Elsevier Biomedical Press
A N T I G E N - S P E C I F I C E L E C T R O P H O R E T I C C E L L S E P A R A T I O N ( A S E C S ) : I S O L A T I O N O F H U M A N T A N D B L Y M P H O C Y T E
S U B P O P U L A T I O N S B Y F R E E - F L O W E L E C T R O P H O R E S I S A F T E R R E A C T I O N W I T H A N T I B O D I E S
ERNIL HANSEN and KURT HANNIG
M a x - P l a n c k - I n s t i t u t f i i r B i o c h e m i e , A m K l o p f e r s p i t z , D - 8 0 3 3 M a r t i n s r i e d , F . R . G . (Received 21 October 1981, accepted 2 December 1981)
The electrophoretic mobility of human l y m p h o c y t e s can be reduced by incubation with surface antigen specific antibodies under non-capping conditions. This renders sub- populations of human peripheral blood l y m p h o c y t e s accessible to separation by free-flow electrophoresis.
After reaction of l y m p h o c y t e preparations with anti-IgM a n t i b o d y and a fluorescent second a n t i b o d y , B l y m p h o c y t e s showed a considerable shift in position in preparative cell electrophoresis and could be separated with high yield, purity and vitality. Similarly, a T cell s u b p o p u l a t i o n reactive with the monoclonal a n t i b o d y T811 could be isolated, even though only small amounts of this a n t i b o d y were bound, by using a double-sandwich method.
Non-specific a n t i b o d y uptake via Fc-receptors did not contribute to the observed shift of antibody-labelled cells to lower electrophoretic mobility. Flow c y t o m e t r i c analysis showed that cells were separated according to their antigen density.
Thus cell electrophoresis can be used to separate antibody-labelled cells. With a flow rate of 100,000 cells/sec this m e t h o d has a much higher separation capacity than fluores- cence-activated cell sorting. The described m e t h o d should be applicable to the separation of a wide range of cell populations for which specific antibodies are available.
Key words: h u m a n l y m p h o c y t e s - - f r e e - f l o w e l e c t r o p h o r e s i s - - c e l l s e p a r a t i o n - - m o n o - c l o n a l a n t i b o d y - - f l o w c y t o m e t r y
INTRODUCTION
F r e e - f l o w e l e c t r o p h o r e s i s h a s p r o v e n t o b e a p o w e r f u l m e t h o d f o r t h e s e p a r a t i o n o f T a n d B c e l l s , a n d o f l y m p h o c y t e s i n v a r i o u s s t a g e s o f d i f f e r e n - t i a t i o n a n d a c t i v a t i o n , f o r m o u s e a n d r a t ( Z e i l l e r e t a l . , 1 9 7 1 ; Z e i l l e r e t a l . , 1 9 7 6 ; f o r r e v i e w s e e : S h e r b e t , 1 9 7 8 ; P r e t l o w a n d P r e t l o w , 1 9 7 9 ) . A t t e m p t s t o s e p a r a t e h u m a n l y m p h o c y t e s , h o w e v e r , h a v e b e e n u n s a t i s f a c t o r y ( S t e i n e t al., 1 9 7 3 ; H / i y r y e t a l . , 1 9 7 5 ; A u l t e t al., 1 9 7 6 ; C h o l l e t e t al., 1 9 7 8 ) .
D i f f e r e n c e s i n t h e e l e c t r o p h o r e t i c m o b i l i t y o f T a n d B c e l l s f r o m h u m a n 0 0 2 2 - 1 7 5 9 / 8 2 / 0 0 0 0 - - 0 0 0 0 / $ 0 2 . 7 5 Q 1982 Elsevier Biomedical Press
peripheral blood have been described in a n u m b e r of analytical investiga- tions, b u t have been questioned b y other authors (for review see Pretlow and Pretlow, 1979). Even if differences in surface charge density do exist, they seem to be t o o small to allow separation of human T and B l y m p h o c y t e s by preparative cell electrophoresis.
The surface charge density of cells can be altered by reaction with anti- bodies. Changes in the electrophoretic mobility after incubation with appro- priate antisera have been observed for erythrocytes, t u m o u r cells and lym- p h o c y t e s in various analytical systems (reviewed by Sherbet, 1978).
We describe here the use of conventional or monoclonal antisera for effi- cient preparative separation of human l y m p h o c y t e subpopulations by free- flow electrophoresis.
