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Sensitive and convenient quantitation of antibody binding to cellular antigens using glutaraldehyde preserved cells
Journal o f l m m u n o l o g i c a l Methods, 26 (1979) 3 6 9 - - 3 8 0
369
© Elsevier/North-Holland Biomedical Press
SENSITIVE AND CONVENIENT QUANTITATION OF ANTIBODY BINDING TO CELLULAR ANTIGENS USING GLUTARALDEHYDE PRESERVED CELLS
S. D A V I D R O C K O F F , K. R O B E R T M c I N T I R E , A H - K A U NG, G E R A L D L. P R I N C L E R , R O N A L D B. H E R B E R M A N and J O H N N. L A R S O N
Department o f Radiology, The George Washington University Medical Center, 901 23rd Street, N.W., Washington, DC 20037, and the Laboratory o f lmmunodiagnosis, National Cancer Institute, National Institutes o f Health, Bethesda, MD 20014, U.S.A. (Received 2 May 1978, a c c e p t e d 17 O c t o b e r 1978)
As a preliminary step in the identification and isolation o f antibodies to h u m a n cancers, we have developed a sensitive and c o n v e n i e n t assay for a n t i b o d y binding to cellular antigens. The basis for the m e t h o d is a n t i b o d y binding to glutaraldehyde-fixed cells ( A b G f C ) and q u a n t i t a t i o n with r a d i o i o d i n a t e d staphylococcal protein A (SPA). Glutarald e h y d e fixation of intact cells, which does n o t appear to affect the ability to form antigen-antibody complexes, provides a c o n v e n i e n t and standard supply of target cells which m a y be stored at 4°C and used in the assay over a period of several months. The a m o u n t o f a n t i b o d y specifically b o u n d to the cells is q u a n t i t a t e d by the addition o f 12sIlabeled SpA. The sensitivity of the m e t h o d was c o m p a r e d with t w o c o m p l e m e n t - d e p e n d e n t c y t o t o x i c i t y m e t h o d s (trypan blue exclusion and SlCr release assays) and tested with t w o antisera to h u m a n lung cancer and one antiserum to a m e m b r a n e antigen o f a m u r i n e l y m p h o m a . These comparisons indicated m u c h greater sensitivity when c o m p a r e d with the trypan blue exclusion assay and equivalent sensitivity with greater dose response characteristics w h e n c o m p a r e d with the s 1Cr release assay.
INTRODUCTION
Over the years there has been a great interest in looking for antibodies directed against human tumor-associated antigens as well as other cell surface antigens. Using available techniques, it has been very difficult to docum e n t the existence of such antibodies in the sera o f cancer patients or to define completely the specificity of heterologous antibodies to tumorassociated antigens on cell surfaces. Part of the problem has been related to the reproducibility, reliability, and ease o f application of existing assay methods. There are many methods for measuring the reactivity of antibody with cell surface antigens and these have been reviewed by Ting and Herberman (1976). Recently, Rosenberg et al. (1977) compared multiple assays for detecting human antibodies directed against surface antigens. They determined that the visual end point cytolysis assay and the SlCr release methods
370 were equally sensitive in measuring complement-mediated antibody cytotoxicity and that both were far more sensitive than the trypan blue exclusion assay. A hemadsorption technique was 10 times more sensitive than the c y t o t o x i c i t y assays but exact quantitation was difficult because of the visual end point. The SlCr release assay was unstable and they were unable to correct for day-to-day variations. The current work in this laboratory is directed toward the identification and isolation of antibodies to human cancers, with special interest in cancer of the lung. Our research requires the availability of a convenient and sensitive assay m e t h o d of antibody-antigen binding. We were, therefore, interested in the m e t h o d for the rapid quantitation of membrane antigens using 12SI-labeled protein A, derived from S t a p h y l o c o c c u s aureus, described by Welsh et al. (1975). The assay they described offered a complement-independent radioimmunoassay applicable to a wide range of mammalian membrane antigens. Their assay has a sensitivity similar to that of the trypan blue exclusion and SlCr assays but they believed it to be more linear and thus more precise than these two tests. A major disadvantage of their assay, however, was that it depended upon the availability of fresh target cells, which could vary considerably from one experiment to another. The recent description of glutaraldehyde fixation of mouse l y m p h o m a (EL4BU and P388) cells onto concanavalin A-coated Sephadex beads by Sela and Edelman (1977), for the purpose of isolation of immunoglobulins specific for cell surface carbohydrates by cell column chromatography, led us to explore the development of a ~2SI-labeled protein A assay utilizing glutaraldehyde-fixed target cells. The purpose of this communication is to describe this assay which has the properties of sensitivity, convenience, rapidity and reproducibility. MATERIALS AND METHODS A n tisera
Pooled normal rabbit serum (NRS) was prepared from 5 normal, adult New Zealand white rabbits. Antisera against h u m a n lung cancer extract were prepared by the intramuscular injection of the extract (approximately 3 mg protein), prepared in 1.2 ml of complete Freund's adjuvant (Difco Lab., Detroit, MI), into adult New Zealand white rabbits. Antisera against cultured h u m a n lung cancer cells were prepared by the injection of 5--10 million Chago cells (Rabson et al., 1973), emulsified in complete Freund's adjuvant. Blood was taken from the ear veins beginning approximately 1 m o n t h after injection. Sera were centrifuged at 30,000 × g for 30 min in a refrigerated centrifuge to remove small particular matter before use, and stored at --20°C. In most experiments, 1 : 80 dilutions of antisera and NRS were used. This dilution was selected after it was observed to be the lowest dilution which could be consistently discriminated from background. Use of this
371 end point dilution facilitated the complete adsorption of antibody with a moderate number of cells.
