Antibodies to Proteins from Yolk of Immunized Hens

transcript

IMMUNOLOGICAL COMMUNICATIONS, 9 ( 5 ) , 495-514 (1980)

ANTIBODIES TO PROTEINS FROM YOLK OF I M M U N I Z E D HENS

A. P o l s o n , M . B a r b a r a von Wechmar and Gina F a z a k e r l e y

Department o f Microbio logy

U n i v e r s i t y o f Cape Town

Rondebosch

South A f r i c a

ABSTRACT

Immunoglobulins IgY, e x t r a c t e d from y o l k s of h e n s immunized a g a i n s t

severa l p r o t e i n s and n a t u r a l m i x t u r e s o f p r o t e i n s w e r e exam.ined by

t h e Ouchter loney g e l d i f f u s i o n and L a u r e l 1 immuno-e lec t rophores i s

t e c h n i q u e s . The m o l e c u l a r w e i g h t s o f t h e p r o t e i n s ranged from

8 x l o 6 t o less t h a n 2 x 10 The hens produced IqY a n t i b o d i e s

r e a d i l y when i n j e c t e d w i t h t h e h i g h m o l e c u l a r a n t i g e n s >150 0 0 0

b u t d i d n o t react as well t o a n t i g e n s of r e I . a t . i v e l y low m o l e c u l a r

weight 4 0 0 0 0 . Optimal p r e c i p i t i n r e a c t i o n s w e r e o b t a i n e d w i t h

low m o l e c u l a r a n t i g e n s when t h e a g a r o s e gel c o n t a i n e d 1 . 5 M NaC1.

S a l t c o n c e n t r a t i o n ( 0 . 1 5 - 1 . 5 M N a C 1 ) h a s no e f f e c t on t h e p r e c i p i -

t i n r e a c t i o n i n g e l s on t h e sys tems o f h i g h m o l e c u l a r a n t i g e n s and

t h e i r IgY a n t i b o d i e s a p a r t f rom a displacement . o f t h e p r e c i p i t i n

l i n e . From t h e r e l a t i v e p o s i t i o n s o f t h e p i - e c i p i t i n l i n e s t o t h e

w e l l s c o n t a i n i n g t h e r e a c t a n t s it would a p p e a r t h a t t h e IgY formed

d imers when t h e c o n c e n t r a t i o n o f N a C l i s 1 . 5 PI and remain as mono-

m e r s i n g e l s c o n t a i n i n g 0 .15 M N a C l . An e x p l a n a t i o n of t.he s a l t

e f f e c t i s o f f e r e d .

INTRODUCTION

I t h a s been shown‘’

yolks of h e n s which

t h a t a n t i b o d i e s c o u l d be i s o l a t e d from t h e

w e r e immunized w i t h a v a r i e t y o f p l a n t v i r u s e s

Copyright 0 1980 by Marcel Dekker, Inc

by t h e use of po lye thy lene g l y c o l o f MW 6 0 0 0 ( P E G ) . A concent ra -

t i o n of 3 .5% of t h e polymer caused t h e l i p i d s and v i t e l l i n t o se -

p a r a t e when t h e yolk was d i l u t e d wi th phosphate b u f f e r of m o l a r i t y

0 . 0 1 and pH 7 . 5 . The immunoglobulin IgY w a s p r e c i p i t a t e d wi th

1 2 % peg. t h e t i t r e of t h e i s o l a t e d a n t i b o d i e s remained a t a mo-

d e r a t e l y h igh leve l i n t h e y o l k s a f t e r c e s s a t i o n of t h e hyper -

immunization of t h e b i r d s . The o b s e r v a t i o n made by s e v e r a l au tho r s

t h a t a h igh s a l t c o n c e n t r a t i o n ( 1 . 5 M N a C l ) enhances fowl serum

a n t i b y t i t r e s , could no t be confirmed wi th yolk a n t i b o d i e s d i r e c -

t e d a g a i n s t t h e p l a n t v i r u s e s . I t was f u r t h e r shown t h a t t h e pro- d u c t i o n of a n t i b o d i e s i n r a b b i t s d i d no t d i f f e r from t h e produc-

t i o n i n hens a s r e f l e c t e d i n t h e i r yo lk IgY t i t r e s when t h e two

s p e c i e s w e r e immunized and hyperimmunized s imul t aneous ly .

POLSON, VON WECHMAR, AND FAZAKGlUEY

The fo l lowing communication i s an account of work done on t h e

p roduc t ion of IgY a n t i b o d i e s d i r e c t e d t o s e v e r a l p r o t e i n s and

n a t u r a l mix tu res of p r o t e i n s and t h e e f f e c t o f c o n c e n t r a t i o n o f

sodium c h l o r i d e ( N a C 1 ) on t h e p r e c i p i t i n r e a c t i o n o f immune IgY's

and t h e i r cor responding a n t i g e n s . It is a l s o shown t h a t t h e mo-

l e c u l a r weight of t h e a n t i g e n i s an impor t an t f a c t o r f o r t h e s t i - mula t ion of p r e c i p i t a t i n g an t ibody p roduc t ion a s w e l l as i n t h e

v i s i b i l i t y of t h e r e a c t i o n between a n t i g e n and an t ibody .

The molecular weights ( m w ' s ) o f t h e " s i n g l e " p r o t e i n s used a s

a n t i g e n s a r e w e l l documented b u t on ly of some of t h e components i n

t h e mixture of a n t i g e n s used. I n t h e g e l p r e c i p i t i n tes ts on t h e

n a t u r a l mix tu res and IgY a n t i b o d i e s , t h e l i n e s formed c l o s e t o t h e

an t ibody w e l l s and were concave towards t h e s e . Th i s behaviour of

t h e a n t i g e n s i s i n d i c a t i v e of a n t i g e n s wi th m w ' s a p p r e c i a b l y lower

t h a n t h a t o f t h e IgY ant ibody.

MATERIALS AND METHODS

Antigens

The fo l lowing p r o t e i n s and " n a t u r a l " mix tu res of p r o t e i n s w e r e used

a s a n t i g e n s :

6 ( a ) Haemocyanin of Burnupena c i n c t a (g. c i n c t a ) , mw 8 x 10 ,

SZOw = 95 and 1 0 0 .

ANTIBODIES TO PROTEINS FROM YOLK 49 7

Baemocyanin of J a s u s l a l a n d i i ( J . lal.a,?dL i-) , major cor.ponent 5 mw 5 x 10 and S 2 0 w = 16 w i t h minor coinponents w i t h s v a l u e s

2 2 , 1 1 , 7 and 4 . 0 .

I-Iuman IgG, p u r i f i e d by i o n exchanging ,on DEAE c e l l u l o s e , mw

1 . 5 x 1 0 . 5

5 Amandin - a p r o t e i n e x t r a c t e d from almonds, mw 3 . 3 x 10 . Tetanus toxoj.d, composed of a minimum o f e ight . a n t i g e n ? .

