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Polyglutaraldehyde: A new reagent for coupling proteins to microspheres and for labeling cell-surface receptors
Journal of Immunological Methods, 2,1 ( 1978 ) 239--250 © Elsevier/North-Holland Biomedical Press
239
P O L Y G L U T A R A L D E H Y D E : A NEW R E A G E N T F O R C O U P L I N G PROTEINS TO MICROSPHERES AND FOR LABELING CELL-SURFACE RECEPTORS
A. REMBAUM, S. MARGEL * and J. LEVY ** Jet Propulsion Laboratory, California lnstitute of Technology, Pasadena, CA 91103, U.S.A. (Received 18 May 1978, accepted 30 May 1978)
Glutaraldehyde polymerized in basic aqueous solutions was found to react with low molecular weight amines, immunoglobulins and hemoglobin. The polyglutaraldehyde was covalently bound to hydrophilic microspheres. The rate of addition of proteins to the polyglutaraldehyde-derivatized microspheres was investigated spectrophotometrieally as a function of pH and temperature. The reaction of polyglutaraldehyde was found to be faster than that of the monomer. The findings led to successful labeling of human lymphocyte subpopulations. INTRODUCTION A m o s t i m p o r t a n t area in which m i c r o s p h e r e s (beads)-conjugates and o t h e r particles have f o u n d a p p l i c a t i o n is in the d e t e c t i o n o f cell-surface r e c e p t o r s b y light and scanning e l e c t r o n m i c r o s c o p y ( R o t h , 1977). The cell t y p e t h a t has been m o s t extensively s t u d i e d with b o t h f l u o r e s c e n t and nonf l u o r e s c e n t markers is the l y m p h o c y t e {Brown and Greaves, 1975). This m e t h o d o l o g y has been i n s t r u m e n t a l to bringing us to the c u r r e n t state o f a p p r e c i a t i n g multiple s u b p o p u l a t i o n s a m o n g peripheral b l o o d l y m p h o c y t e s . T h e r e c e p t o r s w h o s e d e t e c t i o n has m a d e this d i f f e r e n t i a t i o n into s u b p o p u l a tions possible include surface i m m u n o g l o b u l i n , Fc receptors, C' r e c e p t o r s and r e c e p t o r s for sheep red b l o o d cells. It was r e c e n t l y established t h a t i m m u n o g l o b u l i n s c o u l d be m o r e efficiently b o u n d to beads b y m e a n s o f g l u t a r a l d e h y d e , t h a n with o t h e r reagents ( M o l d a y et al., 1977). It was also s h o w n t h a t b e a d s - i m m u n o g l o b u l i n conjugates c o n s t i t u t e s a t i s f a c t o r y markers o f cell m e m b r a n e r e c e p t o r s for light m i c r o s c o p y ( G o r d o n et al., 1977). T h e p r e s e n t p a p e r describes the use o f p o l y g l u t a r a l d e h y d e for the same p u r p o s e . L o w m o l e c u l a r weight amines, f l u o r e s c e n t i m m u n o g l o b u l i n s and h e m o g l o b i n were used to investigate the reactivity o f p o l y g l u t a r a l d e h y d e , and it was f o u n d t h a t b e a d s - h u m a n IgG * Research Fellow at JPL from Weizmann Institute (Rehovot, Israel). ** UCLA School of Medicine, Los Angeles, CA 90024, U.S.A.
2.10 conjugates prepared by means of polyglutaraldehyde successfully labeled Fc receptors of human l y m p h o c y t e s . Glutaraldehyde in m o n o m e r form is used extensively for the tanning of leather, fixation of living cells, immobilization of enzymes, etc. ( t t o p w o o d , 1972). The reaction mechanism of proteins with glutaraldehyde is difficult to ascertain since its structure is still not clear and it has been reported to be in equilibrium with cyclic and hydrated forms (Itardy et al., 1969; Korn et al., 1972). Aqueous solutions of glutaraldehyde contain variable amounts of polyglutaraldehyde, a p o l y m e r which is also formed during monoglutaraldehyde reaction with proteins. The e x t e n t of p o l y m e r formation during these reactions varies considerably because the rate of polymerization is d e p e n d e n t on m o n o m e r concentration, pH, and temperature (Gillet and Gull, 1972; Jones, 1974; Rasmussen and Albrechtsen, 1974). Because of the large number o f applications of glutaraldehyde to biological systems, there is a need to examine the reactivity of the glutaraldehyde p o l y m e r with proteins and to compare it with that of the m o n o m e r . We have therefore carried out a preliminary study o f the polymerization kinetics, the structure and the solubility o f the polymer. These results will be reported separately. In the present paper, the reactivity of a water-insoluble polyglutaraldehyde fraction with immunoglobulins and hemoglobin will be discussed. Most of the reactions were carried out heterogeneously with polyglutaraldehyde suspended in water or with polyglutaraldehyde bound to the surface of polymeric microspheres (beads). In contrast to glutaraldehyde, the p o l y m e r contains conjugated aldehyde groups which impart stability to the Schiff bases formed after reaction with proteins (Mensan et al., 1975). In addition, the hydrophilic polyglutaraldehyde has relatively long chains extending from the microspheres into the surrounding aqueous medium, facilitating the heterogeneous reaction with proteins, e.g., immunoglobulins. The described experiments support these theoretical concepts. MATERIALS AND METHODS
Polymerization Commercial aqueous solutions of glutaraldehyde were purified by means o f activated carbon and were shown to be free of p o l y m e r by absence of absorption at 235 nm (Cary 14 s p e c t r o p h o t o m e t e r ) . A 25% solution of glutaraldehyde was polymerized at 22°C by adjusting the pll to 11.5 with NaOH. At the end of 2 h, the mixture was stirred for 8 h at 50°C. The precipitated p o l y m e r was filtered, washed with water and then with acetone and dried in vacuum for 24 h (yield, 60%). Assuming the repeating unit to be --CII.,--CH,--CH=~--CttO the molal extinction coefficient e2.~ measured in ethanol was found to be equal to 1.53 x l 0 s l/mole/era. The molar extinction coefficient of glutaraldehyde at 280 nm, e2~s (extracted from aqueous
241 solutions with ether and dried with magnesium perchlorate) was found to be equal to 4.2 1/mole/cm.
Syn thesis o f rn icrospheres Poly (4-vinylpyridine) acrylamide (PVP) and poly (2-hydroxy ethylmethacrylate) (Polyhema) mierospheres were synthesized by copolymerization of the monomers in presence of bisaerylamide as previously described (Rembaum et al., 1976, 1978). Microspheres were prepared with a relatively uniform distribution of sizes (S.D. 5--10%) varying in diameter from 0.05 to 10 ~m. In the present work PVP microspheres of 0.7 ~m in diameter were coupled with hydrazine to yield hydrazide groups on the surface (Inman and Dintzis, 1969). Reactions o f polyglutaraldehyde with amines The reactivity of polyglutaraldehyde with low molecular weight amines was investigated in a one-phase (homogeneous) and two-phase (heterogeneous) system. Homogeneous reactions were carried out at relatively low temperature, for example, polyglutaraldehyde was treated with methylamine and liquid anhydrous ammonia at about --40°C for I h. After raising the temperature and evaporation to dryness, the products were washed with water, dried in vacuo at 60°C, analyzed spectrophotometrically and their nitrogen content was determined. The polymer was also soluble at room temperature in higher boiling amines, e.g., allylamine, butylamine, ethanolamine, hydrazine and ethylene diamine. Its reaction with these amines was carried out at 22°C for 1 h and analyzed in the same way as in the case of the low temperature reactions. The heterogeneous reaction was carried out by suspending the polymer (120 rag) in phosphate-buffered saline (PBS) solution (20 ml) of the amine (1 g) at pH of 7.3--7.4 and stirring in a closed container for at least 30 h. After washing with water and drying, the nitrogen content of the products was determined {Table 1). Reactions with human immunoglobulins Fluorescent human IgG and goat anti-human IgM (Meloy) were purified on DEAE cellulose columns (Levy and Sober, 1960). Their fluorescence intensity at different pHs was measured at room temperature by means of an Aminco Fluorimeter model SPF125. The extent of reaction was determined by measuring the fluorescence of the supernatant after centrifugation of suspensions containing fluorescent IgG and insoluble polyglutaraldehyde. Two series of experiments were performed. In the first series, powdered polyglutaraldehyde was coupled directly with fluorescent IgG and the decrease of fluorescence measured after reaction yielded the a m o u n t of IgG bound to the polymer. Polyhema microspheres were treated under identical conditions and served as control. The second series of experiments consisted of reactions with polyglutaral-
242 TABLE l NITROGEN ANALYSIS OF REACTION PRODUCTS OF POLYGLUTARALDEHYDE WITtt AMINES Amine
Homogeneous reaction
Iteterogeneous reaction
% N found
% of t h e theor, amt.