Target cells Cultured cells were obtained by scraping the culture flasks, followed by washing 2 times in cold (4°C) PBS with vigorous vortexing between washes to separate the cell clumps. After each wash, the cells were centrifuged at 500 × g for 10 min. The cells to be fixed in glutaraldehyde after the final wash and centrifugation were vigorously vortexed, poured through a double layer of c o t t o n gauze into a new centrifuge tube with 10 ml of PBS, and then enough 3% glutaraldehyde (Practical grade, Eastman Kodak Co., Rochester, NY) was added to make a total volume of 50 ml. The cells in the glutaraldehyde solution were then continuously and gently mixed with an electric rotator (6 rev/min) at room temperature for 30 min, after which they were centrifuged for 10 min at 500 × g, washed 2 times with PBS and filtered through a double layer of gauze into a fresh tube, to be stored at 4 ° C until needed. Identical assay results were obtained with Chago target cells which had been fixed in glutaraldehyde for intervals ranging from 30 min to 48 h, when tested with an antiserum against human lung cancer extract (R-149) and with NRS. For storage purposes, 0.02% sodium azide solution (in PBS) was used to retard bacterial growth. The cell lines used in the experiments included Chago cultured human lung cancer, K-562 cultured myeloid leukemia cells (Lozzio and Lozzio, 1975) and MBL-2 mouse l y m p h o m a cells (Ng et al., 1977). Preparation o f [~2SI]protein A Purified, lyophilized protein A from Staphylococcus aureus Cowan strain I (Pharmacia, Uppsala, Sweden) was radioiodinated by a modification o f the m e t h o d of Bolton and Hunter (1973). Briefly, protein A (100 pg) in 0.1 ml of 0.01 M phosphate-buffered saline (PBS), pH 7.2, was added to 0.5 mCi of dried Bolton-Hunter reagent (New England Nuclear Corp., Bostot~, MA) and the reaction mixture was vortexed every 2 min for 15 min at 2 5 ° ( ', The reaction was stopped by addition of 1 ml of 0.2 M glycine in PBS to react with the unchanged Bolton-Hunter reagent for 5 min at 25 ° C. The 12sIlabeled protein A was separated from the other products and the reagent by gel filtration on Sephadex G-25 (PD-10, Pharmacia, Uppsala, Sweden) equilibrated with PBS containing 0.25% (w/v) gelatin. The first peak of radioactivity eluted from the Sephadex G-25 column in the void volume of the column was pooled, aliquoted and stored at --20°C. Specific activity of [12SI]protein A, assuming 100% recovery of protein A, was approximately 2 pCi/pg and subsequent preparations of [a2SI]protein A were adjusted to achieve this specific activity. Precipitability of [~2SI]protein A with 10% TCA was at least 85%.
