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Chromatographic Separation and Purification of Secretory IgA from Human Milk H. Khayam-bashi Schwartz
a b
a
, R. M. Blanken & C. L.
a
a
Ames Research Laboratory, Ames company Division , Miles Laboratories, Inc. , Elkhart, Indiana, 46514, U.S.A. b
Department of Laboratory Medicine , University of California School of Medicine, San Francisco, at the Clinical Laboratories, San Francisco General Hospital , San Francisco, California, 94110, U.S.A. Published online: 05 Dec 2006.
To cite this article: H. Khayam-bashi , R. M. Blanken & C. L. Schwartz (1977) Chromatographic Separation and Purification of Secretory IgA from Human Milk, Preparative Biochemistry, 7:3-4, 225-241, DOI: 10.1080/00327487708061640 To link to this article: http://dx.doi.org/10.1080/00327487708061640
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PREPARATIVE BIOCHEMISTRY, 7(3&4), 225-241 (1977)
CHROMATOGRAPHIC SEPARATION AND PURIFICATION OF SECRETORY IgA FROM HUMAN MILK R.M. Blanken, and C.L. Schwartz Ames Research Laboratory, Ames company Division Miles Laboratories, I n c . , E l k h a r t , Indiana 46514 U.S.A.
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d . Khayam-Bashi’,
ABSTRACT Defatted and decaseinated human milk was concentrated and w a s f r a c t i o n a t e d on a p r e p a r a t i v e DEAE c e l l u l o s e column.
E l u t i o n with
various c o n c e n t r a t i o n s of sodium c h l o r i d e i n T r i s - H C 1 b u f f e r (pH 8.0, 0.01 M) r e s u l t e d i n f r a c t i o n s t h a t were r i c h i n e i t h e r secre-
t o r y immunoglobulin A (SIgA) ( 0 . 1 M NaC1) or f r e e s e c r e t o r y component (SC) (0.05 M N a C 1 ) .
The f r a c t i o n s , which were e l u t e d w i t h
0.10 M N a C l from t h e p r e p a r a t i v e column, were f u r t h e r f r a c t i o n a t e d on a G-200 Sephadex column.
Repeated f r a c t i o n a t i o n on t h i s
column r e s u l t e d i n a s i n g l e p u r i f i e d f r a c t i o n , which contained very high SIgA a c t i v i t y and showed immunological c r o s s - r e a c t i o n with both SC and sexum IgA.
Additional s t u d i e s i n d i c a t e d t h a t
t h i s f r a c t i o n was homogeneous as shown by immunoprecipitin and d i s c g e l electrophoresis.
I n j e c t i o n of t h i s p u r i f i e d SIgA i n t o
r a b b i t s r e s u l t e d i n the production of monospecific a n t i s a - 1 . INTRODUCTION
Various s t u d i e s have shown t h e predominance of IgA-type immunoglobulins i n colostrum and lachrymal s e c r e t i o n s .
They
225 Copyright 0 1977 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means. electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
226
KHAYAM-BASHI, BLANKEN, AND SCHWARTZ
suggest t h a t these secretions contain a s p e c i a l c l a s s of IgA which i s d i f f e r e n t f r a n IgA found i n the serum.
Studies by
Tomasi, e t a12 cited s p e c i f i c d i f f e r e n c e s which indicated t h a t these p r o t e i n s a r e d i s t i n c t from, and y e t r e l a t e d t o , the serum IgA proteins.
Evidence obtained i n t h e p a s t t e n years suggests
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t h a t secretory IgA (SIgA) c o n s i s t s of two IgA molecules, p l u s a secretory component (SC)
and J-chain polypeptide’
l4
1 6 .
Various techniques have been applied f o r s t r u c t u r a l s t u d i e s on SIgA.
Electron microscopic s t u d i e s have shown SIgA t o
possess a wishbone-type s t r u c t u r e .
Molecular weight s t u d i e s
i n d i c a t e t h a t SIgA has a sedimentation c o e f f i c i e n t of 11.5S, and a molecular weight of approximately 305 ,0007 I a
.
The p u r i f i c a t i o n of SIgA from human milk has been performed previously by s e v e r a l i n v e s t i g a t o r s 7 1 ’ 1 ’ .
However, since these
procedures are lengthy and r e q u i r e e l e c t r o p h o r e s i s , a s w e l l a s various types of chromatographic treatments, they r e s u l t i n low y i e l d s and a f i n a l product t h a t i s associated with contaminating proteins.
In order t o study the biochemical and b i o l o g i c a l
p r o p e r t i e s of SIgA, a l a r g e amount of highly p u r i f i e d p r o t e i n
i s essential.
The study presented here applied only chromato-
graphic procedures.
This simple and rapid technique r e s u l t e d
i n a homogeneous SIgA product with a r e l a t i v e l y high y i e l d . MATERIALS AND METHODS Preparation of t h e Whey from Human Milk Milk was collectedframtwo normal, healthy women i n l a t e l a c t a t i o n (14 and 15 months postpartum), and w a s frozen a t - 2 O O
SECRETORY IgA FROM HUMAN MILK C.
227
Of t h i s milk, 600 g w a s thawed and then c e n t r i f u g e d a t 30,000
rpm (105,000 x g ) i n a Model L , Beckman p r e p a r a t i v e u l t r a c e n t r i fuge (Beckman Instruments, F u l l e r t o n , CA hours.
92631) a t - 2 O
The d e f a t t e d m i l k was d i l u t e d 1:l with 0.15 M N a C 1 , and
t h e pH w a s a d j u s t e d t o 4.6 using an a c e t i c acid concentrate.
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C f o r two
-
sodium acetate
The mixture was kept a t room temperature f o r two
hours, then was t r a n s f e r r e d t o t h e cold room b e f o r e being rec e n t r i f u g e d a t 30,000 rpm (105,000 x g ) f o r t w o hours t o remove the precipitate.
The c l e a r s u p e r n a t a n t was a d j u s t e d t o 0.01 M
i n T r i s - H C 1 and pH of 8.0 a t room temperature by using a T r i s HC1 c o n c e n t r a t e .
The defatted-decaseinated milk serum obtained
by t h i s method was concentrated t o 40 m l (30X c o n c e n t r a t i o n ) over
a UM-10 membrane (Amicon Corp., Waltham, MA 60-80 P.S.I.
nitrogen i n t h e cold room.
0.1% (13.6 mM) with sodium a z i d e . t h e source f o r SC and SIgA.