% N found
% of t h e theol', a m t .
5.6 14.0 2.9 5.6 6.5 5.0 4.6
38.1 47.4 16.8 48.6 63.1 44.6 31.9
0.6 0.5 7.5 0.9
5.3 3.5 52.1 8.9
.
Methylamine Hydrazine Ammonia Allylamine Butylamine Ethanolamine a Dia m i n o h e p t a n e Hydroxylamine b Glycine
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a Average of t w o d e t e r m i n a t i o n s . b H y d r o x y l a m i n e h y d r o c h l o r i d e was used at pH 7.3 and 2.4. T h e n i t r o g e n c o n t e n t was 7.5 a n d 6.6% respectively.
dehyde covalently bound to hydrazide groups on the surface of beads 0.7 pm in diameter. The chemical binding was carried out in the following way: PVP hydrazide beads (25 mg) were stirred with polyglutaraldehyde (25 mg) dissolved in dimethylsulfoxide (DMSO, 50 ml) for not less than 5 h. After centrifugation and washing with DMSO and water the beads (5 ml) were then stirred with glycine (80 mg) for 4 h. After washing, fluorescent immunoglobulins were added (Tables 2 and 3). This mixture was stirred at a controlled temperature
TABLE 2 REACTION OF POWDERED POLYGLUTARALDEHYDE of f l u o r e s c e n t IgG in 10 ml o f p h o s p h a t e b u f f e r , pH 7.4) Polyglutaraldehyde
Polyhema
(mg/ml)
(mg/ml)
0.2 2.0 20.0
---0.2 2.0 20.0
WITH H U M A N IgG (l m g
Bound IgG/polymer (w/w) pH 7.4
pH 6.0
0.160 0.030 0.004 0 0 0
0.200 0.040 0.006 0 0 0
243 TABLE 3 COMPARISON OF THE REACTIVITY OF GLUTARALDEHYDE AND POLYGLUTARALDEHYDE-DERIVATIZED BEADS Effect of mono- and polyglutaraldehyde concentration (0.5 mg of fluorescent IgG in 10 ml of phosphate buffer, pH 7.4). Control a (mg/ml) .
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Polyglutaraldehyde a (mg/ml) .
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Glutaraldehyde a (mg/ml) .
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Bound IgG/polymer (w/w × 103)
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2.5 7.5 20.0 2.5 7.5 20.0 2.5
l:l .0 4.0 1.4 6.0 2.0 0.7 0
a 25 mg/ml of beads used in all experiments.
and the loss o f f l u o r e s c e n c e with t i m e was d e t e r m i n e d using c e n t r i f u g e d aliquots.
Reaction with hemoglobin P o l y g l u t a r a l d e h y d e or P V P - p o l y g l u t a r a l d e h y d e m i c r o s p h e r e s w e r e susp e n d e d in h e m o g l o b i n (2 X crystallized, f r o m C a l b i o c h e m ) dissolved in 0.01 M p h o s p h a t e b u f f e r . T h e p H was a d j u s t e d with s o d i u m p h o s p h a t e and t h e o p t i c a l d e n s i t y at 4 1 2 n m o f c e n t r i f u g e d aliquots was r e c o r d e d with time.
Application of polyglutaraldehyde conjugated beads H u m a n l y m p h o c y t e s w e r e s e p a r a t e d f r o m red b l o o d cells b y m e a n s o f a F i c o l l - H y p a q u e g r a d i e n t freed f r o m m o n o c y t e s b y m e a n s o f iron particles ( H o r w i t z and L o b o , 1 9 7 5 ) and s u s p e n d e d in a m e d i u m at pH 7 . 2 - - 7 . 4 to which 20% o f fetal calf s e r u m (FCS) was a d d e d . T h e m e d i u m ( R P M I / H ) consisted of RPMI 1640 (Gibco) + 0.025 M Hepes (N-2-hydroxethylp i p e r a z i n e - N - 2 - e t h a n e sulfonic acid f r o m C a l b i o c h e m ) . M i c r o s p h e r e s to which h u m a n IgG was b o u n d b y m e a n s o f p o l y g l u t a r a l d e h y d e (0.5 ml o f m i c r o s p h e r e s u s p e n s i o n c o n t a i n i n g 0.5 m g o f m i c r o s p h e r e s in PBS b u f f e r at p H 7.4) w e r e i n c u b a t e d f o r 1 h at 4°C with 5 X 106 l y m p h o c y t e s in R P M I / H b u f f e r (1 ml) c o n t a i n i n g 0.1% s o d i u m azide. A f t e r a d d i t i o n o f FCS (1.5 m l ) + 0.1% s o d i u m azide, the s u s p e n s i o n was c e n t r i f u g e d at 1 5 0 0 r e v / m i n ( 5 1 5 X g ) . T h e r e c o v e r e d cells were r e s u s p e n d e d in 50% R P M I / H and 50% FCS c o n t a i n i n g 0.1% s o d i u m azide and r e c e n t r i f u g e d . It was f o u n d n e c e s s a r y to r e p e a t this washing p r o c e d u r e 3 t i m e s in o r d e r to r e m o v e p r a c t i c a l l y all u n b o u n d m i c r o s p h e r e s . A f t e r t h e third c e n t r i f u g a t i o n the cells were r e s u s p e n d e d in R P M I / H c o n t a i n i n g 1% b o v i n e s e r u m a l b u m i n
2 44 and 0.1% s o d i u m azide. T h e labeled cells were t h e n c o u n t e d u n d e r the microscope. In the c o n t r o l e x p e r i m e n t s t h e l y m p h o c y t e s were i n c u b a t e d with aggregated IgG or u n c o a t e d b e a d s for 1 h. T h e aggregated IgG was p r e p a r e d b y dissolving h u m a n IgG (50 m g / m l ) in PBS at pH 7.4 and h e a t i n g t h e solution at 63°C for 30 min. T h e colloidal s u p e r n a t a n t l a y e r was used for cell i n c u b a t i o n p r i o r to labeling. M i c r o s p h e r e s c o u p l e d to goat a n t i - h u m a n IgM were also used to test the r e a c t i v i t y o f p o l y g l u t a r a l d e h y d e . RESULTS
Reaction of polyglutaraldehyde with low molecular weight amines P r e l i m i n a r y i n f o r m a t i o n was o b t a i n e d on the reactivity o f the p o l y m e r with a m i n o g r o u p s b y c a r r y i n g o u t the r e a c t i o n with a series of amines. T a b l e 1 s h o w s the results o f e l e m e n t a l analysis o f the p r o d u c t s . T h e t h e o r e t ical a m o u n t o f n i t r o g e n was c a l c u l a t e d on t h e a s s u m p t i o n t h a t each unit; - C H 2 - - C H 2 C H = ~ - - C H O will b i n d o n e m o l e c u l e o f amine. In the case o f hyd r o x y l a m i n e h y d r o c h l o r i d e , h y d r a z i n e a n d b u t y l a m i n e the e x t e n t o f reaction was o f t h e o r d e r o f 5 0 - - 6 0 % o f the t h e o r e t i c a l a m o u n t . S p e c t r o p h o t o m e t r i c e x a m i n a t i o n revealed t h a t in m o s t p r o d u c t s t h e a b s o r p t i o n at 285 n m , a t t r i b u t e d to the a l d e h y d e g r o u p , was c o n s i d e r a b l y reduced.
