ELECTROPHORETIC STUDIES OF BOVINE SERUMS WITH RESPECT TO BRUCELLA by C h a rle s Leonard San Clemente A THESIS S u b m itted t o t h e G raduate School o f M ichigan S t a t e C o lle g e o f A g r i c u l t u r e and A p p lied S c ie n c e i n p a r t i a l f u l f i l m e n t o f th e r e q u ire m e n ts f o r t h e degree o f DOCTOR OF PHILOSOPHY Department o f B a c te r i o l o g y March, 19^4-2 ProQuest Number: 10008421 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008421 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346 E l e c t r o p h o r e t i c S t u d i e s o f t h e P r o t e i n s o f Bovine Serums Yfith R esp ect t o B r u c e l l a I, II. III. In tro d u c tio n V. V I. V II. 1 M a t e r i a l s and M e t h o d s ........................................................................................ . ,1|. E x p e rim e n ta l and D is c u s s io n ................................ ..6 A. E l e c t r o p h o r e t i c s t u d i e s o f norm al c a l f serums b e f o r e and a f t e r i n g e s t i o n o f c o lo s tr u m . ................................................6 B. E l e c t r o p h o r e t i c p a t t e r n s of serum from an i n f e c t e d c a l f drawn a t i n t e r v a l s • 11 C. E l e c t r o p h o r e t i c p a t t e r n s o f h e i f e r serums a f t e r v a c c i n a t i o n w i t h t h e U. S . B. A. I . B r u c e l l a s t r a i n 1 9 - .. 1 3 D. E l e c t r o p h o r e t i c p a t t e r n s of a g e n e r a l s e r i e s o f bovine ........................ seru m s. 15 E. M o b il i t y - pH r e l a t i o n s h i p o f t h e p r o t e i n s i n a serum c o n t a i n i n g B r u c e l l a a g g l u t i n i n s i n a h ig h t i t e r and i n one c o n t a i n i n g no a g g l u t i n i n s . .......................... 20 F. E l e c t r o p h o r e t i c diagram s o f a h o rs e and cow serum a f t e r i n j e c t i o n w i t h a w a t e r - s o l u b l e , c ru sh e d c e l l f r a c t i o n from B r u c e l l a . ........... G. IV• ........................................................... E l e c t r o p h o r e t i c s t u d i e s of B r u c e l la a n tis e r u m a f t e r a g g l u t i n i n a b s o r p t i o n . . . * .V. .................... 23 ....2 8 H. E l e c t r o p h o r e t i c s t u d i e s of B r u c e l l a a n tis e r u m f r a c ­ t i o n a t e d w i t h ammonium s u l f a t e . ............ . . . . . . . . 3 2 I. E l e c t r o p h o r e t i c s t u d i e s o f B r u c e l l a a n tis e r u m f r a c ­ t i o n a t e d w it h e t h a n o l - w a t e r m ix tu re s a t low t e m p e r a t u r e . *35 Summary ............ .................................................................................. Acknowledgment ................ J42 L i t e r a t u r e C i t e d ....................................................................................................1 )3 E l e c t r o p h o r e t i c P a t t e r n s ............................................... I4.9 ELECTROPHORETIC STUDIES OF TEE PROTEINS OF BOVINE SERUMS WITH RESPECT TO BRUCELLA I t h as lon g b een known t h a t when an im als o r humans a r e i n j e c t e d , o r become i n f e c t e d w i t h B r u c e l l a , t h e r e f o llo w s an ap p e a ra n c e o f demon­ s t r a b l e a n t i b o d i e s , o f t e n i n h ig h t i t e r , i n t h e bloo d serum . The r o l e p la y e d by t h e s e a n t i b o d i e s in s h o r t e n i n g t h e d u r a t i o n o f t h e d i s e a s e or i n t h e d e s t r u c t i o n o f t h e in v a d in g organism s i s as y e t unknown. So f a r no jin v i t r o method has b een d e v ise d w hich would i n d i c a t e a n t i b a c t e r i a l a c t i v i t y on th e p a r t o f B r u c e l l a a n t i s e r u m s . There have b een o b t a i n e d , however, i n c e r t a i n e x p e rim e n ts by Huddleson and P e n n e ll (19) d a t a w hich s u g g e s t t h a t a p r o p e r l y p re p a re d a n t is e r u m p la y s an im p o rta n t r o l e in t h e n e u t r a l i z a t i o n o f t h e t o x i c components of t h e b a c t e r i a l c e l l * I t w as, t h e r e f o r e , t h e o r i g i n a l pu rp o se o f t h i s s tu d y t o d ev elo p a p u r i f i e d and c o n c e n tr a t e d B r u c e l l a a n t i s e r u m . A general but n o t e x h a u s t iv e i n v e s t i g a t i o n in c lu d e d t h e employment o f t h e more common methods o f serum p u r i f i c a t i o n , such as t h e u se of v a r io u s s a l t s l i k e t h e s u l f a t e s o f ammonium ( 1, 11, 12, 15, 16) , and sodium ( l l |, 17, 21) , t h e c h l o r i d e s o f z i n c , antim ony, and aluminum ( 7 , 19)5 v a r i o u s o rg a n ic s o l v e n t s l i k e a l c o h o l (9)> a c e to n e (1)6), and e t h e r ( 32) 5 o f v a r i o u s p ro c e d u re s l i k e h e a t i n g o f t h e serum; or th e e f f e c t o f c e r t a i n enzymes ( 3 6 ,3 8 , 3 9 >k-0) , u n d e r c o n t r o l l e d c o n d i t i o n s o f c o n c e n t r a t i o n , pH and t e m p e r a t u r e . Most o f t h e s e methods proved f r u i t l e s s i n th e f i r s t i n s t a n c e , o r s e c o n d ly , * T his s tu d y was made p o s s i b l e by g r a n t s - i n - a i d from t h e Bureau o f Animal I n d u s t r y , U. S. D epartm ent o f A g r i c u l t u r e . -2 a growing r e a l i z a t i o n c o n c e rn in g t h e ig n o ra n c e o f t h e n a t u r e o f t h e a n t i ­ body so u g h t made even t h e a p p r e c i a t i o n o f any p r o g r e s s d i f f i c u l t t o a p p r a i s e . At about t h i s t i m e , f o r t u n a t e l y , t h e h ig h ljr improved e l e c t r o ­ p h o r e s i s a p p a r a t u s o f T i s e l i u s (1+7 ) b e g in n in g t o come t o th e g e n e r a l a t t e n t i o n of s c i e n t i f i c w ork ers i n Am erica. I t s v e r s a t i l i t y and a p p l i c a ­ b i l i t y has made i t one o f t h e g r e a t e s t t o o l s t h a t t h e b io c h e m ist p o s s e s s e s i n a t t a c k i n g t h e enigma of t h e p r o t e i n s . I t seems n a t u r a l t h a t t h e b lo o d plasm a p r o t e i n s sh o u ld have been s u b j e c t e d t o e l e c t r o p h o r e s i s p r e f e r e n t i a l ­ l y s i n c e a n t i b o d i e s a r e found p r i m a r i l y i n t h e b lo o d . S tu d ie s on h o rs e serum (1+,5 *1+8 *50 *51*5 2 *53 *51-1-) were f o llo w e d im m ediately by e l e c t r o p h o r e t i c o b s e r v a t i o n s o f t h e serums o f th e human ( 5 *2 0 , 21 , 28, 3 0 ) * t h e g u in ea p ig (1+5)* t h e r a b b i t (2+5 *14-9 *5 0 , 51*52 ) , and th e b o v in e (i+,1+5). I n g e n e r a l a n t i b o d i e s have been found t o be a s s o c i a t e d w i t h t h e i K - g l o b u l i n (1+9*51*53) • However, i n one c a s e (52-1-) an tipn eu m o co ccal a n t i ­ body was found as a new component, T^, i n h o r s e serum s h o r t l y a f t e r b e ­ g in n in g h y p e rim m u n iz a tio n . A n t i t o x i n a p p e a rs e n t i r e l y as a s e p a r a t e component, T, hav ing a m o b i l i t y c a . 2 .0 x 10” 5 em2 , s e c . \ v o l t ^ ( 5 5 )* The p r e s e n t e l e c t r o p h o r e t i c s t u d i e s were u n d e r ta k e n i n o r d e r t o o b t a i n a c l e a r c o n c e p tio n o f t h e fu nd am en tal d i f f e r e n c e s t h a t e x i s t betw een t h e p r o t e i n s i n normal b o v in e serum and th o s e i n serums from an im a ls which had been e i t h e r t r e a t e d o r exposed t o l i v e B r u c e l l a c e l l s , o r t o some f r a c t i o n th e r e f r o m ; t o i d e n t i f y t h e c o n s t i t u e n t o f th e serum w i t h w hich B r u c e ll a a n tib o d y was a s s o c i a t e d ; t o l e a r n som ething about t h e p h y s ic o - c h e m ic a l n a t u r e of t h e a n t ib o d y ; and w i th t h i s i n f o r m a t i o n t o de­ v e lo p a s u i t a b l e method o f p u r i f y i n g and c o n c e n t r a t i n g B r u c e ll a a n t i s e r u m . S in ce l i t t l e i s known about t h e p r o t e i n changes t h a t o c c u r i n t h e serum of th e b o vin e from b i r t h t o m a t u r i t y or un d er v a r i o u s c o n d i t i o n s , i t was c o n s id e r e d a d v i s a b l e t o b e g i n t h e e l e c t r o p h o r e t i c s t u d i e s on blood serums from new -born c a l v e s t a k e n b e f o r e t h e i n g e s t i o n o f c o lo s tr u m from norm al and i n f e c t e d an im als and t o f o l l o w t h e changes t h a t o c c u rre d a f t e r t h e i n g e s t i o n o f c o lo s t r u m . T h is p r o c e d u re was l i k e w i s e f o llo w e d i n c e r t a i n i n s t a n c e s i n a d u l t an im als b e f o r e and a f t e r i n j e c t i o n w i t h B r u c e l l a . -b MATERIALS AND METHODS Serum S am p les; - The b lood specimens w ere drawn from t h e a n im a ls , p e r m i t t e d t o c l o t a t room t e m p e r a t u r e , and t h e n s t o r e d o v e r n ig h t a t 3 °C* On t h e f o ll o w i n g day th e serum was removed a f t e r c e n t r i f u g a t i o n . I t was d i a l y z e d a g a i n s t a p r e l i m i n a r y volume o f b u f f e r i n t h e c o ld room f o r one day b e f o r e b e in g t r a n s f e r r e d t o a two l i t e r volume o f b u f f e r and allo w ed t o d i a l y z e f o r a t l e a s t one more day b e f o r e b e in g p la c e d i n th e e l e c t r o ­ p h o re sis c e l l . A f t e r d i a l y s i s t h e serum sample was a d j u s t e d t o a p r o t e i n c e n c e n t r a t i o n of one p e r c e n t by t h e r e f r a c t o m e t r i c method. A nother cen­ t r i f u g a t i o n was u s u a l l y n e c e s s a r y t o in s u r e com plete c l a r i t y . The l e t t e r nCT* b e f o r e t h e number o f a serum i n d i c a t e s c a l f , w h ile t h e sm a ll l e t t e r a f t e r t h e number r e p r e s e n t s t h e o r d e r i n w h ich t h e blo o d specimen was drawn. B u f f e r : - The c o m p o sitio n o f t h e b u f f e r used th ro u g h o u t t h i s s tu d y was 0 .0 5 M d i e t h v l b a r b i t u r i c a c i d , 0 .0 25 M NaOH, and 0.075 M NaCl p e r l i t e r w hich g iv e s a pH o f a b o u t 7*9 an d an i o n i c s t r e n g t h of 0 . 