T h e i r molecular w e i g h t s w e r e assumed t o be ].ow w i t h t h e

e x c e p t i o n o f t h e n e u r o - t o x o i d which i s known to have a m a p p r o x i m a t e l y :L50 0 0 0 (2) .

P u f f a d d e r ( B i t i s a r i e t a n s ) venom. Thi!.; venom. c o n t a i n s a minimum of n i n e t e e n a n t i g e n s as shown by t w o d i m e n s i o n a l

L a u r e l 1 e l e c t r o p h o r e s i s ( 2 x L e ) i n whi.ch hyperimmune a n t i -

venirie p r e p a r e d i n a h o r s e w a s used (tic be publ i shed . ) . The

m o l e c u l a r w e i g h t s o f t h e venom components; are all l e s s t h a n

45 O O O ( ~ ) .

Cobra (Naja n i v e a ) venom. Cobra venom i s composed o f a

minimum o f 20 a n t i g e n s as shown by 2 x L e on hyperimmune

h o r s e a n t i v e n i n e ( t o be p u b l i s h e d ) . The venom components

have m o l e c u l a r w e i g h t s less t h a n 20 O O O ( " ) . Serameba a n t i g e n . T h i s a n t i g e n i s a m;.xt:ure o f a minimum

of n i n e t e e n a n t i g e n s as shown by 2 x Lc: w i t h r a b b i t immune

serum. The mo.lecular w e i g h t s w e r e e s t r m a t e d t o b e well

below 2 0 0 0 0 as no component i n t h e mj-xture had a sedimenta-

t i o n c o e f f i c i e n t g r e a t e r t h a n 2 S when t h e a n t i g e n s w e r e u l t r a c e n t r i f u g e d i n t h e Model E . Addit: i o n a l e v i d e n c e f o r

t h e i r m o l e c u l a r w e i g h t s w a s s u p p l i e d by O u c h t e r l o n y tes ts .

I n t h i s t es t the p r e c i p i t i n l i n e s of Serameba w i t h i t s r a b b i t

immune serum formed v e r y c l o s e t o the a n t i b o d y w e l l s and

w e r e concave towards t h e s e ; a n i n d i c a t i o n of l o w m o l e c u - l a r w e i g h t .

IgY as A n t i g e n . R a b b i t s w e r e immunized w i t h I g Y t o g a i n

knowledge o f i . t s a n t i g e n i c c o m p o s i t i o n . The IgY w a s homo-

geneous i n t h e Model E and had a sedlimc?niration c o e f f i i c i e n t

of 7 . 1 s .

Hens. Hybr ids of %ode I s l a n d r e d and w h i t ( - Leghorns, 2 0 weeks

o l d , w e r e k e p t i n I s o l a t i o n f o r immunizat io? and egg p r o d u c t i o n .

49 8 POLSON, VON WECHMAR, AND FAZAKERLEY They showed

o f t h e e x p e r i m e n t . no s i g n o f o f d i s c o m f o r t o r i l l n e s s d u r i n g t h e p e r i o d

Chemica ls . P o l y e t h y l e n e g l y c o l o f m o l e c u l a r weight 6 0 0 0 (PEG)

w a s used i n t h e f r a c t i o n a t i o n o f t h e e g g y o l k . Sodium a z i d e

( N a N 3 )

t i v e o f t h e washed y o l k s immunoglobul in I g Y ( 4 ) .

d e n o t e s immunoglobul in i n h e n serum as w e l l as i n y o l k .

a t a c o n c e n t r a t i o n o f 0 . 1 g / l i t r e w a s .used as a p r e s e r v a -

The t e r m IgY

B u f f e r . The y o l k s w e r e d i l u t e d i n 0 . 0 1 M phosphate b u f f e r o f

pH 7 . 5 .

o f t h e same c o m p o s i t i o n w a s used as t h e d i s p e r s i o n medium fo r

t h e IgY when f i n a l l y p u r i f i e d .

The b u f f e r c o n t a i n e d 0 . 1 M N a C l and 0 . 0 1 % NaN3. B u f f e r

Immuniza t ion . The p u l l e t s r e c e i v e d a n i n i t i a l i n j e c t i o n i n t r a -

m u s c u l a r l y w i t h e a c h o f t h e a n t i g e n s o r n a t u r a l m i x t u r e o f a n t i -

g e n s . The a n t i g e n s were d i s s o l v e d i n p h o s p h a t e b u f f e r and emul- s i f i e d w i t h a n e q u a l volume o f F r e u n d ' s i n c o m p l e t e . The concen-

t r a t i o n o f t h e a n t i g e n w a s n o t c r i t i c a l and v a r i e d from one an-

t i g e n t o a n o t h e r . C o n c e n t r a t i o n s o f 1 t o 5 mg/ml w e r e commonly

used . A f t e r t h e i n i t i a l i n j e c t i o n t h e young h e n s r e c e i v e d a minimum of t h r e e f u r t h e r i n j e c t i o n s a t a p p r o x i m a t e l y weekly i n -

t e rva ls ; t h i s a p p l i e d t o h i g h l y immunogenic a n t i g e n s . To e l i c i t

a n t i b o d i e s o f a d e q u a t e t i t res w i t h a n t i g e n s o f l o w e r a n t i g e n i c i t y

several m o r e i n j e c t i o n s a t weekly i n t e r v a l s w e r e g i v e n . Eggs

l a i d l a t e r t h a n t e n t o f o u r t e e n days a f t e r t h e i n i t i a l immunizing

dose w e r e l a b e l l e d and s t o r e d a t 4OC u n t i l p r o c e s s e d f o r e x t r a c -

t i o n o f IgY.