Reaction of polyglutaraldehyde and polyglutaraldehyde-derivatized microspheres with immunoglobulins T a b l e 2 s h o w s the a m o u n t o f IgG b o u n d to p o l y g l u t a r a l d e h y d e a f t e r stirring the s u s p e n s i o n s o f the p o l y m e r for 2.25 h at r o o m t e m p e r a t u r e . A c o n s t a n t a m o u n t o f f l u o r e s c e n t IgG was used in these e x p e r i m e n t s . It is o b v i o u s t h a t increased a m o u n t s o f p o l y m e r resulted in a l o w e r I g G / p o l y m e r
TABLE ,1 COMPARISON OF THE REACTIVITY OF GLUTARALDEHYDE AND POLYGLUTARALDEHYDE-DERIVATIZED BEADS (effect of IgG concentration) Control a (mg/ml)
Polyglutaraldehyde a (mg/ml)
Human IgG (mg/ml)
Glutaraldehyde (mg/ml) .
2.5 2.5 2.5 2.5 2.5 2.5 2.5
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Bound IgG polymer (W/W X 103 ) .
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0.025 0.050 0.100
5.7 9.5 14.0
0.025 O.050 0.100
5.3 4.5 9.1 0
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245
L Z
Z w
z_ Z u O D
'°°o
~®
~o
3~o
I
,00
5®
TIME (MIN)
Fig. 1. Rate of reaction of polyglutaraldehyde (60 mR) with human IgG (4.5 mR) in PBS (50 ml) at 22°C. e, pH 6;~", pH 7.4.
ratio. The same result was obtained with microspheres derivatized with either the polymer or m o n o m e r (Table 3). Table 2 indicates that larger amounts of IgG are bound to the polymer at pH 6 than at pH 7.4. This pH effect was confirmed in subsequent studies. It is also apparent that somewhat larger amounts of IgG become bound to polyglutaraldehyde than to the polyglutaraldehyde
20 |
"~
I
l
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[
[ ~
0
l
10
~2o
40
4O
•
-
~6o ~
I00
200
300 TIME (MIN)
400
500
600
_ _ - -
_-..-~
70 8O
~
I
I
3o0
6oo
1 90o
I 1200
I L~oo
! 18~
TiME (MIN)
Fig. 2. Rate of reaction of PVP beads with fluorescent human IRG at 22°C and pH 7.4. $, polyglutaraldehyde bound to PVP hydrazide beads; :3, monoglutaraldehyde bound to PVP hydrazide beads; ×, PVP hydrazide beads (control);/~, IgG in PBS (control).
FiR. 3. Rate of reaction of PVP beads with fluorescent human IgG at 4°C. e, polyglutaraldehyde bound to PVP hydrazide beads; c., monoglutaraldehyde bound to PVP hydrazide beads; x, PVP hydrazide beads (control);/,,, IgG in PBS (control).
246 2C
I
~
!
---
I
:
30 Z >-
40
~
50
60
z ~ 7o z u m
80
© -~
90 ~ 100
10
20
30
~
40 50 T~ME (HOURS)
×-
60
70
80
90
Fig. 4. Rate of reaction of PVP beads with fluorescent goat anti-human IgM at 4°C and pH 7.4. '.% polyglutaraldehyde bound to PVP beads; r,, monoglutaraldehyde bound to PVP beads; x, PVP hydrazide beads (control). than by the m o n o m e r . I n f o r m a t i o n on the necessary reaction time was obtained through kinetic experiments. In Fig. 1, the decrease of fluorescence of the supernatant is pl ot t e d against time. It is obvious that at pH 7.4 the reaction is incomplete after 2.25 h (the time used in previous experiments). F u r t h e r m o r e , at pH lower than 7.4 the reaction is faster. Figs. 2 and 3 confirm the faster reaction of the p o l y m e r over the m o n o m e r and also show the effect o f temperature. Similar results were obtained with fluorescent goat anti-human IgM (Fig. 4).
Reactions with hemoglobin Because hemoglobin has a high extinction coefficient at 412 n m ( 6 r a M " 125 based on a molecular weight o f 17,000), it is a convenient model for our studies o f protein immobilization. It can be calculated from the 412 nm absorption peak at pH 6.0, 7.4 and 8.4 that the polyglutaraldehyde immobilized 8.8, 6.6 and 2.8% of hemoglobin respectively after 8.3 h of reaction time. In control experiments (Polyhema) the hemoglobin c o n t e n t was of the order of 1% based on nitrogen analysis (%N in the hemoglobin used was f o u n d to be 15.2%). The hemoglobin c o n t e n t of the polyglutaraldehydehemoglobin products obtained at pH 6.0, 7.4 and 8.4 was calculated to be 10.4, 7.8 and 5.3% respectively (based on nitrogen analysis) and agrees reasonably well with the s p e c t r o p h o t o m e t r i c results (Fig. 5). In contrast to the heterogeneous reaction, when polyglutaraldehyde reacted with an excess of hemoglobin h o m ogeneous l y in aqueous DMSO the product was found to contain 76% o f hemoglobin. T he kinetics of hemoglobin binding to polyglutaraldehyde-derivatized microspheres are shown in Fig. 6. The trend of increasing reaction rate a t p H 6 as compared with pH 7.4 is evident.
247
0.1
- -
0.2
OZ 0.4 0.5
o.e Z
0.7
~
0.8
/I
~ .------'v-'---~" ~ ~
~'
"~ I
0.9 1.0
1130
200 300 TIME ( M I N )
400
500
Fig. 5. Rate of reaction at 22°C of polyglutaraldehyde ( 1 0 0 m g in 5 0 m l of FBS) with hemoglobin (7.5 rag) as a function of pH. c'., pH 6; /', pH 7.4; X, pH 8.4; e, Polyhema beads, pH 6 (control).
i
0 2:
' '
' : ' ' 'o i
.J 0.3 0 Z O. ~ •~ >-
0.5
•
0.6
o.7 0.8
0.9 1.0
200
400
600
800
I000
1200
1400
16(X)
1800
!000
TIME (MIN)
Fig. 6. Rate of reaction of PVP beads with hemoglobin at pH 6 and 22°C. o, derivatized with polyglutaraldehyde ; ~, derivatized with monoglutaraldehyde; $, Polyhema (control).