1 . P r o t e i n D e t e r m i n a t io n :- T o t a l p r o t e i n was d eterm in ed by t h e d i f f e r e n t i a l r e f r a c t o m e t r i c method of Siebenmann (14-2,14-3) * Per a d ju stm e n t o f t h e serum sam ples t o one p e r c e n t , t h e g ra p h ic method was used b ased upon a c h a r t p r e p a r e d from a serum of known p r o t e i n c o n c e n t r a t i o n and d i a l y z e d a g a i n s t b u f f e r u n d e r com parable c o n d i t i o n s . A g g lu tin a tio n T i t r a t i o n : - A ll serum specimens were t i t r a t e d w i t h a smooth c u l t u r e o f B r u c e l l a a b o r tu s d i l u t e d t o a t u r b i d i t y of 1 M cFarland s c a l e i n s a l t s o l u t i o n . The f i r s t serum d i l u t i o n was 1 :20 e x c e p t i n t h e c a s e o f new b o r n c a l v e s when th e f i r s t d i l u t i o n was 1 *5 * R eadings were made a f t e r i n c u b a t i o n f o r I4.8 hours a t 37°C. -5E le c tro p h o re sis :- I n t h e s e ex p e rim e n ts t h e m o d ifie d T i s e l i u s a p p a r a t u s d e s c r i b e d by Longsworth and Maclnnes (26) was u s e d . P h o to ­ g rap h s o f t h e m i g r a t i n g b o u n d a r ie s w ere ta k e n by t h e ”s c h l i e r e n scan n in g m ethod” (2lj.,30) of Longsw orth. Tfith t h e e x c e p t i o n o f a few i n i t i a l ru ns t h e dou ble l e n g t h s e c t i o n o f t h e e l e c t r o p h o r e s i s c e l l ( 29) was employed i n o r d e r t o avoid t h e o b s c u rin g e f f e c t i n t h e m id dle o f th e f i e l d by t h e h o r i z o n t a l g l a s s p l a t e s o f t h e o r i g i n a l tw o s e c t i o n a p p a r a t u s . The e l e c ­ t r o p h o r e t i c runs were c a r r i e d o u t a t 0 .5 °C . and a t a p o t e n t i a l g r a d i e n t o f 5 t o 6 v o l t s p e r c e n t i m e t e r f o r ab o u t two h o u r s . m easured p o t e n t i o m e t r i c a l l y . The c u r r e n t was C o n d u c tiv ity measurements were made on b o t h t h e b u f f e r and p r o t e i n s o l u t i o n w i th a S hedlovsky c e l l ( l |l ) i n a Dewar v e s s e l c o n t a i n i n g c ru sh e d i c e and w a t e r . made a t 25° w i t h a g l a s s e l e c t r o d e . The pH measurements were M o b i l i t i e s and a r e a s of t h e diagram s w ere d eterm in e d from t h e d escen d in g b o u n d a rie s i n t h e manner d e s c r i b e d by L ongsw orth, S h ed lo v sk y , and Maclnnes ( 2 6 ,2 7 ,3 0 )* The arrow s i n d i c a t i n g t h e d i r e c t i o n of m i g r a t i o n j u s t below a l l e l e c t r o p h o r e t i c diagram s i l l u s ­ t r a t e d i n t h i s p a p e r a r e p reced ed by th e l e t t e r s ”d” and ”a ” w hich r e p r e ­ s e n t t h e d escend ing and a sc e n d in g b o u n d a rie s r e s p e c t i v e l y . - 6- EXPERIMENTAL AND DISCUSSION A. E l e c t r o p h o r e t i c s t u d i e s o f normal c a l f serums b e f o r e and a f t e r in g e s tio n of co lo stru m . Howe (18) had o b s e rv e d t h a t t h e serum o f t h e c a l f a t b i r t h c o n t a i n e d n e i t h e r e u g l o b u l i n n o r p s e u d o g lo b u lin I i n a p p r e c i a b l e q u a n t i ­ t i e s , b u t t h a t t h e i n g e s t i o n o f c o lo s tr u m by t h e an im al a t any tim e w i t h ­ i n two days a f t e r b i r t h r e s u l t e d i n th e r a p id a p p earan ce o f t h e s e p r o t e i n f r a c tio n s in th e b lo o d . E a r l e (8) found a s i m i l a r s i t u a t i o n w i t h r e g a r d t o young f o a l s , k i d s , lambs and p i g s . T his a u t h o r a d v is e s t h a t any p r o ­ gram f o r f e e d in g new -born anim als sh o u ld in c lu d e e i t h e r homologous co­ l o s t r u m , o r some s u b s t i t u t e from w h ich t h e new-born an im als may a b s o rb e i t h e r t h e p r o t e c t i v e s u b s ta n c e s o r d i n a r i l y s u p p l i e d i n t h e c o lo s tr u m , or t h e i r eq u iv alen t. A ccording t o L i t t l e and O r c u tt (23) t h e b lo o d of t h e new -born c a l f , w i t h r a r e e x c e p t i o n s , i s f r e e from a g g l u t i n i n s tow ard s B r u c e l l a u n t i l t h e anim al has su c k le d and t a k e n i n t h e c o lo s tru m even when t h e b lo o d and c o lo s tr u m of t h e dam have a h ig h a g g l u t i n i n c o n t e n t . I t seemed l o g i c a l t o b e g in our e l e c t r o p h o r e t i c s t u d i e s f i r s t upon t h e serum o f c a l v e s i n o rd e r t o o b serv e t h e serum p r o t e i n p a t t e r n a t b i r t h , and t o f o ll o w t h e changes t h a t o c c u rre d a f t e r t h e i n g e s t i o n of c o lo s tr u m and as t h e anim al app roached m a t u r i t y . r e s u lts of th is in v e stig a tio n . Table I r e v e a l s t h e A ll c a lv e s excep t numbers 200, 101 and 100 w ere b o rn t o dams i n h e rd s f r e e from b r u c e l l o s i s . The custom ary p r a c t i c e i n t h i s h erd was t o p e rm it new-born c a lv e s t o i n g e s t o n ly one " b e l l y - f u l l ” o f c o lo s tr u m , b e f o r e b e in g s e p a r a t e d from t h e i r dams and t h e n f e d m ilk from a p a i l . The o u t s t a n d i n g c h a r a c t e r i s t i c o f c a l f serum o b t a i n e d b e f o r e t h e i n g e s t i o n o f c o lo s tru m was t h e h ig h c o n c e n t r a t i o n o f -7& - g l o b u l i n w hich m ig h t a lm o st e q u a l o r even exceed t h e c o n c e n t r a t i o n o f alb u m in . On t h e o t h e r hand, g l o b u l i n was d i s t i n g u i s h e d by i t s e x tr e m e ly s m a ll peak o r com p lete a b s e n c e . The u s u a l p o s i t i v e e r r o r c o n t r i b u t e d by t h e £ -b o u n d ary was e a s i l y av o id ed i n t h i s s e r i e s by v i r t u e o f t h e n a t u r e of t h e d iag ram s. In t h e c a s e o f c a l f 100 from an i n f e c t e d dam, t h e plasma was o b serv ed as w e l l as t h e serum i n o r d e r t o d e te rm in e t h e e f f e c t of fib rin o g en . F i g . l b o f t h e plasm a shows t h a t t h e f i b r i n o g e n has c o m p le te ly masked t h e low 'T'-peak -whichwas j u s t d i s c e r n a b l e I n t h e serum o f t h e same b lo o d specim en ( F i g . l a ) . A second specim en ( F i g . l c ) of b loo d was drawn from c a l f 100 s i x days a f t e r b i r t h and a f t e r i t had i n g e s t e d colostru m * An a g g l u t i n a t i o n t i t e r o f 1 :8 0 was now d i s c l o s e d . c r e a s e d from LlLp• 2 t o The h i g h ^ - p e a k had de­ p e r c e n t o f t h e t o t a l a r e a w h ile t h e '7 '- p e a k has r i s e n from 2*3 t o 20*7 pe** c e n t . The album in as w e l l a s t h e t o t a l p r o t e i n had i n c r e a s e d and t h e a r e a o f t h e /3 -p e a k had now d o ub led. Twenty-two days l a t e r t h e diagram ( F i g . I d ) began t o ap p ro ach t h a t o f t h e a d u l t cow. The a g g l u t i n a t i o n t i t e r had i n c r e a s e d t o l : l 6 o b u t t h e ^ - p e a k had d im in ish e d t o 1 0 .3 p e r c e n t . The CC- a n d /^ - b o u n d a r ie s c o n tin u e d t o be q u i t e pronounced and t h e albu m in was a t t a i n i n g i t s norm al c o n c e n t r a t i o n . I t m ight be n o te d t h a t t h e r e was no c o r r e l a t i o n betw een a g g l u t i n a t i o n t i t e r a n d 'X - g l o b u l i n co n c e n tra tio n . An e x p l a n a t i o n w i l l be o f f e r e d l a t e r i n t h i s p a p e r . The re m a in in g specim ens seem t o le a d t o t h e g e n e r a l i z a t i o n t h a t t h e i n g e s t i o n of c o lo s tr u m by c a l v e s from norm al, o r more p r o p e r l y cows f r e e from b r u c e l l o s i s cau sed an i n c r e a s e i n t h e ' / - p e a k from a few p e r c e n t t o ab o u t 30 Pe r c e n t , and a d o u b lin g o f t h e / ^ - p e a k , w h ile t h e i n v e r s e h e ld t r u e f o r t h e a b n o rm a lly h i g h - p e a k . The album in ap p eared t o d e c re a s e w i t h r e s p e c t t o t h e t o t a l a r e a ; y e t e x a m in a tio n of t h e normal i n c r e a s e i n t o t a l p r o t e i n , a s t h e c a l f grew o l d e r day by day, showed t h a t t h e alb um in l i k e ­ w is e was i n c r e a s i n g when e x p r e s s e d i n grams p e r 100 c c . of serum . T his r e l a t i o n s h i p betw een t h e v a r i o u s components a p p e a re d t o p e r s i s t f o r a t l e a s t th r e e o r fo u r w eeks. However, t h e second specim en, b , o f c a l f l]Bh ( F i g . I f ) drawn hjo days a f t e r b i r t h ( F i g . l e ) r e v e a l e d t h a t by t h i s tim e t h e r e l a t i v e c o n c e n t r a t i o n o f t h e serum components began t o ap­ p ro x im a te t h o s e of th e normal h e i f e r . P r o b a b ly b ecau se of t h e ab sence as y e t o f some s o r t o f a n t i g e n i c s t i m u l a t i o n t h e ' / - p e a k , 9*7 Pe r c e n t , was s t i l l q u i t e low. C lose e x a m in a tio n o f T able I shows t h a t r a p i d and pro fo u n d changes o c c u r re d i n t h e serum p r o t e i n components o f t h e c a l f a f t e r t h e i n g e s t i o n of c o lo s tr u m . I t was prop o sed t o f o ll o w t h e s e a l t e r a t i o n s a t s u i t a b l e i n t e r v a l s i n a c a l f b o rn o f a normal dam. C a lf CR2 was o b t a in e d from t h e same b r u c e l l o s i s - f r e e h e r d p r e v i o u s l y m en tio n ed . T his c a l f en jo y ed t h e rem ark ab le d i s t i n c t i o n o f b e in g t h e f i r s t r e ­ co rded o f f s p r i n g o f a cow w i t h a rumen f i s t u l a . In a d d i t i o n , t h e dam o f t h i s c a l f b ein g a member o f a g roup upon w hich f a t m etab o lism s t u d i e s w ere b e in g made was g iv e n 5 6 gms. o f h e x y l b lu e 10 days b e f o r e c a l v i n g . F e e l i n g t h a t t h i s f a c t m ight have m a t e r i a l l y a f f e c t e d t h e c a l f , a n o t h e r c a l f CA22, was a l s o t a k e n fro m t h e same h e r d . The p r o t e i n d i s t r i b u t i o n o f t h e serum o f c a l f CR2 o b ta in e d a t b i r t h as c a l c u l a t e d from e l e c t r o ­ p h o r e t i c a n a l y s i s , T ab le I I , compared f a v o r a b l y w i t h s i m i l a r c a s e s i n T a b le I . The e l e c t r o p h o r e t i c diagram o f a sample o b t a in e d f o u r hours a f t e r t h e i n g e s t i o n o f c o lo s tru m c o u ld be alm o st superim posed upon t h e f i r s t one i n d i c a t i n g t h a t v e r y l i t t l e a b s o r p t i o n of g l o b u l i n from co­ l o s t r u m o c c u r re d i n t h a t s h o r t i n t e r v a l . T w en ty -fo u r hou rs l a t e r a m e a s u ra b le s h i f t i n r e l a t i v e p r o t e i n c o n c e n t r a t i o n c o u ld be d e m o n s tra te d . The alb um in and ^Z-peaks began t o d im in is h a t t h e expense o f t h e rem ain in g g lo b u lin s. However, i n te rm s o f grams p e r 100 c c . t h e album in c o n c e n t r a - -9 to • *H o Pi p; © rH < \x X PI o •H -P •rt CO O B-i \ l o \ o W •P Pi CD Pi O Pi 3 p -p PI CO •H o © rH O -P O O d P h O CO © W to d Pi o l—l •H 1 ® © P Pi - P -P Pi P . CD © O -P P i Cm O < i! O d d PI P i erf © © to p . CD o • H cm © -P • H PQ i—1 • H Cj P o erf Pi 3 Q o •H w d -P Q 3 d u O © p m O O h W P Eh erf © P. w <*{ d r*< O i-l •h erf -P -P erf O Pi Em -P d © "If Pi O ® Pi O o O Pi © PL, P> i—1 o rH 'd r—1 US US • o o KS • o rH CO » o d> vC • o KS CM • O CM KS • o -qh sO * o - if CO • o 00 o • o o CM * o us sO • o 00 o • rH t-’ * o CM CM • r—I t—1 KS • o rH O •. -H sO _ph 7h -Ph • Os CM sO • us -ph US • o 1—1 c— • KS KS CO • rH US KS O ♦ o -p F i—i • o “S oo * o KS o • rH o o • o OS o ♦ o CM Os • o rH • o - p!' ■—i • o o o • rH £> Os » o - it cc • o o o • o -Pt • o co o • 1—I CVI CO • o sO• H o • o KS • CM o • OS OJ • ts- c— • -Ph vO• sO • us US * Q -Ph o5 « o US SO• LfS -Ph US * us -Hh c •us CM • HTj" 3 ds • rH H • ■Pt xj- • o KS s6• O' KS us o • rH a> H' 9 o us sO • o sO -ph « o o • KS KS KS • US rH KS • o CM -ph « rH KS co o • o sO rH • o Os IS• o c— O'. • o CM CO • o o sO • o CM US • o CM US • o KS O • o KS i—! + o US c~• o o rH • rH CO • KS CO • IS— CM -Ph • o CM CO • c— rH o * -PT KS sO• ■—1 ■—1 • CO OS • CO KS KS • Os -Ph CO • sO KS * OS KS KS • CM us ISsO • o *-0 -ph • o Os H* • o CM sO• o Os • l-CS CM •H • ISr—1 IS• CO i—i KS • ■ao KS © rH rH o o • o r— 1 rH • o _dsC • o KS KS * ■—t CO i—! • o CM CM • O US — Ph * o CO 1—1 • I—1 o • o 1S• OS 1—1 • sO Os • iH i—1 CM • _qt CM O • -Ph LIS 00 • sO KS «H • f— us w o •H PI • • O P o rH o <1 O rH « s ft o « Eh V N CO/■—©© •H O {M -P rM ■1“I rH K *3 p t© Q v_/ m Pa Oa US sO• -Ph KS • rH us CM • KS Os CM • rH US CM • KS sO • rH -Ph CO • Os sO• CM rH Os • US CM i—I • CM US CM • -Ph i—1 KS • CM US sO• D— CM rH • CM _ch ts• -ph OCM • CM KS CO • sO co ao • i—i o sO• KS CM sO• CM OS Os • NO OS C\I • CM O i—1 • -ph -p s ■H -p CO CD CD to CO • -1 CM • KS 00 • -Ph CM « vO KS US • c— KS • KS C— • -ph KS • vO CO • rH ts• CM US • -Hh |H • vO O * CM O • KS us • -ch o « sO us • rH C"• CM KS • -ph CO ♦ us KS • iH KS • OJ KS • -Ph us • us -ph • i—i us • CM Os • KS KS • US OS • ■—1 C"• CM D— • -Ph KS • M0 • i—1 O * KS 00 ♦ -Ph OJ * MO r— • rH s0« CM O• US sO• sO os • r—i o • KS C'— • -Ph CM • M3 as • rH ■—1 “ w “ • • KS KS Os IS• • -Pi' -Ph CM sO• « sO SO KS » CM C— » KS US • US -ph • L— O Os • r-- KS CO • o- KS CO • KS r— • E T — o OS r— rH CO • C— CM CO • C"- t*00 • r— CM CO • o- sO CO • [— CU CD • S- co~ o •c— US co • (S- OC m (D P _p d •H CD d PI r— O Pi •iH -P o ph CD i> •rH -p a5 !---i © P3 Pi © rS d d pP o o erf © rH Pi hQ pq P i < •H -P © to rd w I5*! © ■xi W >» © t3 -i-t -P Pi ■H sO CM CM -P w © ■d Os pi o o rH o O O r—1 © KS CO -ph o pi KS CO -Ph o > < © •r> H -p © to © id © > •rH -P © to © Pi +5 Pi »H PI -P © rH to CM O © l> -P © to © id W r^S © d O r— t P 1—1 co CM o © > ■P © to © Pi -P P, •rH -P © KS IS­ © > •iH -P © to © rd U © d us rH P KS S - © > •H -p © to © td Pi -p © »H p © to © iH < CO © d sO -Ph © _ph CO -Ph p -Ph CO -ph T, •rH p p> . •H -P © to © !d p P CO d © •r> H -p erf to © Id O Pi •rH •H © P! o •iH -P erf P. 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Ph « t= r rt EH 0 1—t 1— 1 • 0 _H - O J • t ON * NO UN UN O ON * rH rH ON * rH VQ CO « rH NO ON • O J O J sO O • UN O J rH • K N rH • - r t c o r t UN • 0 KN K N - r t UN - r t to ­ e s • O ON • 1— I O J L fN vO • KN O ' • - r t O J 0 • NO rH • O J r t r IN- O J 1— 1 * rH 0 O J # 1— ! KN NO • <—t O J 0 0 0 • ON UN • KN UN * CO CO * ON ON • • NO GO NO UN UN • NO O J • r—[ X CO © *H P r—" to O rH O CO * rH ' ON • rH o • rH O * 0 0 • O J K N rH CO « 1— 1 O J • rH O J • rH • KN • K N • O n • UN • NO • ON • O n • KN KN KN O J O J OJ O J O J O J C "• rH • c— • rH • i— ! • UN • _H • - r t ■ rt O • O O J O J 0 J 0 • KN KN • O J • rH • - r t • NO • NO • -= t - r t - r t - r t r r CO • UN 00 * C'— • 1— 1 f— t CO O J • k Q • 1— 1 n « r t • 1— 1 NO • rH O ' • 1— 1 rH •rH K r t O £3 V__ r f rH •H P O fv 1 NJ 6 KN • o \ < rt P i UN t r t r t r t r r t t * ON • O J • O ! • rH • UN • O n • tN» NO • CT n • ON • UN UN NO NO NO UN UN UN UN UN UN 0 • 'UN t 1 ia XS © p to o •rH rt 1— 1 CO • 1— I rH CO CO • CO • co O' [— 0 O - 0 1— 1 d r t P h > rt - O l CO O J CO • O J CO • K N CO • 00 • • 0 - t— O - IN ' C— 0 - • • • - 0 - K N 0 0 CO O• O ' CO C— • CO O • c— O ' © i— I rt o i—i aS o © rt •pH S>s rt 0 •rH © rt o r t p Sh •rH Ph O rt P o P rt rt © P •rt 6h 1— 1 P0 © > •H © > •H P d hO © P d P d rt rH fa n fa n © © > •H fao © faO © © © •H •rH •H P d fa O p p d fa n fan © © fp {Si © © r t d r t © > •H P d faO © © > • rH P d fan © is ; © !> •H © l> •H P d faO © !rt P d faO © 0 0 0 0 0 0 •1 *1 0 0 0 0 0 0 0 1—t ** Ol rH rH rH 0 99 0 rH •• rH © <-{ 03 0 0 rH fa o m rj E d rt Pt 1 -P rt • rH r t to rt to rt O P i 0 r t £S Pt rt © rt d R O rt rt CO r t d r t d XJ xs p - r t CO O J 0 OJ P=J r c — < 4 rH © to O - -r rt t •H P P <4 CJ © rt rt rt rt © 0 0 rt rt d X ) P rt r t CO O J c ET l> - Of -4 O r i d xs NO 1— 1 r t p t- t •H rt rt o P © © u to © d d X 5 ri rt 0 p rt xs p c o O J l> - r Z. r *4 c O- C0 0 c r t © d rt rt p & to rt rt rt •H p rt to rt XS 0 rt rt to £ rt rt rt o o 0 © rt rt rt p 0 xs © to rt 0 d P< 0 rt © © rt to rt © d «—1 0 Q, to d to •rH © xJ rt O © 0 r t 1—1 Eh r t 01 t 1— 1 CO rH P 0) X) rt 0 r t fap d rt •H P rt gs p d rt P Pt rt © W NO OJ n to xl 0 o •H P o <4 rt © rf! s * P © to © •H r t rt xJ •1 0 1— 1 *• rt 0 0 aS P t rt HH fan rt O p © -p d t CO rH 43 O •rH © rt rH P •H 0 p O KN M d © u © to art rt r* rt • o © P o © a •H n •H [£ h o 0 1 • 1— 1 0 © o art •H rt d P rt © o rt o rt © P i rt ccS p rt 0 0 o rt P rH d p O Eh P rt © 0 o rH rt * 4 -1 1 t i o n c o n tin u e d s u r p r i s i n g l y c o n s t a n t even over a two week p e r i o d w h ile t h e t o t a l p r o t e i n i n c r e a s e d slow ly* At l e a s t i n t h i s c a s e t h e i n c r e a s e i n t o t a l p r o t e i n was a c c o u n te d f o r e n t i r e l y by t h e two s lo w er moving g lo b u lin s. The d e g re e o f a b s o r p t i o n was u n u s u a l l y slow o v e r t h e e n t i r e p e r i o d and even t h e t o t a l p r o t e i n had i n c r e a s e d o n ly t o I4 .13 from 3*^5 gm p e r 100 c c . The s i g n i f i c a n c e i n t h i s c o n n e c tio n of t h e h e x y l b lu e dye i s p r o b l e m a t i c a l . The a g g l u t i n a t i o n t i t r a t i o n was n e g a t i v e th r o u g h ­ o ut , C a lf CA22, ( F i g , 2 a ) n orm al i n ap p earan ce and from a normal dam, showed enhancement of th e T ^ -p e a k ( F ig ,2 b ) t o 13*8 p e r c e n t w i t h i n f o u r h ou rs a f t e r t h e i n g e s t i o n of c o lo s tr u m (T ab le I I * ) . W ith in 2I4. ho urs t h e y - g l o b u l i n ( F i g . 2 c ) i n c r e a s e d t o a maximum of 3^*5 Pe r c e n t , and t h e # - g l o b u l i n d e c r e a s e d from i t s o r i g i n a l c o n c e n t r a t i o n o f 38*8 t o 19*1 p er c en t. C a lf CR2, on t h e o t h e r hand, showed p r a c t i c a l l y no a b s o r p t i o n o f g l o b u l i n even a f t e r two w eeks. In a week t h e # - and - g l o b u l i n con­ c e n t r a t i o n o f t h e serum o f c a l f CA22 was alm ost norm al; t h e 'T '-g lo b u lin a f t e r i t s i n i t i a l r a p i d i n c r e a s e was now l e v e l i n g o f f , an d i n l 6 days ( F i g . 2d) a l l components were d i s t r i b u t e d i n t h e manner u s u a l l y found i n young h e i f e r s * B. E l e c t r o p h o r e t i c p a t t e r n s o f serum drawn a t i n t e r v a l s from a B r u c e l l a in fe c te d c a l f . Im m ed iately a f t e r co m p letin g t h e stu d y o f t h e serum changes i n t h e c a l v e s , numbers CA22 and CR2, b o rn o f normal dams, an o p p o r t u n i t y a p p e a re d t o f o l l o w p a r a l l e l changes i n a c a l f b o rn o f an i n f e c t e d dam. C a l f 876 , a f u l l - t e r m c a l f , was from an i n f e c t e d dam t h a t had been ex­ p osed t o B r u c e ll a f o u r months b e f o r e p a r t u r i t i o n . I t was u n f o r t u n a t e t h a t t h i s c a l f was born d u rin g t h e n i g h t ; t h a t i t may have n u r s e d some- -1 2 tim e b e f o r e i t was d is c o v e r e d l y i n g by i t s dam's s i d e t h e n e x t m orning was q u i t e l i k e l y . N o n e th e le s s , t h e f i r s t b lo o d sample (F ig .3 a.) t h a t was o b ta in e d im m e d ia te ly proved t o have t h e lo w e s t album in and t h e h i g h e s t 'T '- g l o b u l i n c o n c e n t r a t i o n o f any c a l f serum s t u d i e d . The h ig h t o t a l p r o t e i n , 6 .5 9 gnu P@** 100 c c . (T a b le I I ) a lo n g w i t h t h e a g g lu ­ t i n a t i o n t i t r a t i o n of 1 : 1 0 ,0 0 0 , f u r t h e r s u p p o rte d t h e s u s p i c i o n t h a t c o lo s tr u m had b een p r e v i o u s l y i n g e s t e d . t i t e r was 1:14-0,000. The c o lo s tr u m a g g l u t i n a t i o n By t h e n e x t day d u r in g which c o lo s tr u m was v o lu n ­ t a r i l y i n g e s t e d t h e t o t a l p r o t e i n r o s e t o 8 .5 3 t h e alb um in rem ained unchanged. Pe r 100 c c . w h ile T h e - g l o b u l i n w hich had i t s g r e a t e s t c o n c e n t r a t i o n im m ed iately a t b i r t h had a l r e a d y d e c r e a s e d from 3 2 .2 p er c e n t o f t h e t o t a l a r e a t o 2 3 .8 ( F i g . 3 b ) ; album in had reach ed a minimum o f II4. . I w h ile t h e '7/- g l o b u l i n had a t t a i n e d a maximum o f I48. I p e r c e n t . W ith in a week t h e album in had begun t o r i s e slo w ly i n s p i t e o f a r e ­ d u c t i o n o f t o t a l p r o t e i n t o 6 .8 8 gm. p e r 100 c c . ( F i g . 