MATERIALS AND METHODS Cells
1 0 0 - - 2 0 0 ml of h u m a n peripheral blood from healthy donors were defibri- nated b y means of glass beads. L y m p h o c y t e s were prepared b y Ficoll-Hypa- que density centrifugation (MSL, d = 1.077 kg/1, Mediapharm, Aschaffen- burg) as described by B 5 y u m (1968) and washed 3 times in Puck-G (Difco, Detroit, MI). A p p r o x i m a t e l y 1 X 106 l y m p h o c y t e s were recovered per 1 ml of blood. Contamination b y m o n o c y t e s was 7.3 + 2.3%, as determined by non-specific esterase staining according to Yam et al. (1970).
A n t i b o d y treatment
For immunofluorescence and for electrophoretic separation B lympho- cytes were stained with rabbit-anti-human-IgM antiserum (Behringwerke, Marburg), followed by incubation with FITC-labelled goat-anti-rabbit-Ig immunoglobulin (Behringwerke, Marburg). In another set of experiments monoclonal a n t i b o d y T811, which reacts with a subpopulation of human T l y m p h o c y t e s (Rieber et al., 1981), was used. Treatment with the monoclonal a n t i b o d y was followed b y incubation with TRITC-labelled rabbit-anti-mouse- Ig immunoglobulin and TRITC-labelled goat-anti-rabbit-Ig immunoglobulin (Nordic I m m u n o l o g y , Tilburg).
Before use all antisera were centrifuged at 1 5 , 0 0 0 X g for 30 min to remove protein aggregates. Antisera concentrations were adjusted to give an optimal u p t a k e of antibodies b y h u m a n l y m p h o c y t e s w i t h o u t significant non-specific binding or cell aggregation. The anti-IgM antiserum was used in a 1/4 dilution, and the monoclonal a n t i b o d y in a 1/4 dilution of the culture supernatant. The fluorescent second antibodies were adsorbed with 1/10 volume of human m o n o n u c l e a r cells isolated from b u f f y coat preparations by Ficoll-Hypaque centrifugation, and used in a 1/4 dilution. 50--100 X 106 cells were stained when labelling was p e r f o r m e d before cell electrophoresis, and 1 X 106 cells were stained in the fractions labelled after cell electrophore-
sis. T w e n t y microlitres o f the respective antiserum were used per 1 X 106 cells. I n c u b a t i o n s were f o r 30 min at 4°C and were followed by 3 washes in Puck-G m e d i u m . I m m u n o f l u o r e s c e n c e was evaluated with a Zeiss fluores- cence m i c r o s c o p e u n d e r incident light. By keeping the cells strictly at 3--6°C t h r o u g h o u t the e x p e r i m e n t s capping o f antibodies on the cell surface could be avoided, and it was n o t necessary t o add sodium azide t o the media.
In c o n t r o l e x p e r i m e n t s the anti-IgM antiserum was replaced by a rabbit- anti-Thy-1 antiserum, and t h e m o n o c l o n a l a n t i b o d y T811 replaced b y a non- relevant m o n o c l o n a l a n t i b o d y .
Cell electrophoresis
Free-flow electrophoresis according t o Hannig (1972) was p e r f o r m e d on an E l p h o r VAP5 apparatus (Bender and Hobein, Munich). Electrophoresis buffers and c o n d i t i o n s were those described earlier (Zeiller et al., 1975). The separation b u f f e r consisted o f 0.0187 mol/1 t r i e t h a n o l a m i n e , 0.276 mol/1 glycine and 5 mmol/1 potassium acetate, and was adjusted to pH 7.2 with acetic acid. T h e c o n d u c t i v i t y was 8.4--9.0 X 10 -2 S/m, and the o s m o l a r i t y was 0.30 osmol/1. A 3-fold m o r e c o n c e n t r a t e d solution ( w i t h o u t glycine) was used as electrode buffer. U n t r e a t e d or a n t i b o d y - t r e a t e d cells were washed once in a m i x t u r e o f electrophoresis b u f f e r and Puck-G and once in electro- phoresis b u f f e r alone. Cells were adjusted to a c o n c e n t r a t i o n o f 30--60 X 106 cells/ml and subjected to electrophoresis at a flow rate o f 5--6 ml/h. The b u f f e r flow rate was 550 ml/h, resulting in an exposition time in the electric field for each cell o f a b o u t 210 sec. Electrophoresis was p e r f o r m e d at 200 m A and 8 0 0 - - 1 1 0 0 V. The effective field strength (=measured field strength X 0.8) was 6 0 - - 8 6 V/cm. Fractions were collected in tubes containing 1 ml o f TC-medium Puck-G s u p p l e m e n t e d with 1% BSA ( A r m o u r Pharmaceutical, Phoenix, AZ).