372 Assay p r o c e d u r e using glutaraldehyde-fixed target cells The target cells were taken from storage and observed under the microscope after vortexing. If clumping had occurred, additional washing and filtration as described above were p e r f o r m e d until single cells predominated. The cells were then c o u n t e d in a h e m o c y t o m e t e r . Centrifuge tubes containing known numbers o f cells were centrifuged at 500 X g for 10 min, then supernatant fluid withdrawn and the cells resuspended in 1% bovine serum albumin (BSA) (Reheis Chem. Co., Phoenix, AZ) in PBS at 5 X 10 ~ cells/ml. Into 12 mm X 75 m m glass culture tubes (Coming Glass Works, Coming, NY) were placed 100 pl of antiserum, NRS or PBS (as a background control) and then 100 pl of the cell suspension were added. Random i zat i on consisted o f adding coded antisera and cells and then treating all tubes alike, w i t h o u t reference to sequence. The tubes containing the antiserum, NRS or PBS and the cells in BSA were v o r t e x e d and incubated at r o o m t e m p e r a t u r e for 90 min, vortexing each t ube every 30 min. Then, 1 ml of cold PBS was added to each tube, the tubes were vor t e xe d, an additional 1 ml was added to each tube, v o r t e x e d again, and the tubes were centrifuged at 1800 X g for 10 min. T h e tubes were decanted, bl ot t ed and the washing procedure was repeated with PBS. After the second wash, 100 pl of [12SI]protein A (adjusted so t hat each aliquot contained 10 s c ount s / m i n) was added to each t ube and incubated for 90 min, vortexing initially and then every 30 min during the incubation. At the end o f the incubation, 1 ml of 1% BSA in P B S was added to each tube and, with vortexing, 1 ml of PBS was added and the tubes were centrifuged at 1800 X g for 10 min, decanted and blotted. The washing, centrifugation, decanting and blotting were repeated, after which the cont ent s of each tube were transferred to a fresh tube by adding 1 ml of 1% BSA, vortexing, pipetting, and then rinsing with 2 ml o f 1% BSA and pipetting, for a total volume o f 3 ml o f transfer solution. A final centrifugation, decantation and blotting were p e r f o r m e d as above and the pellets were c o u n t e d for 1 min. Trypan blue exclusion assay The t r y p a n blue exclusion c y t o t o x i c i t y assay, used to compare with the assay m e t h o d being described in this report, is essentially the same as t hat described previously (Takahashi et al., 1971; Princler and McIntire, 1975). After an incubation of the cultured Chago cells with the sera for 45 rain at 37 ° C, 0.4% t r y p a n blue in normal saline (Grand Island Biological Co., Grand Island, NY) was added. Fresh-frozen normal guinea pig serum (NGPS, prepared in o u r labor at or y) was diluted 1 : 4 as a c o m p l e m e n t source. Control tubes contained cells and diluent (Medium-199, Grand Island Biological Co.), cells and NGPS, and cells and antiserum. s 1Cr release cy to toxic i ty assay The StCr release assay used was described previously by Herberman (1972) and modified as follows. Fifty microliters of SlCr-labeled target cells, con-
373 taining 1.0--2.5 × 104 cells, were a d d e d to 25 pl o f serial, 2-fold dilutions o f serum. A f t e r i n c u b a t i o n at 37 ° C f o r 45 min, 50 pl o f I : 3 dilution o f r a b b i t serum ( G r a n d Island Biological Co.), which had b e e n p r e s c r e e n e d for lack o f c y t o t o x i c i t y , were a d d e d as the s o u r c e o f c o m p l e m e n t . T h e i n c u b a t i o n was t h e n c o n t i n u e d f o r 90 min at r o o m t e m p e r a t u r e . T h e r e a c t i o n was termin a t e d b y adding 2 ml o f Roswell Park Memorial I n s t i t u t e M e d i u m 1 6 4 0 (RPMI) c o n t a i n i n g 10% fetal bovine serum (FBS) ( G r a n d Island Biological Co.) at 4 °C. T h e cells were s e d i m e n t e d b y c e n t r i f u g a t i o n and the supernat a n t fluid was c o u n t e d f o r released r a d i o a c t i v i t y . C y t o t o x i c activities o f the antisera were calculated as follows: % c y t o t o x i c i t y = [ ( ~~Cr released f r o m a n t i s e r u m - t r e a t e d cells -- ~' Cr released f r o m c o m p l e m e n t c o n t r o l ) / ( T o t a l SlCr i n c o r p o r a t e d b y target cells)] × 100. RESULTS
Optimization o f assay procedures Selection o f optimum number o f cells per assay tube. Table 1 lists t h e c o u n t s / m i n ( c p m ) o b t a i n e d using Chago c u l t u r e d h u m a n lung c a n c e r cells as t h e target cells, varying the n u m b e r o f cells f r o m 104 to 107 cells per assay tube. T h e a n t i s e r u m t e s t e d was R-149, d e v e l o p e d in rabbits against fresh h u m a n lung c a n c e r e x t r a c t . As a c o n t r o l f o r non-specific binding o f i m m u noglobulin, NRS was used and, as a b a c k g r o u n d c o n t r o l , PBS was tested. As can be seen in Table 1, the c p m increased c o n t i n u o u s l y with increasing n u m b e r s o f cells for t h e a n t i s e r u m , the N R S and f o r t h e PBS. H o w e v e r , the ability to discriminate the a n t i s e r u m f r o m the N R S was d e t e r m i n e d b y a 'specific binding i n d e x ' , d e t e r m i n e d b y the f o r m u l a : ( a n t i s e r u m -- PBS) -- (NRS -- PBS) ( a n t i s e r u m -- PBS) This 'specific binding i n d e x ' expresses the p r o p o r t i o n o f t h e t o t a l i m m u n o globulin in the R-149 a n t i s e r u m b o u n d to t h e