3 c ) . The g r a d u a l d im in u tio n o f d '- g l o b u l i n c o n tin u e d w h ile t h a t of th e 'T '-g lo b u lin was more a b r u p t ; y e t t h e t i t r a t i o n had clim bed t o 1 :2 0 ,0 0 0 . In two weeks th e alb u m in peak had a lm o st a t t a i n e d i t s cu sto m ary prominence as th e v a r io u s peaks now assumed t h e o u t l i n e of t h e g e n e r a l b o v in e e l e c t r o p h o r e t i c p a tte rn (F ig .3d). I t m ight be n o te d t h a t t h e f t - g l o b u l i n s e p a r a t e d from t h e m a s s iv e ‘T '- g l o b u l i n w it h d i f f i c u l t y . The s e p a r a t i o n between t h e s e two b o u n d a r ie s was improved when n e i t h e r was i n g r e a t e x c e s s . The t i t r a t i o n c o n tin u e d a t 1 :1 0 ,0 0 0 The m o b i l i t i e s of t h e v a r i o u s b o u n d a rie s were u n ifo rm a l b e i t a v e r a g in g s l i g h t l y l e s s t h a n t h o s e o f a d u l t cows. -1 3 C. E l e c t r o p h o r e t i c p a t t e r n s of h e i f e r serums a f t e r v a c c i n a t i o n w i t h t h e U. S . B. A. I* B r u c e l l a S t r a i n 19* The l i t e r a t u r e i s r e p l e t e w i t h e x p e r im e n ta l d a ta b ased upon S t r a i n 19* As f a r as can be d e te rm in e d no one had f o llo w e d t h e re sp o n s e o f t h e serum p r o t e i n s t o t h e a c t i o n of s t r a i n 19 v a c c i n a t i o n . The f i r s t two c a s e s o f T a b le I I I were about a y e a r o ld when v a c c i n a t e d ; a n o th e r y e a r l a t e r b lo o d was w ith d raw n f o r e l e c t r o p h o r e t i c a n a l y s i s . re se m b le d t h o s e o f t h e v a c c i n a t e d h e i f e r s o f T a b le IV. The p a t t e r n s The d i s t r i b u t i o n o f t h e serum p r o t e i n s was a p p r o x im a te ly norm al as c o u ld be d e m o n stra te d from t h e f i g u r e s o f th e c o n c e n t r a t i o n or c o m p o sitio n column of T a b le I I I ; how ever, t h e t o t a l p r o t e i n o f c a l f C73 "was somewhat h i g h e r t h a n would be e x p e c te d i n a c a l f of i t s a g e . In o r d e r t o d e te rm in e t h e im m ediate e f f e c t o f an i n j e c t i o n of 6 c c . o f B r u c e l l a s t r a i n 19 v a c c i n e , two c a l f s , A98 and B9I4* about f i v e months o f age w ere s e l e c t e d ( F ig s .i i a and i+d). C a l f &9Q showed no a g g lu ­ t i n a t i o n i n a 1 :20 d i l u t i o n when v a c c i n a t e d , b u t c a l f B9I4 p o s s e s s e d a t i t e r o f 1 : 320; t h e t o t a l serum p r o t e i n i n e a c h was abo ut t h e same. E ig h te e n days a f t e r t h e i n j e c t i o n b o th showed an i n c r e a s e i n a g g l u t i n a ­ t i o n t i t e r , and an enhancement i n a l l g l o b u l i n s w i t h t h e T '-peak showing t h e most marked change (Figs.i+b and i+e) . reduced. The alb um in , o f c o u r s e , was By t h e end o f 25 days t h e a g g l u t i n a t i o n t i t e r i n each had f a l l e n t o 1 :2 0 a lo n g w i t h a d e c r e a s e o f t h e '/'-p e a k , w h ile t h e album in r o s e t o i t s norm al c o n c e n t r a t i o n o f b e t t e r t h a n 50 p e r c e n t (Fig.i^c and ip f). The c o m p o s itio n column c l e a r l y p r e s e n t s t h e r e l a t i v e l y r a p i d r e t u r n o f t h e serum p r o t e i n s t o a norm al d i s t r i b u t i o n . Here i n t h i s s tu d y , as w e l l a s i n t h e p r e v io u s o n e s , th e change i n t h e 9^-peak p la y e d t h e most -X U - tO * •H O fa rt' NO o in- o CVJ O KN I—I » o o fat o KN KN o rH NO i— I • GO o o o i— I • CO OJ NO OJ UN KN V o" OJ OJ KN UN ON co O KN nO CO • o NO• o- KN KN Ol 00 o OJ 00 NN OJ 00 NO rt 0J o ON rH o OJ • UN inUN NO NO 00 NO KN NN KN NO IN- & c— IN- NO OJ 0J OJ UN NO o o o NO GO ON NO NO Ol 0 UN OJ i—I o OJ t— KN co o o KN • OJ OJ o o UN c-~ KN rt 7? ON ON CO CO OJ CO fat rH UN CO UN CO NO UN CO CO O KN KN KN CO UN UN KN NO KN c— cvi • o KN u- NO NO NO NO ON oo KN KN ~j* * « • UN NO r— I • KN NO on -p •H £ O ON KN o- 0J iH OJ KN Ol . KN UN * KN OJ OJ KN KN -rt KN UN UN CO ON UN KN -rt -rt fa t fat KN KN fat -rt o ON • UN KN NO NO UN CO * UN NO NO CO • 0- ON ON • D~- ON On 0 I— NO CO 0 IN- NO CO 0 IN- CO ON 0 N- NO CO 0 CN- NO CO • IN- o fat • to © o -rt NO •« 1—1 o OJ *« rH o OJ KN •• rH o NO 1—I •• 1—1 o CVJ •• rH r— I •rH r-i NO ■— i X r© f a o > Ol • rt rH © fcJO -P foO *H EH ri fe erf U xs o o i—i PQ fa rH o OJ KN •0 rH rH < rt rH < rt UN UN -rt rH not rt © —. © © •H O + ) -rt rH 6 .0 cd Sh -P CO NO >* 6 .1 C o n c e n tra tio n s ^ o f \ a •H and M o tilitie s E le c tro p h o re tic TABLE I I I i— ^ rt ■ — i rt -r t UN .OJ OJ OJ UN .OJ o I—1 o r—1 o rH o o o — rt ,c r t UN .OJ UN OJ ON 5 3 rt -rt On PQ fat H ON X) © -p © rt •H O o ri— tI r-QtT o, erf On O- ON .KN ON .KN ON ON U rn . rt t rt pq © r© erf KN rfa r—1 co fa ^ rt o KN ^rH^ NO UN cS CO ON <*} e s tim a tio n s co OJ >co rt * T3T IN- 1N- o o o o rH iH fat UN th e s e C o m p o n e n ts ON o O 0 rt o o S e ru m o -rt KN in Sh © P-. O rH € -b o u n d a ry C a lv e s of O o NO c -- rt -p rt © O c erf -P - P Crf O Jh E-t -p © o -rt NN OJ OJ o o UN • th e to erf © Jh OJ KN rt fo r V a c c in a te d rt -rt -3- m ade 11 o -it rQ co ON < o co ON <3 erf fat ON PQ o ON PQ w as § fat XI -rt C o rre c tio n 3 O •H -p •H W 0 erf fa t- -1 5 im p o r ta n t r o l e . V a c c i n a t i o n w i t h s t r a i n 19 cau sed o n ly a m ild and te m p o ra ry s h i f t i n t h e e q u i l i b r i u m o f t h e serum p r o t e i n s . In l e s s t h a n a month serum a g g l u t i n i n s had p r a c t i c a l l y d is a p p e a r e d and a more o r l e s s normal serum p r o t e i n d i s t r i b u t i o n was r e s t o r e d . F i g . Jj. shows t h a t , u n l i k e c a l f serum p a t t e r n s a f t e r t h e i n g e s t i o n o f c o lo s tr u m , t h e ^ - b o u n d a r y d id s e p a r a t e e a s i l y from th e " /-p e a k u n d e r t h e p r e s e n t con­ d itio n s. The d escen d in g p a t t e r n s ( F i g s , 14b and ij.e) o f c a l v e s A98b and B9I1I) of t h e f i r s t serum samples drawn a f t e r i n j e c t i o n showed a d o u b le­ p eaked ^ - g l o b u l i n . T his phenomenon was not o b serv ed i n t h e p a t t e r n s o b ta i n e d p r e v io u s t o v a c c i n a t i o n o r t h o s e o b ta in e d s e v e r a l weeks sub­ sequent to in je c tio n . D. E l e c t r o p h o r e t i c p a t t e r n s o f a g e n e r a l s e r i e s of b ov ine seru m s. S e v e r a l w o rkers (lj.,ij.5) have perform ed e l e c t r o p h o r e t i c s t u d i e s upon b o v in e serum . In g e n e r a l most of t h e specimens were p u r p o r t e d t o come from s o - c a l l e d normal cows. In T a b le IV t h e r e a r e c o l l e c t e d d a ta b a se d on e l e c t r o p h o r e t i c a n a l y s e s from a number o f an im als u n d e r v a r io u s co n d itio n s. S in c e ou r c h i e f i n t e r e s t i n t h i s e n t i r e p a p e r was b r u c e l l o s i s , a l l serum specimens were t i t r a t e d f o r B r u c e l l a a g g l u t i n i n s . O b v io u sly , i t i s n o t i n te n d e d t o c o n s id e r an anim al normal i f B r u c e l l a a g g l u t i n i n s w ere a b s e n t . I t must be remembered t h a t a n t i g e n s o t h e r th a n B r u c e l l a m ig h t a f f e c t t h e d i s t r i b u t i o n and amount of serum p r o te in s * To make such e x h a u s tiv e t e s t s i n o r d e r t o prove t h e norm alcy o f t h e anim al would have e n l a r g e d t h e scope o f t h i s s tu d y f a r beyond t h e f a c i l i t i e s a v a i l ­ ab le. T h is s e r i e s r e p r e s e n t s a c o m p ila tio n o f a n a ly s e s g a t h e r e d over a y e a r Ts tim e n o t o n ly from a n e x p e r im e n ta l h e r d , b u t from anim als th r o u g h o u t t h e s t a t e o f M ich igan. T his s tu d y was u n d e r ta k e n t o d e te rm in e - 16 - O 1—! -P •H W O O o o o §• o o O O O O O KN 00 OJ 6n P t KN P t O 0 0 O * 0 OJ O On O pf Bovine of Components P rotein of C o n c e n tra tio n s and M o b ili tie s 0 o- ON KN On LT\ ON co CO OJ OJ CO rH NO KN CO CO O O o* o NO OJ OJ 0'S KN o OJ UN C <— I rH ON 0J• CO KN 00 o 0J * NO KN On • 0J 0j rH 0J p f R5T • KN KN UN 1—1 • <—1 On • rH uN • 1—1 • OJ KN * OJ rH" * KN nD ♦ O• P f ~~Z> P t C— * KN CM • NO "N 'T • UN ' oj » NO 0 —r • UN O co ♦ O- UN KN On * 0- 1—1 co • 0 - ’P © ts; © Ip i OJ « OJ • nS • iH O P4 -P 5=1 © 2 bO -P 2 cj to © © Ph Ph U u aj to ©s* K I^— H CD g Q ■P 5=1 Aj 1 5=1 O 1—T- P-. 0 ,Q d— ON OJ C O Or P t — On OJ UN UN 0 — • KN CO nD • o- “s r o o NO KN 00 KN co 00 U\ CO I— 1 NO • OJ O R CD © U 1 Pi O r^t a 1—1 1—1 UN NO rH [•— UN UN Pf• OJ C— rH NO OJ ON CO UN KN t—1 • KN M? O NO KN KN UN CO UN UN O•N O O- I— I Ol NO 00 » Pf• KN OJ Pt UN rH KN KN KN NO UN OJ • Ol * OJ 1—J P t • KN KN KN O NO NO CO O OJ p f ON ON 7? UN :3 h- KN P t tp iH KN ON • OJ P t IN• Ol UN OJ • -—-J • NO NO NO c—- ON r— KN pt iH C— • OJ UN ON • OJ K\ 0 On • UN co NO KN NO KN P t • NO IN- P f ON • NO c~—• 1—1 cb • ■—1 CO • «—1 UN • 1—1 CO • 1—i NO • 1—1 KN • 1—t c ■ • 1—1 NO • 1—1 t> • ■—1 ■ C>'"• KN • OJ • KN "O n • KN • KN & On • OJ • KN < Si rH • KN NO • P t <—i • P t CO • P t UA • P t OJ • P t u OJ « CO • P t NO • P t 'c 6 ‘ NO • * 1—1 r—i ■ -6j • • KN KN * OJ • • KN '0j • • KN OJ '• J w J 6 n • * P t P t © KN • KN • OJ P£ • A • • • p t • <5n • PT (if • P t 0J * NO rH • NO a^v • NO iS « UN • NO r~i NO • NO US' • NO © * UN Ua • NO rH « NO <5 T • UN ♦ NO — Zf NO • NO KN • NO On On « 0- NO CO • c— OJ CO • UN O• ro• O- o- OJ GO • IN- CO CO • r- 00 • IN- O CO • D— 1—1 CO * IP UN 00 • c— 0J co • IN- IP NO CO • o- UN CO « O- © 0 CO OJ iH O CO OJ i—1 O NO UN OJ •H -P © bO O O O 0 0 0 O O O 1—1 H r-H • -C f 1 © > -P 2 © Pi NO © u • t--J OJ O KN OJ NO Pt -t O - - _j * p t O CO 0 CO 0 OJ — — J O 0 NO rH CO P t 0 CO OJ 1—1 rH rH 1—1 rH i--1 1--1 1—1 *• A, g CD _J OJ rH KN OJ • ♦ OJ u 01 KN CO Kv • ON • I— I • On U\ IN• OJ CO KN • 0 « KN * to U CO • 0J • O ON UN © $ 0- NO bO © vO UN ON UN © CO KN pt rH •H -P KN IN- O oo- KN co Oi > o co O CO 1— 1 © 0 p f KN CO •H -P 0 t— KN OJ KN Ol • © > 00 a1 • • OJ c— 0 O- rH » • K\ 0 KN CO •rH -p © hO UN CO KN rH pf 0 O NO © > O 0 KN 1—I •H -p O 0 1—1 o CO CO • OJ C~— o OJ• • 1— 1 0 NO 0 OJ • KN • ON UN UN -= 0Jt • O KN • On OJ 0* 0J C— UN NO <—1 • O I—1 CO • NO Pt cb 00 0 rH O' • |H KN OJ s • 1—1 P t O CO • ON O * O iH * rH CO • O IN- Pt « • OJ Oi • rH OJ & OJ • 0 rH C '• O KN 0J ON • NO Pt UN t— * o • 0j o• vO KN © R elativ e E lec tro p h o re tic 0 OJ • co r n£> H OJ C1—1 00 CO • • CO EH • Jh rH CD txO -P h0 •H OJ ON O KN • P -4 CO UN o * ON NO * O UN OJ • o CO 0 ON K* • O OJ OJ O 0 0 1—I txj CM NN O UN • O o P t < K p t UN o O -N—- O t*— • O 01 OJ 01 ^ CD 10 •HI O +3 rH •H rH Kj •rH &2 KN Ov • O KN • 0 0 iH rH p f * rH UN On • • UN nC NO 1 Pi O Pi *, w ** -P W < cn O UN to ps -p pi O S Ni © -P Pi C $ Pi bO © Ph pi bO © Ph Ah Ph -p Pi -R Pi aS ccS Pi b0 -P Pi U Ph Ph P-H Ph aj 1—1 CO co co 00 r© O 00 00 o- p^ oPt Pi fcp © pH CL* * aj * aS © * * pi H bO © Ph -p gj Pi bO © rP KN OJ <*H »• © W C/D t—1 1—1 UN NO ■—I C~~ •• • © 3 •• b£) © Ph Ph *. to 0 •H © •H A •• *• CO © •H Ph © P O P5 O *H O •H PM W p *9 KN INCO O KN UN aS O CO PT > • * bO © Ph PM *1 © © vQ W UN NO Pt O KN •* O P t J--I c—I H !>> rH 1—1 © Ph 2 -P © 1—1 I—1 © Ph 2 -p © rH 1—1 © Ph 2 -P © 2 99 a • © W P t NO ■k 0 P t © •H w X •1 O OJ <101 © 4=» PM 0 OJ rH O .© PM aj O * © •rH > •* O © -p 0 © 120 ( F ig . 6b) t o B r u c e l l a by way o f t h e eye c a u se d a trem endous i n c r e a s e i n t h e " /-p e a k , t h e h i g h e s t i n t h i s p a r t i c u l a r s e r i e s ; y e t t h e a g g l u t i n a t i o n t i t e r was o n ly 1:2 56 0. Cow 883 ( F i g . 6 c ) d i s p l a y e d rem ark ab le i n d i v i d u a l i t y by f a i l i n g t o d ev elo p a g g lu ­ t i n i n s and c a l v i n g n o r m a lly a f t e r exp osu re by way o f t h e eye t o 6 ,0 0 0 ,0 0 0 l i v e o rg a n ism s. The serum of t h i s anim al had rem ained c o n s i s t e n t l y n eg a­ ti v e to th e a g g lu tin a tio n t e s t . -1 9 I n c o n t r a s t , t h e l a s t t h r e e members o f t h e s e r i e s a c q u ir e d in fe c tio n n a tu ra lly . T h e ir a g g l u t i n a t i o n t i t e r s ( 1 :4 0 ,0 0 0 ) and t o t a l p r o t e i n c o n c e n t r a t i o n (8 t o 9 gnu p e r 100 c c . ) were e x t r a o r d i n a r i l y h ig h . In t h e c a s e of cow 143993b ( F ig . 