Journal o f Immunological Methods, 7 ( 1975) 237--250
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
A RADIOIMMUNOASSAY OF CELLULAR SURFACE ANTIGENS ON LIVING CELLS USING IODINATED SOLUBLE PROTEIN A FROM STAPHYLOCOCCUS A UREUS
G. DORVAL*, K.I. WELSH** and H. WIGZELL*** * Department o f Tumor Biology, Karolinska Institute, 104 01 Stockholm 60, Sweden ** McIndoe Research Unit, Queen Victoria Hospital, East Grinstead, Sussex, England *** Department o f Immunology, Uppsala University, Box 562, 751 22 Uppsala 1, Sweden
(Received 29 November 1974, accepted 1 December 1974)
Soluble protein A from Staphylococcus aureus does carry great promise as a marker for cellular surface determinants, due to its specific reaction with, and high affinity for, most subclasses of mammalian IgG. In this article we present the different parameters involved in a radioimmunoassay using 1 2 5i.labelled protein A. Using such an approach the actual technical procedures involved are reported in detail together with tests for mammalian alloantigens, including HL-A in the human, H-2 in the mouse and AgB antigenic sites in the rat, as this presents an unique opportunity to compare them with already widely used assays for transplantation antigens. The different parameters of the assay are analysed in view of measuring with precision quantities of cell-surface IgG molecules, thereby allowing possible determinations of antigenic site numbers in a new and simplified manner.
INTRODUCTION T h e s p e c i f i c r e a c t i o n o f S t a p h y l o c o c c u s a u r e u s p r o t e i n A w i t h t h e Fc r e g i o n o f m o s t m a m m a l i a n I g G m o l e c u l e s ( K r o n v a l l et al., 1 9 7 0 a ) has r e c e n t ly b e e n u s e d as a n e w i m m u n o l o g i c a l p r o b e in t h e a n a l y s i s of cell s u r f a c e m a r k e r s ( D o r v a l e t al., 1 9 7 4 ; G h e t i e et al., 1 9 7 4 ) . N o r m a l l y , cells are first c o m b i n e d w i t h an a n t i s e r u m d i r e c t e d a g a i n s t a given s u r f a c e d e t e r m i n a n t a n d then allowed to react with f l u o r e s c e i n - o r radioiodine-labelled soluble prot e i n A. C e l l - b o u n d I g G m o l e c u l e s , a c t i v e l y p r o d u c e d b y m a m m a l i a n l y m p h o c y t e s a n d c u l t u r e d cell lines, c a n also be r e v e a l e d w i t h t h e use o f fluoresc e i n a t e d p r o t e i n A ( F - p A ) ( D o r v a l et al., 1 9 7 4 ; G h e t i e et al., 1 9 7 4 ) ; this is c o n f i r m e d h e r e b y t h e use o f i 2 s I - l a b e l l e d p r o t e i n A ( I - p A ) . In t h e p r e s e n t a r t i c l e , t h e i m p o r t a n c e o f t h e v a r i o u s p a r a m e t e r s i n v o l v e d in t h e I - p A assay are r e p o r t e d in d e t a i l . T h e e f f e c t o f t h e n u m b e r o f w a s h e s , t e m p e r a t u r e a n d d u r a t i o n o f i n c u b a t i o n a t e a c h s t e p are a n a l y s e d . T h e d i r e c t assay (cells + a n t i s e r u m + I - p A ) is c o m p a r e d t o t h e i n d i r e c t assay w h e r e cells, a f t e r i n c u b a t i o n w i t h an a n t i s e r u m , are o v e r l a y e r e d w i t h an a n t i - I g G antis e r u m d i r e c t e d a g a i n s t t h e species a n t i b o d y c o m b i n e d w i t h t h e cells, a n d finally sandwiched with I-pA.
238 Also, the conventional 'multiple individual test tubes' radioimmunoassays require much time and material. Here, such assays are compared to a new fast procedure performed in microplates. Examples of applications of the present radioimmunoassays in mouse, rat, guinea pig and human systems are reported.
MATERIALS AND METHODS The purification of protein A has been described previously (Dorval et al., 1974). 12 s I-labelling o f p r o t e i n A : I-pA
Radioiodination of protein A was performed by the chloramine-T m e t h o d (Hunter and Greenwood, 1962). To 1 mCi 12SI-labeled sodium iodide (Amersham, IMS-30) were added 0.05 ml protein A (50 pg), and 0.005 ml chloramine-T (0.8 mg/ml) in 0.15 M sodium phosphate buffer, pH 7.5. It is important that the chloramine-T is not added in large excess of protein A as this might greatly impair the Fc binding capacity of protein A. The mixture reaction was allowed for 1 min at 23 °C with constant mixing, and terminated by the addition of 0.005 ml sodium metabisulfite (1 mg/ml). 0.25 ml of 5% ovalbumin solution in phosphate buffer, pH 7.1 (OA), was then added to the mixture, which was separated from free iodine by Sephadex G-25 chromatography and stored at --20 °C until use. Se ra
All sera were heat-inactivated for 30 min at 56 °C and kept at --20 °C until use; this complement deactivation is necessary since the IgG site of complement binding is close to that of protein A binding. A n t i - I g G sera
Sheep anti-human IgG has been described elsewhere (Dorval et al., 1974). Rabbit anti-mouse Ig and anti-rat Ig sera were obtained by immunization with I mg/ml of mouse or rat IgG in Freund's complete adjuvant. A lloan tisera
Rat and mouse alloantisera were raised using spleen cells by cross-immunization of animals maintained at the Department of Tumor Biology, Karolinska Institute, Stockholm. A n t i - H L - A sera
The Monkey anti-HL-A 2 serum is a well characterized monospecific reagent (Sanderson and Welsh, 1974).
239 Cells
H u m a n l y m p h o c y t e s were prepared f r om defibrinated peripheral blood using gradient centrifugation ( B o y u m , 1968), followed by iron carbonyl and magnetism. The four h u man lymphoblastoid cell lines (LCL:s) used here had been previously described, but in the present c o n t e x t it should only be recalled that the cell line 1 in table 3 is HL-A 2-negative, whereas the three others are HL-A 2-positive. T hey were kindly provided by Kenneth Nilsson. L y m p h o c y t e s f r om rat, mouse and guinea pig were prepared by gentle teasing of the freshly excised organ through a metal net, followed by one wash in PBS with 0.004 M EDTA, pH 7.4 (PBS-T), and two washes in BSS before use. Only cell preparations that contained m ore than 90% viable cells, as judged by T r y p a n blue exclusion, were used.
Cell-bound a n t i g e n - a n t i b o d y - - 12 Si_labele d p r o t e i n A assay: I-pA assay
The following scheme was f o u n d suitable for 10 6 cells per tube, or 0.1 × 10 6 cells per microwell. It should be n o t e d that the I-pA assay can function down to a few thousand cells provided the antigen density is high enough. Aliquots o f cells in 10--50 pl were added to I ml plastic tubes, or to individual 'U-shaped' microwells f r om 8 × 12 microtiter plastic plate (Cooke Instruments, A.G., Switzerland). When indicated, tubes or microwells were precoated with 10% ovalbumin (OA) or fetal calf serum, (FCS), to prevent non-specific sticking. When possible, antigen-saturating (according to pre-tests) amounts of antisera (1--10 pl) were added and the mixture incubated for 45 min at 4 °C, unless otherwise mentioned. 250 pl ice-cold medium (RPMI 1640) was then added and the tubes centrifuged for 3--5 min at 600 g before removal of supernatant. This washing step was repeated once. Cells on plates were washed by centrifugation in an International Centrifuge, model PRJ, 1000 rpm 6 rain followed by shaking out the supernatant with one, firm movement. Ten microliters of I-pA in k n o w n excess with regard to IgG binding in 5% OA was added per t ube or microwell and a further 30 min incubation at 4 °C carried out. This was followed by three washings as described above, but using PBS-T with or w i t h o u t 5% FCS as washing buffer. The cells were then transferred to tubes for counting o f radioactivity. When an indirect assay (cells + antiserum + anti-IgG + I-pA) was performed, the following steps were done after washing away the first antiserum. Ten microliters of antiserum to the initial a n t i b o d y was added per tube/microwell, and incubated for 30 min at 4 °C, followed by two washes, whereafter I-pA was added in the usual manner. For controls see the Results section.