E x t r a c t i o n and p u r i f i c a t i o n o f IqY. I n d i v i d u a l y o l k s w e r e s e p a r a t e d

from t h e albumen ( w h i t e ) and t h o r o u g h l y washed i n a j e t o f w a t e r t o remove as much as p o s s i b l e o f a d h e r i n g albumen. The t o t a l volume

of t h e c o l l e c t e d y o l k s w a s measured and b u f f e r , e q u i v a l e n t t o two

v o l u m e s o f y o l k w a s added and t h o r o u g h l y mixed. P o l y e t h y l e n e g l y -

co l , which h a d b e e n p u l v e r i z e d , w a s added t o a f i n a l c o n c e n t r a t i o n of 3.5% by w e i g h t o f polymer t o volume o f d i l u t e d y o l k . The mix-

t u r e w a s s t i r r e d u n t i l a l l t h e polymer w a s d i s s o l v e d . The m i x t u r e

w a s c e n t r i f u g e d i n a S o r v a l l c e n t r i f u g e a t 14 000 g f o r 1 0 m i n u t e s . T h i s o p e r a t i o n c a u s e d t h e s e p a r a t i o n o f t h r e e p h a s e s i n t h e cen-

t r i f u g e tubes. These w e r e , a y e l l o w l i p o i d a l l a y e r on t h e s u r f a c e

ANTIBODIES TO PROTEINS FROM YOLK

o f t h e f l u i d , a c lear l a y e r and a s e m i - s o l i d p l i a b l e phaise o f

c a s e i n - l i k e v i t e l l i n r e p r e s e n t i n g a b o u t .L/3 o f t h e t o t a l volume

of s u b s t a n c e i n t h e c e n t r i f u g e t u b e s . The SNF w i t h t h e f a t t y

l a y e r w a s c a r e f u l l y d e c a n t e d i n t o a f u n n e l c o n t a i n i n g a lose p l u g

o f a b s o r b e n t c o t t o n i n t h e neck of t h e f u n n e l . 'This p l u g f i l -

t e r e d o f f t h e l i p i d t h a t became d e t a c h e d an83 w a s d e c a n t e d w i t h

t h e SNF. The volume o f t h e c lear f i l t r a t e was measured and, more

p u l v e r i z e d PEG w a s added by g e n t l e s t i r r i n g t O a d j u s t t h e f i n a l

polymer c o n c e n t r a t i o n t o 1 2 9 PEG i n 100 ml ,SN:F. A t t h i s con-

c e n t r a t i o n t h e polymer c a u s e d comple te dj-splacement o f t h e IgY.

The p r e c i p i t a t e w a s c e n t r i f u g e d o f f a t 1 4 0130 g f o r 10 m i n u t e s .

The p e l l e t s w e r e r e d i s s o l v e d i n phosphate b u f f e r t o t h e o r i g i n a l

volume and t h e IgY once m o r e p r e c i p i t a t e d w i t h 1 2 % PEG. The

p e l l e t s w e r e compacted by c e n t r i f u g a l f o r c e a t 1 4 O O O g and t h e

exuded s o l u t i o n o f PEG removed by s u c t i o n . 'The f i n a l p e l l e t s

i n t h e IgY w e r e d i s s o l v e d i n a volume o f b u f f e r e q u i v a l e n t t o

h a l f t h a t of t h e o r i g i n a l y o l k . The p r o t e i n c o n c e n t r a t i o n i n

t h e f i n a l p r o d u c t i o n w a s o f t h e o r d e r o f lO-l2mg/ml.

i n t h e b u f f e r w a s a d e q u a t e t o p r e s e r v e t h e Ig'Y.

The N a N 3

I f comple te removal o f PEG i s d e s i r e d t h e IagY may b e p r e c i p i t a t e d

w i t h 2 M (NH4)2S04 f o l l o w e d by c e n t r i f u g a t i o n .

t h e PEG w i l l form a l i q u i d phase on t h e (NH4)2S04 s o l u t i o n . PEG may a l s o be removed by p r e c i . p i t a t i o n o f t h e IgY w i t h 4 0 %

e t h a n o l a t s u b z e r o d e g r e e s ; t h e PEG wi l l . b e so1ubl.e i n t h e e t h a -

n o l .

1.n t h i s i n s t a n c e

C a r b a m i l a t i o n . Human IgG and y o l k IgY w e r e carbami l a t e d a c c o r d i n g

t o t h e s t a n d a r d p r o c e d u r e . ( 5 )

t e c h n i q u e . The t i t r e s of t h e immune

IgY w a s a s s e s s e d by p l a c i n g s e r i a l l y two-fo ld d i l u t . i o n s o f t h e IgY i n t h e c e n t r a l w e l l s punched i n t o a g a r o s e p l a t e s f o r Ouchter -

l o n y tes ts and i n s e r t i n g d o u b l i n g d i l u t i o n s of t h e a n t i g e n i n t o

t h e f o u r p e r i p h e r a l w e l l s . The t i t r a t i o n e , i d p o i n t w a s t a k e n as

t h e h i g h e s t d i l u t i o n o f t h e immune IgY which showed a v j . s i b l e

p r e c i p i t i n l i n e .

One and two d i m e n s i o n a l L a u r e l 1 e l e c t r o p b E s L s t es t s . The num-

b e r o f a n t i g e n s i n m i x t u r e s w e r e b e s t d e t e r m i n e d by one or two

500 POLSON, VON WECHMAR, AND FAZAKERLEY

dimensional Laure l1 e l e c t r o p h o r e s i s experiments (1 x Le and 2 x L e r e s p e c t i v e l y ) . I n 1 x L e exper iments t h e a n t i g e n s were p laced

i n w e l l s i n t h e c e n t r e of a g l a s s s l i d e covered wi th agarose

con ta in ing t h e IgY ant ibody t o a l low t h e a n t i g e n s which migra te

t o t h e anode and cathode t o be p r e c i p i t a t e d a s immune complexes.

I n t h e 2 x L e t h e s t r i p of agarose i n which t h e p re l imina ry e l e c -

t r o p h o r e s i s was done was p l aced between two s l a b s of agarose

con ta in ing IgY type of an t ibody. S i m i l a r l y a s wi th 1 x Le, t h e

a n t i g e n s w e r e t hen migra ted accord ing t o t h e i r r e s p e c t i v e mobi l i -

t i e s t o e i t h e r t h e anode o r ca thode , t o form immune p r e c i p i t a t e s

i n t h e g e l . A f t e r thorough washing of t h e g e l s i n s a l i n e t h e

s l i d e s w e r e s t a i n e d wi th Coomassie b lue .

RESULTS

a ) and ( b )

r e a c t i o n s

une IgY wh

B. c i n c t a haemocyanin. I n F igu res 1

Ouchterlony g e l d i f f u s i o n p r e c i p i t i n

haemocyanin of B . c i n c t a wi th i t s i m

are p resen ted

Ogd) of t h e

n t h e NaCl con-

c e n t r a t i o n s i n t h e g e l s w e r e 0 . 1 5 and 1.5 M r e s p e c t i v e l y . The

concen t r a t ions of t h e r e a c t a n t s (haemocyanin and IgY) were t h e

same i n t h e two sets , t h e on ly d i f f e r e n c e be ing t h e concen t r a t ion

of N a C l i n t h e g e l , The c e n t r a l w e l l s w e r e f i l l e d wi th IgY a t

doubl ing d i l u t i o n s and t h e sur rounding f o u r w e l l s con ta ined t h e

haemocyanin a t concen t r a t ions 1 , 0 . 5 , 0 . 2 5 and 0.125 mg/ml. The

d i l u t i o n endpoin t of IgY i n d i c a t e d a t i t r e of 5 1 2 i n both low