6 d ) , album in a c c o u n te d f o r o n ly 35-3 p e r c e n t w h ile - g l o b u l i n com prised l\2 .6 p e r c e n t of t h e t o t a l p r o t e i n ; / ? - g l o b u l i n was a b o u t a v e r a g e . Looking a t t h e s e r i e s i n i t s t o t a l i t y i t i s s e e n t h a t un d er t h e g iv e n c o n d i t i o n s of e l e c t r o p h o r e s i s t h e m o b i l i t y f o r each p r o t e i n component was f a r from c o n s t a n t ; however, i t d id f a l l w i t h i n a c e r t a i n range. T i s e l i u s and o t h e r s (6,149) have p o in te d out t h a t t h e m o b i l i t y o f a g iv e n p r o t e i n , b e s id e s depending upon t h e pH and io n ic s t r e n g t h o f t h e b u f f e r s o l u t i o n , i s a l s o a f u n c t i o n of i t s own c o n c e n t r a t i o n as w e l l a s t h a t o f o t h e r c o n s t i t u e n t s b ecau se o f t h e c o n s ta n t i n t e r ­ a c tio n th e re w ith . I f t h e l i s t g iv e n may be c o n s id e re d a f a i r r e p r e s e n ­ t a t i o n o f b o v in e serum, t h e a v e ra g e m o b i l i t y f o r albu m in , y - g l o b u l i n i s 6 . 2 , 4*4* 3*0* 1*6 r e s p e c t i v e l y . and C lo s e r i n s p e c t i o n o f t h e p a t t e r n s w i l l r e v e a l t h a t t h e c l a s s i f i c a t i o n by T i s e l i u s o f t h e serum p r o t e i n s i s an o v e r s i m p l i f i c a t i o n . a p p e a r w ith two o r more p e a k s . A ll t o o o f t e n t h e an d/ ? - b o u n d a rie s The s i g n i f i c a n c e of t h e s e added sub­ d i v i s i o n s has been a p p r e c i a t e d b u t n o t i n v e s t i g a t e d p a r t i c u l a r l y i n t h i s paper. A gain, b e in g a r b i t r a r y and ig n o r in g th e i n d i v i d u a l c h a r a c t e r ­ i s t i c s o f t h e serum sp ecim ens, t h e a v e ra g e r e l a t i v e c o n c e n t r a t i o n i n p e r c e n t o f e a c h component w i t h r e s p e c t t o t o t a l p r o t e i n i s 4 2 .4 f ° r A, 1 8 .3 f o r df, 8 . 3 f o r JB , and 31*1 f o r y . A r a t i o g r e a t e r t h a n 0 .8 f o r 7 / A would seem t o be s i g n i f i c a n t im m u n o lo g ic a lly . -2 0 E. M o b i l i t y - pH r e l a t i o n s h i p o f t h e p r o t e i n s i n a serum c o n t a i n i n g B r u c e l l a a g g l u t i n i n s i n a h ig h t i t e r and i n one c o n t a i n i n g no a g g l u t i n i n s . Knowing t h a t t h e pH o f a b u f f e r c o u ld a f f e c t t h e r a t e o f e l e c ­ t r o p h o r e t i c s e p a r a t i o n o f p r o t e i n components o f d i f f e r e n t i s o e l e c t r i c p o i n t s , a s e r i e s o f b u f f e r s o f d i f f e r e n t pH were p r e p a re d i n t h e hope t h a t a n t i b o d y m ight be s e p a r a t e d . I f i t be assumed t h a t a serum of v e r y h ig h a g g l u t i n a t i o n t i t e r would c o n t a i n an a p p r e c i a b l e q u a n t i t y o f a n t i ­ body, t h e n a c o n co m itan t e l e c t r o p h o r e t i c ru n w ith a n e g a t i v e , or soc a l l e d norm al serum would be e x p e c te d t o produce diagram s s u f f i c i e n t l y d i f f e r e n t t o d i s t i n g u i s h t h e a n tib o d y . Another o b j e c t of t h i s s t u d y was t o l e a r n i f t h e p H -m o b ility cu rve and t h e I s o e l e c t r i c p o i n t s o f th e f o u r components d i f f e r e d s i g n i f i c a n t l y i n a s p e c i f i c a n t i s e r u m and a n e g a t i v e serum . T i s e l i u s (49) d eterm in e d t h e p H -m o b ility curve o f h o rse serum b o th on i s o l a t e d components and on whole serum. p o i n t o f alb u m in , re sp e c tiv e ly . He found t h e i s o e l e c t r i c - , and y - g l o b u l i n t o be 4 *6 4 * 5 *06 , 5*12 and 6 .0 Cohn (5 ) a l s o has d is c u s s e d t h e im po rtance o f t h e s e v a lu e s w i t h r e g a r d t o t h e s e p a r a t i o n and p u r i f i c a t i o n o f t h e serum p r o t e i n s . The c h o ic e o f b u f f e r s f o r t h e wide pH range p r e s e n t e d a problem b e c a u se o f t h e d e s i r e t o u s e u n i - u n i v a l e n t b u f f e r s . M ic h a e lis ( 34) showed t h a t a b u f f e r sy stem from pH 2 .6 2 t o 9 . 6I4 oould be p re p a re d by u s in g p r o ­ p e r c o n c e n t r a t i o n s of t h e f o ll o w i n g : sodium b a r b i t u r a t e , sodium a c e t a t e , sodium c h l o r i d e , and h y d r o c h lo r i c a c i d . A c c o rd in g ly , b u f f e r s o lu t i o n s a l l o f 0 .1 i o n i c s t r e n g t h and d e s i r e d pH were p r e p a r e d . Cow H4.3993 was n a t u r a l l y i n f e c t e d ; h e r serum c o n ta in e d t h e h ig h t o t a l p r o t e i n o f 9 .2 0 gm. p e r 100 c c . and showed an a g g l u t i n a t i o n t i t e r o f 1 :4 0 ,0 0 0 . The seru m o f cow 881, s u p p o se d ly n o rm a l, c o n ta in e d a t o t a l -2 1 p r o t e i n o f 7*68 gm. p e r 100 c c . and showed a t i t e r o f 1:20 w hich is r a t h e r common b e c a u se most cow serums a t tim e s show a t l e a s t a s l i g h t r e a c tio n to B ru c e lla . F i g s . 7& dnd 7b i l l u s t r a t e t h e s t r i k i n g s i m i l a r i t y betw een t h e diagram s o f th e two serum s. At pH 7*82 cow 881 showed t h e more complex ^ - g l o b u l i n . At o t h e r pH v a l u e s t h e d i f f e r e n c e , i f an y, betw een t h e two sam ples was s l i g h t . One u n u s u a l phenomenon, common t o b o t h , was t h e a p p e a ra n c e i n t h e a s ­ cen d in g boun dary a t pH 6 . 1 4 o f a sm all peak m ig r a tin g j u s t ahead of t h e t a l l a lb u m in peak ( F i g . 7 °)* W ith i n c r e a s i n g a c t i v e a c i d i t y and a c l o s e r a p p ro a c h t o t h e i r i s o e l e c t r i c p o i n t s , t h e degree o f s e p a r a t i o n between t h e g l o b u l i n s became i n c r e a s i n g l y d i f f i c u l t t o d i s t i n g u i s h . The most g r o te s q u e d iag ram and t h e one w it h t h e g r e a t e s t d i s s i m i l a r i t y betw een t h e a s c e n d in g and d escen d in g b o u n d a r ie s was t h e one o b ta in e d a t pH 3*53 ( F ig .7 ^ ) * A f t e r d i a l y s i s an a p p r e c i a b l e amount o f p r e c i p i t a t e was n o t i c e d i n t h e sam ples a t pH 6 . l 4 , 5*18 and 4*65* t h e serum a t pH 3*65 was s i i g h t l y t u r b i d and p o s s e s s e d a more pronounced y e llo w t i n g e t h a n t h e o th e rs. T a b le V r e c o r d s t h e r e s u l t s o f t h e a g g l u t i n a t i o n t e s t upon t h e d i a l y z e d a n tis e r u m s a f t e r a d ju s tm e n t t o a p r o t e i n c o n c e n t r a t i o n o f one per c e n t. At pH v a l u e s o f 5*18 and 4*65 t h e serum showed com plete a g g lu ­ t i n a t i o n a t one d i l u t i o n l e s s t h a n t h e o t h e r s , and t h e one a t pH 3*53 showed a prozone phenomenon. The r e l a t i v e a r e a s o f each component a t t h e s e v e r a l pH v a lu e s i s a l s o r e c o r d e d i n T able V. The i n c o n s i s t e n t v a lu e s o b t a in e d i n d i c a t e th e o bviou s i m p o s s i b i l i t y of a t t a i n i n g c o r r e c t d e te r m in a tio n s by e l e c t r o p h o r e ­ t i c a n a l y s i s i f t h e b o u n d a rie s a r e n o t s u f f i c i e n t l y separated from each o th e r. T h is f a c t was always n o te d when t h e w orking pH v a lu e was c l o s e t o t h e i s o e l e c t r i c p o i n t of t h e p r o t e i n s i n v o lv e d . -2 2 - Serums rD O (0 no d O O E H© CO OJ KN kn 00 MO o CO OJ OJ OJ S+ KN KN kn KN • ON CO ON LfN o -d d -d -d KN -d KN Ol 1—! • o kn CO ON r- CO KN 1 KN OJ • O! —d • r-H + CO i—t rH LTN • i—1 1 r-H O • + O -d O KN j C m • < rH 1 _ d + + TTV Tn =?■ 73T Tp- -d _l_ VO CVI NO KN OJ O O OJ o o o CO o CvJ I O \ o Kn «h o I O CO o • + JL LTN 3* k n I CO o 1 o KN + o o o KN •t rH rH OJ vt ON o• + • • (— i o i 1 rH s+ LTN rH a! • KN 1 O', -d _L_ _d LTN KN OJ o o o o n o < —1 o o —i~ •t co rH 1—1 OJ ON • -d KN • LfN c— —d • -d -d -d ♦ -d co KN • CO rH • LfN LfN NO • -d KN o o o o O O O LfN «• i—1 LIN OJ 4 ON LTN i—1 _d -d - • + CM si NO NO LTN o vD OJ • LTN * OJ NO • l—I CO I LfN CO *J • • i— 1 o 1 * o I _l_ c^- o NO -d NO ON KN \ + NO LTN • LTN + KN OJ I CO •t o 1—I I 03 0 1 o -? o O O rH o i— i ON r- LfN LfN • CO H* • -d - KN IXN « KN o O OJ -d w afte r rH CCS •rH -P a (D -P O Ph * -Cf I •k © S ♦H ON t-- t W ON -P d © •H n0 cd d o g o i> rH NO # -=t id Ph u • U rH © bO - P hO -H Eh o P u © CO sj rH CO • C— • NO Eh Eh O O o LTN i—1 £ o o OJ • ltn vO d*— • -d ltn si Ol CO • IN- _^+ i—* • NO CO rH • LfN nC CD © ♦rl -P aS ao •r1 -P ffj ao © © J> •H •P cd to © £3 was C o ncentratio n C h a r a c t e r i s t i c s D iffe re n t pH V a lu e s and P hysico-C hem ical Some ■rt rH ON LIN rH vO KN OJ • KN *H i— 1 i— I vO• O rH rH 1 •H ID -P O KN f?N o- CO O J ON • w CO rH UN ir\ Oo done © Ph of Two Bovine © Q ® C lO o O P o- ON co p ro te in T=“ ri Eh KN si cent -P ON p - N O KN per ai o ON kn ks r— one ■vzr TfT - P -P CM in­ T itratio n at _ o i— I •H CCt P • KN a c~- "CCT 00 w © r- to 3 “CET" rO s o lu tio n s t>- of the O ph ^ adjustment fcuO • ♦H O d KN ON ON KN ~d * —I O O O LfN *• 1—1 LTN OJ •• 1--1 LfN OJ •• rH ltn • KN o S t oi rH co 00 > LfN • -d ’ © {> •H -P aJ hD © © > •rt P> cd w © -2 3 The m o b i l i t y v a l u e s as o r d i n a t e s a r e p l o t t e d a g a i n s t pH v a lu e s as a b s c i s s a s i n F i g . 8a f o r cow 110993 &nd i n F i g . 