cells which is in excess o f t h e non-specific binding o f i m m u n o g l o b u l i n in NRS, s u b t r a c t i n g the noni m m u n o l o g i c a l a d h e r e n c e o f r a d i o a c t i v i t y as d e t e r m i n e d b y the i n c u b a t i o n o f cells with PBS. T h e i n d e x increased u p t o 5 × l 0 S Chago cells per assay t u b e , a f t e r which n o c o n s i s t e n t increase was o b s e r v e d with increasing n u m bers o f target cells. T h u s 5 × 10 s cells per assay t u b e a p p e a r t o r e p r e s e n t the smallest n u m b e r o f cells for o p t i m a l discrimination o f a n t i b o d y and nonspecific i m m u n o g l o b u l i n binding t o t h e Chago cells. Selection o f o p t i m u m incubation time o f cells with sera In the [~2sI]p r o t e i n A assay d e s c r i b e d b y B r o w n et al. ( 1 9 7 7 ) t h e first and s e c o n d incubations were p e r f o r m e d f o r 60 min. T o d e t e r m i n e the o p t i m u m i n c u b a t i o n t i m e for the a n t i s e r u m or c o n t r o l serum with t h e cells f o r t h e A b G f C - S p A
374 TABLE 1 EFFECT OF NUMBER T A I N E D IN A S S A Y N u m b e r o f Chago cells in assay t u b e
104 5 X 104 10 s 5 X l0 s 106 5 X 106 107
OF CHAGO
CELLS IN A S S A Y
TUBE
Counts/min
ON R E S U L T S
OB-
1 : 8 0 dilution R--149
1 : 8 0 dilution NRS
PBS
Specific binding i n d e x (AS--PBS)--(N RS--PBS ) (AS--PBS)
2 3 3 5 6 18 24
1458 1765 2021 2451 3087 6263 9387
645 711 783 1151 1379 1530 1941
0.44 0.55 0.50 0.68 0.66 0,73 0.68
083 037 248 154 409 748 946
assay, an antiserum (R-170) which had been developed against the Chago cell line was tested, with NRS serving as the control serum, using 5 × 10 s Chago cells as target cells in each assay tube. As is shown in Fig. 1, a plateau of binding was reached for the antiserum and the NRS within 30 rain. Selection of optimum incubation time of cells with 12SI-labeled protein A. Having shown that 90 min was an adequate incubation time for cells with sera, we then determined the time of incubation of the cells with the 12sIlabeled protein A needed for maximal binding. As shown in Fig. 2, 90--120 KINETICS OF 1251-PROTEIN A INCUBATION
KINETICS OF ANTIBODY INCUBATION
4
4
/
o O
x E~
3
o
2
/
o R-170
x
•
#
NRS
/
o
1
3
o R 170 • NRS
2
1
I
I
1
l
I
I
30
60
90
120
150
180
INCUBATION TIME (rain.)
30
60
90
120
150
180
INCUBATION TIME (min.)
Fig. 1. D e t e r m i n a t i o n o f o p t i m u m i n c u b a t i o n t i m e for cells w i t h a n t i b o d y at a dilution o f 1 : 80. Chago cells at 5 × l 0 s cells per tube were i n c u b a t e d at 2 5 ° C w i t h R - 1 7 0 and N R S . I n c u b a t i o n w i t h [ 1 2 s I ] S p A was 90 rain at 25°C. Fig. 2. D e t e r m i n a t i o n o f o p t i m u m i n c u b a t i o n time for cells w i t h [ 1 2 s I ] S p A , after 90 min i n c u b a t i o n w i t h antisera R - 1 7 0 and N R S . The same cells and c o n d i t i o n s were used as in Fig. 1.
375
min appeared to be optimal for this second incubation. Incubation for less than 60 min seemed to give lower values for both antiserum and NRS, while incubation for 180 min resulted in a d i s p r o p o r t i o n a t e decrease in antiserum binding. Selection o f a m o u n t o f ['2SI]protein A to be added to each assay tube. In the [12SI]protein A assays described by Welsh et al. (1975) and by Brown et al. (1977), 10 s cpm per assay t ube was used. We, therefore, began our studies with this a m o u n t o f radioactivity present. After exploring the variables described above, we d e t e r m i n e d that l 0 s cpm was usually needed to adequately discriminate between the binding o f the antisera and NRS. We, t h er ef o r e, selected 10 s cpm/assay tube, based upon our own experience and t h at o f o t h e r investigators.
Determination o f effect o f glutaraldehyde fixation o f target cells on use o f assay. The applicability o f glutaraldehyde-fixed target cells, relative to the use o f the same n u m b e r of fresh cells, was studied by comparing the reactivity o f glutaraldehyde-fixed and unfixed cultured human lung cancer cells (Chago), using antisera developed against Chago cells (R-170) or against h u man lung cancer cells derived from a cancer pat i ent (R-149). The results o f this comparison are shown in Fig. 3. The immunoglobulin binding due to NRS was subtracted f r om the data presented for both antisera. In Fig. 3, it can be n o t e d t ha t the glutaraldehyde-fixed cells give greater or equivalent a n t i b o d y binding with R-170 through 1 : 640 dilution and with R-149 to a dilution of 1 : 160. Above a dilution o f 1 : 160 o f R-149 the unfixed cells appear to bind a n t i b o d y s om e w hat bet t er than the fixed cells. Specificity o f assay Results after adsorption o f immunoglobulin by glutaraldehyde-fixed cells. The application of the assay to quantitate the a m o u n t of specific a n t i b o d y ANTIBODY BINDING WITH FRESH AND FIXED CHAGO TARGET CELLS
3
A Fixed Ceils
8
\
6
~
Fixed Cells • Fresh Cells o
4
1,80
1 /160
1 ' 320
1 '640
ANTISERUM DILUTION R 170
1:80
1, 160
1,320
1 '640
ANTISERUM DILUTION R 149
Fig. 3. C o m p a r i s o n o f a n t i b o d y b i n d i n g to fresh and g l u t a r a l d e h y d e - f i x e d cells. Chago cells, 5 × 10 5 cells per t u b e , were r e a c t e d w i t h t w o d i f f e r e n t anti-lung t u m o r antisera. The c p m b o u n d by NRS at each d i l u t i o n have b e e n s u b t r a c t e d f r o m the total c p m b o u n d , for b o t h antisera.