240 RESULTS Experimental procedure Effect o f the n u m ber o f washes on the ~ 2 Si_labele d p A bound Preliminary e x p e r i m e n t s were carried o u t in o r d e r to o b t a i n o p t i m a l reaction c o n d i t i o n s t o g e t h e r with a minimal b a c k g r o u n d . D i f f e r e n t washing buffer solutions were tested. It was f o u n d t h a t 1) l y m p h o c y t e s were best washed t h r e e times prior to the test so as to avoid the presence o f loosely a t t a c h e d c y t o p h i l i c IgG which interferes with the test; 2) the viability o f the cells plays an i m p o r t a n t role because some dead cells can be s h o w n to take up I-pA in a non-specific m a n n e r to a larger e x t e n t t h a n do live cells; 3) the best b u f f e r solutions used for washings o f cells were Ca 2+/Mg 2+-free buffers c o n t a i n i n g a chelating agent (we used PBS, w i t h o u t Ca 2. and Mg 2÷, pH 7.4, with 4 mM E D T A ) and 2--5% ovalbumin. Such buffers w o u l d give the lowest b a c k g r o u n d values and the highest n u m b e r s of r e c o v e r e d cells; 4) the t r a n s f e r of cells to new tubes at the end o f the test caused a significantly lower b a c k g r o u n d , d u e to the a t t a c h m e n t of serum IgG a n d / o r I-pA to the wall o f the t u b e during the assay; 5) finally, if cell n u m b e r s at t h e end of the actual test or in parallel run controls c o u l d be assessed (we used a Celloscope, Linson I n s t r u m e n t s , S t o c k h o l m , Sweden), this w o u l d f u r t h e r r e d u c e the variations due t o t h e possible loss of cells during t h e e x p e r i m e n t . Table 1 presents t h e e f f e c t o f the n u m b e r o f washes o n the I-pA b o u n d o n DA rat l y m p h n o d e cells, using Lewis anti-DA alloantiserum. The specificity ratio o b t a i n e d r e a c h e d m a x i m u m with one c o m p l e t e removal o f s u p e r n a t a n t after the first i n c u b a t i o n , f o l l o w e d b y t w o washes after the s e c o n d incubation (table 1A). E x p e r i m e n t s were also d o n e using the indirect assay system. As s h o w n in table 1B, a single wash a f t e r alloantiserum f o l l o w e d b y t w o washes a f t e r each o f the t w o o t h e r stages p r o v e d necessary.
Effect o f time and temperature on the incubation with antiserum, and effect o f time on the incubation with I-pA Table 2 presents the results o b t a i n e d w h e n i n c u b a t i n g DA × Lewis F1 rat l y m p h n o d e cells with either Lewis anti-DA a n t i s e r u m or n o r m a l Lewis serum at varying t i m e and t e m p e r a t u r e , f o l l o w e d b y i n c u b a t i o n with I-pA at 4 °C f o r 30, 60, or 90 min. This e x p e r i m e n t was p e r f o r m e d in d u p l i c a t e t u b e s and the specific c o u n t s is the average o f a n t i s e r u m - t r e a t e d cells (a) minus n o r m a l serum (N). T w o washes were p e r f o r m e d using m e d i u m ( R P M I - 1 6 4 0 ) prior t o the a d d i t i o n o f I-pA, and t h r e e washes with PBS-T were d o n e a f t e r the i n c u b a t i o n with I-pA. In brackets are the specific cps o b t a i n e d with the indirect assay, w h e r e cells prior t o the a d d i t i o n o f I-pA were i n c u b a t e d for 30 rain at 4 ° C with a rabbit anti-rat Ig, f o l l o w e d by t w o washes. Optimal
241 TABLE 1 Effect of numbers of washes on the 128Icounts bound (cpm/106 cells). A. Direct assay Number of pre-washes:
1 2 3 4
Number of post-washes: 1 2
3
4
13,887 (387) 12,113 (812) 9624 (237) 8561
9865 (120) 8872 (130) 6077 (121) 4421
9804 (123) 8566 (101) 5765 (98) 4376
3
4
10,009 (148) 9019 (139) 6271 (162) 4899
B. Indirect assay Number of pre-washes: 1 2 3
Number of post-washes: 1 2 23,092 (13111) 18,011 (7211) 15,201 (6866)
19,820 (4760) 17,772 (4552) 13,981 (4277)
19,620 (3998) 17,521 (4443) 13,700 (4144)
19,799 (4421) 17,641 (4420) 13,977 (4019)
- - T h e first column (number of pre-washes) gives the number of washes prior to the addition of protein A. Thus in A, the number of washes after the incubation with the alloantiserum, and in B, the number of washes after the anti-IgG, the number of washes after the alloantiserum being here one. - - T h e first row (number of post-washes) gives the number of washes after the protein A incubation.
b i n d i n g was a l r e a d y a c h i e v e d using 30 m i n p e r i o d s o f i n c u b a t i o n , w i t h t e m perature being of relatively little importance. In d o i n g e x p e r i m e n t s u n d e r c o n d i t i o n s n o t i n v o l v i n g a n t i b o d y a n d / o r I - p A excess, i n c u b a t i o n p e r i o d s s h o u l d be i n c r e a s e d t o 6 0 - - 9 0 m i n t o a c h i e v e optimal binding.
Comparison o f the direct (I-pA-alloantibody--cell) assay with the indirect (I-pA--anti-IgG--alloantibody--cell) assay P r e l i m i n a r y c o m p a r i s o n s o f t h e d i r e c t a n d i n d i r e c t I - p A a s s a y s have b e e n r e p o r t e d e l s e w h e r e . As p r o t e i n A will b i n d t o a v a r y i n g d e g r e e t o I g G sub-
242 TABLE 2 Effect of time and temperature of incubation with antiserum and effect of time of incubation with I-pA (cps/0.2 × 106 cells). Effect of
Time of Inc.
T ~ of inc.
10 (min)
4°C
38 41
15 13
26
53 50
13 11
40
42 45
8 7
59 61
14 13
42
56 53
12 13
41(55)a
61 64
13 17
48(55) a
59 54
13 12
44
59 53
15 12
43
70 73
13 18
51
42 42
12 12
30
50 58
10 16
41
58 55
11 12
46
53 56
13 12
42
54 54
14 12
41(56) a
54 57
12 12
44(56) a
53 53
11 8
43
63 61
10 15
50
80 65
19 15
56
45 43
12 9
34
45 48
7 9
39
46 46
10 11
36
51 51
12 10
40
52 54
12 12
41(57) a
59 59
12 12
47(59)a
63 64
11 13
52
64 54
13 16
46
79 75
19 15
62
~
N
spec ~
N
spec
~
N
spec
22°C
37°C
With antiserum
Time of inc. at 4°C 30 (min)
60 (min)
(min) 36
30 60 9O 3O 60 9O 30 60 90 With I-pA
a Specific cps in indirect assay, with anti-Ig incubation time of 30 min at 4°C.
classes o f d i f f e r e n t m a m m a l s ( K r o n v a l l a n d W i l l i a m s , 1 9 6 9 ; L i n d e t al., 1 9 7 0 ; K r o n v a l l e t al., 1 9 7 4 ) , t h e o u t c o m e o f d i r e c t a n d i n d i r e c t I - p A a s s a y w o u l d largely depend on the species providing the IgG molecules to be tested. Thus such c o m p a r i s o n s are of m o r e practical t h a n t h e o r e t i c a l i m p o r t a n c e . Table 3 s h o w s h o w t h e a m o u n t o f I - p A b o u n d t o h u m a n cells is o n l y i n f l u e n c e d t o a m i n o r e x t e n t b y t h e i n c l u s i o n o f a n e x t r a l a y e r o f Ig m o l e c u l e s . I t is also noted that the values obtained with control normal monkey serum do not c h a n g e in t h e i n d i r e c t a s s a y in p a r t A , as t h e h u m a n c e l l l i n e s u s e d w e r e s u r f a c e I g G n e g a t i v e . T h e m o u s e s y s t e m is c o n s i d e r a b l y a f f e c t e d b y t h e a d d i t i o n o f a n a n t i - I g s e r u m b u t t h i s is d u e t o t h e f a c t t h a t t h e n o r m a l m o u s e l y m p h n o d e cel l s c o n t a i n a p p r o x i m a t e l y 2 0 % B cells.