and h igh s a l t concen t r a t ion . The on ly d i f f e r e n c e between t h e two Ogd diagrams i s t h a t t h e p r e c i p i t i n l i n e s formed c l o s e r t o

t h e IgY w e l l s when 1.5 M N a C l w a s p r e s e n t t han i n t h e diagrams

wi th N a C I a t 0 . 1 5 M i n t h e g e l . An a d d i t i o n a l p r e c i p i t i n l i n e

i s seen i n t h e diagram which, judging from i t s r e l a t i v e p o s i t i o n

t o t h e a n t i g e n and an t ibody w e l l s , appear t o be formed by an an-

t i g e n which h a s a molecular weight of t h e o r d e r of t h a t of IgY. The B . c i n c t a haemocyanin w a s a l s o s u b j e c t e d t o 2 x Le t h e re-

s u l t of which i s shown i n F igure 2 . Two major a n t i g e n i c a l l y

d i s t i n c t components are e v i d e n t t o g e t h e r w i th a minor a t l o w e r concen t r a t ion . The main peaks w e r e poss ib ly formed by t h e com-

ponents of S = 95 and 100 r e s p e c t i v e l y . The minor component was

no t d e t e c t e d by u l t r a c e n t r i f u g a t i o n i n t h e Model E .

F I G U R E 1 [a) and (1)) Ouchter lony qel d i i f u s i o n d iagrams o f liaernocyaniii o f B. !:incta and IgY a n t i b o d y , ( a ) w i t h 0 . 1 5 and (13 ) w i t h 1 . 5 M N a C l Ln g e l . Two-fold d i l u t i o n s of haemocyanin i n p e r i p h e r a l ,and IgY Ln c e n t r a l w e l l s . C o n c e n t r a t i o n ; haemocymln and IgY in t h e two s e t s ( a ) and (b) i d e n t i c a l . Note d i s p l a c e m e n t cf p r e c i p t t i n l i n e s away from a n t i q e r , w e l l s when NaCL F r e s c n t i n h i g h concen- t r a t i o n .

502 POLSON, VON WECHMAR, AND FAZAKERLEY

FIGURE 2 Two d i m e n s i o n a l L a u r e l 1 e l e c t r o p h o r e s i s d iagram of g. c i n c t a haemocyanin and i t s IgY a n t i b o d y . The c o - a x i a l major components p o s s i b l y t h e components S = 95 and 100 . The minor component w a s n o t e v i d e n t d u r i n g a n i q y t i c a l u l t r a c e n t r i f u g a t i o n . 1%, s t a g e (1) p h o s p h a t e b u f f e r p H 7 . 5 ; s t a g e ( 2 ) McLlvane b u f f e r p H 5 . 0 ; i o n i c s t r e n g t h 0 . 0 2 .

Agarose

J . l a l a n d i i haemocyanin

Diagrams of Ogd o f t h e s y s t e m J . l a l a n d i i and i t s IgY a n t i b o d y w i t h low ( 0 . 1 5 M I and h i g h ( 1 . 5 M ) N a C l i n t h e g e l are p r e s e n t e d

i n F i g u r e s 3 ( a ) and ( b ) . The e x p e r i m e n t a l c o n d i t i o n s w e r e s i m i -

l a r t o ’ t h o s e o f t h e e x p e r i m e n t w i t h B. c i n c t a haemocyanin. The

t i t r a t i o n e n d p o i n t w a s 1 / 2 5 6 and “ s h i f t “ i n p o s i t i o n o f t h e p r e -

c i p i t i n l i n e s towards t h e w e l l s c o n t a i n i n g t h e a n t i b o d y w a s a l so

e v i d e n t when N a C l a t h i g h c o n c e n t r a t i o n w a s p r e s e n t i n t h e g e l .

The e x p e r i m e n t i n which t h e haemocyanin w a s s u b j e c t e d t o 2 x L e

( F i g u r e 4 ) i n d i c a t e d t h a t , a p a r t f rom t h e main components , a d d i -

t i o n a l f o u r minor a n t i g e n i c components w e r e p r e s e n t i n agreement

w i t h t h e r e s u l t s o b t a i n e d b y a n a l y t i c a l u l t r a c e n t r i f u g a t i o n .

Amandin. . T h i s p r o t e i n p r o v e d t o b e a p o o r a n t i g e n f o r l a y i n g

h e n s . F o u r h e n s w e r e u s e d i n t h e rest and none produced IgY of t i t r e g r e a t e r t h a n 6 4 . I n c r e a s e i n s a l t c o n c e n t r a t i o n i n t h e

FIGURE 3 ( a ) and (b) Ouchter lony g e l di.f f u s i o n d iagrams o f 2 . I .a l .andi i haemocyanin and i t s IgY a n t i b o d y , ( a ) w i t h 0 . 1 5 and (b) w i . t h 1 . 5 M NaCl i n g e l . Two f o l d d i l u t i o n s of haemocyanin i n p e r i p h e r a l . and I g Y i n cen- t r a l w e l l s . C o n c e n t r a t i o n s of a n t i g e n and I q Y i n ( a ) and (b) i d e n t i c a l . Note d i s p l a c e m e n t of p r e c i p i t i - n l i n e s away frsom a n t i - gen w e l l s when N a C l p r e s e n t i n h i g h c o n c e n t r a t i o n .

504 POLSON, VOW WECHMAR, AND FAZAKERLEY

F I G U R E 4 Two dimensional L a u r e l l e l e c t r o p h o r e s i s diagram of J. l a l a n d i i haemocyanin and i t s IgY ant ibody. I n a d d i t i o n t o t h e main, t h e presence of 4 a d d i t i o n a l a n t i g e n s i s e v i d e n t . A n a l y t i c a l u l t r a - c e n t r i f u g a t i o n showed main component of 1 6 S ( 8 0 % ) and minor components o f 2 2 , 11, 6 and 4 S i r , con f i rma t ion of two dimentio- n a l L a u r e l l e l e c t r o p h o r e s i s . Agarose 1%, s t a g e (1) phosphate b u f f e r pH 7 . 5 ; s t a g e ( 2 ) McLlvane pH 5 . 0 , i o n i c s t r e n g t h 0 . 0 2 .

g e l d i d no t i n f l u e n c e t h e endpo in t . A f t e r c e s s a t i o n of t h e hyper-

immunization t h e an t ibody t i t r e remained a t t h e 6 4 l e v e l f o r

t h e next t h r e e months, when t h e experiment was t e rmina ted .

Humai? IqG. The p r e c i p i t i n r e a c t i o n between IgY and i t s IgG a n t i -

gen i n Ogd tes ts was n o t n o t i c e a b l y d i f f e r e n t when conducted i n

low and h igh N a C l c o n c e n t r a t i o n wi th t h e excep t ion t h a t t h e r e l a -

t i v e p o s i t i o n s of t h e p r e c i p i t i n l i n e s , no t i ced wi th t h e haemocya-

n i n s , changed a s expec ted when IgY forms dimers i n h igh s a l t con-

c e n t r a t i o n .