376 p r e s e n t in an a n t i s e r u m to a p a r t i c u l a r h u m a n c a n c e r was t e s t e d b y a d s o r b i n g t w o p r e v i o u s l y d e s c r i b e d a n t i s e r a against h u m a n lung c a n c e r ( R - 1 7 0 a n d R - 1 4 9 ) w i t h m y e l o i d l e u k e m i a ( K - 5 6 2 ) tissue c u l t u r e cells. T h e a d s o r p t i o n s o f t h e s e a n t i s e r a a n d o f N R S were p e r f o r m e d using 1 : 80 d i l u t i o n s o f all sera, a d s o r b i n g w i t h 4 × 106 o f g l u t a r a l d e h y d e - f i x e d K - 5 6 2 cells/ml o f s e r u m . T h e i n c u b a t i o n t i m e o f t h e K - 5 6 2 cells w i t h t h e sera was 16 h, p e r f o r m e d w i t h gentle r o t a t i o n o f t h e sera a n d cells at 4°C o n an electric r o t a t o r at 6 rev/ m i n . T h e a d s o r b e d a n d u n a d s o r b e d sera w e r e t h e n a s s a y e d w i t h K - 5 6 2 a n d w i t h Chago t a r g e t cells, using t h e m e t h o d d e s c r i b e d in this p a p e r with t w o e x c e p t i o n s . T o e c o n o m i z e on t h e a m o u n t o f a d s o r b e d s e r u m , o n l y 25 pl o f s e r u m w e r e used in e a c h assay t u b e instead o f t h e 100 pl s t a n d a r d u s e d in o u r assay. T h e results o f this e x p e r i m e n t are s h o w n in Fig. 4. A d s o r p t i o n o f b o t h a n t i s e r a gave similar results w i t h t h e t w o antisera ( R - 1 7 0 a n d R - 1 4 9 ) . B e f o r e a d s o r p t i o n , b o t h a n t i s e r a r e a c t e d s t r o n g l y against K - 5 6 2 , w h e r e a s t h e a d s o r b e d sera gave similar results as N R S . On t h e o t h e r h a n d , m u c h o f t h e r e a c t i v i t y o f t h e a n t i s e r u m against C h a g o lung c a n c e r r e m a i n e d a f t e r a d s o r p t i o n . T h e results o f this e x p e r i m e n t i n d i c a t e t h a t t h e g l u t a r a l d e h y d e - f i x e d cells are c a p a b l e o f b i n d i n g t h e a n t i b o d y in t h e antis e r u m w h i c h is r e a c t i v e w i t h t h e antigens o f t h e a d s o r b i n g cell a n d t h a t selec-
EFFECT OF ADSORPTION ON ANTIBODY BINDING
34~ F 32~
K 562
CHAGO TARGET CELLS
Fig. 4. Effect o t adsorption of two anti-lung tumor antisera and NRS with cultured myeloid leukocytes, K-562, on antibody binding activity with the adsorbing cells and a lung tumor cell line. The horizontal lines indicate the cpm before adsorption and the downward pointing arrows show decrease in binding after adsorption. Bar graphs show post-adsorption data. Note retention of activity by antisera against the lung tumor cells, when compared with NRS, while activity of the adsorbed antisera against K-562 approximates the NRS control.
377 rive r e a c t i v i t y w i t h a s e c o n d c a n c e r cell can b e i d e n t i f i e d a f t e r such a d s o r p tion.