Flow cytornetry
Cells were analysed for f l u o r e s c e n c e intensity and narrow-angle (2--10 ° ) light scatter intensity according to H e r z e n b e r g and Herzenberg (1978), using a flow c y t o m e t e r developed in o u r laboratories, which is c o m p a r a b l e t o com- mercially available fluorescence activated cell sorters (FACS).
RESULTS
T h e aim o f o u r studies was t o investigate the distribution o f a n t i b o d y - labelled cells in free-flow electrophoresis, and t o see t o what e x t e n t a n t i b o d y u p t a k e would allow preparative isolation o f the cells. H u m a n blood l y m p h o - cytes were incubated with specific antibodies and t h e n subjected t o cell electrophoresis. F l u o r e s c e n t second antibodies were used so t h a t the position o f antigen-positive cells could be traced easily b y i m m u n o f l u o r e s c e n c e m i c r o s c o p y . Each individual e x p e r i m e n t was p r e c e d e d by an electrophoresis o f t h e u n t r e a t e d l y m p h o c y t e s as a c o n t r o l .
Separation of IgM-positive cells
The first set o f experiments dealt with B cells, detected by a rabbit-anti- human-IgM antiserum and FITC-labelled goat-anti-rabbit-Ig immunoglobulin.
Results from a representative experiment out of 12 can be seen in Fig. 1.
Fig. 1A shows the electrophoretic distribution of human blood lymphocytes and o f IgM-positive cells. Only a slight difference in mean electrophoretic mobility (EPM) was observed between B cells and total lymphocytes. Some enrichment o f B cells was seen in fractions of l o w EPM. These, however, comprised only a minute proportion of the IgM-positive cells.
A dramatic change in the distribution profile was observed, when cells were incubated with antibody prior to cell electrophoresis (Fig. 1B). A pro- nounced shoulder appeared in the region of l o w EPM. This shoulder was shown by immunofluorescence microscopy to contain the IgM-positive lym- phocyte subpopulation. It was found to be shifted by about 5 fractions after antibody treatment, corresponding to a reduction of 15--20% in mean EPM.
Electrophoretic migration path [mm]
4?,3 39,71 34,0 28,3j
20 A nucl. cells
d g M ÷ cells
j / , ~ , ~ ~ .
//2
' \
/ \
3,5 3'7 3'9 4'1 4'3 4,5 4'7 49 51
Fraction number
Fig. 1. Effect of anti-lgM antiserum on the electrophoretic mobility of human B cells. A:
electrophoretic distribution of human blood lymphoeytes. B cells were detected by stain- ing with rabbit-anti-human-IgM and FITC-labelled goat-anti-rabbit-Ig antisera. B: electro- phoretic distribution o f antibody-treated cells. L y m p h o c y t e s were incubated with rabbit- anti-human-IgM and FITC-labelled goat-anti-rabbit-Ig antisera (at 4°C) before cell electro- phoresis. Effective field strength: 71 V / c m . The arrow marks the position o f formalde- hyde-fixed h u m a n erythrocytes.
, ~ t O
20
"6
m
\
• 1 0 -
z
TABLE 1
Enrichment of IgM-positive cells by free-flow electrophoresis. Data expressed as average
± S.D. of 12 experiments.
Nucleated cells IgM ÷ cells
Recovery (%) Purity (%) R e c o v e r y (%) d Lymphocyte preparation before
cell electrophoresis a
Sum of all fractions after electro- phoresis of lymphocytes Sum of all fractions after electro-
phoresis of antibody-treated lymphocytes b
Fractions pooled after electro- phoresis of antibody-treated lymphocytes (pool L) c
100 15.2±2.1 100
8 1 . 2 ± 5 . 0 14.8±1.7 79
6 5 . 5 ± 6 . 9 1 9 . 2 ± 3 . 8 82
7 . 1 ± 0 . 3 8 9 . 2 ± 3 . 7 41
a Lymphocytes were prepared from defibrinated human blood by Ficoll-Hypaque centri- fugation. Monocyte contamination was 7.2 -+ 2.1%.
b Lymphocytes were incubated with rabbit-anti-human-IgM and FITC-goat-anti-rabbit-Ig antisera before free-flow electrophoresis.
c Fractions were pooled in the region of low electrophoretic mobility so to contain half of the IgM-positive cells.
d Values were calculated from total cell recovery and percentage of IgM-positive cells.