243 TABLE 3 Comparison of the direct (I-pA-alloantibody-cell) assay with the indirect (I-pA-anti-Igalloantibody-cell) assay. A. Binding to human cell lines (cps/10~ cells) Cell line Direct assay Indirect assay 1 250 (10) 305 (10) 2 785 (10) 1050 (65) 3 1200 (10) 1300 (10) 4 800 (15) 915 (15) The results are obtained with a monkey anti-HL-A 2 antiserum compared to results obtained with normal human serum (in brackets). Cell line 1 is HL-A 2 negative. Sheep anti-human IgG was used as the second antiserum. B. Binding to mouse lymph node cells (cps/106 cells) Direct assay Indirect assay 145 (54) 270 (150) The results are obtained with an anti-H-2 b antiserum compared to results obtained with syngeneic normal mouse serum, on H-2 b lymph node cells. Rabbit anti-mouse Ig was used as the second antiserum.
The I-pA assay done in wells of microtiter plates in comparison with the conventional multiple individual test tubes radioimmunoassay Time and material are limiting f a c t o r s in multiple i m m u n o a s s a y s . Originally, the c o n v e n t i o n a l multiple individual test t u b e s r a d i o i m m u n o a s s a y was f o u n d to require c o m p a r a t i v e l y long m a n i p u l a t i o n s w h i c h impaired the rep r o d u c i b i l i t y . Also, relatively large a m o u n t s o f cells and sera were used. This p r o m p t e d us to t u r n to the use o f m i c r o t i t e r plates in w h i c h several plates, each c o n t a i n i n g up t o 96 samples, c o u l d be c e n t r i f u g e d at o n c e and washed b y m e r e l y shaking o u t the s u p e r n a t a n t . This is in sharp c o n t r a s t to the ' t u b e s ' assay w h e r e the pellets had to be carefully avoided during the removal o f the s u p e r n a t a n t . Preliminary e x p e r i m e n t s were carried o u t in view o f assessing the possible increase o f cell losses using t h e plate s y s t e m . Surprisingly, w h e n the original cell suspension had a g o o d viability, o n l y a few per c e n t o f t h e cells were lost d u r i n g t h e e x p e r i m e n t a l p r o c e d u r e . Table 4 presents t h e results o f a ' p l a t e ' assay run in parallel with a ' t u b e ' assay, and u n d e r similar e x p e r i m e n t a l c o n d i t i o n s e x c e p t f o r the n u m b e r o f cells being d i f f e r e n t in the t w o systems. L y m p h n o d e cells f r o m a H-2 m o u s e were first t r e a t e d for 45 min at 4 °C with either n o r m a l H-2 ~ m o u s e serum {first r o w ) , or with an H-2 f anti-H-2 k a l l o a n t i s e r u m ( s e c o n d row). Vertical c o l u m n s c o m p a r e the e f f e c t o f p r e c o a t i n g the t u b e s or t h e wells o f m i c r o titer plates with 100% fetal calf serum (FCS), or with 1% O A , or w i t h o u t p r e c o a t i n g (O), as well as the e f f e c t o f a d d i n g FCS in the washing b u f f e r after the i n c u b a t i o n with I-pA. A l t h o u g h a t t e m p t i n g t o d o the tests in paral-
+ NMS
Sera a d d e d :
+ anti-H-2
+ NMS
n.d. = n o t d e t e r m i n e d .
In wells cells o f 0.2 x 10 (' (corrected for 106)
(corrected for 106 ) + anti-H-2
In tubes cells 0.5 × 106
Washes after I-pA Precoated with
169 1.901 (9.505)
4.432 (8.864)
4.900 (9.800) 246 2.520 (12.600)
870
695
PBS-T100% FCS 1% OA-RPMI
10Xl :5
I-pA assay: in tubes vs. m i c r o p l a t e wells (cpm).
TABLE 4
191 2.539 (] 1.695)
3.861 (7.722)
739
261 2.739 (13.685)
3.270 (6.540)
562
208 2.259 (11.295)
3.847 (7.694)
640
PBS-T- + 7.5% FCS 100% FCS 1% OA-RPMI
250 2.789 (13.945)
5.613 (11.226)
868
n.d.
ll.d.
4.781
714
PBS-T + 7.5% FCS 0
lOx undil.
d~
b~
245 lel t h e t e c h n i c a l l y t e n u o u s ' t u b e ' assay r e q u i r e d m u c h m o r e t i m e f o r t h e individual washing p r o c e d u r e s resulting in a m a r k e d d e l a y in p e r f o r m i n g t h a t assay in c o m p a r i s o n w i t h t h e p l a t e assay. I - p A was k n o w n to have b e e n a d d e d in excess, w h e r e a s the last c o l u m n s h o w s t h a t increasing t h e c o n c e n t r a t i o n o f a l l o a n t i s e r u m b y 5-fold did n o t cause an increase o f the c o u n t s b o u n d . T h u s it was a d d e d also in excess. T h e a p p a r e n t d i s c r e p a n c y b e t w e e n the results o b t a i n e d w i t h t h e t w o tests c a n n o t be fully e x p l a i n e d . F r o m several e x p e r i m e n t s , h o w e v e r , we can c o n c l u d e t h a t b o t h w i t h regard to t h e t o t a l I - p A b o u n d to I g G - c o a t e d cells a n d with regard to l o w b a c k g r o u n d values t h e m i c r o p l a t e assay w o u l d s e e m s u p e r i o r t o t h e t u b e assay.
Application of the technique Direct binding of I-pA on normal, uncoated mammalian lymphocytes T h e direct b i n d i n g o f I - p A to n o r m a l , u n c o a t e d guinea pig, h u m a n , rat, a n d m o u s e l y m p h o i d cells has b e e n investigated {table 5). I n c r e a s i n g n u m bers o f l y m p h o c y t e s , 1 × 106 , 2 X 106 , and 5 × 106 cells w e r e r e a c t e d with I - p A in t u b e s f o r 45 m i n at 4°C. T w o washes w i t h PBS-T were s u b s e q u e n t l y d o n e b e f o r e t h e cells w e r e t r a n s f e r r e d t o o t h e r t u b e s a n d w a s h e d o n c e m o r e Binding
of
l-pA on mammalian
lymphocytes
cpm
~G.p.
1500
i /
1000
500
/ 1°
~
i
/
"
"
~ M o u s e
-__
.._.-o R~t
1 2 3 Number of cells x 10 6 : e without SRBC _ o. . . . .
--o w i t h
150 x IO b SRBC
Fig. 1. H = human peripheral blood lymphocytes. G.p. = Guinea pig lymph node cells. Mouse and rat = lymph node cells of respectivespecies.
246 TABLE 5 Direct binding o f I-pA on m a m m a l i a n l y m p h o c y t e s ( c p m ) Cells:
N u m b e r × 105 + S R B C × 106(a)
Guinea pig-spleen Guineapig-LNC(c) Human-periph. Rat-LNC Mouse-LNC No ceels: 206 (a) S R B C a l o n e : × 106 (cpm)
1 0 610 270 362 204 280
2 0 900 366 550 228 258
40 198
50 196
5 0 1720 598 822(b) 214 388
60 216
1 150 872 258 416 234 270
75 228
2 5 150 150 1250 1918 328 570 574 n.d. 258 286 240 386
100 248
120 150 256 258
(b) only 3 × 106 l y m p h o c y t e s . (c) LNC = l y m p h n o d e cells. n.d. = n o t d e t e r m i n e d .
with the s a m e buffer. In o r d e r to avoid possible increase o f n o n - s p e c i f i c t r a p p i n g o f I - p A with an increase of t h e cell mass, parallel t u b e s were r u n in the s a m e fashion with a c o n s t a n t high n u m b e r o f s h e e p e r y t h r o c y t e s (SRBC). C o n t r o l s with increasing n u m b e r s o f SRBC alone s h o w a l m o s t no d i f f e r e n c e w h e n c o m p a r e d to t u b e s which had received o n l y I - p A and no cells ( b a c k g r o u n d ) . When b a c k g r o u n d binding values w e r e s u b t r a c t e d , a very nice c o r r e l a t i o n c o u l d be seen b e t w e e n the binding a n d the increasing n u m ber of guinea pig a n d h u m a n l y m p h o c y t e s (fig. 1). In c o n t r a s t , values obtained with m o u s e a n d rat l y m p h o i d cells s h o w o n l y m a r g i n a l increase a b o v e the b a c k g r o u n d .