02154) and under
The s o l u t i o n was made
The concentrated whey was
A flow c h a r t f o r t h i s p r e p a r a t i o n
i s shown i n Figure 1. Chromatographic Procedures A.
DEAE c e l l u l o s e :
A column, 5
x 65 cm ( r e s i n h e i g h t ) , was
packed w i t h 800 g of DE52 (Whatman Co., England) i n T r i s - H C 1 b u f f e r (0.01 M, pH 8.01,
which w a s prepared as reconmended by t h e
manufacturer.
Larger columns, 8 x 93 cm, were used i n subsequent
preparations.
The column w a s washed with 0.01 M, pH 8.0 T r i s - H C 1
b u f f e r (200 m l ) , a t a r a t e of 30 ml/hr, a t room temperature.
The
whey c o n c e n t r a t e (35 m l ) w a s a p p l i e d t o t h e column b e f o r e s t e p -
w i s e elution was initiated.
The e l u t i o n was performed w i t h T r i s -
KHAYAM-BASHI, BLANKEN, AND SCHWARTZ
228
Diluted Human Milk 1: 1 with 0.15 M NaCl
1
Centrifuge
Defatted Milk
I5.
1. Acetic Acid (pH 4.6) 2. Centrifuge
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Decaseinated Whey Concentrated Whey
I
1. Dialyze 2. Concentrate
DEAE Cellulose
Other Proteins (e. g., Lactoferrin)
SIgA Rich
S.C. Rich Fraction
Fraction
1. Concentrate 2. Sephadex G-200 (3x1
I
SIgA FIGURE 1 Schematic p r e s e n t a t i o n of v a r i o u s s t e p s f o r t h e p u r i f i c a t i o n of SIgA from human milk.
HC1 b u f f e r (0.01 M, p H 8.0) which contained 0.03 M, 0.05 M, o r
0.10 M sodium c h l o r i d e , according t o need.
E l u t i o n w a s maintained
by g r a v i t y with 1200 m l of each b u f f e r a t 30 m l / h r .
A n automated
f r a c t i o n c o l l e c t o r (Fractomette 200, Buchler Instruments, F o r t
Lee, N J ) t h a t was a t t a c h e d t o a recording Beclanan DBG Spoctrophotometer w a s used throughout t h e s t u d i e s .
Optical d e n s i t y a t 280
nm eerved ae a means € o r the e s t i m a t i o n of p r o t e i n c o n c e n t r a t i o n .
229
SECRETORY IgA FROM HUMAN MILK
B.
Sephadex G-200:
(Pharmacia Fine Chemicals, Upsala, Sweden)
A column (Pharmacia) was packed (2.7 x 92 an) with Sephadex G-200 ( 4 0 g ) suspended in borate
borate 0.15 M, pH 8.0).
-
sodium chloride buffer (0.1 M NaCl in
The same buffer was used for the final
elution of SIgA from the column (35 ml/hr).
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The estimation of proteins in the column fraction was based on W light absorption at 280 nm, as measured with a DBG-Beckman Recording Spectrophotometer. Protein content of the concentrates or other samples was determined by (a) Lowry's method"
using a
Beckman DU Spectrophotometer, and (b) the measurement of W light abeorption ratio at 260 and 280 nm". Acrylamide Disc Gel Electrophoresis A Canalco disc gel electrophoresis instrument (Canalco, Rockville, MD) was ussd. a 7 % acrylamide gel.
The sample (100 pg) was applied over
Current was applied for 45 min.
The
procedure was performed in the manner described by Davis"
and
by the manufacturer's bulletin. Immunological Procedures Antiserum IgA was supplied by Hyland Laboratories (Costa Mesa, CA) Cappel Laboratories (Philadelphia, P A ) , and from Miles-Yeda (Israel). Immunodouble Diffusion (ID) was performed according to Crowle".
A drop of suitable dilutions of antigens and antisera
(20 ~ 1 was ) placed on adjacent discs.
The precipitin band
between the two discs in the agar gel was observed and photographed.
KHAYAM-BASHI, B L A " ,
2 30
AND SCHWARTZ
Immunoelectrophoresis (IEP): The basic technique described by Grabar and Burtin"
was employed.
ma was applied to each slide. continue for up to 60 min. serum.
A constant current of 2
Electrophoresis was allowed to
The trough was filled with anti-
The slide was incubated overnight (16-20 hr) in a
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moist chamber at room temperature. Inrmunoelectroosmophoresis (IEOP) or Counter Immunoelectrophoresis:
The following pattern was used to monitor the
SIgA activity in each column fraction:
Four parallel columns of
wells ( 2 nun diameter, 5 mm apart) were punched into the agar slide (5.0 x 7.5 an). Each of the two columns formed one pair
of wells, with 10 well-pairs in each column (total of 21 pairs). The antigen samples ( 5 pl) were placed in the column of wells near the cathode; the specific antiserum was placed in the wells near the anode.
Electrophoresis was carried out up to 60 min.
The current per plate was held conatant at 20 ma. The precipitate formed between the antibody and antigen wells indicated the presence of IgA activity.
The intensity of the
precipitate was judged visually, and values from +4 to 0 were assigned, depending on the amount of precipitate formed.
Photo-
graphs were taken for permanent records, and the plates were stained for protein (Ponceau S) and stored. Preparation of Antisera against Purified Secretory IgA in Rabbits Two rabbits were injected for the preparation of each anti-
serum.
Each rabbit received 0.4 ml per injection, consisting of
0.0 mg of purified SIgA in complete Freund'a adjuvant.
The
SECRETORY IgA FROM HUMAN MILK
231
a n t i g e n was given i n two i n j e c t i o n s , each 10 days a p a r t , i n t o t h e f o u r footpads of t h e animals.
A f t e r t h e f i r s t 10 days, b l e e d i n g
w a s performed a t weekly i n t e r v a l s f o r f o u r weeks (50 m l blood
.
each time) l 6 Reference Antiserum
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S p e c i f i c antiserum was donated by WHO I n t e r n a t i o n a l Reference Center f o r Immunoglobulins (Lausanne, S w i t z e r l a n d ) .
According t o
t h e i r d e s c r i p t i o n , t h i s antiserum t o SC (M 33 A ) w a s prepared by immunization of a sheep with human SIgA t h a t had been prepared from colostrum by f r a c t i o n a t i o n of an SIgA-rich f r a c t i o n of DEAEc e l l u l o s e and G-200 Sephadex.
This a n t i g e n w a s i n j e c t e d i n t r a -
muscularly with complete Freund's adjuvant.