248 TABLE
5
LABELING OF IIUMAN LYMPttOCYTES WITH MICROSPHERE-IgG CONJUGATES Experiment
Type of beads
Antibody (mg)
Number of heads ( x 1 0 ' l {))
% Labeled cells
1
IgG conjugates lgG conjugates Unreacted beads (control
0.18 0.29
1.25 2.5 2.5
13.6 20.7 6.7
2
IgG conjugates Unreaeted heads (control ARgregated IgG (control) IgG eonjmlates Unreacted beads (control
l 7.9
3
4
IgG conjugates Unreacted beads (control)
0.,15
2.5
--
2.5
,1.6
0.,15 --
2.5 2.5 2.5
0 17.2 2.9
0.19
2.5
1.9
"-
8.82 0
Labeling of Fc receptors P u r i f i e d l y m p h o c y t e s (5 ~' 106), as d e s c r i b e d in t h e e x p e r i m e n t a l p a r t , w e r e i n c u b a t e d w i t h I g G - b e a d c o n j u g a t e s . T h e r e s u l t s a r e s h o w n in T a b l e 5. T h e n o n - s p e c i f i c a l l y l a b e l e d c e l l s in c o n t r o l e x p e r i m e n t s ( e x p . 1 a n d 2) h a d a m a x i m u m o f o n l y 4 b e a d s o n t h e cell s u r f a c e . T h e a p p e a r a n c e o f s p e c i f i c a l l y l a b e l e d cells is s h o w n ill Fig. 7a. CONCI,USIONS AND DISCUSSION The examination of the results leads to the following conclusions: (1) P o l y g l u t a r a l d e h y d e e x h i b i t s a h i g h r e a c t i v i t y t o w a r d s l o w m o l e c u l a r
Fig. 7. a: SEM photomicrograph of a lymphocyte labeled with beads with human IgG bound to the surface, h: control from exp. 3 (see Table 5).
249 weight amines under mild experimental conditions (Table 1). The remarkable reactivity of hydroxylamine indicates that it may constitute a superior reagent to glycine which was used in our experiments for the inactivation of aldehyde groups on glutaraldehyde or polyglutaraldehyde-activated reagents for cell labeling. The highest theoretical yields of reaction products, based on the assumption that the repeating unit in the polymer is a substituted acrolein moiety, amounted to about 50--60%. Spectrophotometric studies showed presence of unreacted aldehyde functions in the reaction products. Therefore it is likely that Schiff base formation reached an equilibrium stage, and by changing the experimental conditions higher yields than those recorded in Table 1 may be expected. (2) The presence of acrolein structure in the polymer was confirmed by the reaction of the latter with rn-aminophenol which yielded a highly fluorescent polymer with fluorescence emission maximum at 470 nm. (3) The reactivity of the polymer with human IgG is about twice that of the m o n o m e r {Tables 2, 3, 4 and Figs. 2, 3, 4 and 6). The number of IgG molecules bound per bead (0.7 pm in diameter) after 10 h of reaction at 22°C (Fig. 2) was calculated to be equal to 3.8 × 103 and 2.3 r 103 for the polyglutaraldehyde and monoglutaraldehydc procedures, respectively. Larger yields and higher reaction rates were observed in the reactions of immunoglobulins with polyglutaraldehyde than with microspheres (beads) derivatized with polyglutaraldehyde (compare Tables 2, 3 and 4 and Figs. 1 and 2). The same conclusions apply to the reactions with hemoglobin {Figs. 5 and 6). (4) The lowering of pH from 7.4 to 6 enhances the reaction rate. This phenomenon applies to human IgG (Fig. 1) as well as to hemoglobin {Fig. 5). To interpret this effect we assume that the reaction of polyglutaraldehyde takes place between free amino groups of the protein, e.g., lysine residues, and aldehyde groups of the polymer to form Schiff bases. It might be expected that carbonyl addition reactions would be powerfully acidcatalyzed for, after a proton attack on oxygen, the carbon atom will become positively charged and hence readier to react with a nucleophile: C = O + H ÷ ~ C*---OH. When the nucleophile is R-NH2, an acid converts the amine to the unreactive species R-NH3. We should thus expect to find that the rate of addition will show a maximum at a moderately acid pH falling off sharply at each side. This is indeed observed in practice (Jencks, 1969). (5) The small extent of non-specific binding of immunoglobulins and hemoglobin (Figs. 2 and 5) to substrates free of glutaraldehyde and polyglutaraldehyde is attributed to physical adsorption and can be reduced by lowering the reaction temperature (Fig. 3). (6) The high reactivity of polyglutaraldehyde, its stability, ease of administration, and the retention of physiolo~,dcal activity of human immunoglobulins bound to the polymer make it a desirable new reagent for protein binding. The experimental results demonstrate the successful application of polyglutaraldehyde beads for quantitation of human l y m p h o c y t e subpopulations.
250 'Fable 5 d e m o n s t r a t e s the ability o f beads c o a t e d with h u m a n IgG to bind to the Fc r e c e p t o r on B and null cells with the a p p r o p r i a t e c o n t r o l o f inhibition o f this binding when free aggregated IgG is i n c u b a t e d in the system. In a similar fashion, beads c o u p l e d to a goat a n t i - h u m a n IgM were able to d e m o n strate a p o p u l a t i o n o f i m m u n o g l o b u l i n - b e a r i n g B cells. O u r ability to observe these beads u n d e r the m i c r o s c o p e with or w i t h o u t f l u o r e s c e n c e has the a d d e d advantage o f p e r m i t t i n g the s i m u l t a n e o u s m e a s u r e m e n t o f a n u m b e r o f cell-surface receptors, either b y using d i f f e r e n t l y c o l o r e d dyes i n c o r p o r a t e d into beads with light m i c r o s c o p y or by simultan e o u s assessment o f d i f f e r e n t size beads relating to d i f f e r e n t receptors. The i n c o r p o r a t i o n o f m a g n e t i c particles into p o l y g l u t a r a l d e h y d e beads also raises the future possibility o f being able to r e m o v e magnetically particular cell s u b p o p u l a t i o n s by the a d h e r e n c e o f magnetic m i c r o b e a d s {conjugated to a n t i b o d y or antigen) to particular cell-surface receptors. F u t u r e applications, t h e r e f o r e , m a y lie n o t o n l y in a m o r e rapid and efficient t e c h n i q u e for m e a s u r e m e n t o f cell types, b u t "also in providing novel a p p r o a c h e s t o w a r d s the m a n i p u l a t i o n or removal o f individual cell p o p u l a t i o n s . ACKNOWLEDGEMENTS We wish to t h a n k Z. T i m o r for assistance in labeling o f h u m a n l y m p h o cytes. This investigation was s u p p o r t e d by G r a n t No 1 R 0 1 - C A 2 0 6 6 8 - 0 1 a w a r d e d by National C a n c e r I n s t i t u t e , DHEW. REFERENCES Brown, G. and M.F. Greaves, 1975, Eur. d. Immunol. 4, 302. Gillet, R. and K. Gull, 1972, Histochemie 30, 167. Gordon, I.L., W.L. Dreyer, S.P.S. Yen and A. Rembaum, 1977, Cell. Immunol. 28,307. tlardy, P.M., A.C. Nichols and H.N. Rydon, 1969, Chem. Comm. 565 and 1976, JCS Perkin I 958. Hopwood, D., 1972, Histochem. J. 4,267. Horwitz, D.-A. and P.I. Loho, 1975, J. Clin. Invest. 56, 1464. Inman, J.K. and H.M. Dintzis, 1969, Biochemistry 8, 4074. Jencks, W.P., 1969, Catalysis in Chemistry and Enzymolo~y (McGraw-Hill, New York). Jones, G.J., 1974, J. Histochem. Cytochcm. 22,911. Korn, A.H., S.H. Fearheller and E.M. Filanchione, 1972, J. Mol. Biol. 65,525. Levy, H.B. and H.A. Sober, 1960, Proc. Soc. Exp. Biol. Med. 130,250. Mensan, P., G. Puzo and H. Mazarguil, 1975, Biochemie 57, 1281. Molday, R.S., W.J. Dreyer, A. Rembaum and S.P.S. Yen, 197.1, Nature 249, 81. Rasmussen, K.E. and d. Albrechtsen, 1974, Histochemistry 38, 19. Rembaum, A., S.P.S. Yen, E. Cheong, S. Wallace, R.S. Molday, I.L. Gordon and W.J. Dreyer, 1976, Macromolecules 9,328. Rembaum, A., S.P.S. Yen and W. Volksen, 1978, Chem. Tech. March, 182. Roth, G., 1977, J. Immunol. Methods 18, 1.