Add i t iona l exper iments w e r e conducted wi th human IgG and i t s

IgY ant ibody. The average i s o e l e c t r i c p o i n t (PI) of human IgG's

i s a t pH 6 . 8 wh i l e t h e average PI of IgY components i s approxi -

mately 1 pH u n i t l o w e r ( 6 ) .

r iments a r e conducted i n agarose g e l s d i s s o l v e d i n b u f f e r of hy-

drogen i o n c o n c e n t r a t i o n cor responding t o pH 8 . 6 t h e m o b i l i t y

The re fo re , when e l e c t r o p h o r e s i s expe-

ANTIBODIES TO PROTEINS FliOPI YOLK 505

of- IyY to t h e p o s i t i v e po lc s h o u l d be g r e a t e r t2ia-i t:hat of IgG. T h i s s u r m i s e was conf i rmed by t h e e v i d e n c e pi:c?seni:etl i n t h e g e l

c1ectrophores:s d iagram ( F i g u r e 5) .

When t h e mobi:.ity of: IgY i s compared to t h a t of c,irhamiLated IgC

( a p r o c e d u r e wnich r - e d u c e s t h e n e t n e g a t i v e c h a r g c on L ( i G ) it

was est.imat:ed t n a t t.1712 mobility o f I g Y i s PL: t o 9 0 2 tha: of c a r -

barn i la ted I g G . Cat-kmmilated I g Y , in t u r n , has ii net negat . ive

c h a r g e g r e a t e r t han t h a t o f c a r b a m i l a t e d IgC (Figi;.re 6) , hence

i t s h i g h e r rnojji1it.f.

F l G U R E 5 E l e c t r o p h o r e s i s d iagrams i n 1% a g a r o s e g e l oi humall IgG an'd 1:gY i n B a r b i t a l g l y c i n e T r i s b u f f e r p H 8 . 8 . On t h e l e f t of cent r l s , d i a - grams o f IgG i n d u p l i c a t e , on r i g h t o f c e n t r e . d iagrams of Igl! i n d u p l i c a t e . T iny c i rc les p o s i t i o n s of o r i g i n . Note t h e n e t f o r - ward movement of IgY and n e t backward movement of I g Y . I o n i c s t r e n g t h b u f f e r 0 . 0 2 .

506 POLSON, VON WECHMAR, AND FAZAKERLN

F I G U R E 6 E l e c t r o p h o r e s i s diagrams i n 1% agarose g e l of ca rbami la t ed IgG and IgY. From l e f t t o r i g h t ; u n t r e a t e d IqY and carbami la ted IgY. B a r b i t a l g l y c i n e T r i s b u f f e r , pH 8 .8 , agarose 1%; i o n i c s t r e n g t h 0 . 0 2 .

One and 2 x Le diagrams on IgG and i t s IgY a n t i b o d i e s i n t h e g e l ,

shown i n F igu re 7(a ) and ( b ) i n d i c a t e d t h a t t h e IgG used f o r t h e

product ion of IgY type a n t i b o d i e s was composed of s e v e r a l a n t i -

gen ic types . N o a t t empt w a s made t o i d e n t i f y t h e s e .

Monospecific an t ibody a g a i n s t human IgG was prepared by a f f i n i t y

chromatography accord ing t o t h e procedure desc r ibed by Hardie and van Regenmortel (7 ) fo r o b t a i n i n g s p e c i f i c IgG a n t i b o d i e s t o

tobacco mosaic v i r u s . I n t h e p r e s e n t work human IgG was bound

t o agarose beads and IgY ( a n t i IgG) absorbed t o t h e immobilized

IgG. The s p e c i f i c IgY was e l u t e d from t h e beads wi th g lyc ine - H C 1 b u f f e r o f mo la r i ty 0 . 0 0 5 and pH 2 . 9 . The pH of t h e e l u t e d

IgY w a s immediately r a i s e d t o n e u t r a l i t y t o minimize dena tura-

t i o n .

Although t h e IgY b e f o r e adso rp t ion and e l u t i o n showed a s i n g l e

component of S = 7 . 1 i n t h e Model E , t h e e l u t e d f r a c t i o n appeared

ANTIBODIES TO PROTEINS FROM YOLK 507

F I G U R E 7 (a) and (b) One ( a ) and two ( b ) d i m e n s i o n a l L a u r e l 1 e l e c t r o p h o r e s i s d iagrams o f human I g G a g a i n s t i t s IgY a n t i b o d y . The e l e c t r o p h o r e s i s con- d u c t e d i n 1% a g a r o s e . N o t e t h e m u l t i component n a t u r e o f IgG. One d i m e n s i o n a l ( a ) i n a c e t a t e b u f f e r pH 5 . 5 . TWO d i m e n s i o n a l , s t a g e s (1) and ( 2 ) i n phosphate b u f f e r p H 6 . 0 . Agarose 1%; i o n i c s t r e n g t h 0 . 0 2 .

t o be p a r t i a l l y polymer ized b e i n g composed o.E t h r e e components

of a p p r o x i m a t e l y e q u a l concent ra t . ion . T h e i r s e d i m e n t a t i o n coef f i-

c i e n t s w e r e 7 , 13 and 1 7 S . The p o l y m e r i z a t i o n was v e r y l i k e l y

due t o e x p o s u r e t o t.he b u f f e r o f l o w pH as s i m i l a r polymer iza-

t i o n of IgG w a s n o t i c e d when t r e a t e d w i t h bu:Efer a t pH 2 . 9 . ( 8 )

Venom of Naja n i v e a - ( C a p e C o e . E'or t h e sake o f comparison i n

t h e i r a b i l i t i e s t o produce a n t i b o d i e s , r a b b i t s and hens w e r e i m -

munized s i m u l t a n e o u s l y w i t h c o b r a venom and izhe p r o d u c t i o n o f

p r e c i p i t a t i n g a n t i b o d i e s fo l lowed by Ogd. It: was o b s e r v e d t h a t

w h i l e t h e r a b b i t s produced s e v e r a l p r e c i p i t i n l i n e s a f t e r 4 i n -

j e c t i o n s of t h e venom, t h e I g Y ' s o f t h e h e n s showed a s i n g l e

f a i n t l i n e when t h e NaCl c o n c e n t r a t i o n i n t h e g e l was 1 . 5 M .

The p r e c i p i t i n l i n e formed apprec iab ly c l o s e r t o t h e a n t i g e n

than t o t h e an t ibody w e l l s .