IgM-positive cells in the original lymphocyte preparation was set as 100%.
T h u s , B cells c o u l d be i s o l a t e d in h i g h y i e l d a n d p u r i t y (Table 1). Frac- t i o n s were p r e s e n t in t h e r e g i o n o f l o w EPM t h a t c o n s i s t e d t o m o r e t h a n 95%
o f IgM-positive cells. A p o o l w h i c h c o n t a i n e d h a l f o f t h e a n t i g e n - p o s i t i v e cells ( p o o l L in T a b l e 1) was still a b o u t 89% p u r e . This p o o l L c o r r e s p o n d s t o f r a c t i o n s 4 7 - - 5 1 in t h e e x p e r i m e n t s h o w n in Fig. 1. Cell vitality, as mea- s u r e d b y t r y p a n - b l u e e x c l u s i o n , was g r e a t e r t h a n 90% in all f r a c t i o n s e x c e p t t h o s e o f h i g h e s t EPM, regardless o f w e t h e r o r n o t cells were t r e a t e d with a n t i b o d y b e f o r e cell e l e c t r o p h o r e s i s .
F o r u n t r e a t e d cells, m e a n cell loss o c c u r r e d d u r i n g t r a n s f e r o f cells t o t h e e l e c t r o p h o r e s i s b u f f e r o f l o w ionic s t r e n g t h . With a n t i b o d y - t r e a t e d cells, t o t a l cell r e c o v e r y was r e d u c e d , m a i n l y b e c a u s e o f t h e a d d i t i o n a l c e n t r i f u g a - t i o n steps. Surprisingly, r e c o v e r y o f IgM-positive cells was u n c h a n g e d at a b o u t 80%, posssibly as a result o f a h i g h e r resistance o f a n t i b o d y - c o a t e d cells t o e l e c t r o p h o r e s i s .
Influence of antibody uptake on electrophoretic mobility
B cells are h e t e r o g e n e o u s in t h e i r q u a n t i t a t i v e e x p r e s s i o n o f s u r f a c e Ig. We t h e r e f o r e i n v e s t i g a t e d t h e r e l a t i o n s h i p o f a n t i g e n d e n s i t y t o t h e s l o w i n g e f f e c t o f a n t i b o d i e s o n t h e EPM.
A
B
m
p!
T
c
u3
Fluorescence i n t e n s i t y - :
Fig. 2. F l o w c y t o m e t r i c analysis o f e l e c t r o p h o r e t i c a l l y s e p a r a t e d h u m a n b l o o d l y m p h o - c y t e s s t a i n e d w i t h r a b b i t - a n t i - h u m a n - I g M a n d F I T C - l a b e l l e d g o a t - a n t i - r a b b i t - I g a n t i s e r a . F r a c t i o n s were t a k e n f r o m t h e e l e c t r o p h o r e t i c s e p a r a t i o n s s h o w n in Fig. 1. A: f r a c t i o n s f r o m t h e e l e c t r o p h o r e s i s o f u n t r e a t e d h u m a n l y m p h o c y t e s . As a c o n t r o l for n o n - s p e c i f i c a n t i b o d y u p t a k e h u m a n l y m p h o c y t e s were i n c u b a t e d w i t h a r a b b i t - a n t i - r a t - b r a i n a n t i - s e r u m a n d F I T C - l a b e l l e d g o a t - a n t i - r a b b i t - I g i m m u n o g l o b u l i n . B: f r a c t i o n s f r o m t h e elec- t r o p h o r e s i s o f a n t i b o d y - t r e a t e d cells. E a c h d o t r e p r e s e n t s t h e values for a n i n d i v i d u a l cell o f n a r r o w - a n g l e l i g h t s c a t t e r (y-axis), as a m e a s u r e o f cell size, a n d o f f l u o r e s c e n c e (x-axis), as a m e a s u r e o f s u r f a c e a n t i g e n c o n t e n t .
Fractions o f the e x p e r i m e n t shown in Fig. 1, were analysed by flow c y t o m e t r y . Fig. 2 shows the 2-dimensional plots of narrow-angle light scatter signal, as a measure o f cell size, and fluorescence signal, as a measure o f anti- gen c o n t e n t , o f individual cells.