239
P O L Y G L U T A R A L D E H Y D E : A NEW R E A G E N T F O R C O U P L I N G PROTEINS TO MICROSPHERES AND FOR LABELING CELL-SURFACE RECEPTORS
A. REMBAUM, S. MARGEL * and J. LEVY ** Jet Propulsion Laboratory, California lnstitute of Technology, Pasadena, CA 91103, U.S.A. (Received 18 May 1978, accepted 30 May 1978)
Glutaraldehyde polymerized in basic aqueous solutions was found to react with low molecular weight amines, immunoglobulins and hemoglobin. The polyglutaraldehyde was covalently bound to hydrophilic microspheres. The rate of addition of proteins to the polyglutaraldehyde-derivatized microspheres was investigated spectrophotometrieally as a function of pH and temperature. The reaction of polyglutaraldehyde was found to be faster than that of the monomer. The findings led to successful labeling of human lymphocyte subpopulations. INTRODUCTION A m o s t i m p o r t a n t area in which m i c r o s p h e r e s (beads)-conjugates and o t h e r particles have f o u n d a p p l i c a t i o n is in the d e t e c t i o n o f cell-surface r e c e p t o r s b y light and scanning e l e c t r o n m i c r o s c o p y ( R o t h , 1977). The cell t y p e t h a t has been m o s t extensively s t u d i e d with b o t h f l u o r e s c e n t and nonf l u o r e s c e n t markers is the l y m p h o c y t e {Brown and Greaves, 1975). This m e t h o d o l o g y has been i n s t r u m e n t a l to bringing us to the c u r r e n t state o f a p p r e c i a t i n g multiple s u b p o p u l a t i o n s a m o n g peripheral b l o o d l y m p h o c y t e s . T h e r e c e p t o r s w h o s e d e t e c t i o n has m a d e this d i f f e r e n t i a t i o n into s u b p o p u l a tions possible include surface i m m u n o g l o b u l i n , Fc receptors, C' r e c e p t o r s and r e c e p t o r s for sheep red b l o o d cells. It was r e c e n t l y established t h a t i m m u n o g l o b u l i n s c o u l d be m o r e efficiently b o u n d to beads b y m e a n s o f g l u t a r a l d e h y d e , t h a n with o t h e r reagents ( M o l d a y et al., 1977). It was also s h o w n t h a t b e a d s - i m m u n o g l o b u l i n conjugates c o n s t i t u t e s a t i s f a c t o r y markers o f cell m e m b r a n e r e c e p t o r s for light m i c r o s c o p y ( G o r d o n et al., 1977). T h e p r e s e n t p a p e r describes the use o f p o l y g l u t a r a l d e h y d e for the same p u r p o s e . L o w m o l e c u l a r weight amines, f l u o r e s c e n t i m m u n o g l o b u l i n s and h e m o g l o b i n were used to investigate the reactivity o f p o l y g l u t a r a l d e h y d e , and it was f o u n d t h a t b e a d s - h u m a n IgG * Research Fellow at JPL from Weizmann Institute (Rehovot, Israel). ** UCLA School of Medicine, Los Angeles, CA 90024, U.S.A.
2.10 conjugates prepared by means of polyglutaraldehyde successfully labeled Fc receptors of human l y m p h o c y t e s . Glutaraldehyde in m o n o m e r form is used extensively for the tanning of leather, fixation of living cells, immobilization of enzymes, etc. ( t t o p w o o d , 1972). The reaction mechanism of proteins with glutaraldehyde is difficult to ascertain since its structure is still not clear and it has been reported to be in equilibrium with cyclic and hydrated forms (Itardy et al., 1969; Korn et al., 1972). Aqueous solutions of glutaraldehyde contain variable amounts of polyglutaraldehyde, a p o l y m e r which is also formed during monoglutaraldehyde reaction with proteins. The e x t e n t of p o l y m e r formation during these reactions varies considerably because the rate of polymerization is d e p e n d e n t on m o n o m e r concentration, pH, and temperature (Gillet and Gull, 1972; Jones, 1974; Rasmussen and Albrechtsen, 1974). Because of the large number o f applications of glutaraldehyde to biological systems, there is a need to examine the reactivity of the glutaraldehyde p o l y m e r with proteins and to compare it with that of the m o n o m e r . We have therefore carried out a preliminary study o f the polymerization kinetics, the structure and the solubility o f the polymer. These results will be reported separately. In the present paper, the reactivity of a water-insoluble polyglutaraldehyde fraction with immunoglobulins and hemoglobin will be discussed. Most of the reactions were carried out heterogeneously with polyglutaraldehyde suspended in water or with polyglutaraldehyde bound to the surface of polymeric microspheres (beads). In contrast to glutaraldehyde, the p o l y m e r contains conjugated aldehyde groups which impart stability to the Schiff bases formed after reaction with proteins (Mensan et al., 1975). In addition, the hydrophilic polyglutaraldehyde has relatively long chains extending from the microspheres into the surrounding aqueous medium, facilitating the heterogeneous reaction with proteins, e.g., immunoglobulins. The described experiments support these theoretical concepts. MATERIALS AND METHODS
Polymerization Commercial aqueous solutions of glutaraldehyde were purified by means o f activated carbon and were shown to be free of p o l y m e r by absence of absorption at 235 nm (Cary 14 s p e c t r o p h o t o m e t e r ) . A 25% solution of glutaraldehyde was polymerized at 22°C by adjusting the pll to 11.5 with NaOH. At the end of 2 h, the mixture was stirred for 8 h at 50°C. The precipitated p o l y m e r was filtered, washed with water and then with acetone and dried in vacuum for 24 h (yield, 60%). Assuming the repeating unit to be --CII.,--CH,--CH=~--CttO the molal extinction coefficient e2.~ measured in ethanol was found to be equal to 1.53 x l 0 s l/mole/era. The molar extinction coefficient of glutaraldehyde at 280 nm, e2~s (extracted from aqueous
241 solutions with ether and dried with magnesium perchlorate) was found to be equal to 4.2 1/mole/cm.
Syn thesis o f rn icrospheres Poly (4-vinylpyridine) acrylamide (PVP) and poly (2-hydroxy ethylmethacrylate) (Polyhema) mierospheres were synthesized by copolymerization of the monomers in presence of bisaerylamide as previously described (Rembaum et al., 1976, 1978). Microspheres were prepared with a relatively uniform distribution of sizes (S.D. 5--10%) varying in diameter from 0.05 to 10 ~m. In the present work PVP microspheres of 0.7 ~m in diameter were coupled with hydrazine to yield hydrazide groups on the surface (Inman and Dintzis, 1969). Reactions o f polyglutaraldehyde with amines The reactivity of polyglutaraldehyde with low molecular weight amines was investigated in a one-phase (homogeneous) and two-phase (heterogeneous) system. Homogeneous reactions were carried out at relatively low temperature, for example, polyglutaraldehyde was treated with methylamine and liquid anhydrous ammonia at about --40°C for I h. After raising the temperature and evaporation to dryness, the products were washed with water, dried in vacuo at 60°C, analyzed spectrophotometrically and their nitrogen content was determined. The polymer was also soluble at room temperature in higher boiling amines, e.g., allylamine, butylamine, ethanolamine, hydrazine and ethylene diamine. Its reaction with these amines was carried out at 22°C for 1 h and analyzed in the same way as in the case of the low temperature reactions. The heterogeneous reaction was carried out by suspending the polymer (120 rag) in phosphate-buffered saline (PBS) solution (20 ml) of the amine (1 g) at pH of 7.3--7.4 and stirring in a closed container for at least 30 h. After washing with water and drying, the nitrogen content of the products was determined {Table 1). Reactions with human immunoglobulins Fluorescent human IgG and goat anti-human IgM (Meloy) were purified on DEAE cellulose columns (Levy and Sober, 1960). Their fluorescence intensity at different pHs was measured at room temperature by means of an Aminco Fluorimeter model SPF125. The extent of reaction was determined by measuring the fluorescence of the supernatant after centrifugation of suspensions containing fluorescent IgG and insoluble polyglutaraldehyde. Two series of experiments were performed. In the first series, powdered polyglutaraldehyde was coupled directly with fluorescent IgG and the decrease of fluorescence measured after reaction yielded the a m o u n t of IgG bound to the polymer. Polyhema microspheres were treated under identical conditions and served as control. The second series of experiments consisted of reactions with polyglutaral-
242 TABLE l NITROGEN ANALYSIS OF REACTION PRODUCTS OF POLYGLUTARALDEHYDE WITtt AMINES Amine
Homogeneous reaction
Iteterogeneous reaction
% N found
% of t h e theor, amt.