POLSON, VON WECHMAR, AND FAZAKERLEY

From t h e r e s u l t s ob ta ined wi th v i r u s e s (’) and t h e haemocyanins,

which a l l have h igh molecular weights , i t was dec ided t o i n c r e a s e

t h e s i z e of t h e venom immunogens by c r o s s - l i n k i n g t h e components

i n t h e venom wi th 0 . 6 % g lu t a ra ldehyde . It was assumed t h a t t h e

c ros s - l inkage occurred i n a random manner and t h a t a l l t h e a n t i - gens would be p re sen t on t h e s u r f a c e s as “ a n t i g e n i c s i t e s “ . A hen which w a s a l r e a d y s e n s i t i z e d t o cobra venom was g iven a

s i n g l e i n j e c t i o n of t h e c ros s - l inked o r aggrega ted venom whi le ano the r s e n s i t i z e d hen w a s g iven u n t r e a t e d venom. This hen

a c t e d a s c o n t r o l . Eggs l a i d t e n days a f t e r r e c e i v i n g t h e i n -

j e c t i o n s w e r e e x t r a c t e d and assayed by Ogd. The hen which re- ce ived t h e c ros s - l inked venom components showed some improve-

ment o f i ts Ogd p a t t e r n wh i l e t h e c o n t r o l hen showed none. The

Ogd p a t t e r n s cou ld be demonstrated i n g e l s con ta in ing 1 .5 M N a C l

bu t n o t i n g e l s i n 0 . 1 5 M N a C 1 . A t t h e p r e s e n t s t a g e o f immuni-

z a t i o n t h e IgY showed no p r o t e c t i v e a c t i v i t y a g a i n s t t h e venom i n an a s say performed i n m i c e .

Tetanus t o x i n . A s t h e main i n t e r e s t i n a n t i - t e t a n u s IgY w a s i n i t s a b i l i t y t o n e u t r a l i z e t h e neu ro tox ic component of t h e

tox in : t h e presence o f p r e c i p i t i n l i n e s i n t h e Ogd tes t could no t be used as a r e l i a b l e index f o r t h e presence o f a n t i - t e t a n u s

a c t i v i t y , as t h e crude t e t a n u s toxo id used i n t h e immunizing programme w a s composed of a minimum of e i g h t components as shown by e l e c t r o p h o r e s i s i n polyacrylamide g e l . An t i - t e t anus a c t i v i - t y w a s , t h e r e f o r e , expressed as t h e h i g h e s t d i l u t i o n of IgY which n e u t r a l i z e d 2 rnld of t o x i n . The r e s u l t s ob ta ined on f o u r hens used i n t h e experiment i n d i c a t e d t h a t t h e a n t i - t e t a n u s a c t i v i t y o f t h e i r yo lk IgY ranged from a t a c o n c e n t r a t i o n o f 6 mg/ml. The a n t i - t e t a n u s a c t i v i t y remained a t t h i s l e v e l t h r e e months a f t e r t h e hens r ece ived t h e i r l a s t i n j e c t i o n .

t o lo-’ when t h e IgY was used

A s i n g l e broad p r e c i p i t i n l i n e wi th an i n t e n s e c e n t r a l r eg ion w a s found i n Ogd both when 0 .15 M and 1 .5 M N a C l w a s p r e s e n t i n t h e g e l . As t h e most i n t e n s e r eg ion i n t h e band w a s approximately midway between t h e a n t i g e n and an t ibody w e l l s it was concluded

t h a t t h e " l i n e " was t h a t o f t h e n e u r o t o x o i d and i t s IgY a n t i b o d y .

T h i s c o n c l u s i o n w a s b a s e d on a v a i l a b l e d a t a on t h e m o l e c u l a r

w e i g h t s o f t h e n e u r o t o x i n and IgY which have m o l e c u l a r w e i g h t s

a p p r o x i m a t e l y 1 5 0 0 0 0 and 170 000 r e s p e c t i v e 1.y and t - h e r e f o r e

s i m i l a r d i f f u s i o n c o e f f i c i e n t s . The ant igen1.c c o m p o s i t i o r o f

t h e t o x o i d i s i n d i c a t e d by i t s 1 x L e d iagrar? p r e s e n t e d i n F i g u r e

Serameba a n t i g e n . Serameba a n t i g e n , e x M i l e s , Cape Town, used

i n t h e d i a g n o s i s o f a m o e b i a s i s proved t o b e a weak a n t i q c n f o r

f o w l s . Whereas a n t i s e r a o b t a i n e d from r a b b i t s which w e r e immu-

n i z e d s i m u l t a n e o u s l y w i t h t h e h e n s showed t h c p r e s e n c e of 20

a n t i g e n s by 2 x L e t h e IgY c o u n t e r p a r t showed only two f a i n t

l i n e s i n Ogd when immune IgY i n d i l u t i o n s 'of 1 / 4 was u s e d . These

l i n e s d i s a p p e a r e d i n Ogd tests a t d i l u t i o n s o f t h e 1gY h i g h e r

t h a n 1 / 4 . The two l i n e s which were v i s i b l ' e were o b t a i n e d when

h i g h N a C l c o n c e n t r a t i o n w a s p r e s e n t i n t h e g e l and t h e y w e r e

c h a r a c t e r i s t i c of a n t i g e n s of l o w m o l e c u l a r weight..

F I G U R E 8 One d i m e n s i o n a l L a u r e l 1 e l e c t r o p h o r e s i s d iagram o f t e t a n u s t o x o i d a g a i n s t i t s IgY t y p e a n t i b o d y . The heavy p r e c i p i t i n l i n e porjs ibly formed by t e t a n u s t o x o i d and i t s IgY a n t i t o x i n . Agarose 1%, MC- L lvane b u f f e r pH 5 . 5 , i o n i c s t r e n g t h 0.02.

B i t i s a r i e t a n s venom. Hens r e a c t e d poor ly t o t h e a n t i g e n s i n

g. a r i e t a n s venom. A f t e r n i n e immunizing doses f a i n t p r e c i p i t i n

l i n e s were n o t i c e d i n t h e Ogd t e s t . The l i n e s w e r e on ly appa ren t

when NaCl a t 1 . 5 M was p r e s e n t i n t h e g e l . A f t e r 3 a d d i t i o n a l

i n j e c t i o n s of t h e immunizing doses more v i s i b l e p r e c i p i t i n l i n e s

appeared i n t h e g e l b u t on ly when NaCl a t h igh c o n c e n t r a t i o n was

p r e s e n t . When t e s t e d by 2 x L e a t s u i t a b l e c o n c e n t r a t i o n s of

t h e venom and IgY, approximately 18 components could be d i sce rned .