When l y m p h o c y t e s were stained for surface IgM after cell electrophoresis (A in Fig. 2), all fractions were f o u n d t o c o n t a i n a m a j o r i t y o f small cells with only b a c k g r o u n d fluorescence signals, and a small p r o p o r t i o n o f cells with bright fluorescence. The fluorescence intensities o f these antigen-posi- tive cells were widely distributed, b u t were clearly separate f r o m antigen- negative cells. A m i n o r p o p u l a t i o n o f larger cells with low fluorescence inten- sities was observed in fractions o f low EPM.
When electrophoresis was p e r f o r m e d after a n t i b o d y t r e a t m e n t (B in Fig. 2) fractions were o b t a i n e d containing cells with b a c k g r o u n d fluorescence only, or containing antigen-positive cells. T h e IgM-positive cells showed a distinct d i f f e r e n c e in fluorescence intensity b e t w e e n fractions o f m e d i u m and of low EPM. The brightest fluorescence was seen on cells o f lowest EPM. As anti- b o d y u p t a k e o f cells correlates with their antigen c o n t e n t this indicates that
those B cells with the highest a m o u n t of surface IgM also experienced the m o s t extensive reduction in their EPM. The cell volume of the IgM:positive l y m p h o c y t e s was rather homogeneous. Thus electrophoresis separated the antibody-labelled cells according to their surface antigen density.
On average the observed fluorescence intensities were similar for cells stained before or after electrophoresis, indicating t h a t no significant loss of antibodies from the cells occurred during the separation. The few larger cells with faint fluorescence were f o u n d in the same position (centred around fraction 45 in the experiment represented in Fig. 1) regardless of whether a n t i b o d y incubation was performed before or after electrophoresis.
Separation o f a T lymphocyte subpopulation reacting with monoclonal anti- body T811
The described separation m e t h o d should be applicable to any antibody- defined l y m p h o c y t e subpopulation. Because of their high significance and value for the classification of h u m a n l y m p h o c y t e s , it was of interest to see, whether also monoclonal antibodies could be used in antigen-specific electro- phoretic cell separation (ASECS).
The monoclonal a n t i b o d y T811 (Rieber et al., 1981), which we chose as an example, reacts with a T cell subpopulation and consistently stained about 25% of the cells in our l y m p h o c y t e preparations. It was used in a con- centration known from flow c y t o m e t r i c analysis to guarantee a n t i b o d y saturation. However, the cells showed considerably lower intensity of stain- ing in indirect immunofluorescence t h a n with conventional antisera to l y m p h o c y t e surface antigens. No efficient separation by cell electrophoresis was achieved u n d e r these conditions.
We therefore applied a double-sandwich m e t h o d , using TRITC-labelled rabbit-anti-mouse-Ig and goat-anti-rabbit-Ig immunoglobulins. TRITC-con- jugates were chosen in preference to FITC-conjugates, because fluorescein significantly elevates the isoelectric point of immunoglobulin. When h u m a n e r y t h r o c y t e s and FITC-labelled, TRITC-labelled and unlabelled goat-anti- rabbit-Ig immunoglobulins were subjected to free-flow electrophoresis (effec- tive field strength: 74 V/cm), the observed mean electrophoretic migration paths were 47.1 m m , 20.2 m m , 6.7 m m and 2.2 m m , respectively. Thus the difference in charge density b e t w e ~ the l y m p h o c y t e cell surface and bound immunoglobulins can be better maintained by using r h o d a m i n e label.
The n u m b e r of cells stained by the m o n o c l o n a l a n t i b o d y T811 was the same whether a single second a n t i b o d y or the double-sandwich technique was used. Antigen-positive cells could be distinguished unambiguously from antigen-negative cells and from cells with faint fluorescence.
Fig. 3 shows the electrophoretic distribution of all nucleated cells and of the T811-defined T cell subpopulation before and after incubation with anti- b o d y . One representative experiment o u t of 7 is shown. The EPM of antigen- positive cells before incubation was similar to the mean EPM of all cells.