% N found
% of t h e theol', a m t .
5.6 14.0 2.9 5.6 6.5 5.0 4.6
38.1 47.4 16.8 48.6 63.1 44.6 31.9
0.6 0.5 7.5 0.9
5.3 3.5 52.1 8.9
.
Methylamine Hydrazine Ammonia Allylamine Butylamine Ethanolamine a Dia m i n o h e p t a n e Hydroxylamine b Glycine
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
a Average of t w o d e t e r m i n a t i o n s . b H y d r o x y l a m i n e h y d r o c h l o r i d e was used at pH 7.3 and 2.4. T h e n i t r o g e n c o n t e n t was 7.5 a n d 6.6% respectively.
dehyde covalently bound to hydrazide groups on the surface of beads 0.7 pm in diameter. The chemical binding was carried out in the following way: PVP hydrazide beads (25 mg) were stirred with polyglutaraldehyde (25 mg) dissolved in dimethylsulfoxide (DMSO, 50 ml) for not less than 5 h. After centrifugation and washing with DMSO and water the beads (5 ml) were then stirred with glycine (80 mg) for 4 h. After washing, fluorescent immunoglobulins were added (Tables 2 and 3). This mixture was stirred at a controlled temperature
TABLE 2 REACTION OF POWDERED POLYGLUTARALDEHYDE of f l u o r e s c e n t IgG in 10 ml o f p h o s p h a t e b u f f e r , pH 7.4) Polyglutaraldehyde
Polyhema
(mg/ml)
(mg/ml)
0.2 2.0 20.0
---0.2 2.0 20.0
WITH H U M A N IgG (l m g
Bound IgG/polymer (w/w) pH 7.4
pH 6.0
0.160 0.030 0.004 0 0 0
0.200 0.040 0.006 0 0 0
243 TABLE 3 COMPARISON OF THE REACTIVITY OF GLUTARALDEHYDE AND POLYGLUTARALDEHYDE-DERIVATIZED BEADS Effect of mono- and polyglutaraldehyde concentration (0.5 mg of fluorescent IgG in 10 ml of phosphate buffer, pH 7.4). Control a (mg/ml) .
.
.
.
.
Polyglutaraldehyde a (mg/ml) .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Glutaraldehyde a (mg/ml) .
.
.
.
.
.
.
.
.
.
.
.
Bound IgG/polymer (w/w × 103)
.
2.5 7.5 20.0 2.5 7.5 20.0 2.5
l:l .0 4.0 1.4 6.0 2.0 0.7 0
a 25 mg/ml of beads used in all experiments.
and the loss o f f l u o r e s c e n c e with t i m e was d e t e r m i n e d using c e n t r i f u g e d aliquots.
Reaction with hemoglobin P o l y g l u t a r a l d e h y d e or P V P - p o l y g l u t a r a l d e h y d e m i c r o s p h e r e s w e r e susp e n d e d in h e m o g l o b i n (2 X crystallized, f r o m C a l b i o c h e m ) dissolved in 0.01 M p h o s p h a t e b u f f e r . T h e p H was a d j u s t e d with s o d i u m p h o s p h a t e and t h e o p t i c a l d e n s i t y at 4 1 2 n m o f c e n t r i f u g e d aliquots was r e c o r d e d with time.
Application of polyglutaraldehyde conjugated beads H u m a n l y m p h o c y t e s w e r e s e p a r a t e d f r o m red b l o o d cells b y m e a n s o f a F i c o l l - H y p a q u e g r a d i e n t freed f r o m m o n o c y t e s b y m e a n s o f iron particles ( H o r w i t z and L o b o , 1 9 7 5 ) and s u s p e n d e d in a m e d i u m at pH 7 . 2 - - 7 . 4 to which 20% o f fetal calf s e r u m (FCS) was a d d e d . T h e m e d i u m ( R P M I / H ) consisted of RPMI 1640 (Gibco) + 0.025 M Hepes (N-2-hydroxethylp i p e r a z i n e - N - 2 - e t h a n e sulfonic acid f r o m C a l b i o c h e m ) . M i c r o s p h e r e s to which h u m a n IgG was b o u n d b y m e a n s o f p o l y g l u t a r a l d e h y d e (0.5 ml o f m i c r o s p h e r e s u s p e n s i o n c o n t a i n i n g 0.5 m g o f m i c r o s p h e r e s in PBS b u f f e r at p H 7.4) w e r e i n c u b a t e d f o r 1 h at 4°C with 5 X 106 l y m p h o c y t e s in R P M I / H b u f f e r (1 ml) c o n t a i n i n g 0.1% s o d i u m azide. A f t e r a d d i t i o n o f FCS (1.5 m l ) + 0.1% s o d i u m azide, the s u s p e n s i o n was c e n t r i f u g e d at 1 5 0 0 r e v / m i n ( 5 1 5 X g ) . T h e r e c o v e r e d cells were r e s u s p e n d e d in 50% R P M I / H and 50% FCS c o n t a i n i n g 0.1% s o d i u m azide and r e c e n t r i f u g e d . It was f o u n d n e c e s s a r y to r e p e a t this washing p r o c e d u r e 3 t i m e s in o r d e r to r e m o v e p r a c t i c a l l y all u n b o u n d m i c r o s p h e r e s . A f t e r t h e third c e n t r i f u g a t i o n the cells were r e s u s p e n d e d in R P M I / H c o n t a i n i n g 1% b o v i n e s e r u m a l b u m i n
2 44 and 0.1% s o d i u m azide. T h e labeled cells were t h e n c o u n t e d u n d e r the microscope. In the c o n t r o l e x p e r i m e n t s t h e l y m p h o c y t e s were i n c u b a t e d with aggregated IgG or u n c o a t e d b e a d s for 1 h. T h e aggregated IgG was p r e p a r e d b y dissolving h u m a n IgG (50 m g / m l ) in PBS at pH 7.4 and h e a t i n g t h e solution at 63°C for 30 min. T h e colloidal s u p e r n a t a n t l a y e r was used for cell i n c u b a t i o n p r i o r to labeling. M i c r o s p h e r e s c o u p l e d to goat a n t i - h u m a n IgM were also used to test the r e a c t i v i t y o f p o l y g l u t a r a l d e h y d e . RESULTS
Reaction of polyglutaraldehyde with low molecular weight amines P r e l i m i n a r y i n f o r m a t i o n was o b t a i n e d on the reactivity o f the p o l y m e r with a m i n o g r o u p s b y c a r r y i n g o u t the r e a c t i o n with a series of amines. T a b l e 1 s h o w s the results o f e l e m e n t a l analysis o f the p r o d u c t s . T h e t h e o r e t ical a m o u n t o f n i t r o g e n was c a l c u l a t e d on t h e a s s u m p t i o n t h a t each unit; - C H 2 - - C H 2 C H = ~ - - C H O will b i n d o n e m o l e c u l e o f amine. In the case o f hyd r o x y l a m i n e h y d r o c h l o r i d e , h y d r a z i n e a n d b u t y l a m i n e the e x t e n t o f reaction was o f t h e o r d e r o f 5 0 - - 6 0 % o f the t h e o r e t i c a l a m o u n t . S p e c t r o p h o t o m e t r i c e x a m i n a t i o n revealed t h a t in m o s t p r o d u c t s t h e a b s o r p t i o n at 285 n m , a t t r i b u t e d to the a l d e h y d e g r o u p , was c o n s i d e r a b l y reduced.