The r e s u l t i n g
antiserum was absorbed with both l a c t o f e r r i n and t w o s e r a cont a i n i n g IgA myeloma p r o t e i n s of t h e monomer and polymer form. Upon inmunoelectrophoresis and Ouchterlony t e s t s a g a i n s t colostrum and normal serum, t h e r e s u l t i n g antiserum appeared t o be s p e c i f i c f o r SC and SIgA. RESULTS Separation of SIgA from Milk by DEAE C e l l u l o s e Chromatography The concentrated defatted-decaseinated milk contained 22 mg/ml p r o t e i n .
T h i r t y m l of t h e c o n c e n t r a t e w a s used f o r a DEAE-
c e l l u l o s e column. u r e 2.
A t y p i c a l f r a c t i o n a t i o n p a t t e r n is shown i n Fig-
E l u t i o n with 0.01 M T r i s - H C 1 b u f f e r c o n t a i n i n g 0.03 M
sodium c h l o r i d e caused t h e e l a b o r a t i o n of three t o f o u r t y p e s of p r o t e i n s .
These p r o t e i n s d i d n o t c o n t a i n any d e t e c t a b l e IgA
a c t i v i t y , as measured by IEOP a n a l y s i s a g a i n s t a n t i s e r a f o r human
serum IgA.
KHAYAM-BASHI, BLANREN, AND SCHWARTZ
2 32
.OIN NoCI.
0.03N NoCI.
0.1N NoCI.
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2.4
'0
1.2
0
0.8 0.4
10
1
1
I
I
I
1
I
I
1
30
50
70
90
110
130
150
170
190
FRACTIONS (20 ml each) FIGURE 2 Fractionation of whey p r o t e i n s on DEAE c e l l u l o s e . The e l u t i o n b u f f e r Elution performed a t 4 0 m l / h r . was h i s - H C 1 , 0.1 M, pH 8 . 0 , containing indicated amounts of NaCl as shown i n t h e graph. Each f r a c t i o n is 20 ml/tube. Arrows i n d i c a t e b u f f e r changes.
Elution with t h e same b u f f e r containing 0.05 M NaCl r e l e a s e d
a small peak containing free SC.
Depending on t h e conditions,
e s p e c i a l l y those r e l a t e d t o t h e donor (e.g.,
h e a l t h , d i e t , and
postpartum p e r i o d ) , the p r o t e i n content of t h i s peak was v a r i a b l e . Similar r e s u l t s have been described by o t h e r i n v e s t i g a t o r s working W i t h human cQlostrum"'s.
A change of b u f f e r t o one that contained
SECRETORY IgA FROM HUMAN M I L K
233
0.10 M sodium c h l o r i d e i n 0.01 M T r i s - H C 1 caused SIgA t o be e l u t e d from t h e column.
The IgA a c t i v i t y of each f r a c t i o n from
0.05 M and 0 . 1 M e l u t i o n peaks w a s t e s t e d by IEOP as d e s c r i b e d
above.
F r a c t i o n s w i t h hi jn SIgA c o n t e n t were used f o r subsequent
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p u r i f i c a t i o n of t h e s e p r o t e i n s . P u r i f i c a t i o n of SIgA The f r a c t i o n s t h a t had been e l u t e d with 0.1 M N a C l i n T r i s HC1 b u f f e r (pH 8.0,
0.01 M) f r m DEAE c e l l u l o s e column (Figure 2 )
were used a s a source f o r SIgA.
The pooled and concentrated
f r a c t i o n was applied t o a G-200 Sephadex column.
The f i r s t f r a c -
t i o n a t i o n (Figure 3-top) showed a major p r o t e i n peak which contained IgA a c t i v i t y .
Following t h e IgA a c t i v e f r a c t i o n s , a minor
contaminating band (Figure +top)
was e l u t e d .
The f r a c t i o n s t h a t contained t h e h i g h e s t IgA a c t i v i t y were pooled, concentrated, and rechromatographed on t h e same column. E l u t i o n r e s u l t e d i n two major p r o t e i n bands, of which o n l y t h e f i r s t contained IgA a c t i v i t y .
The IgA a c t i v e p r e p a r a t i o n w a s
f u r t h e r f r a c t i o n a t e d over t h e same column.
The r e s u l t s are
shown i n Figure 3 (bottom), i n which a s i n g l e p r o t e i n band cont a i n i n g IgA a c t i v i t y is i l l u s t r a t e d . I d e n t i f i c a t i o n and E s t a b l i s h i n g Homogeneity The p u r i t y of t h e IgA f r a c t i o n (Figure 3-bottom) w a s es-
t a b l i s h e d by polyacrylamide d i s c g e l e l e c t r o p h o r e s i s , which showed
that no migrating contaminants were p r e s e n t .
Purity also was
demonstrated by IEP a g a i n s t anti-whey p r o t e i n s (Figure 4 ) .
The
antiserum used i n this t e s t w a s prepared from a n t i g e n s t h a t had
KHAYAM-BASHI, BLANKEN, AND SCHWARTZ
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234
I
.Or
ELUATE VOLUME ( 5 ml/Fraction) FIGURE 3 Top: F i r s t p u r i f i c a t i o n of SIgA on Sephadex G-200 e l u t e d from DEAE c e l l u l o s e column shown i n Fig. 2. Bottom: F i n a l p u r i f i c a t i o n of SIgA on Sephadex column. A l l donditions are s i m i l a r t o those i n Fig. 2. Elution r a t e 20 m l k . Shaded a r e a i s a measure of IgA a c t i v i t y on a scale of 0-4 a s described i n t h e t e x t measured as a c t i v i t y a g a i n s t anti-IgA and anti-SC.
been i s o l a t e d previously.
This SIgA preparation produced a s i n g l e
arc, which w a s i n d i c a t i v e of the hanogeneity of the p r o t e i n s i n
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SECRETORY IgA FROM HUMAN MILK
S1gA I
q
R
0 0 a
b
235
b
i
t 10-2 Anti-Whey
Whey P r o t e i n s
FIGURE 4 Innnunoelectrophoretic p a t t e r n of p u r i f i e d bottom) and whey SIgA ( a s shown i n Fig. 3 p r o t e i n s against r a b b i t anti-whey serum.
-
t h i s procedure.
I n c o n t r a s t t o o t h e r i n v e s t i g a t o r s who have
used f u r t h e r p u r i f i c a t i o n on Sepharose 6B", a d d i t i o n a l s t e p was not e s s e n t i a l .
we found #at
this
Further inmunochemical analy-
sis by Ouchterlony i.nmunodiffusion p l a t e s also indicated that t h i s SIgA preparation was pure, and t h a t it contained common determinants with SC. On an agar p l a t e (Figure 51, t h e p u r i f i e d SIgA reacted with anti-SIgA, and showed t y p i c a l l i n e s of i d e n t f t y with SC ( l e f t portion of Figure 5 ) .