After reaction with a n t i b o d y the antigen-positive cells possessed a signifi-
Electrophoretic migration path [mm]
39,7 34,0L 28,3
A nucl. cells
/ \ ~ TS11" cells
~_
~ 20/// ~i~ ~
-~ 10
~_
20 B
\
"'° , ././
~0
4'2 Fraction number ~ 4'6 ~;s so s'2Fig. 3. Effect of monoclonal antibody T811 on the electrophoretic mobility of a human T cell subpopulation. A: electrophoretic distribution of human blood lymphocytes. The T cell subpopulation was detected by labelling with monoclonal antibody T811 followed by TRITC-labelled rabbit-anti-mouse-Ig and TRITC-labelled goat-anti-rabbit-Ig immuno- globulins. B: electrophoretic distribution of antibody-treated cells. Lymphocytes were incubated with monoclonal antibody T811 and with TRITC-labelled rabbit-anti-mouse- Ig and goat-anti-rabbit-Ig immunoglobulins (at 4°C) before cell electrophoresis. Effective field strength: 62 V/cm. The arrow marks the position of formaldehyde-fixed human erythrocytes.
c a n t l y l o w e r EPM, c a u s i n g a b i m o d a l i t y in t h e e l e c t r o p h o r e t i c profile. Cell r e c o v e r y a n d p u r i t y g r e a t l y paralleled t h a t o b t a i n e d w i t h t h e anti-IgM anti- s e r u m , as c a n be seen f r o m T a b l e 2. Again, r e c o v e r y o f a n t i b o d y - l a b e l l e d cells f r o m e l e c t r o p h o r e s i s was b e t t e r t h a n t h a t o f t o t a l cells, a n d cell viability was h i g h e r t h a n 90% in all f r a c t i o n s , e x c e p t t h o s e at t h e high EPM e n d o f t h e d i s t r i b u t i o n s .
Contribution of non-specific antibody uptake
As a c o n t r o l f o r n o n - s p e c i f i c b i n d i n g o f a n t i b o d i e s via F c - r e c e p t o r s a n d a test o f its i n f l u e n c e o n t h e e l e c t r o p h o r e t i c s e p a r a t i o n , l y m p h o c y t e s were i n c u b a t e d w i t h n o n - r e l e v a n t c o n v e n t i o n a l or m o n o c l o n a l a n t i b o d i e s against T h y - 1 a n t i g e n a n d w i t h t h e r e s p e c t i v e f l u o r e s c e n t s e c o n d a n t i b o d i e s . A f t e r this t r e a t m e n t 9 a n d 7% o f cells r e s p e c t i v e l y s h o w e d faint f l u o r e s c e n c e . N o e f f e c t o n t h e e l e c t r o p h o r e t i c profiles o f t o t a l l y m p h o c y t e s or o f these
TABLE 2
Enrichment of cells reactive with monoclonal antibody T811 by free-flow electrophoresis.
Data expressed as average -+ S.D. of 7 experiments.
Nucleated cells T811-reactive cells
Recovery (%) Purity (%) Recovery (%) d
• Lymphocyte preparation before cell electrophoresis a
Sum of all fractions after electro- phoresis of lymphocytes Sum of all fractions after electro-
phoresis of antibody-treated lymphocytes b
Fractions pooled after electro- phoresis of antibody-treated lymphocytes (pool L) c
100 2 3 . 2 ± 3 . 2 100
8 2 . 4 ± 4 . 0 2 3 . 2 ± 2 . 8 84 6 7 . 1 ± 7 . 2 3 0 . 0 ± 4 . 3 87 1 1 . 2 ± 0 . 6 9 0 . 1 ± 4 . 9 43.5
a Lymphocytes were prepared from defibrinated human blood by Ficoll-Hypaque centri- fugation. Monocyte contamination was 7.5 -+ 2.7%.
b Lymphocytes were incubated with monoclonal antibody T811 followed by TRITC- labelled rabbit-anti-mouse-Ig and TRITC-labelled goat-anti-rabbit-Ig immunoglobulins before free-flow electrophoresis.
c Fractions were pooled in the region of low electrophoretic mobility so to contain half of the T811-positive chain.
d Values were calculated from total cell recovery and percentage of T811-positive cells.
T811-positive cells in the original lymphocyte preparation was set as 100%.
f l u o r e s c e n t cells was o b s e r v e d . F l o w c y t o m e t r y d e m o n s t r a t e d t h a t t h e cells w i t h n o n - s p e c i f i c u p t a k e o f a n t i b o d i e s were m o s t l y larger ( ' c o n t r o l ' in Fig. 2), a n d t h a t t h e y were also p r e s e n t in t h e e x p e r i m e n t s w i t h specific anti- sera (A a n d B in Fig. 2). T h e s e cells m a i n l y c o n s i s t e d o f m o n o c y t e s , w h i c h were s h o w n t o have a 7% l o w e r m e a n EPM as c o m p a r e d t o l y m p h o c y t e s in several e l e c t r o p h o r e t i c s e p a r a t i o n s w i t h o r w i t h o u t specific a n t i b o d y treat- m e n t .