The 2 x Le diagram d i s p l a y e d i n F igu re 9 i n d i c a t e d t h e complexity

of t h e venom. The 2 x L e diagram ob ta ined wi th ho r se a n t i v e n i n e

i n d i c a t e d t h e presence of approximately t h e same number of a n t i -

gens ( t o be p u b l i s h e d ) . Although t h e ho r se a n t i v e n i n e a f fo rded

good p r o t e c t i o n of mice a g a i n s t t h e venom, t h e IgY d i d not neu-

t r a l i z e t h e l e t h a l components a t a l e v e l of 2 mld of venom.

POLSON, VON WECHMAR, AND FAZAKERLEY

I t i s i n t e r e s t i n g t o note t h a t t h e p r e c i p i t i n l i n e s i n Ogd w e r e v i s i b l e only when s a l t a t h igh c o n c e n t r a t i o n was p r e s e n t i n the

F I G U R E 9 Two d imens iona l Laure l1 e l e c t r o p h o r e s i s diagram of t h e venom of B i t i s a r i e t a n s and i t s IgY an t ibody . Approximately 18 a n t i g e n s w e r e recognized . Agarose 1%, b u f f e r , s t a g e (1) G e l m a n h igh re- s o l u t i o n b u f f e r pH 8 .8 , s t a g e ( 2 ) McLlvane b u f f e r pH 5 . 0 , i o n i c s t r e n g t h 0 . 0 2 .

ANTIBODIES TO PROTEINS FKOM YOLK 511

g e l , t h e a n t i g e n s m u s t have r e a c t e d w i t h t h e i r ipmune I g Y com-

p o n e n t s as t h e complexes formed w e r e w e l l d ispl .ayed i n t.he 2 x Le d iagram a f t e r s t a i n i n g w i t h Coomassie bluc!.

- IgY as a n t i g e n . The a n t i g e n i c complexi ty of I g Y i s e v i d e n t from

t h e 2 x L e d iagram, ( F i g u r e 10). A minimum of 5 a n t i g e n s maly be

d i s c e r n e d i n t h e photograph . The m a t e r i a l , IgY, which s e r v e d as

a n t i g e n t o e l i c i t a n t i b o d i e s i n t h e r a b b i t and f o r t h e s u b s e q u e n t

2 x L e t e s t was homogeneous i n p a r t i c l e s i z e as shown by u l t r a -

c e n t r i f u g a t i o n i n t h e Model E .

F I G U R E 10 Two d i m e n s i o n a l L a u r e l 1 e l e c t r o p h o r e s i s diag.ram of IgY a s a n t i - gen a g a i n s t i t s r a b b i t IgG a n t i b o d y . F i v e a n t i g e n i c components are r e c o g n i z e d . Agarose 1%. GelnJan h i g h r e s o l u t i o n b u f f e r pH 8 .8 , i o n i c s t r e n g t h 0.02 f o r b o t h 1st and 2nd s t a g e s .

512 POLSON, VON WECHMAR, AND FAZAKERZEY

N o a t t e m p t w a s made t o i d e n t i f y t h e components d i s p l a y e d i n t h e

2 x L e d iagram.

DISCUSSION AND CONCLUSIONS

Laying h e n s w e r e immunized w i t h a v a r i e t y of p r o t e i n s r a n g i n g i n

m o l e c u l a r w e i g h t s ( m w ’ s ) f rom 8 x l o 6 t o les t h a n 2 x l o 4 and

t h e a n t i b o d i e s , IgY, e x t r a c t e d from t h e y o l k s o f e g g s l a i d by

t h e s e b i r d s .

The m o l e c u l a r w e i g h t (mw) o f t h e a n t i g e n w a s a n i m p o r t a n t f a c t o r

e l i c i t i n g a n t i b o d i e s i n h e n s as r e f l e c t e d i n t h e t i t r e o f t h e

a n t i b o d i e s i n t h e i r y o l k s . A n t i g e n s w i t h m w ’ s e q u a l t o or h i g h e r

t h a n t h a t o f human IgG a p p e a r t o produce good r e s p o n s e s i n h e n s

whereas a n t i g e n s o f lower m w ’ s and less a p p e a r t o be p o o r a n t i -

g e n s . The dependency o f a n t i g e n i c r e s p o n s e o f h e n s on m o l e c u l a r

s i z e of t h e a n t i g e n i s n o t un ique b u t i s a well-known phenomenon

which i s f r e q u e n t l y o b s e r v e d when mammalian s p e c i e s w e r e hyper im

munized w i t h a n t i g e n s o f l o w mw; t h e o n l y d i f f e r e n c e b e i n g t h a t

t h e r e s p o n s e t o a n t i g e n s o f l o w mw i s p o o r e r i n h e n s .

The e f f e c t o f s a l t c o n c e n t r a t i o n on t h e t i t re by t h e O u c h t e r l o n y

g e l d i f f u s i o n (Ogd) t e c h n i q u e w a s n o t o b s e r v e d when d e a l i n g w i t h

a n t i g e n s o f m w ’ s e q u a l t o o r g r e a t e r t h a n t h a t of human IgG. The

sys tems examined f o r t h e “ s a l t e f f e c t ” w e r e immune I g Y ’ s and t h e i r

a n t i g e n s which i n c l u d e d s e v e r a l p l a n t v i r u s e s ( t h i s J o u r n a l ) h a v i n g

m w ’ s o f several m i l l i o n s , t h e haemocyanins of t h e whelk and c r a y - f i s h o f m w ’ s 8 x l o 6 and 5 x l o 5 r e s p e c t i v e l y and human IgG, mw 150 0 0 0 . High s a l t c o n c e n t r a t i o n s a p p e a r t o b e a p r e r e q u i s i t f o r o b t a i n i n g v i s i b l e p r e c i p i t i n r e a c t i o n s i n g e l s when a n t i g e n s

o f l o w m w ’ s are u s e d . I n t h e p r e s e n t work t h e “ n a t u r a l “ m i x t u r e

o f a n t i g e n s i n t h e venom o f N a j a n i v e a , i n c r u d e t e t a n u s t o x i n

and i n serameba a n t i g e n w e r e d e p e n d e n t upon h i g h s a l t c o n c e n t r a -

t i o n f o r v i s ib l e g e l p r e c i p i t i n r e a c t i o n s . B e n e d i c t and Yamaga

i n a r e v i e w on a n t i b o d y p r o d u c t i o n i n a v i a n s p e c i e s d i s c u s s e d

h y p o t h e s e s by several a u t h o r s i n a t t e m p t s a t e x p l a i n i n g t h e s a l t e f f e c t . I t a p p e a r e d t h a t t h e r e w a s no s a t i s f a c t o r y e x p l a n a t i o n

f o r t h e phenomenon. There w a s g e n e r a l c o n s e n s u s t h a t t h e pheno-

menon i s i n s o m e manner r e l a t e d t o t h e tendency of IgY t o form

d i m e r s i n media c o n t a i n i n g s a l t a t h i g h c o n c e n t r a t i o n . Format ion

o f d imers o f IgY a n t i b o d y e x p l a i r x j t h e s h i f t (-~f t h e p o s i t i o n s of

t h e p r e c i p i t i n l i n e r e l a t i v e t o t h e wells o f the r e a c t a i i t s i n Ogd

tests when t h e e x p e r i m e n t s w e r e per formed j.n t h e p r e s e n c e Of