This p u r i f i e d SIgA also produced a s i n g l e
arc a g a i n s t WHO reference anti-SC.
The reason f o r t h e r e a c t i o n
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2 36
KHAYAM-BASHI, BLANKEN, AND SCWARTZ
FIGURE 5
Reaction between anti-SIgA (Rabbit 1) produced a g a i n s t t h e p u r i f i e d SIgA; as shown i n Fig. 3 bottom! SC; and p u r i f i e d SIgA ( 2 2 h r . incubation a t 31° c ) .
between SC and commercial anti-IgA is nbt clear.
I t might be due
t o the presence of anti-SIgA contamination i n t h e antiserum. These experiments provided evkdence that t h i s p u r i f i e d inrmuno-
globulin is of t h e s e c r e t o r y type (Figure 5 ) .
SECRETORY IgA FROM HUMAN MILK
237
For further substantiation of purity, antibody production in goats was also undertaken. Purified SIgA was injected into goats, which resulted in the production of a single homogeneous antibody, when tested against whey proteins and by purified SIgA. All tests for homogeneity and for identification suggested that the SIgA
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fraction prepared in our laboratory cross-reacts with both serum IgA and
SC.
This SIgA fraction also was free of contaminants,
as shown by disc electrophoresis. DISCUSSION The concentration of SIgA in human milk varies in relation to the length of lactation. In early days postpartum, the colostrum contains relatively large amounts of s ~ g ~ ~ ~AS , ~ , ~ ~ . lactation continues, the level of IgA drops to the lowest point between the fourth ts fiftieth weeks of lactation".
This level
increases after the fiftieth week, but dmes not reach the levels apparent in the immediately postpartum stage. During the initial DEAE-cellulose chromatography (Figure 2 )
,
it was noted that protein fractions eluted with 0.05 M NaCl in his-HC1 (0.01 M I pH 8.0) contained SC activity. Because of the lack of any reference material or specific antisera, the SC activity was assigned to this fraction temporarily, based on the previous data described by Newcomb, Normansell and Stanworth' O'Daly and Cebra",
and Tomasi and his associates'Br'.
,
This
initial assignment was confirmed when compared with a sample of specific antisera for SC supplied by WHO Reference Laboratories. Further purification of SC has been accanplished, and is reported elsewhere".
KHAYAM-BASHI, BLANKEN, AND SCHWARTZ
238
The nature of the major contaminating protein that is associated with SIgA and SC has been examined by several investigators2
It has been shown that this protein might be
associated with lactoferrins, which are commonly found in most body fluids.
Masson, Heremans and Divez2 have reported that as
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much as 1 mg/ml of this protein is available in milk, whereas its concentration in saliva and tears is about 0.1 mg/rnl.
Lippes,
et a1.23 were able to show that this protein, as well as IgA and SC, also are present in the female genital tract.
Lacto-
ferrin appeared to be absent f r m our final preparation. However, SIgA preparations indicated the positive existence of homogeneity when they were tested against anti-whey serum.
In
addition, anti-SIgA that was produced against our purified SIgA also resulted in a single arc, when tested against whey protein concentrates. The purification of SIgA resulted in a homogeneous protein, and disc electrophoretic assay did not show any fast-moving contaminants.
Successive Sephadex G-200 chromatography finally
resulted in a homogeneous protein.
Antibodies formed with this
protein in rabbits and goats showed that the protein was also immunologically homogeneous in both species.
This antiserum has
been used successfully for the evaluation of IgA and SIgA, since it contains determinant sites for both of these proteins, as well as for the SC in bound form. The evidence presented by several investigators indicates that measurable quantities of these proteins are present in the
SECRETORY I g A FROM HUMAN MILK
239
urine24r25and in the urogenital
and that the urinary
output of SIgA is increased in experimental pyelonephritis and in urinary infection in rabbits26. Waldman, Cruz and RoweZ7 show evidence for this rise in the presence of Candida albicans. Studies with Escherechia
& infection
in pigs has shown that
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the level of the SIgA also can rise in the milk produced by these animalsz6. It is also shown that salivary IgA is involved in protection against microorganisms in the oral cavity”
and
could perhaps play a role in susceptibility or resistance to tooth decay.
In light of the clinical utility of SIgA levels
in various body fluids, the quantitative measurement of SIgA in the biological f1uidsZ9 can provide meaningful and significant information for diagnostic, prognostic or investigative purposes. ACKNOWLEDGEMENTS The scientific cooperation and encouragement of Dr. R.C. Boguslasky and Dr. C.L.
Sutula throughout this work, as well as
the editorial assistance of Susan Eastwood (UCSF) and secretarial cooperation of Brenda Sharp, are gratefully acknowledged. REFERENCES 1.
Present address of Dr. Khayam-Bashi:
Department of Labora-
tory Medicine, University of California School of Medicine, San Francisco, at the Clinical Laboratories, S a n Francisco General Hospital, San Francisco, California 2.
Tomasi, T.B., Jr., W. Tan, A. Solomon and R. Prendergast. J. Exp. Med.
3.
94110 U.S.A.
121,101-124
Svehag, S and B. Bloth.
(1965).
Science 168, 847 (1970).
KHAYAM-BASHI, BLANKW, AND SCHWARTZ
240 4.
Tmasi, T., Jr. and A. Yurchak.
J. Immunol.
108,1132-
1135 (1972). 5.
Mestecky, J., J. Zikan and W. Butler.
Science 171, 1163-
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1165 (1971). 6.
Halpern, M. and
7.
Kobayashi, K.
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Mach, J.
9.
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10.
M.
Koshland.
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Immunochemistry g, 785-800 (1971).
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905-914 (1968).
Lowry,
O.,
N. Rosebrough, A. Farr and R. Randell.
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(1951).
11.
Warburg, 0. and W. Christian.
12.
Davis, B.
13.
Cmwle,
A.
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Biochem. 2 . 310, 384 (1942).
Ann. N.Y. Acad. Sci.
121,404-427
(1964).
in "Immunodiffusion," Acadaic Press, New York,
1961, p. 332. 14.
Grabar, P. and P. Burtin in "Inmunoelectrophoretic Analysis", Elsevier Publishing Co., New York, 1964, p. 302.