8b f o r cow 881. C o n s id e r in g t h e d i f f i c u l t y i n o b t a i n i n g t h e s e v a lu e s on whole serum, t h e i s o e l e c t r i c p o i n t s found f o r t h e p r o t e i n components of t h e two serums compared f a v o r a b l y w i t h th e m s e lv e s and w i t h t h o s e found by T i s e l i u s (14-9 ) f o r h o r s e serum . Over t h e ra n g e o f pH i n v e s t i g a t e d no a n tib o d y b oundary was d i f ­ fe re n tia te d . I t was u n f o r t u n a t e t h a t serum 881 had an u n u s u a l l y l a r g e ‘T'-peak i n s p i t e of a n e g l i g i b l e t i t e r . At about t h i s tim e an i n v e s t i ­ g a t i o n upon t h e a b s o r p t i o n of an a n tis e r u m o f h ig h t i t e r was co m p leted . I t was found t h a t a g g l u t i n i n a n t i b o d y was a s s o c i a t e d w i t h t h e ' / - g l o b u l i n ( S e c t i o n G ). In t h e l i g h t o f t h i s f a c t i t i s u n d e r s ta n d a b le why serum 881 w i t h a ' / - p e a k a lm o st as l a r g e a s t h a t o f serum lii.3993 produced p a tte rn s in s ig n ific a n tly d iffe re n t. I t must be remembered t h a t th e i n ­ c r e a s e d p r o d u c t io n of " / - g l o b u l i n i s n o t th e f u n c t i o n o f any p a r t i c u l a r a n t i g e n alo ne* Van d e r S c h e e r, W yckoff, and C larke (53) found t h a t pneumococcus, m eningococcus and p a s t e u r e l l a hyperimmune serums from h o r s e s c o n t a i n e d i n c r e a s e d amounts o f " / - g l o b u l i n . F. E l e c t r o p h o r e t i c diagram s o f a h o rs e and a cow serum a f t e r i n j e c t i o n wi t h a w a t e r - s o l u b l e c ru sh e d c e l l f r a c t i o n from Bruce l l a . A w a t e r - s o l u b l e c ru s h e d c e l l f r a c t i o n * has been p re p a re d i n t h i s l a b o r a t o r y which has s u c c e s s f u l l y immunized g u in ea p ig s a g a i n s t Bruce l l a . Having found t h a t t h i s f r a c t i o n s t i m u l a t e d th e p r o d u c t io n o f a n tib o d y * The manner of p r e p a r a t i o n and r e s u l t s o f t h i s f r a c t i o n w i t h g u in e a p ig s i s t o a p p e a r i n a f o rth c o m in g p u b l i c a t i o n . -2l+a g a i n s t B r u c e l l a , an e x p e rim e n t was p la n n e d t o i n j e c t b o t h a h o rse and a cow a t i n t e r v a l s w i t h t h i s f r a c t i o n and t o f o l l o w t h e e f f e c t upon t h e s erum p r o t e i n s e l e c t r o p h o r e t i c a l l y , and i f p o s s i b l e t o d e te rm in e t h e l o c a t i o n o f t h e a n tib o d y i n th e e l e c t r o p h o r e t i c p a t t e r n . E l e c t r o p h o r e t i c p a t t e r n s were made and t h e a g g l u t i n a t i o n t i t e r s d e te rm in e d on b o t h a n im a ls b e f o r e t h e f i r s t i n j e c t i o n o f m a t e r i a l . "Where­ as t h e cow was n e g a t i v e , t h e h o r s e showed an a g g l u t i n a t i o n t i t e r o f 1 :8 0 , i n d i c a t i n g p o s s i b l e p r e v io u s i n f e c t i o n w i t h B r u c e l l a . The o r i g i n a l p a t t e r n s t a k e n b e f o r e i n j e c t i o n a r e i l l u s t r a t e d i n F i g s . 9a 10a. The n e x t sam­ p le from t h e cow was n o t t a k e n u n t i l i t had r e c e i v e d t h r e e i n j e c t i o n s each o f 5 mg* m a t e r i a l a t tw o day i n t e r v a l s . A s h a rp e n in g of t h e / - p e a k o c c u r re d as i t r o s e from 2 2 .9 t o 2 5 .2 p e r c e n t of t h e t o t a l a r e a , Table V I. An a g g l u t i n a t i o n t i t e r o f 1:1280 was now n o te d . was made j u s t p r i o r t o t h e f o u r t h i n j e c t i o n . A t h i r d b l e e d in g A lthough t h e t o t a l p r o t e i n was r e t u r n i n g t o t h e o r i g i n a l c o n c e n t r a t i o n a f t e r an i n i t i a l drop a t t h e b e g in n in g o f t h e e x p e r im e n t, t h e / - p e a k d e c re a s e d s l i g h t l y w h ile t h e a g g l u t i n a t i o n t i t e r c o n tin u e d t o r i s e t o 1:5000 ( F ig . 9 b ) . Two more i n j e c t i o n s w ere g iv e n and t h e f o u r t h b le e d in g w as perform ed one week a f t e r th e t h i r d . The t o t a l p r o t e i n , / - g l o b u l i n , and a g g l u t i n a t i o n t i t e r ( 1 : 1 0 , 0 0 0 ) . a l l a t t a i n e d a maximum f i g u r e . w ith o u t a f u r th e r i n j e c t i o n . d e c lin e ( ^ ig . 9 c ). A nother b le e d in g was made two days l a t e r The same t h r e e f a c t o r s a l l showed a s l i g h t A s i x t h and l a s t i n j e c t i o n was made a t which t i m e , 21 days a f t e r t h e f i r s t , th e a g g l u t i n a t i o n t i t e r was 1 :5000. The anim al was p e r m i t t e d t o r e s t s i x weeks b e f o r e a f i n a l serum specimen was examined (F ig . 9 d ). The t o t a l p r o t e i n was p r a c t i c a l l y r e s t o r e d t o t h e o r i g i n a l c o n c e n t r a t i o n b u t t h e a g g l u t i n a t i o n t i t e r was now o n ly 1:61+0. T h e /-p e a k -2 5 ho • O Ph £5 •H Lf\ • o OJ O J o o p 0 Ph I—I UN o o PS o aJ o OJ TT Pi -P Q > Pi O C\j o ft NO o CO p r OJ OJ 4 CP o KN OJ UN CO 4 4 OJ rH CO rH ON I-- \ KN • 1— I _4 • to UN CD KN KN OJ KN T T NO I" ON UN OJ NO NO C"— TTT ~U\ nO O n0 n • NO CO CO W OJ •H O Oi o ON KN KN KN OJ O ♦ -4* UN UN OJ rH •H 9 4 4 4 "0T " rJ KN n0 NO NO UN CO 9 w CP • p rH © fcO -p £x0*H <£l EH On CO co co Q0 o rH CO 0- o- o- c*~~ t— IN- K\ 0 t> •H -P CD U) CD o CO OJ rH «• rH O o o UN •• i—1 o • O o o9v o rH 9* rH o o o o o o UN UN rH 1--1 & rH ** 99 4 pci o O i—I 1-T> i—1 LT\ LP\ o 4 NO *• rH i—\ < NO "X n O* Pi o •H -P •H -P CD CD CD CD CD 4 -P Pi •H T3 © •x} p rH © Pi •H 9 § ctJ P rd 4 -P r’ © P © •xi o o r—I CD rP t>5 Ch o P aJ "O d 3 o 41 VJ © EH to p © p •rH i—1 © © P _r~| Eh - 26 had m a i n t a i n e d i t s i n c r e a s e d a r e a and even g a in e d a l i t t l e more* The alb u m in p a t t e r n had l o s t a b o u t 5 Pe ** c e n t o f t h e t o t a l area* The h o r s e r e a c t e d l o c a l l y and s y s t e m i c a l l y t o a l l i n j e c t i o n s each o f 5 mg* o f m a t e r i a l g iv e n a b o u t 3 days a p a r t . A b lo o d specim en (F ig* 10b) o b ta in e d 10 days a f t e r t h e f i r s t i n j e c t i o n d is p l a y e d a r i s e o f t o t a l p r o t e i n from 6*03 "bo 7*66 gm. p e r 100 c c ; s in c e t h e c o n c e n t r a ­ t i o n o f albu m in rem ain ed c o n s t a n t , 2*36 t o 2*35* "the e n t i r e i n c r e a s e was due t o g l o b u l i n a l o n e . F u r t h e r i n j e c t i o n s were made and t h e i r e f f e c t i s r e c o r d e d i n T a b le V I I . The h i g h e s t a g g l u t i n a t i o n t i t e r o b ta in e d was 1:50 00 w i t h a ^ / A r a t i o of 1*38. The b lo o d specim en ( F ig . 10c) a n a ly z e d a f t e r t h e f o u r t h i n j e c t i o n showed t h a t , b ased upon p r o p o r t i o n o f t o t a l p r o t e i n , a l l f r a c t i o n s had d e c r e a s e d e x c e p t t h e 'T - g l o b u l i n . L e ttin g a lb u m in e q u a l 100, a f - g l o b u l i n had f a l l e n from 36 t o 3 1 *^*-fro m i+2 t o i+0, b u t t h e 'T '- g lo b u lin r o s e from 77 t o 138* R e g a rd le s s of th e mode of ex­ p r e s s i o n , t h e 'T'-peak a lo n e showed a c o n s i s t e n t enhancem ent. The t o t a l p r o t e i n showed an i n c r e a s i n g l y a c c u m u la tiv e e f f e c t w i t h e a c h s u c c e e d in g i n j e c t i o n , an o b s e r v a t i o n t h a t has b een commonly known i n im m u n izatio n p r o c e s s e s . The t o t a l p r o t e i n r o s e from 6 .0 3 t o 8*60 gm. p er 100 c c . a f t e r s i x i n j e c t i o n s o v e r a p e r i o d of 35 days. An e x a m in a tio n of t h e alb um in c o n c e n t r a t i o n o v e r t h i s p e r io d revedLed t h e s t r i k i n g f a c t t h a t w i t h t h e e x c e p t i o n o f a sm a ll drop i n t h e i n t e r i m , and i n s p i t e o f a 30 p e r c e n t i n c r e a s e i n t o t a l p r o t e i n , t h e c i r c u l a t i n g album in p er u n i t volume o f serum m a in ta in e d p r a c t i c a l l y a c o n s ta n t c o n c e n t r a t i o n , a b o u t 2 .8 5 gm. p e r 100 c c . Even a f t e r a r e s t o f alm ost seven weeks d u rin g w hich tim e t h e t o t a l p r o t e i n c o n c e n t r a t i o n dropped t o 7*3-3 snd -the a g g l u t i n a t i o n t i t e r t o 1:61+0, t h e c i r c u l a t i n g album in was 2 .9 2 . ( F ig . lO d ) . This o b s e r v a t i o n would seem t o i n d i c a t e t h a t album in p la y e d no d i r e c t p a r t i n t h e immune A- I Pl o •H -P •H © to d (D a] U o NO KN • O o d w LA OJ * CO o © si o <4 EH ho -P CD - H O CO •V P-. o N O LA OJ «« 1-- 1 CO OJ rH •« rH LA •• 1--1 OJ rH •• rH NO LA to •H Pi P. OJ ** rH Pl d 0 d 1 vu co F©4 © d I—! o Pl (4 * u I—1 © to *H •H CO • to © On rc\ d *P © to *rrr 75d <4 •H NO OJ KN d T " o ON « o -d •P LA to OJ TvT" H C5N O J ■vZT •rH © -P O * H i—! o KN LA OJ « OJ Kr O O CO 9 o CO H KN OJ KN OJ LA KN CO KN • O OJ KN • E— KN O d KN LA i— I ON CO o rH vO O KN KN o co kn o LA OJ • A © © kn —5” to •H LA ITT * Pi \— 1 *H © -op Pl KN i— ! © Ph O O © *A> Pl Pl O o o LA Pi O . © Hw vO rH O d PQ 5-. KN © fl g > LA LA "S •H O f-H o ■ ^r U Eh *P CO O • (—J d O o 1 —1 OJ © 0 H ON KN. • < O -P KN nO 5h (4 "3~ A d d co A • O o o O A~ • O OJ d 10b bjO • O Ph Id •H 10a -2 7 - XJ Pi o £ O <4 •PP — I SdP. Pi o •H -P d CO _d d LA rH d ^d O CM • ON • A £d nG NO KN • KN CM • KN CM • KN T5T NO CO CO Pt KN • nC pt CM • NO Pf NO • vO Pt A• NO UN CO • A co U\ • A NO CO • A NO cu • A Q\ i— I CO ©10 rH • KN •H O P rH ■H rH d •H •H p£? O -p d Pf ON X Xo ■>X — Pt d o © M Pi d •rH d •H -P d Pn • d i—i rH -4 -4 Eh hD tO © to p t o *H P O -P o © Oh •H P © to © X Pl o o o o P t «* © d «h <4 pq o © X (4 1—1 o CM > p <4 to © 1—I d p © © KN ON ON KN ■pi" d © p % -3 1 a c c o u n t ed f o r 1+.16 p e r c e n t o f t h e t o t a l p r o t e i n o r 7 .8 p e r c e n t o f t h e t o t a l g lo b u lin . In a n t i s e r u m ll|.3993b ( F i g s , 11c and l i d ) , t h e a n t ib o d y a c c o u n te d f o r lj.,7 o f th e t o t a l p r o t e i n , o r 7*7 po** c e n t o f th e t o t a l g lo b u lin . These v a l u e s a r e c o r r e l a t i v e w i t h t h e a g g l u t i n a t i o n t i t e r o f t h e two a n t i serum s. Having d e m o n stra te d c o n c l u s i v e l y t h e l o c a t i o n o f B r u c e l l a a n t i ­ body i n t h e a n t i s e r u m p a t t e r n , some o f t h e u n c e r t a i n t i e s o f t h e p r e c e d in g s t u d i e s can now be e x p l a i n e d . I f i t i s remembered t h a t / - g l o b u l i n may be enhanced by any one o f s e v e r a l a n t i g e n s , and t h a t a f t e r i n i t i a l s t i m u l a ­ t i o n t h e s u b s e q u e n t i n c r e a s e need n o t be p r o p o r t i o n a l t o t h e number o r amount o f a n t i g e n i n j e c t e d , i t can now be su rm ised t h a t i n t h e p re c e d in g stuojr ( S e c t i o n F ) , t h e cow p a t t e r n showed no p r o g r e s s i v e o r s t a r t l i n g i n ­ c r e a s e i n t h e / - p e a k b ecau se i t s / - p e a k was i n i t i a l l y h ig h p o s s i b l y from some p r i o r a n t i g e n i c s t i m u l a t i o n . The p r e v io u s h i s t o r y o f t h i s h e i f e r was n o t known c o m p le te ly . I n s e c t i o n s A and B o f t h i s p a p e r m en tio n was made t h a t no d i r e c t c o r r e l a t i o n e x i s t e d betw een t h e B r u c e l l a a g g l u t i n a t i o n t i t e r and a r e a o f th e /-p e a k . Hew i t i s c l e a r t h a t B r u c e l la a n tib o d y i s o n ly a sm a ll p a r t o f t h e / - p e a k and t h a t t h e re m a in d e r o f t h e / - g l o b u l i n may be n o rm a l, or a n t ib o d y g l o b u l i n due t o some a n t i g e n o t h e r th a n B r u c e l l a ; and hence n o t d e t e c t a b l e i n a B r u c e l l a a n tib o d y t i t r a t i o n p ro c e d u re . T hat t h e / - g l o b u l i n i s n o t homogeneous i s e v id e n t from t h e d i f f u s e and s p r e a d in g c h a r a c t e r o f i t s p e a k . When more f u n c t i o n a l t e s t s a r e made upon t h i s f r a c t i o n , i t s c o m p le x ity w i l l be r e a l i z e d . P i l le m e r e t a l . (37) r e c e n t l y have found t h a t one o f t h e components o f complement composing o n ly 0 . 