0 . 1 5 and 1 . 5 M NaCl. D i m e r s o f I q Y w i l l have j 3 w e r d i f f u s i o n

c o e f f i c i e n t s t h a n t h o s e of I g Y i n t h e monomeric stii1.e; hence

t h e f o r m a t i o n of t h e p r e c i p i t i n bands c l o s e r t o t h e L g Y w e : L l s

t h a n t h e p o s i t i o n o f t h e prec ip i t . i .n bands j.11 T-h'a g e l conta- in ing

t h e s a l t o f l o w e r c o n c e n t r a t i o n . The d i f f e r e n t - i a l s h i f t 0 - E t h e

p r e c i p i t i n l i n e s was n o t i c e d w i t h a l l t h e syst.i:nis i ~ n v e s t i g a t e d .

The e f f e c t o f h i g h s a l t c o n c e n t r a t i o n s on tihc 1:reci.piti n bands

may w e l l be l i n k e d t o t h e presenc t? cE more p r o b e i n ~n t h e p r e c i -

p i t a t e s as a r e s u l t o f t h e d i m e r i : : a t ~ i o n of t h r I g Y . . Al~though

it i s unknown which (chains o f t h e Ig'l a r e ~ . i i ~ o l ~ i - ? d in ?.he mutua l

l i n k a g e , a t l e a s t two o f t h e f o u r a n t i g e n k>.indinq s l t e s o f t h . e

d imer remain r e a c t i v e and are t h u s a b l e t o b i n d a n t i g e n s t o

form a l a t t i c e . When a n IgY dimeir r e a c t s w i t h a n a n t i g e n o f

low m o l e c u l a r weight t h e t o t a l m a s s o f t h e prot:cins 2.nvolved i n

t h e p r e c i p i t a t e would be n e a r l y doub Le t h a t $her iroriomeric I g Y

reacts w i t h i t s a n t i g e n , hence more v i s i b l e

Although n o t ment ioned i n t h e r e s u l t s sect?.on i t may be s t a t e d

t h a t d e s p i t e t h e a p p a r e n t absence of g e l p r c c i p i t i n l i n e s i n

t h e t w o d i n e n s i o n a l Laure 11 e l e c t r o p h o r e s i s cl i a q r am i n s p e c t e d

p r i o r t o washing and s t a i n i n g , t h e s e p r e c i p i t i n iiiie:; a r e made

v i s i b l e a f t e r s t a i n i n g w i t h Coomassie b l u e .

The f a i l u r e of t h e imnune I g Y t o n e u c r a l i z c the l e t h a l components

i n snake venom may p o s s i b l y be due t o weak f u r c e s b i n d i n g the an-

t i g e n s t o t h e i r s p e c i f i c IgY a n t i b o d . i e s . Whrn t h e complexes o f

IgY and t h e i r s p e c i f i c a n t i g e n s are i n j e c t e d into mice the com-

p l e x e s may d i s s o c i a t e and t h e t o x i c a n t i g e n s woula be f r e e t-n

per form t h e i r b i o c h e m i c a l f u n c t i o n s . I n t h e react j .on between

h o r s e a n t i v e n i n e and t h e venom components t h c b i n d i n q may i n v o l v e

t h e weak van d e r Waal's as w e l l a s Coulomb fcsrces forming q3 c o m -

p l e x which does n o t d i s s o c i a t e r e a d i l y , hence t h c s u p e r i o r . i t y

o f t h e h o r s e ant i -venom f o r n e u t r a l i z i n g venom cornponents .

I t i s f e a s i b l e t h a t more a v i d a n t i b o d i e s t o s i i a k e venom components

may be e l i c i t e d i n hens by u s i n g c r o s s - l i n k e d venom t o b a c t e r i a

o r t o g i a n t molecules such a s haemocyanin as a n t i g e n s .

514 POLSON, VON WECHMAR, AND FAZAKERLN

The advantages of t h e yolk IgY system have been d i scussed i n

some d e t a i l i n a prev ious paper ' ' ) i n t h i s Jou rna l .

ACKNOWLEDGEMENTS

G r a t e f u l acknowledgements a r e extended t o M i s s Margaret Donnelly

of t h e M.R.C. V i r u s Research Un i t , whose a s s i s t a n c e w a s i nva luab le

du r ing t h e i n i t i a l s t a g e s o f t h e work. D r s . P . Chr i s t ensen and

D . Botes of t h e South Af r i can I n s t i t u t e f o r Medical Research and

t h e Council f o r S c i e n t i f i c and I n d u s t r i a l Research r e s p e c t i v e l y

a r e thanked f o r t h e k ind donat ion of t e t a n u s t o x i n and snake

venom .

P r o f e s s o r C . von Hol t i s thanked f o r h i s cont inued i n t e r e s t i n

t h e work.

The t e c h n i c a l a s s i s t a n c e of M r s . F . Har r i son , M r . D . du P l e s s i s

and M r . P . Buckton i s g r a t e f u l l y acknowledged.

REFERENCES

Polson , A . , von Wechmar, M . Barbara and van Regenmortel,

M . H . V . This J o u r n a l .

Murphy, S.G. and M i l l e r , K . D . , J . Bact. 94: 5 8 0 , 1967.

Botes , D . P . and Strydom, D . J . , J . B i o l . Chem. 244: 4 1 4 7 , 1 9 6 9 .

Botes , D . P . Pe r sona l communication.

A Manual of Q u a n t i t a t i v e Immunoelectrophoresis. Methods

and AEpl ica t ion . e d i t e d by N . H . Axelson, J . K r a l l and B .

Weeke. U n i v e r s i t s t s f G r l a q e t , Os lo , Bergen, Tromsb. 1973.

Benedic t , A .A. and Yamaga, K . Chapter 13 i n lmmunoqlobulins

and Antibody Product ion i n Avian Spec ie s , e d i t e d by J .J .

Marchalonis, Comparative Immunology, Blackwell , Oxford, 1 9 6 6 .

Hard ie , G . and van Regenmortel, M.H.V. J . Immunological

Methods, 15: 305, 1 9 7 7 .

Polson , A . , P rep . Biochem. 2: 327, 1 9 7 3 .

Nossa l , G . J . V . and Ada, G . L . Ant igens , Lymphoid C e l l s and

t h e Immune Response, Academic P r e s s , N e w York and London, 1 9 7 1 .

Antibodies to Proteins from Yolk of Immunized Hens

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