15.
Smith, I. in "chromatographic and Electrophoretic Technique," Vol 11, 2nd ed., Interacience Publishing Co., New York, 1968, p. 524.
16.
Khayam-Bashi, H., S. Slaughter and R. Blanken.
Fed. Proc. 32,
4211 (1973). 17.
Cebra, J. and J. Robbina.
18.
Tanaai, T., Jr.
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976-989 (1971).
449-455 (1971).
SECRETORY IgA FROM HUMAN MILK 21.
T o u r v i l l e , D., Med. 129, --
22.
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J. Exp.
411 (1969). C l i n . Chim. Acta
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(1966).
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-1, 163 24.
R. Adler, J. Bienenstock and T. To m asi .
Messon, P., J. Heremans and C . Dive. 735-739
23.
241
Contraception
(1970).
Bienenstock, J. and T. T a n a s i , Jr. i n " P r o t e i n s i n Normal and P a t h o l o g i c a l Urine," Manuel and Yves, E d s . ,
U n i v e r s i t y Park
P r e s s , Baltimore, 1970, p. 59. 25.
Kaufrnan, D . ,
R. Katz and R. McIntosh.
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463 (1970). 26.
Waldman, R., 427-434
J. Cruz and D.
Rowe.
C l i n . Exp. I m u n o l .
lo,
(1972).
108,
27.
Smith, J. and W. Hand.
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Crawford, J., M. Tauinan and D. Smith.
J. I m u n o l .
861-866
(1972).
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1209 (1975). 29.
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Preparative Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lpbb19
Chromatographic Separation and Purification of Secretory IgA from Human Milk H. Khayam-bashi Schwartz
a b
a
, R. M. Blanken & C. L.
a
a
Ames Research Laboratory, Ames company Division , Miles Laboratories, Inc. , Elkhart, Indiana, 46514, U.S.A. b
Department of Laboratory Medicine , University of California School of Medicine, San Francisco, at the Clinical Laboratories, San Francisco General Hospital , San Francisco, California, 94110, U.S.A. Published online: 05 Dec 2006.
To cite this article: H. Khayam-bashi , R. M. Blanken & C. L. Schwartz (1977) Chromatographic Separation and Purification of Secretory IgA from Human Milk, Preparative Biochemistry, 7:3-4, 225-241, DOI: 10.1080/00327487708061640 To link to this article: http://dx.doi.org/10.1080/00327487708061640
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PREPARATIVE BIOCHEMISTRY, 7(3&4), 225-241 (1977)
CHROMATOGRAPHIC SEPARATION AND PURIFICATION OF SECRETORY IgA FROM HUMAN MILK R.M. Blanken, and C.L. Schwartz Ames Research Laboratory, Ames company Division Miles Laboratories, I n c . , E l k h a r t , Indiana 46514 U.S.A.
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d . Khayam-Bashi’,
ABSTRACT Defatted and decaseinated human milk was concentrated and w a s f r a c t i o n a t e d on a p r e p a r a t i v e DEAE c e l l u l o s e column.
E l u t i o n with
various c o n c e n t r a t i o n s of sodium c h l o r i d e i n T r i s - H C 1 b u f f e r (pH 8.0, 0.01 M) r e s u l t e d i n f r a c t i o n s t h a t were r i c h i n e i t h e r secre-
t o r y immunoglobulin A (SIgA) ( 0 . 1 M NaC1) or f r e e s e c r e t o r y component (SC) (0.05 M N a C 1 ) .
The f r a c t i o n s , which were e l u t e d w i t h
0.10 M N a C l from t h e p r e p a r a t i v e column, were f u r t h e r f r a c t i o n a t e d on a G-200 Sephadex column.
Repeated f r a c t i o n a t i o n on t h i s
column r e s u l t e d i n a s i n g l e p u r i f i e d f r a c t i o n , which contained very high SIgA a c t i v i t y and showed immunological c r o s s - r e a c t i o n with both SC and sexum IgA.
Additional s t u d i e s i n d i c a t e d t h a t
t h i s f r a c t i o n was homogeneous as shown by immunoprecipitin and d i s c g e l electrophoresis.
I n j e c t i o n of t h i s p u r i f i e d SIgA i n t o
r a b b i t s r e s u l t e d i n the production of monospecific a n t i s a - 1 . INTRODUCTION
Various s t u d i e s have shown t h e predominance of IgA-type immunoglobulins i n colostrum and lachrymal s e c r e t i o n s .
They
225 Copyright 0 1977 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means. electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
226
KHAYAM-BASHI, BLANKEN, AND SCHWARTZ
suggest t h a t these secretions contain a s p e c i a l c l a s s of IgA which i s d i f f e r e n t f r a n IgA found i n the serum.
Studies by
Tomasi, e t a12 cited s p e c i f i c d i f f e r e n c e s which indicated t h a t these p r o t e i n s a r e d i s t i n c t from, and y e t r e l a t e d t o , the serum IgA proteins.
Evidence obtained i n t h e p a s t t e n years suggests
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t h a t secretory IgA (SIgA) c o n s i s t s of two IgA molecules, p l u s a secretory component (SC)
and J-chain polypeptide’
l4
1 6 .
Various techniques have been applied f o r s t r u c t u r a l s t u d i e s on SIgA.
Electron microscopic s t u d i e s have shown SIgA t o
possess a wishbone-type s t r u c t u r e .
Molecular weight s t u d i e s
i n d i c a t e t h a t SIgA has a sedimentation c o e f f i c i e n t of 11.5S, and a molecular weight of approximately 305 ,0007 I a
.
The p u r i f i c a t i o n of SIgA from human milk has been performed previously by s e v e r a l i n v e s t i g a t o r s 7 1 ’ 1 ’ .
However, since these
procedures are lengthy and r e q u i r e e l e c t r o p h o r e s i s , a s w e l l a s various types of chromatographic treatments, they r e s u l t i n low y i e l d s and a f i n a l product t h a t i s associated with contaminating proteins.
In order t o study the biochemical and b i o l o g i c a l
p r o p e r t i e s of SIgA, a l a r g e amount of highly p u r i f i e d p r o t e i n
i s essential.
The study presented here applied only chromato-
graphic procedures.
This simple and rapid technique r e s u l t e d
i n a homogeneous SIgA product with a r e l a t i v e l y high y i e l d . MATERIALS AND METHODS Preparation of t h e Whey from Human Milk Milk was collectedframtwo normal, healthy women i n l a t e l a c t a t i o n (14 and 15 months postpartum), and w a s frozen a t - 2 O O
SECRETORY IgA FROM HUMAN MILK C.