T h u s n o n - s p e c i f i c b i n d i n g o f a n t i b o d i e s via F c - r e c e p t o r s c a n be d i s r e g a r d e d in t h e e l e c t r o p h o r e t i c s e p a r a t i o n o f a n t i b o d y - l a b e l l e d cells.
DISCUSSION
F r e e - f l o w e l e c t r o p h o r e s i s has b e e n u s e d v e r y s u c c e s s f u l l y in t h e s e p a r a t i o n o f a w i d e v a r i e t y o f cells a n d cell organelles (reviewed b y Hannig, 1 9 7 2 ; S h e r b e t , 1 9 7 8 ) . A m o n g s t t h e a d v a n t a g e s o f this m e t h o d are its high separa- t i o n c a p a c i t y a n d t h a t it is carrier-free, t h u s a v o i d i n g n o n - s p e c i f i c interac- t i o n s o f cells w i t h solid surfaces. Cell e l e c t r o p h o r e s i s has b e e n especially use- ful in t h e s e p a r a t i o n o f m u r i n e T a n d B l y m p h o c y t e s (Zeiller et al., 1 9 7 1 ; f o r review see S h e r b e t , 1 9 7 8 ; P r e t l o w a n d P r e t l o w , 1 9 7 9 ) . U n f o r t u n a t e l y , it has
n o t been f o u n d possible to use preparative cell electrophoresis to separate h u m a n l y m p h o c y t e s (Stein, 1973; H~/yry et al., 1975; Chollet et al., 1978), although a wide d e m a n d for preparative quantities of highly pure and vital h u m a n T and B cells exists in immunological and clinical research.
The m e t h o d we describe here allows the efficient electrophoretic separa- tion of h u m a n l y m p h o c y t e subpopulations on a preparative scale. It makes use of a decrease in the electrophoretic mobility (EPM) of cells after reaction with specific antibodies.
Analytical studies have already shown t h a t antibodies can influence the EPM of cells (reviewed by Sherbet, 1978). A reduction in surface charge den- sity has been observed in l y m p h o c y t e s from various mammals after treat- m e n t with ALS or anti-Ig antisera (Bert et al., 1969; Phondke et al., 1970;
Bert et al., 1971; Bona et al., 1972; Kaplan and Uzgiris, 1976). However, an increase in EPM (Von Boehmer, 1974; Zeiller et al., 1976), or a lack of any change (Bert, 1969; Wioland et al. 1976; Zeiller et al., 1976) has also been described. In all of these studies antibody-induced redistribution of surface antigens has to be considered since incubations at room temperature or 37°C were involved.
We have used antibody-induced changes in surface charge density for the first time in the preparative electrophoretic separation of cells. We observed a regular decrease in the EPM of h u m a n l y m p h o c y t e subpopulations by 15-- 20% after a n t i b o d y t r e a t m e n t .
It is necessary to observe certain precautions in order to apply this techni- que of antigen-specific electrophoretic cell separation (ASECS). Some points m a y be i m p o r t a n t to be stressed. For any combination of cells and a n t i b o d y it is necessary to determine optimal concentrations, resulting in maximal up- take of antibodies w i t h o u t cell aggregation or non-specific staining. Sand- wich and double-sandwich m e t h o d s can then be used to compensate for low antigen density. The low iso-electric point of immunoglobulin is retained best by using r h o d a m i n e instead of fluorescein as fluorescence label. Capping of antibodies on the cell surface should be avoided by use of low tempera- ture or the addition of blocking agents. Under these conditions the observed decrease in EPM appears to be due to covering of surface charge by immuno- globulin.
The m e t h o d described combines the high specificity of antibody-depen- d e n t separation techniques with the high separation power of free-flow elec- trophoresis. As with other m e t h o d s involving antibodies, it should be kept in mind t h a t the functional state of the cells m a y be changed in some way by reaction with a n t i b o d y (Braun and Unanue, 1980). Generally, however, investigations of other cell surface antigens or receptors and most functional assays of l y m p h o c y t e s are n o t impaired by a n t i b o d y t r e a t m e n t . In the pres- ent case the antibodies can easily be removed from the cells by incubating at 37°C which results in capping and shedding of antigen-antibody complexes from the l y m p h o c y t e surface.