Reaction of polyglutaraldehyde and polyglutaraldehyde-derivatized microspheres with immunoglobulins T a b l e 2 s h o w s the a m o u n t o f IgG b o u n d to p o l y g l u t a r a l d e h y d e a f t e r stirring the s u s p e n s i o n s o f the p o l y m e r for 2.25 h at r o o m t e m p e r a t u r e . A c o n s t a n t a m o u n t o f f l u o r e s c e n t IgG was used in these e x p e r i m e n t s . It is o b v i o u s t h a t increased a m o u n t s o f p o l y m e r resulted in a l o w e r I g G / p o l y m e r
TABLE ,1 COMPARISON OF THE REACTIVITY OF GLUTARALDEHYDE AND POLYGLUTARALDEHYDE-DERIVATIZED BEADS (effect of IgG concentration) Control a (mg/ml)
Polyglutaraldehyde a (mg/ml)
Human IgG (mg/ml)
Glutaraldehyde (mg/ml) .
2.5 2.5 2.5 2.5 2.5 2.5 2.5
.
.
.
.
.
.
.
.
.
.
Bound IgG polymer (W/W X 103 ) .
.
.
.
.
0.025 0.050 0.100
5.7 9.5 14.0
0.025 O.050 0.100
5.3 4.5 9.1 0
.
.
.
.
.
.
.
.
245
L Z
Z w
z_ Z u O D
'°°o
~®
~o
3~o
I
,00
5®
TIME (MIN)
Fig. 1. Rate of reaction of polyglutaraldehyde (60 mR) with human IgG (4.5 mR) in PBS (50 ml) at 22°C. e, pH 6;~", pH 7.4.
ratio. The same result was obtained with microspheres derivatized with either the polymer or m o n o m e r (Table 3). Table 2 indicates that larger amounts of IgG are bound to the polymer at pH 6 than at pH 7.4. This pH effect was confirmed in subsequent studies. It is also apparent that somewhat larger amounts of IgG become bound to polyglutaraldehyde than to the polyglutaraldehyde
20 |
"~
I
l
'
'
[
[ ~
0
l
10
~2o
40
4O
•
-
~6o ~
I00
200
300 TIME (MIN)
400
500
600
_ _ - -
_-..-~
70 8O
~
I
I
3o0
6oo
1 90o
I 1200
I L~oo
! 18~
TiME (MIN)
Fig. 2. Rate of reaction of PVP beads with fluorescent human IRG at 22°C and pH 7.4. $, polyglutaraldehyde bound to PVP hydrazide beads; :3, monoglutaraldehyde bound to PVP hydrazide beads; ×, PVP hydrazide beads (control);/~, IgG in PBS (control).
FiR. 3. Rate of reaction of PVP beads with fluorescent human IgG at 4°C. e, polyglutaraldehyde bound to PVP hydrazide beads; c., monoglutaraldehyde bound to PVP hydrazide beads; x, PVP hydrazide beads (control);/,,, IgG in PBS (control).
246 2C
I
~
!
---
I
:
30 Z >-
40
~
50
60
z ~ 7o z u m
80
© -~
90 ~ 100
10
20
30
~
40 50 T~ME (HOURS)
×-
60
70
80
90
Fig. 4. Rate of reaction of PVP beads with fluorescent goat anti-human IgM at 4°C and pH 7.4. '.% polyglutaraldehyde bound to PVP beads; r,, monoglutaraldehyde bound to PVP beads; x, PVP hydrazide beads (control). than by the m o n o m e r . I n f o r m a t i o n on the necessary reaction time was obtained through kinetic experiments. In Fig. 1, the decrease of fluorescence of the supernatant is pl ot t e d against time. It is obvious that at pH 7.4 the reaction is incomplete after 2.25 h (the time used in previous experiments). F u r t h e r m o r e , at pH lower than 7.4 the reaction is faster. Figs. 2 and 3 confirm the faster reaction of the p o l y m e r over the m o n o m e r and also show the effect o f temperature. Similar results were obtained with fluorescent goat anti-human IgM (Fig. 4).
Reactions with hemoglobin Because hemoglobin has a high extinction coefficient at 412 n m ( 6 r a M " 125 based on a molecular weight o f 17,000), it is a convenient model for our studies o f protein immobilization. It can be calculated from the 412 nm absorption peak at pH 6.0, 7.4 and 8.4 that the polyglutaraldehyde immobilized 8.8, 6.6 and 2.8% of hemoglobin respectively after 8.3 h of reaction time. In control experiments (Polyhema) the hemoglobin c o n t e n t was of the order of 1% based on nitrogen analysis (%N in the hemoglobin used was f o u n d to be 15.2%). The hemoglobin c o n t e n t of the polyglutaraldehydehemoglobin products obtained at pH 6.0, 7.4 and 8.4 was calculated to be 10.4, 7.8 and 5.3% respectively (based on nitrogen analysis) and agrees reasonably well with the s p e c t r o p h o t o m e t r i c results (Fig. 5). In contrast to the heterogeneous reaction, when polyglutaraldehyde reacted with an excess of hemoglobin h o m ogeneous l y in aqueous DMSO the product was found to contain 76% o f hemoglobin. T he kinetics of hemoglobin binding to polyglutaraldehyde-derivatized microspheres are shown in Fig. 6. The trend of increasing reaction rate a t p H 6 as compared with pH 7.4 is evident.
247
0.1
- -
0.2
OZ 0.4 0.5
o.e Z
0.7
~
0.8
/I
~ .------'v-'---~" ~ ~
~'
"~ I
0.9 1.0
1130
200 300 TIME ( M I N )
400
500
Fig. 5. Rate of reaction at 22°C of polyglutaraldehyde ( 1 0 0 m g in 5 0 m l of FBS) with hemoglobin (7.5 rag) as a function of pH. c'., pH 6; /', pH 7.4; X, pH 8.4; e, Polyhema beads, pH 6 (control).
i
0 2:
' '
' : ' ' 'o i
.J 0.3 0 Z O. ~ •~ >-
0.5
•
0.6
o.7 0.8
0.9 1.0
200
400
600
800
I000
1200
1400
16(X)
1800
!000
TIME (MIN)
Fig. 6. Rate of reaction of PVP beads with hemoglobin at pH 6 and 22°C. o, derivatized with polyglutaraldehyde ; ~, derivatized with monoglutaraldehyde; $, Polyhema (control).