227
Of t h i s milk, 600 g w a s thawed and then c e n t r i f u g e d a t 30,000
rpm (105,000 x g ) i n a Model L , Beckman p r e p a r a t i v e u l t r a c e n t r i fuge (Beckman Instruments, F u l l e r t o n , CA hours.
92631) a t - 2 O
The d e f a t t e d m i l k was d i l u t e d 1:l with 0.15 M N a C 1 , and
t h e pH w a s a d j u s t e d t o 4.6 using an a c e t i c acid concentrate.
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C f o r two
-
sodium acetate
The mixture was kept a t room temperature f o r two
hours, then was t r a n s f e r r e d t o t h e cold room b e f o r e being rec e n t r i f u g e d a t 30,000 rpm (105,000 x g ) f o r t w o hours t o remove the precipitate.
The c l e a r s u p e r n a t a n t was a d j u s t e d t o 0.01 M
i n T r i s - H C 1 and pH of 8.0 a t room temperature by using a T r i s HC1 c o n c e n t r a t e .
The defatted-decaseinated milk serum obtained
by t h i s method was concentrated t o 40 m l (30X c o n c e n t r a t i o n ) over
a UM-10 membrane (Amicon Corp., Waltham, MA 60-80 P.S.I.
nitrogen i n t h e cold room.
0.1% (13.6 mM) with sodium a z i d e . t h e source f o r SC and SIgA.
02154) and under
The s o l u t i o n was made
The concentrated whey was
A flow c h a r t f o r t h i s p r e p a r a t i o n
i s shown i n Figure 1. Chromatographic Procedures A.
DEAE c e l l u l o s e :
A column, 5
x 65 cm ( r e s i n h e i g h t ) , was
packed w i t h 800 g of DE52 (Whatman Co., England) i n T r i s - H C 1 b u f f e r (0.01 M, pH 8.01,
which w a s prepared as reconmended by t h e
manufacturer.
Larger columns, 8 x 93 cm, were used i n subsequent
preparations.
The column w a s washed with 0.01 M, pH 8.0 T r i s - H C 1
b u f f e r (200 m l ) , a t a r a t e of 30 ml/hr, a t room temperature.
The
whey c o n c e n t r a t e (35 m l ) w a s a p p l i e d t o t h e column b e f o r e s t e p -
w i s e elution was initiated.
The e l u t i o n was performed w i t h T r i s -
KHAYAM-BASHI, BLANKEN, AND SCHWARTZ
228
Diluted Human Milk 1: 1 with 0.15 M NaCl
1
Centrifuge
Defatted Milk
I5.
1. Acetic Acid (pH 4.6) 2. Centrifuge
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Decaseinated Whey Concentrated Whey
I
1. Dialyze 2. Concentrate
DEAE Cellulose
Other Proteins (e. g., Lactoferrin)
SIgA Rich
S.C. Rich Fraction
Fraction
1. Concentrate 2. Sephadex G-200 (3x1
I
SIgA FIGURE 1 Schematic p r e s e n t a t i o n of v a r i o u s s t e p s f o r t h e p u r i f i c a t i o n of SIgA from human milk.
HC1 b u f f e r (0.01 M, p H 8.0) which contained 0.03 M, 0.05 M, o r
0.10 M sodium c h l o r i d e , according t o need.
E l u t i o n w a s maintained
by g r a v i t y with 1200 m l of each b u f f e r a t 30 m l / h r .
A n automated
f r a c t i o n c o l l e c t o r (Fractomette 200, Buchler Instruments, F o r t
Lee, N J ) t h a t was a t t a c h e d t o a recording Beclanan DBG Spoctrophotometer w a s used throughout t h e s t u d i e s .
Optical d e n s i t y a t 280
nm eerved ae a means € o r the e s t i m a t i o n of p r o t e i n c o n c e n t r a t i o n .
229
SECRETORY IgA FROM HUMAN MILK
B.
Sephadex G-200:
(Pharmacia Fine Chemicals, Upsala, Sweden)
A column (Pharmacia) was packed (2.7 x 92 an) with Sephadex G-200 ( 4 0 g ) suspended in borate
borate 0.15 M, pH 8.0).
-
sodium chloride buffer (0.1 M NaCl in
The same buffer was used for the final
elution of SIgA from the column (35 ml/hr).
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The estimation of proteins in the column fraction was based on W light absorption at 280 nm, as measured with a DBG-Beckman Recording Spectrophotometer. Protein content of the concentrates or other samples was determined by (a) Lowry's method"
using a
Beckman DU Spectrophotometer, and (b) the measurement of W light abeorption ratio at 260 and 280 nm". Acrylamide Disc Gel Electrophoresis A Canalco disc gel electrophoresis instrument (Canalco, Rockville, MD) was ussd. a 7 % acrylamide gel.
The sample (100 pg) was applied over
Current was applied for 45 min.
The
procedure was performed in the manner described by Davis"
and
by the manufacturer's bulletin. Immunological Procedures Antiserum IgA was supplied by Hyland Laboratories (Costa Mesa, CA) Cappel Laboratories (Philadelphia, P A ) , and from Miles-Yeda (Israel). Immunodouble Diffusion (ID) was performed according to Crowle".
A drop of suitable dilutions of antigens and antisera
(20 ~ 1 was ) placed on adjacent discs.
The precipitin band
between the two discs in the agar gel was observed and photographed.
KHAYAM-BASHI, B L A " ,
2 30
AND SCHWARTZ
Immunoelectrophoresis (IEP): The basic technique described by Grabar and Burtin"
was employed.
ma was applied to each slide. continue for up to 60 min. serum.
A constant current of 2
Electrophoresis was allowed to
The trough was filled with anti-
The slide was incubated overnight (16-20 hr) in a
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moist chamber at room temperature. Inrmunoelectroosmophoresis (IEOP) or Counter Immunoelectrophoresis:
The following pattern was used to monitor the
SIgA activity in each column fraction:
Four parallel columns of
wells ( 2 nun diameter, 5 mm apart) were punched into the agar slide (5.0 x 7.5 an). Each of the two columns formed one pair
of wells, with 10 well-pairs in each column (total of 21 pairs). The antigen samples ( 5 pl) were placed in the column of wells near the cathode; the specific antiserum was placed in the wells near the anode.
Electrophoresis was carried out up to 60 min.
The current per plate was held conatant at 20 ma. The precipitate formed between the antibody and antigen wells indicated the presence of IgA activity.