Quantitative detection o f DA anti-Lewis and Lewis anti-DA antibody bound to Lewis, DA, or (Lewis × D A ) F1 lymphoid cells T w o a n t i b o d y c o n c e n t r a t i o n s and t w o p r o t e i n A c o n c e n t r a t i o n s , b o t h t h o u g h t to be in excess, were used in a direct assay p e r f o r m e d in individual t u b e s on rat l y m p h n o d e cells. C o l u m n s A a n d B (table 6) in t e r m s of c o u n t s b o u n d are n o t m a r k e d l y d i f f e r e n t s h o w i n g t h a t p r o t e i n A is in excess; t h e c o n t r o l s are, h o w e v e r , b e t t e r in t h e l o w e r excess case. T h e results in g r o u p s 1 and 2 should be identical if D A anti-Lewis is in excess; h o w e v e r , t h e cps are less in r o w 2 s h o w i n g t h a t 10 pl a n t i s e r u m is i n s u f f i c i e n t to s a t u r a t e 106 cells (the t i t e r o f this a n t i s e r u m in the T r y p a n blue test was 1 : 4). T h e results in g r o u p s 3 and 4 are similar showing t h a t the Lewis a n t i - D A was in excess (titer 1 : 40 in t h e T r y p a n blue). It is also n o t i c a b l e t h a t t h e F1 c o u n t s are o n l y half the p a r e n t a l c o u n t s if t h e b a c k g r o u n d is l o w and the a n t i s e r u m in excess. F o r c o m p a r a t i v e tests in antigenic sites d e t e r m i n a t i o n u n d e r n o n excess c o n d i t i o n s , it is necessary to adjust the cell n u m b e r s to give a p p r o x i m a t i v e l y equal n u m b e r s o f c o u n t s b o u n d , as suggested p r e v i o u s l y ( B a t c h e l o r et al., 1973).
247 TABLE 6 Use of the I-pA assay to quantitate the binding of DA anti-Lewis, and Lewis-anti-DA, on DA, Lewis, and (DAxLewis) Fl rat lymph node cells. Antiserum
Cells (10 6 )
cps bound A B
cps specific A B
DA anti-Le (20/11)
Le DA FI Le DA F1
215 86 150 185 21 122
256 39 148 187 12 124
129
217
64 164
109 175
101
112
Le anti-DA (20/21)
Le DA FI
107 316 163
33 324 158
219 59
291 125
Le anti-DA (10 pl)
Le DA FI
43 284 158
13 282 152
246 115
269 139
DA anti-Le (10 pl)
A 1.7 × 106 counts I-pA added. B 2.2 × 106 counts I-pA added.
DISCUSSION P r o t e i n A f r o m S t a p h y l o c o c c u s aureus has affinity for t h e Fc region o f m o s t m a m m a l i a n IgG subclasses (Kronvall et al., 1970a). It can thus be used as a p r o b e for c e l l - b o u n d IgG molecules, and p r e l i m i n a r y d a t a suggest it as a very useful p r o b e in the analysis o f cellular antigens (K.I. Welsh, G. Dorval and H.W. Wigzell, 1975). I n the p r e s e n t article we describe the technical details o f i m p o r t a n c e w h e n using 12 Si_labele d p r o t e i n A as cell surface marker, as t h e t e c h n i q u e has n o t been described to a n y e x t e n t previously. Starting with the labeling p r o c e d u r e s we have f o u n d it o f great i m p o r t a n c e n o t to use t h e c o n v e n t i o n a l r e c o m m e n d e d c o n c e n t r a t i o n s o f C h l o r a m i n e - T ( H u n t e r a n d G r e e n w o o d , 1962), b u t r a t h e r a c o n c e n t r a t i o n n o t above 4 mM. A n increase a b o v e this m o l a r i t y w o u l d t e n d to h a r m p r o t e i n A and m a k e it less f u n c t i o n a l in the assay. Also, trace labeling o f p r o t e i n A with 12 5I aiming at o n e iodine per p r o t e i n - m o l e c u l e should be p e r f o r m e d as binding of iodine t o all f o u r t y r o s y l residues f o u n d in t h e p r o t e i n A m o l e c u l e (SjSquist et al,, 1 9 7 2 ) w o u l d seem to d e s t r o y t h e Fc binding activity ( S j S h o l m et al., 1973). It is i m p o r t a n t , w h e n d o i n g q u a n t i t a t i v e c o m p a r i s o n s as to s u r f a c e - b o u n d IgG o n d i f f e r e n t cells, t h a t excess 1 2 Si_labele d p r o t e i n A be used. Otherwise s o m e w h a t c o m p l i c a t e d binding curves o f t h e p r o b e m i g h t o c c u r , m a y be due to its valence f o r Fc being higher t h a n 1 (Sjoquist et al., 1 9 7 2 ) . N o r m a l l y we r e c o m m e n d t o use excess a m o u n t s o f b o t h t h e a n t i s e r u m against cellular
248 antigens as well as the protein A as this would make standardization easier. In our analysis of the influence of t e m per a t u re during incubation we found little effect, in contrast to reports using 125i.labele d antibodies in similar systems (Shumak et al., 1973). The reason for this is u n k n o w n to us. A stabilizing role of protein A for the surface-bound IgG molecules, however, can be seen from the washing experiments, where washing after alloantiserum but prior to the addition of I-pA would reduce the a m o u n t of I-pA bound. Washing after I-pA addition, on the other hand, indicated stability of the binding as no such further losses were found during repeated washes. Continuing the technical aspects, when using cells as targets in the I-pA assay we could demonstrate that microplate techniques were superior to multiple, individual tube assays. The advantage was n o t only apparent with regard to the time saved but also with regard to increase in specificity ratios obtained. Some examples of applications have been included. I-pA could be shown to bind in significant manner to normal l y m p h o c y t e s from guinea pig and man, but to a much lesser e x t e n t to rat or mouse l ym phocyt es. This difference is in direct agreement with the known variation in the binding capacity of protein A to IgG from these species (Kronvall et al., 1970a; Kronvall et al., 1970b), and would thus merely reflect at the cellular level what was known at the serum level. The results also indicate the fact that B lymphocytes actively producing IgG express enough IgG molecules on their surface to bind I-pA, thus confirming what has already been found for fluoresceinlabeled protein A (Dorval et al., 1974; Ghetie et al., 1974). The example of alloantibodies in the rat system demonstrates two things: a ) I g G antibodies, although derived from a species with ' p o o r ' protein A binding IgG can, when present as antibodies on multivalent structures such as cellular surface antigens, bind protein A in highly significant manner; b) It has long been argued whether cells from F1 -hybrid individuals derived from two parents differing at the major histocompatibility locus will express 50% of the concent rat i on of each of the parental antigens (Klein et al., 1970). The present data using saturating conditions for antibody and probe would argue in favour of the 50% value as the mean specific binding of the F1 -hybrid cells in comparison to the parental in table 6 is 49% versus 100%.
ACKNOWLEDGEMENTS This work was supported by the Canadian Medical Research Council to G.D. and by the Leverholme Trust to K.I.W. The work was supported by grants from the Swedish Cancer Society and by NIH-contract NO1-CB-33859 and NO1-CB-43883 to H.W. We are grateful to Peter Landvall for his generous help with Staph. aureus, and to Miss Berit Olsson and Mrs Ann SjSlund for their very skillful assistance.
249 REFERENCES Batchelor, J.R., K. Shumak and H. Watts, 1973, Transplantation 15, 80. BCyum, A., 1968, Scand. J. Clin. Lab. Invest. 21, 97. Dorval, G., K.I. Welsh and H. Wigzell, 1974, Scand. J. Immunol. 3,405. Ghetie, V., H.~. Fabricius, K. Nilsson and J. Sj6quist, 1974, J. Immunol. 26, 1081. Hunter, W.M. and F.C. Greenwood, 1962, Nature 194,495. Klein, G., U. Gars and H. Harris, 1970, Exptl. Cell Res. 62, 149. Kronvall, G. and R.C. Williams, Jr., 1969, J. Immunol. 103,828. Kronvall, G., U.S. Seal, J. Finstad and R.C. Williams, Jr., 1970a, J. Immunol. 104, 140. Kronvall, G., H.M. Grey and R.C. Williams, Jr., 1970b, J. Immunol. 105, 1116. Kronvall, G., U.S. Seal, S. Svensson and R.C. Williams, Jr., 1974, Acta Pathol. Microbiol. Scand. B, 82, 12. Lind, I., I. Live and B. Mansa, 1970, Acta Pathol. Microbiol. Scand. B, 78,673. Sanderson, A.R. and K.I. Welsh, 1974, Transplantation, in press. Shumak, K.H., J.R. Batchelor and H.G. Watts, 1973, Transplantation 1, 70. Sj6holm, I., A. Bjerken and J. Sj6quist, 1973, J. Immunol. 110, 1562. Sj6quist, J., B. Meloun and H. Hjelm, 1972, Eur. J. Biochem. 29,572. Welsh, K.I., G. Dorval and H. Wigzell, 1975, Nature 254, 67.
© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
A RADIOIMMUNOASSAY OF CELLULAR SURFACE ANTIGENS ON LIVING CELLS USING IODINATED SOLUBLE PROTEIN A FROM STAPHYLOCOCCUS A UREUS
G. DORVAL*, K.I. WELSH** and H. WIGZELL*** * Department o f Tumor Biology, Karolinska Institute, 104 01 Stockholm 60, Sweden ** McIndoe Research Unit, Queen Victoria Hospital, East Grinstead, Sussex, England *** Department o f Immunology, Uppsala University, Box 562, 751 22 Uppsala 1, Sweden
(Received 29 November 1974, accepted 1 December 1974)
Soluble protein A from Staphylococcus aureus does carry great promise as a marker for cellular surface determinants, due to its specific reaction with, and high affinity for, most subclasses of mammalian IgG. In this article we present the different parameters involved in a radioimmunoassay using 1 2 5i.labelled protein A. Using such an approach the actual technical procedures involved are reported in detail together with tests for mammalian alloantigens, including HL-A in the human, H-2 in the mouse and AgB antigenic sites in the rat, as this presents an unique opportunity to compare them with already widely used assays for transplantation antigens. The different parameters of the assay are analysed in view of measuring with precision quantities of cell-surface IgG molecules, thereby allowing possible determinations of antigenic site numbers in a new and simplified manner.
INTRODUCTION T h e s p e c i f i c r e a c t i o n o f S t a p h y l o c o c c u s a u r e u s p r o t e i n A w i t h t h e Fc r e g i o n o f m o s t m a m m a l i a n I g G m o l e c u l e s ( K r o n v a l l et al., 1 9 7 0 a ) has r e c e n t ly b e e n u s e d as a n e w i m m u n o l o g i c a l p r o b e in t h e a n a l y s i s of cell s u r f a c e m a r k e r s ( D o r v a l e t al., 1 9 7 4 ; G h e t i e et al., 1 9 7 4 ) . N o r m a l l y , cells are first c o m b i n e d w i t h an a n t i s e r u m d i r e c t e d a g a i n s t a given s u r f a c e d e t e r m i n a n t a n d then allowed to react with f l u o r e s c e i n - o r radioiodine-labelled soluble prot e i n A. C e l l - b o u n d I g G m o l e c u l e s , a c t i v e l y p r o d u c e d b y m a m m a l i a n l y m p h o c y t e s a n d c u l t u r e d cell lines, c a n also be r e v e a l e d w i t h t h e use o f fluoresc e i n a t e d p r o t e i n A ( F - p A ) ( D o r v a l et al., 1 9 7 4 ; G h e t i e et al., 1 9 7 4 ) ; this is c o n f i r m e d h e r e b y t h e use o f i 2 s I - l a b e l l e d p r o t e i n A ( I - p A ) . In t h e p r e s e n t a r t i c l e , t h e i m p o r t a n c e o f t h e v a r i o u s p a r a m e t e r s i n v o l v e d in t h e I - p A assay are r e p o r t e d in d e t a i l . T h e e f f e c t o f t h e n u m b e r o f w a s h e s , t e m p e r a t u r e a n d d u r a t i o n o f i n c u b a t i o n a t e a c h s t e p are a n a l y s e d . T h e d i r e c t assay (cells + a n t i s e r u m + I - p A ) is c o m p a r e d t o t h e i n d i r e c t assay w h e r e cells, a f t e r i n c u b a t i o n w i t h an a n t i s e r u m , are o v e r l a y e r e d w i t h an a n t i - I g G antis e r u m d i r e c t e d a g a i n s t t h e species a n t i b o d y c o m b i n e d w i t h t h e cells, a n d finally sandwiched with I-pA.
238 Also, the conventional 'multiple individual test tubes' radioimmunoassays require much time and material. Here, such assays are compared to a new fast procedure performed in microplates. Examples of applications of the present radioimmunoassays in mouse, rat, guinea pig and human systems are reported.
MATERIALS AND METHODS The purification of protein A has been described previously (Dorval et al., 1974). 12 s I-labelling o f p r o t e i n A : I-pA
Radioiodination of protein A was performed by the chloramine-T m e t h o d (Hunter and Greenwood, 1962). To 1 mCi 12SI-labeled sodium iodide (Amersham, IMS-30) were added 0.05 ml protein A (50 pg), and 0.005 ml chloramine-T (0.8 mg/ml) in 0.15 M sodium phosphate buffer, pH 7.5. It is important that the chloramine-T is not added in large excess of protein A as this might greatly impair the Fc binding capacity of protein A. The mixture reaction was allowed for 1 min at 23 °C with constant mixing, and terminated by the addition of 0.005 ml sodium metabisulfite (1 mg/ml). 0.25 ml of 5% ovalbumin solution in phosphate buffer, pH 7.1 (OA), was then added to the mixture, which was separated from free iodine by Sephadex G-25 chromatography and stored at --20 °C until use. Se ra
All sera were heat-inactivated for 30 min at 56 °C and kept at --20 °C until use; this complement deactivation is necessary since the IgG site of complement binding is close to that of protein A binding. A n t i - I g G sera
Sheep anti-human IgG has been described elsewhere (Dorval et al., 1974). Rabbit anti-mouse Ig and anti-rat Ig sera were obtained by immunization with I mg/ml of mouse or rat IgG in Freund's complete adjuvant. A lloan tisera
Rat and mouse alloantisera were raised using spleen cells by cross-immunization of animals maintained at the Department of Tumor Biology, Karolinska Institute, Stockholm. A n t i - H L - A sera
The Monkey anti-HL-A 2 serum is a well characterized monospecific reagent (Sanderson and Welsh, 1974).
239 Cells
H u m a n l y m p h o c y t e s were prepared f r om defibrinated peripheral blood using gradient centrifugation ( B o y u m , 1968), followed by iron carbonyl and magnetism. The four h u man lymphoblastoid cell lines (LCL:s) used here had been previously described, but in the present c o n t e x t it should only be recalled that the cell line 1 in table 3 is HL-A 2-negative, whereas the three others are HL-A 2-positive. T hey were kindly provided by Kenneth Nilsson. L y m p h o c y t e s f r om rat, mouse and guinea pig were prepared by gentle teasing of the freshly excised organ through a metal net, followed by one wash in PBS with 0.004 M EDTA, pH 7.4 (PBS-T), and two washes in BSS before use. Only cell preparations that contained m ore than 90% viable cells, as judged by T r y p a n blue exclusion, were used.
Cell-bound a n t i g e n - a n t i b o d y - - 12 Si_labele d p r o t e i n A assay: I-pA assay
The following scheme was f o u n d suitable for 10 6 cells per tube, or 0.1 × 10 6 cells per microwell. It should be n o t e d that the I-pA assay can function down to a few thousand cells provided the antigen density is high enough. Aliquots o f cells in 10--50 pl were added to I ml plastic tubes, or to individual 'U-shaped' microwells f r om 8 × 12 microtiter plastic plate (Cooke Instruments, A.G., Switzerland). When indicated, tubes or microwells were precoated with 10% ovalbumin (OA) or fetal calf serum, (FCS), to prevent non-specific sticking. When possible, antigen-saturating (according to pre-tests) amounts of antisera (1--10 pl) were added and the mixture incubated for 45 min at 4 °C, unless otherwise mentioned. 250 pl ice-cold medium (RPMI 1640) was then added and the tubes centrifuged for 3--5 min at 600 g before removal of supernatant. This washing step was repeated once. Cells on plates were washed by centrifugation in an International Centrifuge, model PRJ, 1000 rpm 6 rain followed by shaking out the supernatant with one, firm movement. Ten microliters of I-pA in k n o w n excess with regard to IgG binding in 5% OA was added per t ube or microwell and a further 30 min incubation at 4 °C carried out. This was followed by three washings as described above, but using PBS-T with or w i t h o u t 5% FCS as washing buffer. The cells were then transferred to tubes for counting o f radioactivity. When an indirect assay (cells + antiserum + anti-IgG + I-pA) was performed, the following steps were done after washing away the first antiserum. Ten microliters of antiserum to the initial a n t i b o d y was added per tube/microwell, and incubated for 30 min at 4 °C, followed by two washes, whereafter I-pA was added in the usual manner. For controls see the Results section.