With m e t h o d s such as i m m u n o a b s o r b e n t c h r o m a t o g r a p h y , where antibodies
bound to particles or solid surfaces are used problems can arise in the disso- ciation of a n t i b o d y - b o u n d cells from the matrix as well as from unspecific interactions with the carrier matrix. Smolka et al. (1979) have recently described a reduction in the EPM of h u m a n e r y t h r o c y t e s after reaction with relevant antibodies and with anti-Ig antibodies coupled to neutral plastic beads. They also observed an effect of the immunomicrospheres on unsen- sitized erythrocytes. In addition, t h e y encountered difficulties in the removal of the b o u n d particles. We consider t h a t it is neither necessary nor desirable to add cell-particle interactions to the cell-antibody reaction for electro- phoretic cell separation, especially when working with nucleated cells.
In the experiments described here the antiserum concentrations used to achieve optimal u p t a k e of antibodies by the cells were a b o u t 10-fold higher than those necessary for the detection of surface antigens by immunofluo- rescence microscopy. The c o n s u m p t i o n of antisera can be lowered by saving the supernatant after each incubation and recycling the antisera, since only a minimal p r o p o r t i o n of a n t i b o d y activity is removed during incubation.
However, a n t i b o d y - d e p e n d e n t cell separation techniques are usually limited n o t by the a m o u n t of a n t i b o d y , b u t by the separation capacity of the m e t h o d . Thus cell recovery, cell vitality and the n u m b e r of cells t h a t can be processed in a reasonable time are the critical factors in i m m u n o a b s o r b e n t c h r o m a t o g r a p h y and fluorescence activated cell sorting (FACS). In FACS, for example, the maximal flow rate is 5000 cells/sec (Herzenberg and Herzen- berg, 1978), whereas free-flow electrophoresis allows separation of at least 100,000 cells/sec. Separation conditions can be held stable for m a n y hours.
Cell recovery in free-flow electrophoresis ranges from 70 to 85% and cell viability in the relevant fractions is over 90%. Dead cells are preferentially removed from the fractions of interest because t h e y tend to acquire higher negative charge (Sherbet, 1978), whereas the antigen-positive cells are col- lected in the region of low EPM. In our experiments the vitality of the separated l y m p h o c y t e s has n o t y e t been verified in functional assays, but the use of electrophoretically separated cells in such tests is well d o c u m e n t e d (Zeiller et al., 1971; Zeiller et al., 1976; Sherbet, 1978), and a n t i b o d y treat- m e n t is k n o w n n o t to interfere with m o s t l y m p h o c y t e functions.
It is clear from the flow c y t o m e t r i c analysis t h a t in our experiments cells were actually separated according to their antigen density. Therefore the m e t h o d should also be applicable to the separation of cells in which only quantitative differences in surface antigens exist.
We also tested the possibility of applying m o n o c l o n a l antibodies to ASECS. Despite of the low uptake of antibodies by the cells, m o n o c l o n a l antibodies could be used to introduce the desired decrease in surface charge density using double-sandwich procedures.
Pretreatment of cells with antibodies greatly enhances the potential of free-flow electrophoresis. As a m e t h o d for the isolation of large quantities of highly pure and viable h u m a n l y m p h o c y t e subpopulations, ASECS meets the requirements for wide application in immunological and clinical research.
M o r e o v e r , t h e m e t h o d m a y b e o f g e n e r a l u s e f o r t h e s e p a r a t i o n o f a n y c e l l p o p u l a t i o n o r s u b c e l l u l a r f r a c t i o n o r m a c r o m o l e c u l e t h a t c a n b e m a i n t a i n e d i n s u s p e n s i o n a n d f o r w h i c h s p e c i f i c a n t i b o d i e s a r e a v a i l a b l e .
ACKNOWLEDGEMENTS
T h e a u t h o r s t h a n k D r . E . P . R i e b e r , I n s t i t u t f i i r I m m u n o l o g i e d e r U n i v e r - sit~/t M f i n c h e n , f o r t h e g e n e r o u s g i f t o f m o n o c l o n a l a n t i b o d y T 8 1 1 .
T h e e x c e l l e n t t e c h n i c a l a s s i s t a n c e o f M r s . C a r o l a E c k e l t a n d M r s . R i t a W i e - m e y e r is g r a t e f u l l y a p p r e c i a t e d .
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