s^-philitic serum. 



CHOICE OF AN ANTIGEN — ITS PREPARATION 



An antigen prepared from wet heart muscle was found to be of insufificient sen- 

 sitivity with syphilitic serum, due undoubtedly to inadecjuate concentration of 

 lipoids. Furthermore, with an antigen of this type most sera required prolonged in- 

 cubation before showing precipitation reactions. An antigen prepared by direct 

 alcoholic extraction of dry heart muscle produced non-dispersive suspensions with 

 saline, and could not be used with serum, especially after the addition of cholesterol. 

 Only by adding an excessive amount of saline to the antigen thus producing an opa- 

 lescent mixture could it be employed with serum. Then, however, the results were 

 of insufficient sensitivity. An alcoholic extract of dry heart muscle following pre- 

 liminary ether extraction (Neyman and Gager)' or an acetone-insoluble extract 

 (Noguchi)^ prepared from dry heart muscle gave more desirable results. Based on 

 comparative studies with serum, the former antigen appeared superior and ultimately 

 formed the basis for the preparation of a desirable antigen for the writer's test. 



In considering the preparation of the antigen, it was observed that the extent of 

 the ether extraction of the beef heart was directly related to the sensitivity of the 

 final product. The ether apparently removes not only the bulk of fat and non- 

 specific antigenic material, but also some specific lipoids. Therefore, when the ether 

 extraction is excessive, the antigen sensitivity is reduced. When the extraction is in- 

 sufficient, the antigen-saline suspension may contain lipoid particles which are non- 

 dispersive when mbced with serum, thus rendering the antigen unfit for use. After a 

 number of experiments, the following method was adopted for antigen preparation. 

 Twenty-five gm. of powdered heart muscle are extracted in a 250-cc. Erlenmeyer 

 flask at ten-minute intervals with 100, 75, 75, and 75 cc. of ether, respectively. After 

 the last extraction, the redried powder is extracted for three days at room temperature 

 with 95 per cent alcohol, in the proportion of 5 cc. per gram of powder. To the alco- 

 holic extract is then added 6 mgm. cholesterol per cubic centimeter. If the four ether 

 extractions were reduced to one, about four times the amount of ether (about 1,200 

 cc.) would be required for similar results. With regard to the alcohol extraction, a 

 period of several hours gives results similar to that of several days except that l)y the 



' Nej'inan, C. A., and Gager, L. T.: /. Immunol., 2, 571. 1917. 



= Noguchi, H.: Serum Diagnosis of Syphilis. Philadelphia: Lippincott, 1912. 