240 RESULTS Experimental procedure Effect o f the n u m ber o f washes on the ~ 2 Si_labele d p A bound Preliminary e x p e r i m e n t s were carried o u t in o r d e r to o b t a i n o p t i m a l reaction c o n d i t i o n s t o g e t h e r with a minimal b a c k g r o u n d . D i f f e r e n t washing buffer solutions were tested. It was f o u n d t h a t 1) l y m p h o c y t e s were best washed t h r e e times prior to the test so as to avoid the presence o f loosely a t t a c h e d c y t o p h i l i c IgG which interferes with the test; 2) the viability o f the cells plays an i m p o r t a n t role because some dead cells can be s h o w n to take up I-pA in a non-specific m a n n e r to a larger e x t e n t t h a n do live cells; 3) the best b u f f e r solutions used for washings o f cells were Ca 2+/Mg 2+-free buffers c o n t a i n i n g a chelating agent (we used PBS, w i t h o u t Ca 2. and Mg 2÷, pH 7.4, with 4 mM E D T A ) and 2--5% ovalbumin. Such buffers w o u l d give the lowest b a c k g r o u n d values and the highest n u m b e r s of r e c o v e r e d cells; 4) the t r a n s f e r of cells to new tubes at the end o f the test caused a significantly lower b a c k g r o u n d , d u e to the a t t a c h m e n t of serum IgG a n d / o r I-pA to the wall o f the t u b e during the assay; 5) finally, if cell n u m b e r s at t h e end of the actual test or in parallel run controls c o u l d be assessed (we used a Celloscope, Linson I n s t r u m e n t s , S t o c k h o l m , Sweden), this w o u l d f u r t h e r r e d u c e the variations due t o t h e possible loss of cells during t h e e x p e r i m e n t . Table 1 presents t h e e f f e c t o f the n u m b e r o f washes o n the I-pA b o u n d o n DA rat l y m p h n o d e cells, using Lewis anti-DA alloantiserum. The specificity ratio o b t a i n e d r e a c h e d m a x i m u m with one c o m p l e t e removal o f s u p e r n a t a n t after the first i n c u b a t i o n , f o l l o w e d b y t w o washes after the s e c o n d incubation (table 1A). E x p e r i m e n t s were also d o n e using the indirect assay system. As s h o w n in table 1B, a single wash a f t e r alloantiserum f o l l o w e d b y t w o washes a f t e r each o f the t w o o t h e r stages p r o v e d necessary.
Effect o f time and temperature on the incubation with antiserum, and effect o f time on the incubation with I-pA Table 2 presents the results o b t a i n e d w h e n i n c u b a t i n g DA × Lewis F1 rat l y m p h n o d e cells with either Lewis anti-DA a n t i s e r u m or n o r m a l Lewis serum at varying t i m e and t e m p e r a t u r e , f o l l o w e d b y i n c u b a t i o n with I-pA at 4 °C f o r 30, 60, or 90 min. This e x p e r i m e n t was p e r f o r m e d in d u p l i c a t e t u b e s and the specific c o u n t s is the average o f a n t i s e r u m - t r e a t e d cells (a) minus n o r m a l serum (N). T w o washes were p e r f o r m e d using m e d i u m ( R P M I - 1 6 4 0 ) prior t o the a d d i t i o n o f I-pA, and t h r e e washes with PBS-T were d o n e a f t e r the i n c u b a t i o n with I-pA. In brackets are the specific cps o b t a i n e d with the indirect assay, w h e r e cells prior t o the a d d i t i o n o f I-pA were i n c u b a t e d for 30 rain at 4 ° C with a rabbit anti-rat Ig, f o l l o w e d by t w o washes. Optimal
241 TABLE 1 Effect of numbers of washes on the 128Icounts bound (cpm/106 cells). A. Direct assay Number of pre-washes:
1 2 3 4
Number of post-washes: 1 2
3
4
13,887 (387) 12,113 (812) 9624 (237) 8561
9865 (120) 8872 (130) 6077 (121) 4421
9804 (123) 8566 (101) 5765 (98) 4376
3
4
10,009 (148) 9019 (139) 6271 (162) 4899
B. Indirect assay Number of pre-washes: 1 2 3
Number of post-washes: 1 2 23,092 (13111) 18,011 (7211) 15,201 (6866)
19,820 (4760) 17,772 (4552) 13,981 (4277)
19,620 (3998) 17,521 (4443) 13,700 (4144)
19,799 (4421) 17,641 (4420) 13,977 (4019)
- - T h e first column (number of pre-washes) gives the number of washes prior to the addition of protein A. Thus in A, the number of washes after the incubation with the alloantiserum, and in B, the number of washes after the anti-IgG, the number of washes after the alloantiserum being here one. - - T h e first row (number of post-washes) gives the number of washes after the protein A incubation.
b i n d i n g was a l r e a d y a c h i e v e d using 30 m i n p e r i o d s o f i n c u b a t i o n , w i t h t e m perature being of relatively little importance. In d o i n g e x p e r i m e n t s u n d e r c o n d i t i o n s n o t i n v o l v i n g a n t i b o d y a n d / o r I - p A excess, i n c u b a t i o n p e r i o d s s h o u l d be i n c r e a s e d t o 6 0 - - 9 0 m i n t o a c h i e v e optimal binding.
Comparison o f the direct (I-pA-alloantibody--cell) assay with the indirect (I-pA--anti-IgG--alloantibody--cell) assay P r e l i m i n a r y c o m p a r i s o n s o f t h e d i r e c t a n d i n d i r e c t I - p A a s s a y s have b e e n r e p o r t e d e l s e w h e r e . As p r o t e i n A will b i n d t o a v a r y i n g d e g r e e t o I g G sub-
242 TABLE 2 Effect of time and temperature of incubation with antiserum and effect of time of incubation with I-pA (cps/0.2 × 106 cells). Effect of
Time of Inc.
T ~ of inc.
10 (min)
4°C
38 41
15 13
26
53 50
13 11
40
42 45
8 7
59 61
14 13
42
56 53
12 13
41(55)a
61 64
13 17
48(55) a
59 54
13 12
44
59 53
15 12
43
70 73
13 18
51
42 42
12 12
30
50 58
10 16
41
58 55
11 12
46
53 56
13 12
42
54 54
14 12
41(56) a
54 57
12 12
44(56) a
53 53
11 8
43
63 61
10 15
50
80 65
19 15
56
45 43
12 9
34
45 48
7 9
39
46 46
10 11
36
51 51
12 10
40
52 54
12 12
41(57) a
59 59
12 12
47(59)a
63 64
11 13
52
64 54
13 16
46
79 75
19 15
62
~
N
spec ~
N
spec
~
N
spec
22°C
37°C
With antiserum
Time of inc. at 4°C 30 (min)
60 (min)
(min) 36
30 60 9O 3O 60 9O 30 60 90 With I-pA
a Specific cps in indirect assay, with anti-Ig incubation time of 30 min at 4°C.
classes o f d i f f e r e n t m a m m a l s ( K r o n v a l l a n d W i l l i a m s , 1 9 6 9 ; L i n d e t al., 1 9 7 0 ; K r o n v a l l e t al., 1 9 7 4 ) , t h e o u t c o m e o f d i r e c t a n d i n d i r e c t I - p A a s s a y w o u l d largely depend on the species providing the IgG molecules to be tested. Thus such c o m p a r i s o n s are of m o r e practical t h a n t h e o r e t i c a l i m p o r t a n c e . Table 3 s h o w s h o w t h e a m o u n t o f I - p A b o u n d t o h u m a n cells is o n l y i n f l u e n c e d t o a m i n o r e x t e n t b y t h e i n c l u s i o n o f a n e x t r a l a y e r o f Ig m o l e c u l e s . I t is also noted that the values obtained with control normal monkey serum do not c h a n g e in t h e i n d i r e c t a s s a y in p a r t A , as t h e h u m a n c e l l l i n e s u s e d w e r e s u r f a c e I g G n e g a t i v e . T h e m o u s e s y s t e m is c o n s i d e r a b l y a f f e c t e d b y t h e a d d i t i o n o f a n a n t i - I g s e r u m b u t t h i s is d u e t o t h e f a c t t h a t t h e n o r m a l m o u s e l y m p h n o d e cel l s c o n t a i n a p p r o x i m a t e l y 2 0 % B cells.