Reproducibility o f assay T a b l e 2 gives t h e assay results o b t a i n e d f r o m 5 i n d e p e n d e n t , c o n s e c u t i v e e x p e r i m e n t s p e r f o r m e d using 1 : 80 d i l u t i o n s o f a n t i s e r a R - 1 7 0 a n d R - 1 4 9 , N R S a n d PBS c o n t r o l , using t h e C h a g o cells as t a r g e t s in t h e assay. T h e s e e x p e r i m e n t s w e r e p e r f o r m e d w i t h n e w l y p r e s e r v e d t a r g e t cells a n d w i t h cells w h i c h h a d b e e n g l u t a r a l d e h y d e - f i x e d a n d s t o r e d f o r as l o n g as 3 m o n t h s . T h e results are p r e s e n t e d as t h e t o t a l r a d i o a c t i v i t y c o u n t s p e r m i n u t e ( c p m ) obt a i n e d f r o m t h e [12SI]protein A b o u n d t o t h e t a r g e t cell surface a n t i g e n s or p r e s e n t in t h e t u b e s as b a c k g r o u n d a n d , also, as t h e 'specific b i n d i n g i n d e x ' d e f i n e d earlier. As is s h o w n in T a b l e 2, a l t h o u g h t h e r e was general a g r e e m e n t in t h e values o b t a i n e d f o r t h e t w o a n t i s e r a in t h e five e x p e r i m e n t s , r a t h e r large s t a n d a r d d e v i a t i o n s o f t h e c p m w e r e o b s e r v e d . T h e v a r i a t i o n is m o s t likely d u e t o diff e r e n c e s in a c t u a l n u m b e r s o f t a r g e t cells p r e s e n t (cell c o u n t i n g error), e x p e r i m e n t a l e r r o r in a d d i t i o n o f r e a g e n t s , a n d a l t e r a t i o n s in t h e specific a c t i v i t y o f t h e 12SI-labeled p r o t e i n A. H o w e v e r , if o n e l o o k s at t h e indices, w h i c h w o u l d t e n d to c o r r e c t f o r t h e s e variations, closer r e p l i c a t i o n was o b t a i n e d f o r b o t h antisera.
Comparison with trypan blue exlusion assay T h e sensitivity a n d q u a n t i t a t i o n capabilities o f t h e A b G f C - S p A assay w e r e c o m p a r e d w i t h t h e t r y p a n b l u e e x c l u s i o n assay b y c o m p a r i n g t h e d i l u t i o n s at w h i c h t h e t w o tests gave d e t e c t a b l e d i f f e r e n c e s w h e n c o m p a r e d w i t h c o n t r o l
TABLE 2 REPRODUCIBILITY OF ASSAY RESULTS IN FIVE CONSECUTIVE, INDEPENDENT EXPERIMENTS WITH CHAGO TARGET CELLS Reagent tested
Counts/rain Experiment 1
R-170 R-149 NRS PBS
33 9 3 3
095 473 229 054
2
3
35 524 9 502 2 148 893
33 10 1 1
4
184 334 804 206
24 8 2 1
Mean
S.D.
29 397 6 722 1 443 606
31 8 2 1
4163 1383 669 958
0.97 0.86
0.98 0.91
5
888 503 054 591
218 907 136 470
Computed index R-170 R-149
0.99 0.97
0.96 0.85
0.98 0.93
0.98 0.93
0.02 0.10
378
TRYPAN BLUE EXCLUSION A S S A Y
Antisera
R-170: R-149:
IO0
~_
ANTIBODY BINDING A S S A Y
/k R-170 o R149 a NRS
Complement Control
o ~
• •
8o
o
8o
30
4O
20
2O
10
1/2
1 ,'4
1/8
1/64
1/256 1/128
DILUTION
1/1024
1/512
1/4096
1/2048
DILUTION
Fig. 5. C o m p a r i s o n o f a n t i b o d y binding with the trypan blue e x c l u s i o n assay o f c y t o t o x icity using R - 1 7 0 and R - 1 4 9 against Chago tissue culture cells.
conditions (e.g., the complement control in the trypan blue exclusion assay and the NRS control in the AbGfC-SpA assay). In Fig. 5 the comparison of antisera R-170 and R-149 with the Chago cultured cell being used as the target cell in each of the assays is shown.
51Cr RELEASE A S S A Y
A N T I B O D Y BINDING A S S A Y
o Anti-MBL-2
• NRS
(J
x 0 o
O Anti-MBL*2
20
• NRS
60
10
50
5 ×
~ 3o
(J
~,-r 20 m 10
1/8
1/32
1/128 DILUTION
1/512 1/2048
. . . . . . . . . . . . . . . . . . . . . I I I I I I I I I 1/8 1/32 1/128 1/512 1/2048 DILUTION
Fig. 6. C o m p a r i s o n o f the SlCr release assay ( A ) w i t h the A b G f C - S p A assay (B) using MBL-2 murine l y m p h o m a cells and anti-MBL-2 antiserum. The assays have similar detection sensitivity to a dilution o f at least 1 : 512. N o t e , h o w e v e r , the e x c e l l e n t doseresponse characteristics o f the A b G f C - S p A assay.
379 In the trypan blue exclusion assay, both antisera gave significant cytotoxicity above the complement controls at dilutions of 1 : 4 or less. In comparison, using the AbGfC-SpA assay, R-170 had a titer of greater than 1 : 4096 while R-149, although less reactive than R-170, had a titer of 1 : 4096, and the results at this dilution were still significantly above the NRS control. Comparison with S~Cr release assay A comparison of the sensitivity and quantitation capabilities of the AbGfC-SpA assay with the SlCr release assay was performed using the MBL-2 mouse l y m p h o m a cell system (Fig. 6). In the 5~Cr release assay, the antiMBL-2 antiserum had a titer of 1 : 512. This antiserum gave a similar titer in the AbGfC-SpA assay. However, note that the AbGfC-SpA assay provided a dose response curve of immunoglobulin binding whereas the 51Cr release m e t h o d gave a plateau until the 'end point' dilution.