The intensity of the
precipitate was judged visually, and values from +4 to 0 were assigned, depending on the amount of precipitate formed.
Photo-
graphs were taken for permanent records, and the plates were stained for protein (Ponceau S) and stored. Preparation of Antisera against Purified Secretory IgA in Rabbits Two rabbits were injected for the preparation of each anti-
serum.
Each rabbit received 0.4 ml per injection, consisting of
0.0 mg of purified SIgA in complete Freund'a adjuvant.
The
SECRETORY IgA FROM HUMAN MILK
231
a n t i g e n was given i n two i n j e c t i o n s , each 10 days a p a r t , i n t o t h e f o u r footpads of t h e animals.
A f t e r t h e f i r s t 10 days, b l e e d i n g
w a s performed a t weekly i n t e r v a l s f o r f o u r weeks (50 m l blood
.
each time) l 6 Reference Antiserum
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S p e c i f i c antiserum was donated by WHO I n t e r n a t i o n a l Reference Center f o r Immunoglobulins (Lausanne, S w i t z e r l a n d ) .
According t o
t h e i r d e s c r i p t i o n , t h i s antiserum t o SC (M 33 A ) w a s prepared by immunization of a sheep with human SIgA t h a t had been prepared from colostrum by f r a c t i o n a t i o n of an SIgA-rich f r a c t i o n of DEAEc e l l u l o s e and G-200 Sephadex.
This a n t i g e n w a s i n j e c t e d i n t r a -
muscularly with complete Freund's adjuvant.
The r e s u l t i n g
antiserum was absorbed with both l a c t o f e r r i n and t w o s e r a cont a i n i n g IgA myeloma p r o t e i n s of t h e monomer and polymer form. Upon inmunoelectrophoresis and Ouchterlony t e s t s a g a i n s t colostrum and normal serum, t h e r e s u l t i n g antiserum appeared t o be s p e c i f i c f o r SC and SIgA. RESULTS Separation of SIgA from Milk by DEAE C e l l u l o s e Chromatography The concentrated defatted-decaseinated milk contained 22 mg/ml p r o t e i n .
T h i r t y m l of t h e c o n c e n t r a t e w a s used f o r a DEAE-
c e l l u l o s e column. u r e 2.
A t y p i c a l f r a c t i o n a t i o n p a t t e r n is shown i n Fig-
E l u t i o n with 0.01 M T r i s - H C 1 b u f f e r c o n t a i n i n g 0.03 M
sodium c h l o r i d e caused t h e e l a b o r a t i o n of three t o f o u r t y p e s of p r o t e i n s .
These p r o t e i n s d i d n o t c o n t a i n any d e t e c t a b l e IgA
a c t i v i t y , as measured by IEOP a n a l y s i s a g a i n s t a n t i s e r a f o r human
serum IgA.
KHAYAM-BASHI, BLANREN, AND SCHWARTZ
2 32
.OIN NoCI.
0.03N NoCI.
0.1N NoCI.
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2.4
'0
1.2
0
0.8 0.4
10
1
1
I
I
I
1
I
I
1
30
50
70
90
110
130
150
170
190
FRACTIONS (20 ml each) FIGURE 2 Fractionation of whey p r o t e i n s on DEAE c e l l u l o s e . The e l u t i o n b u f f e r Elution performed a t 4 0 m l / h r . was h i s - H C 1 , 0.1 M, pH 8 . 0 , containing indicated amounts of NaCl as shown i n t h e graph. Each f r a c t i o n is 20 ml/tube. Arrows i n d i c a t e b u f f e r changes.
Elution with t h e same b u f f e r containing 0.05 M NaCl r e l e a s e d
a small peak containing free SC.
Depending on t h e conditions,
e s p e c i a l l y those r e l a t e d t o t h e donor (e.g.,
h e a l t h , d i e t , and
postpartum p e r i o d ) , the p r o t e i n content of t h i s peak was v a r i a b l e . Similar r e s u l t s have been described by o t h e r i n v e s t i g a t o r s working W i t h human cQlostrum"'s.
A change of b u f f e r t o one that contained
SECRETORY IgA FROM HUMAN M I L K
233
0.10 M sodium c h l o r i d e i n 0.01 M T r i s - H C 1 caused SIgA t o be e l u t e d from t h e column.
The IgA a c t i v i t y of each f r a c t i o n from
0.05 M and 0 . 1 M e l u t i o n peaks w a s t e s t e d by IEOP as d e s c r i b e d
above.
F r a c t i o n s w i t h hi jn SIgA c o n t e n t were used f o r subsequent
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p u r i f i c a t i o n of t h e s e p r o t e i n s . P u r i f i c a t i o n of SIgA The f r a c t i o n s t h a t had been e l u t e d with 0.1 M N a C l i n T r i s HC1 b u f f e r (pH 8.0,
0.01 M) f r m DEAE c e l l u l o s e column (Figure 2 )
were used a s a source f o r SIgA.
The pooled and concentrated
f r a c t i o n was applied t o a G-200 Sephadex column.
The f i r s t f r a c -
t i o n a t i o n (Figure 3-top) showed a major p r o t e i n peak which contained IgA a c t i v i t y .
Following t h e IgA a c t i v e f r a c t i o n s , a minor
contaminating band (Figure +top)
was e l u t e d .
The f r a c t i o n s t h a t contained t h e h i g h e s t IgA a c t i v i t y were pooled, concentrated, and rechromatographed on t h e same column. E l u t i o n r e s u l t e d i n two major p r o t e i n bands, of which o n l y t h e f i r s t contained IgA a c t i v i t y .
The IgA a c t i v e p r e p a r a t i o n w a s
f u r t h e r f r a c t i o n a t e d over t h e same column.
The r e s u l t s are
shown i n Figure 3 (bottom), i n which a s i n g l e p r o t e i n band cont a i n i n g IgA a c t i v i t y is i l l u s t r a t e d . I d e n t i f i c a t i o n and E s t a b l i s h i n g Homogeneity The p u r i t y of t h e IgA f r a c t i o n (Figure 3-bottom) w a s es-
t a b l i s h e d by polyacrylamide d i s c g e l e l e c t r o p h o r e s i s , which showed
that no migrating contaminants were p r e s e n t .
Purity also was
demonstrated by IEP a g a i n s t anti-whey p r o t e i n s (Figure 4 ) .