243 TABLE 3 Comparison of the direct (I-pA-alloantibody-cell) assay with the indirect (I-pA-anti-Igalloantibody-cell) assay. A. Binding to human cell lines (cps/10~ cells) Cell line Direct assay Indirect assay 1 250 (10) 305 (10) 2 785 (10) 1050 (65) 3 1200 (10) 1300 (10) 4 800 (15) 915 (15) The results are obtained with a monkey anti-HL-A 2 antiserum compared to results obtained with normal human serum (in brackets). Cell line 1 is HL-A 2 negative. Sheep anti-human IgG was used as the second antiserum. B. Binding to mouse lymph node cells (cps/106 cells) Direct assay Indirect assay 145 (54) 270 (150) The results are obtained with an anti-H-2 b antiserum compared to results obtained with syngeneic normal mouse serum, on H-2 b lymph node cells. Rabbit anti-mouse Ig was used as the second antiserum.
The I-pA assay done in wells of microtiter plates in comparison with the conventional multiple individual test tubes radioimmunoassay Time and material are limiting f a c t o r s in multiple i m m u n o a s s a y s . Originally, the c o n v e n t i o n a l multiple individual test t u b e s r a d i o i m m u n o a s s a y was f o u n d to require c o m p a r a t i v e l y long m a n i p u l a t i o n s w h i c h impaired the rep r o d u c i b i l i t y . Also, relatively large a m o u n t s o f cells and sera were used. This p r o m p t e d us to t u r n to the use o f m i c r o t i t e r plates in w h i c h several plates, each c o n t a i n i n g up t o 96 samples, c o u l d be c e n t r i f u g e d at o n c e and washed b y m e r e l y shaking o u t the s u p e r n a t a n t . This is in sharp c o n t r a s t to the ' t u b e s ' assay w h e r e the pellets had to be carefully avoided during the removal o f the s u p e r n a t a n t . Preliminary e x p e r i m e n t s were carried o u t in view o f assessing the possible increase o f cell losses using t h e plate s y s t e m . Surprisingly, w h e n the original cell suspension had a g o o d viability, o n l y a few per c e n t o f t h e cells were lost d u r i n g t h e e x p e r i m e n t a l p r o c e d u r e . Table 4 presents t h e results o f a ' p l a t e ' assay run in parallel with a ' t u b e ' assay, and u n d e r similar e x p e r i m e n t a l c o n d i t i o n s e x c e p t f o r the n u m b e r o f cells being d i f f e r e n t in the t w o systems. L y m p h n o d e cells f r o m a H-2 m o u s e were first t r e a t e d for 45 min at 4 °C with either n o r m a l H-2 ~ m o u s e serum {first r o w ) , or with an H-2 f anti-H-2 k a l l o a n t i s e r u m ( s e c o n d row). Vertical c o l u m n s c o m p a r e the e f f e c t o f p r e c o a t i n g the t u b e s or t h e wells o f m i c r o titer plates with 100% fetal calf serum (FCS), or with 1% O A , or w i t h o u t p r e c o a t i n g (O), as well as the e f f e c t o f a d d i n g FCS in the washing b u f f e r after the i n c u b a t i o n with I-pA. A l t h o u g h a t t e m p t i n g t o d o the tests in paral-
+ NMS
Sera a d d e d :
+ anti-H-2
+ NMS
n.d. = n o t d e t e r m i n e d .
In wells cells o f 0.2 x 10 (' (corrected for 106)
(corrected for 106 ) + anti-H-2
In tubes cells 0.5 × 106
Washes after I-pA Precoated with
169 1.901 (9.505)
4.432 (8.864)
4.900 (9.800) 246 2.520 (12.600)
870
695
PBS-T100% FCS 1% OA-RPMI
10Xl :5
I-pA assay: in tubes vs. m i c r o p l a t e wells (cpm).
TABLE 4
191 2.539 (] 1.695)
3.861 (7.722)
739
261 2.739 (13.685)
3.270 (6.540)
562
208 2.259 (11.295)
3.847 (7.694)
640
PBS-T- + 7.5% FCS 100% FCS 1% OA-RPMI
250 2.789 (13.945)
5.613 (11.226)
868
n.d.
ll.d.
4.781
714
PBS-T + 7.5% FCS 0
lOx undil.
d~
b~
245 lel t h e t e c h n i c a l l y t e n u o u s ' t u b e ' assay r e q u i r e d m u c h m o r e t i m e f o r t h e individual washing p r o c e d u r e s resulting in a m a r k e d d e l a y in p e r f o r m i n g t h a t assay in c o m p a r i s o n w i t h t h e p l a t e assay. I - p A was k n o w n to have b e e n a d d e d in excess, w h e r e a s the last c o l u m n s h o w s t h a t increasing t h e c o n c e n t r a t i o n o f a l l o a n t i s e r u m b y 5-fold did n o t cause an increase o f the c o u n t s b o u n d . T h u s it was a d d e d also in excess. T h e a p p a r e n t d i s c r e p a n c y b e t w e e n the results o b t a i n e d w i t h t h e t w o tests c a n n o t be fully e x p l a i n e d . F r o m several e x p e r i m e n t s , h o w e v e r , we can c o n c l u d e t h a t b o t h w i t h regard to t h e t o t a l I - p A b o u n d to I g G - c o a t e d cells a n d with regard to l o w b a c k g r o u n d values t h e m i c r o p l a t e assay w o u l d s e e m s u p e r i o r t o t h e t u b e assay.
Application of the technique Direct binding of I-pA on normal, uncoated mammalian lymphocytes T h e direct b i n d i n g o f I - p A to n o r m a l , u n c o a t e d guinea pig, h u m a n , rat, a n d m o u s e l y m p h o i d cells has b e e n investigated {table 5). I n c r e a s i n g n u m bers o f l y m p h o c y t e s , 1 × 106 , 2 X 106 , and 5 × 106 cells w e r e r e a c t e d with I - p A in t u b e s f o r 45 m i n at 4°C. T w o washes w i t h PBS-T were s u b s e q u e n t l y d o n e b e f o r e t h e cells w e r e t r a n s f e r r e d t o o t h e r t u b e s a n d w a s h e d o n c e m o r e Binding
of
l-pA on mammalian
lymphocytes
cpm
~G.p.
1500
i /
1000
500
/ 1°
~
i
/
"
"
~ M o u s e
-__
.._.-o R~t
1 2 3 Number of cells x 10 6 : e without SRBC _ o. . . . .
--o w i t h
150 x IO b SRBC
Fig. 1. H = human peripheral blood lymphocytes. G.p. = Guinea pig lymph node cells. Mouse and rat = lymph node cells of respectivespecies.
246 TABLE 5 Direct binding o f I-pA on m a m m a l i a n l y m p h o c y t e s ( c p m ) Cells:
N u m b e r × 105 + S R B C × 106(a)
Guinea pig-spleen Guineapig-LNC(c) Human-periph. Rat-LNC Mouse-LNC No ceels: 206 (a) S R B C a l o n e : × 106 (cpm)
1 0 610 270 362 204 280
2 0 900 366 550 228 258
40 198
50 196
5 0 1720 598 822(b) 214 388
60 216
1 150 872 258 416 234 270
75 228
2 5 150 150 1250 1918 328 570 574 n.d. 258 286 240 386
100 248
120 150 256 258
(b) only 3 × 106 l y m p h o c y t e s . (c) LNC = l y m p h n o d e cells. n.d. = n o t d e t e r m i n e d .
with the s a m e buffer. In o r d e r to avoid possible increase o f n o n - s p e c i f i c t r a p p i n g o f I - p A with an increase of t h e cell mass, parallel t u b e s were r u n in the s a m e fashion with a c o n s t a n t high n u m b e r o f s h e e p e r y t h r o c y t e s (SRBC). C o n t r o l s with increasing n u m b e r s o f SRBC alone s h o w a l m o s t no d i f f e r e n c e w h e n c o m p a r e d to t u b e s which had received o n l y I - p A and no cells ( b a c k g r o u n d ) . When b a c k g r o u n d binding values w e r e s u b t r a c t e d , a very nice c o r r e l a t i o n c o u l d be seen b e t w e e n the binding a n d the increasing n u m ber of guinea pig a n d h u m a n l y m p h o c y t e s (fig. 1). In c o n t r a s t , values obtained with m o u s e a n d rat l y m p h o i d cells s h o w o n l y m a r g i n a l increase a b o v e the b a c k g r o u n d .