DISCUSSION The identification and isolation of antibodies" specific for antigens of cancer cells require an antiserum containing such antibodies, an effective means of removing selectively these antibodies from the antiserum, and a means of identifying the nature and a m o u n t of reactive antibody. Existing assays did not fulfill our needs because t h e y were either too insensitive (i.e., the trypan blue exclusion method), were sensitive but semi-quantitative and variable on a daily basis (i.e., the SlCr release assay) or depended upon the ready availability of fresh cells (i.e., the standard 125I-labeled protein A assay). This latter disadvantage we found to be prohibitive when we began dealing with fresh human cancer cells obtained by mincing t u m o r and washing the cells. With existing tests, the cells either had to be used that same day, or a culture line of the t u m o r had to be started, with all of the inherent problems including a possible change in the antigenicity of the cells involved. Recently, Brown et al. (1977) described a monolayer microwell modification of the 12SI-labeled protein A assay using cultured fibrosarcoma cells induc, ed in BALB/c and C57BL/6 mice and alloantisera developed against these t u m o r cells and against spleen cells. They demonstrated that the ~2sIlabeled protein A assay was more sensitive than the conventional isotopic antiglobulin assay, ascribing the increased sensitivity to the lesser binding of the protein A to IgG bound non-specifically to cells. Zeltzer and Seeger (1977) described a microassay using radioiodinated protein A for antibodies bound to cell surface antigens of t u m o r cells which were adherent, thus being a variation of the m e t h o d of Welsh et al. (1975), who developed the radioiodinated protein A assay for suspensions of cells. Both assays depend upon the availability of viable cells, thus presenting a major obstacle to the elective use of the m e t h o d to assay for antibodies binding to the surfaces of h u m a n cancers on days other than when the tissue or cells al~ ~resifly available.
380 O u r assay appears t o o v e r c o m e all o f the disadvantages e n u m e r a t e d for the o t h e r t y p e s o f assays. F o r e x a m p l e , we have s t o c k s o f g l u t a r a l d e h y d e - f i x e d h u m a n lung c a n c e r cells, n o r m a l h u m a n cells o f various organs, and o f a n u m b e r o f c u l t u r e d h u m a n c a n c e r cells r e a d y for use. We have been able to screen 40 antisera d e v e l o p e d against h u m a n lung cancers for reactivity against cell surface antigens on h u m a n lung c a n c e r cells in o n e d a y and have identified t h o s e with high reactivity w h i c h m i g h t be p r o m i s i n g sources o f specific a n t i b o d y . Zeltzer and Seeger ( 1 9 7 7 ) suggested t h a t fetal calf serum (FCS) can act as cell surface antigens in a p r o t e i n A microassay. In this regard, a l t h o u g h the cells used b y us were g r o w n in FCS and s h o w e d reactivity, we also n o t e d a similarly high r e a c t i v i t y o f g l u t a r a l d e h y d e - f i x e d , fresh h u m a n lung c a n c e r cells, w h i c h had never been in c o n t a c t with FCS, with antisera against lung c a n c e r extract. This suggests t h a t the c u l t u r e c o n d i t i o n s are n o t the source o f the m a j o r p o r t i o n o f t h e a n t i g e n - a n t i b o d y i n t e r a c t i o n s being observed. The loss o f r e a c t i v i t y o f t h e antisera with K - 5 6 2 m y e l o i d l e u k e m i a cells a f t e r a d s o r p t i o n with K - 5 6 2 cells (Fig. 4) and the r e t e n t i o n o f activity o f these antisera against a cell line (Chago) derived f r o m lung c a n c e r indicate t h e applicability o f these g l u t a r a l d e h y d e - f i x e d cells t o a d s o r p t i o n steps w h i c h we are using t o m a k e antisera m o r e specific for the antigens o f certain c a n c e r cells. O u r studies t o date indicate the assay permits sensitive, c o n v e n i e n t and rapid q u a n t i t a t i o n o f a n t i b o d y b i n d i n g t o cellular antigens a n d t h e r e f o r e m a y p r o v i d e an i m p o r t a n t t e c h n i q u e in research in this area. ACKNOWLEDGEMENTS T h e a u t h o r s wish t o t h a n k Dr. Grace C a n n o n o f Bionetics, Inc. for her g e n e r o u s gift o f tissue c u l t u r e cell lines and Dr. Alan R a b s o n o f the National C a n c e r I n s t i t u t e f o r the C h a g o cell line and his helpful e n c o u r a g e m e n t . REFERENCES Bolton, A.E. and W.M. Hunter, 1973, Biochem. J. 133, 529. Brown, J.P., J.P. Klitzman and K.E. HellstrSm, 1977, J. Immunol. Methods 15, 57. Herberman, R.B., 1972, J. Natl. Cancer Inst. 48, 265. Lozzio, C.B. and B.B. Lozzio, 1975, Blood 45, 321. Ng, A., K.R. McIntire, J.A. Braatz and R.B. Herberman, 1977, in: Regulatory Mechanisms in Lymphocyte Activation: Proceedings of Eleventh Leukocyte Culture Conference, ed. D.O. Lucas (Academic Press, New York) p. 374. Princler, G.L. and K.R. McIntire, 1975, Cell. Immunol. 