The
antiserum used i n this t e s t w a s prepared from a n t i g e n s t h a t had
KHAYAM-BASHI, BLANKEN, AND SCHWARTZ
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234
I
.Or
ELUATE VOLUME ( 5 ml/Fraction) FIGURE 3 Top: F i r s t p u r i f i c a t i o n of SIgA on Sephadex G-200 e l u t e d from DEAE c e l l u l o s e column shown i n Fig. 2. Bottom: F i n a l p u r i f i c a t i o n of SIgA on Sephadex column. A l l donditions are s i m i l a r t o those i n Fig. 2. Elution r a t e 20 m l k . Shaded a r e a i s a measure of IgA a c t i v i t y on a scale of 0-4 a s described i n t h e t e x t measured as a c t i v i t y a g a i n s t anti-IgA and anti-SC.
been i s o l a t e d previously.
This SIgA preparation produced a s i n g l e
arc, which w a s i n d i c a t i v e of the hanogeneity of the p r o t e i n s i n
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SECRETORY IgA FROM HUMAN MILK
S1gA I
q
R
0 0 a
b
235
b
i
t 10-2 Anti-Whey
Whey P r o t e i n s
FIGURE 4 Innnunoelectrophoretic p a t t e r n of p u r i f i e d bottom) and whey SIgA ( a s shown i n Fig. 3 p r o t e i n s against r a b b i t anti-whey serum.
-
t h i s procedure.
I n c o n t r a s t t o o t h e r i n v e s t i g a t o r s who have
used f u r t h e r p u r i f i c a t i o n on Sepharose 6B", a d d i t i o n a l s t e p was not e s s e n t i a l .
we found #at
this
Further inmunochemical analy-
sis by Ouchterlony i.nmunodiffusion p l a t e s also indicated that t h i s SIgA preparation was pure, and t h a t it contained common determinants with SC. On an agar p l a t e (Figure 51, t h e p u r i f i e d SIgA reacted with anti-SIgA, and showed t y p i c a l l i n e s of i d e n t f t y with SC ( l e f t portion of Figure 5 ) .
This p u r i f i e d SIgA also produced a s i n g l e
arc a g a i n s t WHO reference anti-SC.
The reason f o r t h e r e a c t i o n
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2 36
KHAYAM-BASHI, BLANKEN, AND SCWARTZ
FIGURE 5
Reaction between anti-SIgA (Rabbit 1) produced a g a i n s t t h e p u r i f i e d SIgA; as shown i n Fig. 3 bottom! SC; and p u r i f i e d SIgA ( 2 2 h r . incubation a t 31° c ) .
between SC and commercial anti-IgA is nbt clear.
I t might be due
t o the presence of anti-SIgA contamination i n t h e antiserum. These experiments provided evkdence that t h i s p u r i f i e d inrmuno-
globulin is of t h e s e c r e t o r y type (Figure 5 ) .
SECRETORY IgA FROM HUMAN MILK
237
For further substantiation of purity, antibody production in goats was also undertaken. Purified SIgA was injected into goats, which resulted in the production of a single homogeneous antibody, when tested against whey proteins and by purified SIgA. All tests for homogeneity and for identification suggested that the SIgA
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fraction prepared in our laboratory cross-reacts with both serum IgA and
SC.
This SIgA fraction also was free of contaminants,
as shown by disc electrophoresis. DISCUSSION The concentration of SIgA in human milk varies in relation to the length of lactation. In early days postpartum, the colostrum contains relatively large amounts of s ~ g ~ ~ ~AS , ~ , ~ ~ . lactation continues, the level of IgA drops to the lowest point between the fourth ts fiftieth weeks of lactation".
This level
increases after the fiftieth week, but dmes not reach the levels apparent in the immediately postpartum stage. During the initial DEAE-cellulose chromatography (Figure 2 )
,
it was noted that protein fractions eluted with 0.05 M NaCl in his-HC1 (0.01 M I pH 8.0) contained SC activity. Because of the lack of any reference material or specific antisera, the SC activity was assigned to this fraction temporarily, based on the previous data described by Newcomb, Normansell and Stanworth' O'Daly and Cebra",
and Tomasi and his associates'Br'.
,
This
initial assignment was confirmed when compared with a sample of specific antisera for SC supplied by WHO Reference Laboratories. Further purification of SC has been accanplished, and is reported elsewhere".
KHAYAM-BASHI, BLANKEN, AND SCHWARTZ
238
The nature of the major contaminating protein that is associated with SIgA and SC has been examined by several investigators2
It has been shown that this protein might be
associated with lactoferrins, which are commonly found in most body fluids.
Masson, Heremans and Divez2 have reported that as
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much as 1 mg/ml of this protein is available in milk, whereas its concentration in saliva and tears is about 0.1 mg/rnl.
Lippes,
et a1.23 were able to show that this protein, as well as IgA and SC, also are present in the female genital tract.
Lacto-
ferrin appeared to be absent f r m our final preparation. However, SIgA preparations indicated the positive existence of homogeneity when they were tested against anti-whey serum.
In
addition, anti-SIgA that was produced against our purified SIgA also resulted in a single arc, when tested against whey protein concentrates. The purification of SIgA resulted in a homogeneous protein, and disc electrophoretic assay did not show any fast-moving contaminants.
Successive Sephadex G-200 chromatography finally
resulted in a homogeneous protein.
Antibodies formed with this
protein in rabbits and goats showed that the protein was also immunologically homogeneous in both species.
This antiserum has
been used successfully for the evaluation of IgA and SIgA, since it contains determinant sites for both of these proteins, as well as for the SC in bound form. The evidence presented by several investigators indicates that measurable quantities of these proteins are present in the
SECRETORY I g A FROM HUMAN MILK
239
urine24r25and in the urogenital
and that the urinary
output of SIgA is increased in experimental pyelonephritis and in urinary infection in rabbits26. Waldman, Cruz and RoweZ7 show evidence for this rise in the presence of Candida albicans. Studies with Escherechia
& infection
in pigs has shown that
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the level of the SIgA also can rise in the milk produced by these animalsz6. It is also shown that salivary IgA is involved in protection against microorganisms in the oral cavity”
and
could perhaps play a role in susceptibility or resistance to tooth decay.
In light of the clinical utility of SIgA levels
in various body fluids, the quantitative measurement of SIgA in the biological f1uidsZ9 can provide meaningful and significant information for diagnostic, prognostic or investigative purposes. ACKNOWLEDGEMENTS The scientific cooperation and encouragement of Dr. R.C. Boguslasky and Dr. C.L.
Sutula throughout this work, as well as
the editorial assistance of Susan Eastwood (UCSF) and secretarial cooperation of Brenda Sharp, are gratefully acknowledged. REFERENCES 1.
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