6 p e r c e n t o f t h e t o t a l p r o t e i n has a m o b i l i t y o f about 2 ,9 x 10“ 5. -3 2 O r d i n a r i l y t h i s component i s masked I n t h e / - p e a k . The s u p p o s i t i o n t h a t t h e / - f r a c t i o n may c o n t a i n o t h e r f u n c t i o n a l u n i t s t r a v e l i n g w i t h a s i m i l a r m o b i l i t y i s e n t i r e l y a r e a s o n a b l e one. H. E l e c t r o p h o r e t i c s t u d i e s o f B r u c e l l a a n tis e r u m o f h ig h t i t e r f r a c t i o n a t e d w i t h ammonium s u l f a t e . The pro blem i n t h e p r e p a r a t i o n o f p u r i f i e d and c o n c e n t r a t e d serums i s i n s e p a r a b l y c o n c e rn e d w i t h t h e d i s t r i b u t i o n o f t h e a n t i b o d i e s among t h e v a r i o u s p r o t e i n f r a c t i o n s of a n t i s e r u m . In g e n e r a l , a n tib o d y produced b y b a c t e r i a l e x o to x in and v i r u s i s l o c a t e d i n t h e p s e u d o g lo b u lin f r a c t i o n o r c a n be p r e c i p i t a t e d w i t h t h i s f r a c t i o n (3i-i-~5Q/£ s a t u r a t e d . The a n t i b o d y p ro du ced by b a c t e r i a ( a g g l u t i n i n s , p r e c i p i t i n s , o p s o n in s ) i s l o c a t e d i n t h e e u g l o b u li n f r a c t i o n . T etanus a n t i t o x i n p r e c i p i t a t e s w i t h t h e two f r a c t i o n s , and a c c o rd in g t o K lo b u s itz k y (22) i s i r r e g u l a r l y d i s t r i b u t e d betw een them . For t h i s r e a s o n w a s t e f u l and i n e f f i c i e n t serum p u r i f i c a t i o n can be a v o id e d o n ly by t h e i n d i v i d u a l e x a m in a tio n o f e v e ry l o t o f serum . The u s e o f ammonium s u l f a t e i n serum f r a c t i o n a t i o n i s c l a s s i c a l , and y e t modern. Gibson (12) i n 1907 f i r s t u s e d t h i s s a l t t o p r e c i p i t a t e t h e e n t i r e Immune g l o b u l i n by h a l f s a t u r a t i o n and t h e n s u b s e q u e n tly ex­ t r a c t e d w i t h sodium c h l o r i d e . Banzhaf ( l ) su b d iv id e d immune serums i n t o t h e e u g l o b u l i n , p s e u d o g lo b u lin , and album in f r a c t i o n s . O th er e a r l y w orkers l i k e C hick and M a r tin (3) and Homer (1 5 ,1 ^ ) l a t e r e l a b o r a t e d t h e e n t i r e pro ced u re. T i s e l i u s ih-9) found t h a t t h e serum p r o t e i n s u b d iv i s i o n s o b t a in e d w i t h ammonium s u l f a t e f r a c t i o n a t i o n and o t h e r chem ical p ro c e d u re were n o t i d e n t i c a l w i t h t h e f r a c t i o n s s e p a r a t e d by e l e c t r o p h o r e s i s . The s a l t -3 3 f r a c t i o n s c o n t a i n e d m ix tu r e s o f t h e e l e c t r o p h o r e t i c com ponents. Cohn and cow orkers (!}.) have fo llo w e d t h e f r a c t i o n a t i o n o f h o rs e serum w i t h v a r i o u s s a t u r a t i o n s of ammonium s u l f a t e . Svensson (I4.5 ) more r e c e n t l y h as f o llo w e d e l e c t r o p h o r e t i c a l l y t h e s u p e r n a t a n t s o f serum s from t h e h o r s e , t h e cow, t h e sw ine and t h e r a b b i t p r e c i p i t a t e d w i th v ary in g amounts o f ammonium s u l f a t e . In a l l t h e s e p re c e d in g s t u d i e s t h e serum o f a v e r a g e or norm al a n im als was u se d ; hence no a tte m p t was made t o f o l l o w t h e a n t ib o d y c o n te n t o f t h e f r a c t i o n s . T a b le IX p r e s e n t s th e d a ta o b t a in e d from b o th t h e p r e c i p i t a t e s and s u p e r n a t a n t s from a serum o f h ig h B r u c e l l a a g g l u t i n i n c o n t e n t , and t h e r e s u l t i n g a g g l u t i n a t i o n t i t e r s o f t h e s u p e r n a t a n t s w hich w ere e a s i l y r e s t o r e d t o t h e o r i g i n a l volume. On accou nt o f t h e sm all amount of m a t e r i a l , t h e a g g l u t i n a t i o n t i t e r s of t h e p r e c i p i t a t e s can be approxim ated r e a d i l y by d i f f e r e n c e . The e n t i r e p ro c e d u re and t h e p r e p a r a t i o n o f t h e s a t u r a t e d amnonium s u l f a t e was made a t room te m p e r a tu r e w i th t h e r e a c t i o n a d j u s t e d n e a r pH 6 . The serum 201, 75 c c . , was d i l u t e d once w i t h s a l i n e s in c e s e p a r a t i o n s (U) a p p e a r t o be s l i g h t l y more e f f e c t i v e i f t h e p r o t e i n s a r e l e s s c o n c e n t r a t e d t h a n n o r m a lly found i n serum. The d i l u t e d serum was d iv id e d i n t o t h r e e l o t s o f 50 c c . each and p la c e d i n g r a d u a t e s . A c a l c u l a t e d amount of t h e s a t u r a t e d ammonium s u l f a t e s o l u t i o n was d i a l y z e d i n t o serum th ro u g h r o t a t i n g c e llo p h a n e membranes a c c o r d in g t o t h e method o f McMeekin ( 3 l) « was a t t a i n e d i n a b o u t 2 h, h o u r s . E q u ilib r iu m The p r e c i p i t a t e s a f t e r c e n t r i f u g a t i o n were washed w i t h s m a ll a l i q u o t s of ammonium s u l f a t e o f th e c o n c e n t r a t i o n a t w h ich s e p a r a t i o n was e f f e c t e d . A l l f r a c t i o n s were d i a l y z e d a g a i n s t w a te r t o remove t h e ammonium s u l f a t e , d i l u t e d t o a one p e r c e n t p r o t e i n con- -P 4 NO • •H O fp Cj 0J »—1 P 0J O 0J I—1 nd OJ i—1 CO O J o o o« o Pt I—I OJ rH rO KN rH o KN i—I Ti KN i—1 On vO* KN o dO OJ -p •H 03 0 1 Composition of the S u p e rn a ta n ts and P r e c i p i t a t e s F ra c tio n a te d With Varying Amounts of a High T iter Antiserum of Ammonium S u l f a t e (2 0 1 a ) o t— *- o o Pt VO OJ o o o NO NO • LT\ o KN oJ Q) d KN w d o rH •H ctS P -P CiS O d Eh ' P d -P © d © © d O o o d © Pn KN Pf UN • CO OJ o 01—I KN OJ • I—I Pt • o rH LfN 7% o o o♦ o Pl* nO CJN _ t O J LfN n £> o c? co co • KN KN o O o o o NO N O o- KN • OJ • ON o* o 01• OJ O i—1 KN i— i O • OJ UN o NO N O E"- • ON D— OJ p o •H rH KN •H NO J! i— I K pf pt KN KN O! NO N O NO * • 0- rH aJ © o EH o o on o OvJ co• C1— o o o•V OJ co• p- o LTN OJ KN rH rH rH •* •• ON o o • -d rH rH • o ON OJ INo• o NO NO o • o OJ OJ KN OJ n0 LfN P t ON • d o •H 1~> OJ GO • C-- pf pt 0- NO NO OJ 00• co• c— OJ r— LT\ NO OJ co « o- © !> OJ d © -P OJ 0-+ UN ©-p *H OJ C— • OJ 03 Pt CO P d 00 pt NO • d rH © NO P NO -H < EH CO Pt * i*3' !5i '— o• o 3* C"t-J 00 CO «H tO CO to o P •t r-t i— I UN •* CO • KN *H P oS NO © £ -P 03 © © 03 © P P d •H i—•1 rH LfN OJ NO f • I—1 C— © CH Ph o O i—1 £-i % 'Ssx^ p* a & • KN -d ON • rH o LfN • rH OJ a—1 o i © -P • rH © OJ o» NO H- C^ OJ OJ OJ • o & xi o d ©o ©o -p o 3 •rt d O d © > d CM ,d -P xl ♦H d d XJ 03 © -P -P d d aS d -P o d ♦H d -P d O © d PMd d CO © rd 4? KN NO C O fl) r— > •H ID -P O •H rH rH o CO o KN t s 7 ? O o • O + i s ♦r NO NO no CM TFT TTT m nco KN - g - ON • CO * KN NO KN CO CO CM KN o CM O -j- kn O KN m ON " cm " o o -4 - ON c-~- o ON ON NO NO NO ON o in "nS* 7 3 OJ T T -P t o OJ i— l UN in t? O Xl -d" _d- o• o UN • KN • n- c~ •- rH• UN n- aj TFT ■\ZT ■— i o n- CO O• o © o CM o O d CVJ ON n- in c— KN in -d - UN KN s O- KN CM CM KN UN KN KN — t ON rH • • UN NO -dO NO -d NO NO NO nO ON ON • ON ON ON ON ON ON • ON ON # ON ON • pH — -Pf td PM Oi * d rH © bO-p bO*rH Eh NO NO NO CO • c— o CM rH U+»N rH ON ON • C^ o CM rH U»•N rH t>o aD C M i—I «• rH • c~-~ o CO CM rH rH d o •H -p d rH O TO 00 KN » KN • r- o o NO NO UN C•«M rH UN C♦* M rH no o o U««N rH no NO UN C«•M rH * © d o •H tdJ •rH -P © © © T O © -P d -P rH © ON n• ON rH ON NO 3 in P S d as © -p rt o o EH - p O d . . d CM O f t , o o co -d KN UN 8 CM 00 KN o UN UN NO © CP o -d CM CM O N O in XI © Xl d i—i o d ■H © •H 1--1 NO o 'S' CM CM -P S >3 d d XI d d o r© 1 MJ o S i 01 o cm 0 2 ^O d© rHP rd o ■p UN O © o o rH d ^ © >3 PM r© o CM O KN O -d o © d o •rt -P O d d PH d © © © rH O .A ..! *8 d -P d d d © PM d CO -P d -P •H PM •H O © d P-t o CM O KN O d • d o •rH -P d t—i o TO a> d o NO rH -d d d © PM IP- CO CM o o © -P O d PM O O 00 do •H -P •H ■dH CM CM NO KN CM 2 « < fO -Pt in H ~ f rd s bO •H cm td o Ph o co ON • o O o TFT "T\T “TFT ON X> rH i— I "TFT “TfT i— i H CM • o O UN EH 1-- 1 •P rH d TO )—1 d © •H 6D O i—1 o •iH TO r 'i d! PM © r* -P C m O -P d © n -p TO d , © d o TO d -P •H £ d © -P •H -P Xl © •P d rH •H XI d • rt d -P d rH to bJD < 01 d d © TO •H -P to determ in ation PS. • LTN an electro ph oretic 2 * CM oS UN for i— 1 protein d • o Insufficient hO •H -3 9 t h e # - p e a k was removed f o r t h e f i r s t t im e . P i g . li+d i l l u s t r a t i n g t h e accom panying p r e c i p i t a t e , how ever, shows t h e p r o b a b i l i t y o f d e n a t u r a t i o n b e c a u s e i n s t e a d o f t h e two u s u a l l y w e l l —d e f i n e d ^ — an -peaks, t h e d iagram shows a much skewed peak w i t h a m o b i l i t y i n betw een t h a t o f t h e two g l o b u l i n s . The a g g l u t i n a t i o n t i t e r s of b o th th e p r e c i p i ­ t a t e and t h e s u p e r n a t a n t now a r e alm o st e q u i v a l e n t , i n d i c a t i n g t h a t a l c o h o l p r e c i p i t a t i o n , ev en f o r t h e same component, was i n d i s c r i m i n a t e . U n lik e ammonium s u l f a t e w hich c o m p le te ly p r e c i p i t a t e d a l l o f t h e 7 g l o b u l i n a t I4.O p e r c e n t s a t u r a t i o n , e t h a n o l removed no component com­ p l e t e l y ev en a t t h e h i g h e s t c o n c e n t r a t i o n u n d er t h e c o n d it i o n s men­ t i o n e d ( F i g . 