248 TABLE
5
LABELING OF IIUMAN LYMPttOCYTES WITH MICROSPHERE-IgG CONJUGATES Experiment
Type of beads
Antibody (mg)
Number of heads ( x 1 0 ' l {))
% Labeled cells
1
IgG conjugates lgG conjugates Unreacted beads (control
0.18 0.29
1.25 2.5 2.5
13.6 20.7 6.7
2
IgG conjugates Unreaeted heads (control ARgregated IgG (control) IgG eonjmlates Unreacted beads (control
l 7.9
3
4
IgG conjugates Unreacted beads (control)
0.,15
2.5
--
2.5
,1.6
0.,15 --
2.5 2.5 2.5
0 17.2 2.9
0.19
2.5
1.9
"-
8.82 0
Labeling of Fc receptors P u r i f i e d l y m p h o c y t e s (5 ~' 106), as d e s c r i b e d in t h e e x p e r i m e n t a l p a r t , w e r e i n c u b a t e d w i t h I g G - b e a d c o n j u g a t e s . T h e r e s u l t s a r e s h o w n in T a b l e 5. T h e n o n - s p e c i f i c a l l y l a b e l e d c e l l s in c o n t r o l e x p e r i m e n t s ( e x p . 1 a n d 2) h a d a m a x i m u m o f o n l y 4 b e a d s o n t h e cell s u r f a c e . T h e a p p e a r a n c e o f s p e c i f i c a l l y l a b e l e d cells is s h o w n ill Fig. 7a. CONCI,USIONS AND DISCUSSION The examination of the results leads to the following conclusions: (1) P o l y g l u t a r a l d e h y d e e x h i b i t s a h i g h r e a c t i v i t y t o w a r d s l o w m o l e c u l a r
Fig. 7. a: SEM photomicrograph of a lymphocyte labeled with beads with human IgG bound to the surface, h: control from exp. 3 (see Table 5).
249 weight amines under mild experimental conditions (Table 1). The remarkable reactivity of hydroxylamine indicates that it may constitute a superior reagent to glycine which was used in our experiments for the inactivation of aldehyde groups on glutaraldehyde or polyglutaraldehyde-activated reagents for cell labeling. The highest theoretical yields of reaction products, based on the assumption that the repeating unit in the polymer is a substituted acrolein moiety, amounted to about 50--60%. Spectrophotometric studies showed presence of unreacted aldehyde functions in the reaction products. Therefore it is likely that Schiff base formation reached an equilibrium stage, and by changing the experimental conditions higher yields than those recorded in Table 1 may be expected. (2) The presence of acrolein structure in the polymer was confirmed by the reaction of the latter with rn-aminophenol which yielded a highly fluorescent polymer with fluorescence emission maximum at 470 nm. (3) The reactivity of the polymer with human IgG is about twice that of the m o n o m e r {Tables 2, 3, 4 and Figs. 2, 3, 4 and 6). The number of IgG molecules bound per bead (0.7 pm in diameter) after 10 h of reaction at 22°C (Fig. 2) was calculated to be equal to 3.8 × 103 and 2.3 r 103 for the polyglutaraldehyde and monoglutaraldehydc procedures, respectively. Larger yields and higher reaction rates were observed in the reactions of immunoglobulins with polyglutaraldehyde than with microspheres (beads) derivatized with polyglutaraldehyde (compare Tables 2, 3 and 4 and Figs. 1 and 2). The same conclusions apply to the reactions with hemoglobin {Figs. 5 and 6). (4) The lowering of pH from 7.4 to 6 enhances the reaction rate. This phenomenon applies to human IgG (Fig. 1) as well as to hemoglobin {Fig. 5). To interpret this effect we assume that the reaction of polyglutaraldehyde takes place between free amino groups of the protein, e.g., lysine residues, and aldehyde groups of the polymer to form Schiff bases. It might be expected that carbonyl addition reactions would be powerfully acidcatalyzed for, after a proton attack on oxygen, the carbon atom will become positively charged and hence readier to react with a nucleophile: C = O + H ÷ ~ C*---OH. When the nucleophile is R-NH2, an acid converts the amine to the unreactive species R-NH3. We should thus expect to find that the rate of addition will show a maximum at a moderately acid pH falling off sharply at each side. This is indeed observed in practice (Jencks, 1969). (5) The small extent of non-specific binding of immunoglobulins and hemoglobin (Figs. 2 and 5) to substrates free of glutaraldehyde and polyglutaraldehyde is attributed to physical adsorption and can be reduced by lowering the reaction temperature (Fig. 3). (6) The high reactivity of polyglutaraldehyde, its stability, ease of administration, and the retention of physiolo~,dcal activity of human immunoglobulins bound to the polymer make it a desirable new reagent for protein binding. The experimental results demonstrate the successful application of polyglutaraldehyde beads for quantitation of human l y m p h o c y t e subpopulations.
250 'Fable 5 d e m o n s t r a t e s the ability o f beads c o a t e d with h u m a n IgG to bind to the Fc r e c e p t o r on B and null cells with the a p p r o p r i a t e c o n t r o l o f inhibition o f this binding when free aggregated IgG is i n c u b a t e d in the system. In a similar fashion, beads c o u p l e d to a goat a n t i - h u m a n IgM were able to d e m o n strate a p o p u l a t i o n o f i m m u n o g l o b u l i n - b e a r i n g B cells. O u r ability to observe these beads u n d e r the m i c r o s c o p e with or w i t h o u t f l u o r e s c e n c e has the a d d e d advantage o f p e r m i t t i n g the s i m u l t a n e o u s m e a s u r e m e n t o f a n u m b e r o f cell-surface receptors, either b y using d i f f e r e n t l y c o l o r e d dyes i n c o r p o r a t e d into beads with light m i c r o s c o p y or by simultan e o u s assessment o f d i f f e r e n t size beads relating to d i f f e r e n t receptors. The i n c o r p o r a t i o n o f m a g n e t i c particles into p o l y g l u t a r a l d e h y d e beads also raises the future possibility o f being able to r e m o v e magnetically particular cell s u b p o p u l a t i o n s by the a d h e r e n c e o f magnetic m i c r o b e a d s {conjugated to a n t i b o d y or antigen) to particular cell-surface receptors. F u t u r e applications, t h e r e f o r e , m a y lie n o t o n l y in a m o r e rapid and efficient t e c h n i q u e for m e a s u r e m e n t o f cell types, b u t "also in providing novel a p p r o a c h e s t o w a r d s the m a n i p u l a t i o n or removal o f individual cell p o p u l a t i o n s . ACKNOWLEDGEMENTS We wish to t h a n k Z. T i m o r for assistance in labeling o f h u m a n l y m p h o cytes. This investigation was s u p p o r t e d by G r a n t No 1 R 0 1 - C A 2 0 6 6 8 - 0 1 a w a r d e d by National C a n c e r I n s t i t u t e , DHEW. REFERENCES Brown, G. and M.F. Greaves, 1975, Eur. d. Immunol. 4, 302. Gillet, R. and K. Gull, 1972, Histochemie 30, 167. Gordon, I.L., W.L. Dreyer, S.P.S. Yen and A. Rembaum, 1977, Cell. Immunol. 28,307. tlardy, P.M., A.C. Nichols and H.N. Rydon, 1969, Chem. Comm. 565 and 1976, JCS Perkin I 958. Hopwood, D., 1972, Histochem. J. 4,267. Horwitz, D.-A. and P.I. Loho, 1975, J. Clin. Invest. 56, 1464. Inman, J.K. and H.M. Dintzis, 1969, Biochemistry 8, 4074. Jencks, W.P., 1969, Catalysis in Chemistry and Enzymolo~y (McGraw-Hill, New York). Jones, G.J., 1974, J. Histochem. Cytochcm. 22,911. Korn, A.H., S.H. Fearheller and E.M. Filanchione, 1972, J. Mol. Biol. 65,525. Levy, H.B. and H.A. Sober, 1960, Proc. Soc. Exp. Biol. Med. 130,250. Mensan, P., G. Puzo and H. Mazarguil, 1975, Biochemie 57, 1281. Molday, R.S., W.J. Dreyer, A. Rembaum and S.P.S. Yen, 197.1, Nature 249, 81. Rasmussen, K.E. and d. Albrechtsen, 1974, Histochemistry 38, 19. Rembaum, A., S.P.S. Yen, E. Cheong, S. Wallace, R.S. Molday, I.L. Gordon and W.J. Dreyer, 1976, Macromolecules 9,328. Rembaum, A., S.P.S. Yen and W. Volksen, 1978, Chem. Tech. March, 182. Roth, G., 1977, J. Immunol. Methods 18, 1.