ANTIGENS, HISTOCHEMICAL 



16 



ANTIGENS, HISTOCHEMICAL 



sections with antigenic components 

 other than that under investigation. 

 However, it is possible to remove such 

 interfering antibodies by absorbing the 

 anti-serum with material containing the 

 * antigenic impurities, but not contain- 

 ing the antigen it is desired to study. 

 Pains should be taken to secure as high 

 a titer of antibody as is feasible. Pro- 

 longed courses of immunization and the 

 use of adjuvants may be necessary. 



Concentration of antibodies. It is 

 often advantageous to concentrate the 

 globulin fraction of such immune sera, 

 either by precipitation of the globulins 

 with half-saturated ammonium sul- 

 phate, or the use of low temperature, 

 buffers, and alcohol after the method 

 of Nichol and Deutsch (J. Am. Chem. 

 Soc, 1948, 70, 80). If ammonium 

 sulphate is used, the ammonium ion 

 must be carefully dialyzed from the 

 final globulin solution as otherwise it 

 will interfere seriously with subsequent 

 procedures. 



Conjugation with fluorescein isocyanate. 

 The derivatives of fluorescein leading 

 to the isocyanate are not as yet com- 

 mercially available. Fluorescein amine 

 may be synthesized and converted to 

 the isocyanate for conjugation to pro- 

 tein by the procedures desci-ibed by 

 Coons and Kaplan (J. E.xp. Med., 1950, 

 91, 1). 



Conjugation of antibody solution with 

 fluorescein isocyanate. The protein con- 

 tent of the serum or isolated globulin 

 fraction to be conjugated must be de- 

 termined. It should be at least 1.7%. 

 A convenient amount of protein for one 

 run is from 300 to 600 mgm. The 

 amount must be known. 



Fix a small beaker in an ice-bath and 

 equip it with good mechanical stirring. 

 Put reagents by volume into it in the 

 following order: 

 Saline (0.9% NaCl) to make 100%. 

 Carbonate - bicar- 

 bonate buffer (0.5 



M, pH9.0) 15% 



Dioxane (distilled 



from and stored 



over sodium) 15% 



Acetone 7.5% (minus 2 ml.) 



When the solution is 4°C. or below, 

 and the isocyanate solution is ready, 

 add 



Protein solution, such that the pro- 

 tein concentration in the final mixture 

 is 1%. 



For example, there are 30 ml. of a 

 concentrated antibody solution con- 

 taining 2.0% total protein. It is de- 

 sired to conjugate 500 mgm. (25 ml.), 

 holding the remainder for control pur- 



poses. The reaction mixture: 



Saline 6.75 ml. 



Buffer 7.5 ml. 



Dioxane 7.5 ml. 



Acetone 1.25 ml. 



Protein sol 25 ml. 



Total 48 ml. 



An amount of one of the two fluores- 

 cein amine isomers such that there is 

 0.05 mgm. of amine per mgm. of protein 

 (in the example, 25 mgm.) is dissolved 

 in dry acetone and treated with phos- 

 gene. This procedure should be carried 

 out in a good chemical hood with forced 

 exhaust. Phosgene is led out of the 

 tank through concentrated sulphuric 

 acid, thence to a vessel containing 15 

 ml. of dry acetone and fitted with a 

 dropping funnel, thence to an empty 

 vessel which serves as a trap, thence 

 through 20% sodium hydroxide to 

 destroy the excess phosgene, and finally 

 through a trap with a controlled leak 

 to a water suction pump. The whole 

 reaction train should be maintained 

 at a pressure slightly below atmospheric 

 by means of the controlled leak. Phos- 

 gene is turned on and allowed to bubble 

 through to remove air and to saturate 

 the acetone in the reaction flask. At 

 the end of a few minutes, the amine dis- 

 solved in 5 cc. of dry acetone is added 

 slowly from a dropping funnel to the 

 vessel containing the acetone saturated 

 with phosgene. When all the amine- 

 containing solution has been added to 

 the reaction vessel, phosgene is allowed 

 to continue bubbling through. During 

 this time the color of the solution in the 

 reaction flask slowly changes from a 

 fluorescent green to a pale yellow. A 

 small amount of heat is generated dur- 

 ing the reaction of the amine with 

 phosgene. At the end of about 15 min- 

 utes the reaction flask is transferred to 

 a vacuum still, immersed in a water 

 bath at about 45°C., and the acetone 

 boiled off under reduced pressure. Small 

 pieces of dry anthracite can be used as 

 antibumping chips. This step removes 

 the phosgene still dissolved in the ace- 

 tone. When the reaction flask is warm 

 and dry, a greenish brown gum is vis- 

 ible on the wall; this should be dis- 

 solved in 2 ml. of acetone. This solu- 

 tion is added in toto, drop by drop, to 

 the stirred, chilled, buffered protein so- 

 lution. (This additional acetone brings 

 the total acetone concentration up to 

 7.5%.) Stirring is allowed to continue 

 for 16 hours in the cold. The solution 

 is poured into a cellophane sac and 

 dialyzed against repeated changes of 

 saline buffered with 0.01 molar phoa- 