Quantitative detection o f DA anti-Lewis and Lewis anti-DA antibody bound to Lewis, DA, or (Lewis × D A ) F1 lymphoid cells T w o a n t i b o d y c o n c e n t r a t i o n s and t w o p r o t e i n A c o n c e n t r a t i o n s , b o t h t h o u g h t to be in excess, were used in a direct assay p e r f o r m e d in individual t u b e s on rat l y m p h n o d e cells. C o l u m n s A a n d B (table 6) in t e r m s of c o u n t s b o u n d are n o t m a r k e d l y d i f f e r e n t s h o w i n g t h a t p r o t e i n A is in excess; t h e c o n t r o l s are, h o w e v e r , b e t t e r in t h e l o w e r excess case. T h e results in g r o u p s 1 and 2 should be identical if D A anti-Lewis is in excess; h o w e v e r , t h e cps are less in r o w 2 s h o w i n g t h a t 10 pl a n t i s e r u m is i n s u f f i c i e n t to s a t u r a t e 106 cells (the t i t e r o f this a n t i s e r u m in the T r y p a n blue test was 1 : 4). T h e results in g r o u p s 3 and 4 are similar showing t h a t the Lewis a n t i - D A was in excess (titer 1 : 40 in t h e T r y p a n blue). It is also n o t i c a b l e t h a t t h e F1 c o u n t s are o n l y half the p a r e n t a l c o u n t s if t h e b a c k g r o u n d is l o w and the a n t i s e r u m in excess. F o r c o m p a r a t i v e tests in antigenic sites d e t e r m i n a t i o n u n d e r n o n excess c o n d i t i o n s , it is necessary to adjust the cell n u m b e r s to give a p p r o x i m a t i v e l y equal n u m b e r s o f c o u n t s b o u n d , as suggested p r e v i o u s l y ( B a t c h e l o r et al., 1973).
247 TABLE 6 Use of the I-pA assay to quantitate the binding of DA anti-Lewis, and Lewis-anti-DA, on DA, Lewis, and (DAxLewis) Fl rat lymph node cells. Antiserum
Cells (10 6 )
cps bound A B
cps specific A B
DA anti-Le (20/11)
Le DA FI Le DA F1
215 86 150 185 21 122
256 39 148 187 12 124
129
217
64 164
109 175
101
112
Le anti-DA (20/21)
Le DA FI
107 316 163
33 324 158
219 59
291 125
Le anti-DA (10 pl)
Le DA FI
43 284 158
13 282 152
246 115
269 139
DA anti-Le (10 pl)
A 1.7 × 106 counts I-pA added. B 2.2 × 106 counts I-pA added.
DISCUSSION P r o t e i n A f r o m S t a p h y l o c o c c u s aureus has affinity for t h e Fc region o f m o s t m a m m a l i a n IgG subclasses (Kronvall et al., 1970a). It can thus be used as a p r o b e for c e l l - b o u n d IgG molecules, and p r e l i m i n a r y d a t a suggest it as a very useful p r o b e in the analysis o f cellular antigens (K.I. Welsh, G. Dorval and H.W. Wigzell, 1975). I n the p r e s e n t article we describe the technical details o f i m p o r t a n c e w h e n using 12 Si_labele d p r o t e i n A as cell surface marker, as t h e t e c h n i q u e has n o t been described to a n y e x t e n t previously. Starting with the labeling p r o c e d u r e s we have f o u n d it o f great i m p o r t a n c e n o t to use t h e c o n v e n t i o n a l r e c o m m e n d e d c o n c e n t r a t i o n s o f C h l o r a m i n e - T ( H u n t e r a n d G r e e n w o o d , 1962), b u t r a t h e r a c o n c e n t r a t i o n n o t above 4 mM. A n increase a b o v e this m o l a r i t y w o u l d t e n d to h a r m p r o t e i n A and m a k e it less f u n c t i o n a l in the assay. Also, trace labeling o f p r o t e i n A with 12 5I aiming at o n e iodine per p r o t e i n - m o l e c u l e should be p e r f o r m e d as binding of iodine t o all f o u r t y r o s y l residues f o u n d in t h e p r o t e i n A m o l e c u l e (SjSquist et al,, 1 9 7 2 ) w o u l d seem to d e s t r o y t h e Fc binding activity ( S j S h o l m et al., 1973). It is i m p o r t a n t , w h e n d o i n g q u a n t i t a t i v e c o m p a r i s o n s as to s u r f a c e - b o u n d IgG o n d i f f e r e n t cells, t h a t excess 1 2 Si_labele d p r o t e i n A be used. Otherwise s o m e w h a t c o m p l i c a t e d binding curves o f t h e p r o b e m i g h t o c c u r , m a y be due to its valence f o r Fc being higher t h a n 1 (Sjoquist et al., 1 9 7 2 ) . N o r m a l l y we r e c o m m e n d t o use excess a m o u n t s o f b o t h t h e a n t i s e r u m against cellular
248 antigens as well as the protein A as this would make standardization easier. In our analysis of the influence of t e m per a t u re during incubation we found little effect, in contrast to reports using 125i.labele d antibodies in similar systems (Shumak et al., 1973). The reason for this is u n k n o w n to us. A stabilizing role of protein A for the surface-bound IgG molecules, however, can be seen from the washing experiments, where washing after alloantiserum but prior to the addition of I-pA would reduce the a m o u n t of I-pA bound. Washing after I-pA addition, on the other hand, indicated stability of the binding as no such further losses were found during repeated washes. Continuing the technical aspects, when using cells as targets in the I-pA assay we could demonstrate that microplate techniques were superior to multiple, individual tube assays. The advantage was n o t only apparent with regard to the time saved but also with regard to increase in specificity ratios obtained. Some examples of applications have been included. I-pA could be shown to bind in significant manner to normal l y m p h o c y t e s from guinea pig and man, but to a much lesser e x t e n t to rat or mouse l ym phocyt es. This difference is in direct agreement with the known variation in the binding capacity of protein A to IgG from these species (Kronvall et al., 1970a; Kronvall et al., 1970b), and would thus merely reflect at the cellular level what was known at the serum level. The results also indicate the fact that B lymphocytes actively producing IgG express enough IgG molecules on their surface to bind I-pA, thus confirming what has already been found for fluoresceinlabeled protein A (Dorval et al., 1974; Ghetie et al., 1974). The example of alloantibodies in the rat system demonstrates two things: a ) I g G antibodies, although derived from a species with ' p o o r ' protein A binding IgG can, when present as antibodies on multivalent structures such as cellular surface antigens, bind protein A in highly significant manner; b) It has long been argued whether cells from F1 -hybrid individuals derived from two parents differing at the major histocompatibility locus will express 50% of the concent rat i on of each of the parental antigens (Klein et al., 1970). The present data using saturating conditions for antibody and probe would argue in favour of the 50% value as the mean specific binding of the F1 -hybrid cells in comparison to the parental in table 6 is 49% versus 100%.
ACKNOWLEDGEMENTS This work was supported by the Canadian Medical Research Council to G.D. and by the Leverholme Trust to K.I.W. The work was supported by grants from the Swedish Cancer Society and by NIH-contract NO1-CB-33859 and NO1-CB-43883 to H.W. We are grateful to Peter Landvall for his generous help with Staph. aureus, and to Miss Berit Olsson and Mrs Ann SjSlund for their very skillful assistance.
249 REFERENCES Batchelor, J.R., K. Shumak and H. Watts, 1973, Transplantation 15, 80. BCyum, A., 1968, Scand. J. Clin. Lab. Invest. 21, 97. Dorval, G., K.I. Welsh and H. Wigzell, 1974, Scand. J. Immunol. 3,405. Ghetie, V., H.~. Fabricius, K. Nilsson and J. Sj6quist, 1974, J. Immunol. 26, 1081. Hunter, W.M. and F.C. Greenwood, 1962, Nature 194,495. Klein, G., U. Gars and H. Harris, 1970, Exptl. Cell Res. 62, 149. Kronvall, G. and R.C. Williams, Jr., 1969, J. Immunol. 103,828. Kronvall, G., U.S. Seal, J. Finstad and R.C. Williams, Jr., 1970a, J. Immunol. 104, 140. Kronvall, G., H.M. Grey and R.C. Williams, Jr., 1970b, J. Immunol. 105, 1116. Kronvall, G., U.S. Seal, S. Svensson and R.C. Williams, Jr., 1974, Acta Pathol. Microbiol. Scand. B, 82, 12. Lind, I., I. Live and B. Mansa, 1970, Acta Pathol. Microbiol. Scand. B, 78,673. Sanderson, A.R. and K.I. Welsh, 1974, Transplantation, in press. Shumak, K.H., J.R. Batchelor and H.G. Watts, 1973, Transplantation 1, 70. Sj6holm, I., A. Bjerken and J. Sj6quist, 1973, J. Immunol. 110, 1562. Sj6quist, J., B. Meloun and H. Hjelm, 1972, Eur. J. Biochem. 29,572. Welsh, K.I., G. Dorval and H. Wigzell, 1975, Nature 254, 67.