1 5 )- I t was a l s o n o te d t h a t i n t h e p r e c i p i t a t e s t h e r e was a s l i g h t b u t d i r e c t i n c r e a s e of albu m in w i t h a l c o h o l c o n c e n t r a t i o n ( F i g . II4) . W hile n e i t h e r t h e s a l t n o r th e a l c o h o l method w i l l p r e f e r e n t i a l l y d e ta c h t h e a n t i b o d y from th e " X -p e a k , a co m b in atio n of t h e two methods would p r o b a b l y be e f f e c t i v e i n o b t a i n i n g a 7 - g l o b u l i n e n t i r e l y f r e e o f any o f t h e o t h e r c h i e f p r o t e i n components of s e ru m . Having p r e p a re d a p u r e s o l u t i o n of t o t a l 7 - g l o b u l i n , f u r t h e r s tu d y c o u ld be c o n tin u e d t o s e p a r a t e t h e a n t i b o d y from a m ajor p o r t i o n o f t h e i n e r t X - c o n s t i t u e n t i n an a t t e m p t u l t i m a t e l y t o p r e p a r e a t r u l y p u r i f i e d and c o n c e n t r a te d an tiseru m . -U o- SUT&IARY The e l e c t r o p h o r e t i c p a t t e r n s of1 a s e r i e s o f normal and " b r u c e l l o s i s i n f e c t e d "bovine serums have been o b ta in e d u s in g t h e s c h l i e r e n s c a n n in g method o f Longsw orth. The m o b i l i t i e s and concen­ t r a t i o n s o f e l e c t r o p h o r e t i c a l l y d i s t i n c t p r o t e i n components have been computed from t h e s e p a t t e r n s . The m o b i l i t i e s o f t h e serums f a l l i n t o f o u r -well d e f i n e d groups c o rre s p o n d in g t o albu m in, & s f t - , and Y ~ g l o b u l i n s • The o u t s t a n d i n g c h a r a c t e r i s t i c o f t h e serum o f a new-born c a l f was th e e x tr e m e ly h ig h c o n c e n t r a t i o n o f ^ - g l o b u l i n i n c o n t r a s t t o a n e g l i g i b l e , o r a lm o st co m p lete ab sen ce o f '7 '-g l o b u l i n . W ith in fo u r hours a f t e r t h e i n g e s t i o n of c o lo s tr u m by a norm al c a l f t h e 'T -peak m ight a cco u n t f o r a b o u t 15 p e r c e n t o f t h e t o t a l p r o t e i n , w h ile in an i n f e c t e d d a l f t h e f i g u r e m igh t r i s e t o 30 or ^ 0 p e r c e n t . A g g lu tin in s w ere e v i d e n t l y ab­ s o rb e d a l s o a s m easured by t h e a g g l u t i n a t i o n p ro c e d u re . By t h e end o f two weeks a l l p r o t e i n components were i n t h e r e l a t i v e c o n c e n t r a t i o n u s u a l l y found i n young h e i f e r s . V a c c in a tio n o f c a l v e s w i t h U .S .B .A .I . B r u c e l l a s t r a i n 19 c a u se d o n ly a s l i g h t change i n t h e normal serum d i s t r i b u t i o n ; ev en t h e s l i g h t i n c r e a s e i n T '- g lo b u l i n a lo n g w i t h th e a g g l u t i n i n t i t e r r e t u r n e d t o norm al w i t h i n a month. No s i g n i f i c a n t o r permanent change c o u ld be d e m o n s tra te d e l e c t r o p h o r e t i c a l l y t o a c c o u n t f o r t h e immunizing p r o p e r t y o f s t r a i n 19. The p a t t e r n o f th e v a c c i n a t e d anim al resem bled t h e cu stom ary p a t t e r n o f any young h e i f e r . The a v e ra g e m o b i l i t y from a la r g e number o f v a r i o u s bovine serums f o r album|in, £ ( - , J3 — a n d 'X - g l o b u l i n was 6 . 2 , resp e c tiv e ly . 3*0* and 1 .6 From t h e same group t h e a v e ra g e r e l a t i v e c o n c e n t r a t i o n i n p e r c e n t o f e a c h component w i t h r e s p e c t t o t o t a l p r o t e i n was found -1+1t o b e 1+2.1* f o r a lb u m in , 1 8 .3 f o r / ? - , 8 .3 f o r >3-, and 3 1 .1 f o r y - g l o b u l i n . A r a t i o g r e a t e r t h a n 0 .8 f o r 'T/A a p p e a re d t o be s i g n i f i c a n t im munological l y . U sing a u n i - u n i v a l e n t system o f b u f f e r s a t d i f f e r e n t pH v a lu e s and a t a c o n s t a n t i o n i c s t r e n g t h o f 0 . 1 , t h e i s o e l e c t r i c p o i n t o f th e p r o t e i n com­ p o n e n ts o f a b o v in e a n t i b r u c e l l a serum was found t o be I*.1+3, 1+.98, 5*28 and 6 . 0 2 f o r a l b u m i n , ^ - , / ? - , and 7 - g l o b u l i n r e s p e c t i v e l y . I n j e c t i o n o f a w a t e r - s o l u b l e c ru s h e d c e l l f r a c t i o n a t i n t e r v a l s i n t o a h o r s e and a cow cau sed an a c c u m u la tin g i n c r e a s e i n t o t a l p r o t e i n in b o th s p e c ie s . In c o n t r a s t t o t h e cow t h e h o rse showed a tremendous i n c r e a s e o f t h e ' / - g l o b u l i n w h ich a t t a i n e d a maximum a r e a o f 1+I+.3 p e r c e n t from an i n i t i a l 3 0 .1 p e r c e n t w h ile t h e cow i n c r e a s e d o n ly t o 2 8 .5 from an i n i t i a l 2 2 .9 Pe r c e n t . A p p a re n tly , t h e album in c o n c e n t r a t i o n p e r u n i t volume of b lood rem ained c o n s ta n t d u rin g t h e whole p e r i o d of h y p e rim m u n izatio n i n s p i t e o f a r i s e o f over 25 p e r c e n t i n t o t a l p r o t e i n i n th e case o f th e h o rse. A b s o rp tio n o f an a n ti s e r u m o f h ig h t i t e r showed th e 7 - g l o b u l i n t o be t h e a s s o c i a t e o f B r u c e l l a a n t i b o d y . Ammonium s u l f a t e f r a c t i o n a t i o n a t a pH o f 6 by d i f f u s i o n th ro u g h a r o t a t i n g c e llo p h a n e membrane su cceeded i n p r e c i p i t a t i n g o u t a l l t h e 7 -component in c lu d in g about 99 Pe r c e n t of t h e a n t i b o d y a t a s a l t c o n c e n t r a t i o n of i+0 p e r c e n t s a t u r a t i o n . E th an o l i n t r o ­ duced i n t o a n t i s e r u m i n a s i m i l a r manner b u t a t 0°C. was l e s s d i s c r i m i n a t e in th e fra c tio n s p r e c ip ita te d . Under t h e c o n d i t io n s d e s c r i b e d , an a l c o h o l c o n c e n t r a t i o n o f 1+0 p e r cen t by volume f a i l e d t o p r e c i p i t a t e more t h a n 50 p e r c e n t o f t h e a n t ib o d y , w h ile 30 p e r c e n t of t h e a l c o h o l d id succeed In p r e c i p i t a t i n g o v e r t h r e e q u a r t e r s of t h e a g g l u t i n i n c o n te n t* A com b ination o f t h e two p r o t e i n p r e c i p i t a n t s seemed t o o f f e r t h e p o s s i b i l i t y of f ra c tio n a tin g - 1+2 - serum i n t o components c l a s s i f i e d by T i s e l i u s . A f t e r o b t a in i n g t o t a l 7 - g l o b u l i n i n t h i s way, f u r t h e r e x p e r i m e n t a tio n m ight y i e l d a method t o f u r t h e r p u r i f y and c o n c e n t r a t e B r u c e l la a n tib o d y . ACKNOWLEDGMENT The a u t h o r w ish e s t o e x p re s s h i s deep g r a t i t u d e t o Dr. I . F o r e s t H uddleson u n d e r whose d i r e c t i o n t h i s stu d y vras done f o r h i s c e a s e l e s s i n t e r e s t and i n s p i r i n g exam ple, and t o th a n k Dr. L. M. H u tch in g s a l s o o f t h i s l a b o r a t o r y f o r g e n e r a l t e c h n i c a l and v e t e r i n a r v a id . -U SLIT ERATURE CITED 1, B an z h af, E. J . and R. B« G ibson. a n t i t o x i c serum. 2. The f r a c t i o n a l p r e c i p i t a t i o n o f J . B i o l . Chem., B l i x , G ., A. T i s e l i u s , E. S vensson. 253 (1907) L ip id s and p o ly s a c c h a r i d e s i n e l e c t r o p h o r e t i c a l l y s e p a r a t e d b lo o d serum p r o t e i n s . J. B io l. Chem., 137, U85 (19U1). 3- C h ic k , H ., and C. J . M a r t i n . ammonium s u l f a t e . The p r e c i p i t a t i o n o f egg-album en by A c o n t r i b u t i o n t o t h e t h e o r y of th e " s a ltin g -o u t” of p ro te in s . ij.. C ohn, E . J . , Biochem. J , , 7» 380 (1913)- T . L. M cM eekin, J . 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Med. 6 5 , 62j.l (1 9 3 7 ). 51* T i s e l i u s , A ., and E. A. K abat. E l e c t r o p h o r e s i s o f immune serum. S c ie n c e 87, 2+16 (1 9 3 8 ). 52, T i s e l i u s , A* and E . A , K ab at. An e l e c t r o p h o r e t i c s tu d y o f immune serums and p u r i f i e d a n tib o d y p r e p a r a t i o n s . J . E x p t l . Med. 69, 119 ( 1939). 53. Tan d e r S c h e e r , J . , R. W« G. W y ck o ff and F . E. C l a r k e . The e l e c t r o p h o r e t i c a n a l y s i s o f s e v e r a l hyperimmune h o r s e s e r a . J . Immunol. 39 ( l ) , 65 (192+0). 52+. v®-*1 d e r S c h e e r, J . , J . B. Lags d in , and R. W. G. Ytfyckoff. E le c tro ­ p h o r e t i c and u l t r a - c e n t r i f u g a l a n a ly s e s o f an ti-p n e u m o c o c c a l h o rs e s e r a . 5 5. J . Immunol, 2+1, 209 (I9 2 ;l). van d e r S c h e e r, J . , R. W. G. Wyckoff and F* E. C la r k e . The e l e c t r o p h o r e t i c a n a l y s i s of t e t a n a l a n t i t o x i c h o r s e - s e r a . J . Immunol., 1+0 ( 2 ) , 173 (192+1) • Calf 100b, 6 days Serum, 1«80 Calf ICOa, at birth plasma, negative (* 6) a (a) Calf USl+a, at birth serum, negative (d) CeIf 100c, 22 days serum, l:l6o Fig. 1. Calf l|6i|b, 1|6 days serum, negative Blood Specimens Drawn Before ana After Ingestion of Colostrum with Age of Calf and Agglutination Titer. A d. A Ik. (b) (eO Calf CA 22b hours Calf CA 22a at birth A Ajj L (c) Calf CA 22o 28 hours Fig. 2. l . |A jj[ W Calf CA 22e 16 days Serum Specimens from a Domini Calf Drawn at Intervals with Age of Calf; Agglutinin Titer was Always begative. -5 0 - (b) Calf 876b 26 hours, 1*10,000 1230 ' Heifer 3U0D6120 exposed via eye, pregnant, 1»2560 Cow lu5995b infected naturally, 1>1+0, 000 6. A General Serios of Bovine Serums with Berserks and Agglutination Titer. -5 2 - a') M Cow 381a T iter 1«20, pH 7.82 (c) Cow lit3993b pH 6 .1 * Cow ll>3993b pH 7.81 A Ce) uj Cow 113993b pH ?.13 Fig. 7* C o?/ U +3 9 9 3 H pH U .65 a. (O Cow 12*3993b PH. 3 .5 3 Mobility - pH Kelationship of the Serum Proteins of a Cow with an Agglutinin Titer of lil|0,O0O. M o b ility (D i 10“ ) U .5 7 Fie*_8*. UoblIitr-pH fiolotlrm*hi£ o f Ccht3 a r a n A gr.lutlttVtlon. TltW rlj, liTio.ooo. 11+39 9 3 ^ tlty+pB Roltttlonihlp of C^r £»rt» £6Ijl , Scrum before injection agglutination, negative 53- 2 days after 3rd.injection 1:5000 , (♦s) a d 1+ days a fte r j t h , injection 115000 F ig. 9. Response of II (d) 1*2 daya afte r 6th.injection 1:61+0 the Serum Proteins of -^ ife r 755297 to Injections of a Water-Soluble Crushed Cell Fraction of Brucella. Serum before injection Agglutinrtion,1:30 'c; j ia s after 1+th. injection 1:1230 f i r . 10. “ ( d) >1 days alter 6th. inject ion 1 *&+0 Resnonse of the Scrum Proteins of a horse to Injections of a ’..ater-Soluble ' Crushed Cell fraction of Bruoella. d .— >) (a) Cow 201a before absorption, lti4.0 ,000 A r A (+0 Con 201a a fte r absorption, negative r (*S) ft C L (d) (c) Cow U(.3993b before absorption, 11/4.C,OOO Fig. 11. Cow lu3993b after absorption, negative Effect of Agglutinin Absorption upon the frotein Components of a Brucella Antiserum. Cow 201a whole serum, 1«20,000 d . (c) I4.O % saturation agglutination, l t 320 Fie. 12. (d) 50 % saturation agglutination, negative Supernatants of a Brucella Antiserum Fractionated with Varying Amounts of Ammonium — Sul fat e. -5 5 - ili % saturation Cow 201a wY-ole serun, 1 :20,000 (*) (d) 50 % saturation 1(0 % seiuration Fig. 13. Frecipitates of a Brucella Antiserun Fractionated with Varying Amounts of Smmonium Sulfate. (h) £*0 Yihole serum agglutination, 1:5120 L d,----------------------- ► A u: 30 .3 1-hanol agglutination, 1:5000 Fig. lit. 20 Ethanol a glutination, 1:2560 d.- i n jd) h0 ,3 Ethanol agglutination, 1 :256c Precipitates of a Brucella Antiserum Fractionated with V>rying Amounts of Ethanol at O'C. ▲i 1C 2 Ethanol i-'lutirm tion, ls>120 a r $ d. — (O £0 X Ethanol agglutination, Is 1280 ill (c 30 /c Ethanol agglutination, Is 1280 Fit-. 15. ! i*.0 X Ethanol agglutination, I 12560 Supernatnnts of a Brucella Antiserun Fractionated with Varying Amounts of Ethanol at 0°C.