15, 197. Rabson, A.S., S.W. Rosen, A.H. Tashjian, Jr. and B.D. Weintraub, 1973, J. Natl. Cancer Inst. 50,669. Rosenberg, S.A., S. Schwarz, H. Anding, C. Hyatt and G.M. Williams, 1977, J. Immunol. Methods 17, 225. Sela, B. and G.M. Edelman, 1977, J. Exp. Med. 145,443. Takahashi, T., L.J. Old, K.R. McIntire and E.A. Boyse, 1971, J. Exp. Med. 134,815. Ting, C.C. and R.B. Herberman, 1976, in: International Review of Experimental Pathology, Vol. 15, eds. G.W. Richter and M.A. Eptein (Academic Press, New York) p. 94. Welsh, K.I., G. Dorval and H. Wigzell, 1975, Nature 254, 67. Zeltzer, P.M. and R.C. Seeger, 1977, J. Immunol. Methods 17, 163.
369
© Elsevier/North-Holland Biomedical Press
SENSITIVE AND CONVENIENT QUANTITATION OF ANTIBODY BINDING TO CELLULAR ANTIGENS USING GLUTARALDEHYDE PRESERVED CELLS
S. D A V I D R O C K O F F , K. R O B E R T M c I N T I R E , A H - K A U NG, G E R A L D L. P R I N C L E R , R O N A L D B. H E R B E R M A N and J O H N N. L A R S O N
Department o f Radiology, The George Washington University Medical Center, 901 23rd Street, N.W., Washington, DC 20037, and the Laboratory o f lmmunodiagnosis, National Cancer Institute, National Institutes o f Health, Bethesda, MD 20014, U.S.A. (Received 2 May 1978, a c c e p t e d 17 O c t o b e r 1978)
As a preliminary step in the identification and isolation o f antibodies to h u m a n cancers, we have developed a sensitive and c o n v e n i e n t assay for a n t i b o d y binding to cellular antigens. The basis for the m e t h o d is a n t i b o d y binding to glutaraldehyde-fixed cells ( A b G f C ) and q u a n t i t a t i o n with r a d i o i o d i n a t e d staphylococcal protein A (SPA). Glutarald e h y d e fixation of intact cells, which does n o t appear to affect the ability to form antigen-antibody complexes, provides a c o n v e n i e n t and standard supply of target cells which m a y be stored at 4°C and used in the assay over a period of several months. The a m o u n t o f a n t i b o d y specifically b o u n d to the cells is q u a n t i t a t e d by the addition o f 12sIlabeled SpA. The sensitivity of the m e t h o d was c o m p a r e d with t w o c o m p l e m e n t - d e p e n d e n t c y t o t o x i c i t y m e t h o d s (trypan blue exclusion and SlCr release assays) and tested with t w o antisera to h u m a n lung cancer and one antiserum to a m e m b r a n e antigen o f a m u r i n e l y m p h o m a . These comparisons indicated m u c h greater sensitivity when c o m p a r e d with the trypan blue exclusion assay and equivalent sensitivity with greater dose response characteristics w h e n c o m p a r e d with the s 1Cr release assay.
INTRODUCTION
Over the years there has been a great interest in looking for antibodies directed against human tumor-associated antigens as well as other cell surface antigens. Using available techniques, it has been very difficult to docum e n t the existence of such antibodies in the sera o f cancer patients or to define completely the specificity of heterologous antibodies to tumorassociated antigens on cell surfaces. Part of the problem has been related to the reproducibility, reliability, and ease o f application of existing assay methods. There are many methods for measuring the reactivity of antibody with cell surface antigens and these have been reviewed by Ting and Herberman (1976). Recently, Rosenberg et al. (1977) compared multiple assays for detecting human antibodies directed against surface antigens. They determined that the visual end point cytolysis assay and the SlCr release methods
370 were equally sensitive in measuring complement-mediated antibody cytotoxicity and that both were far more sensitive than the trypan blue exclusion assay. A hemadsorption technique was 10 times more sensitive than the c y t o t o x i c i t y assays but exact quantitation was difficult because of the visual end point. The SlCr release assay was unstable and they were unable to correct for day-to-day variations. The current work in this laboratory is directed toward the identification and isolation of antibodies to human cancers, with special interest in cancer of the lung. Our research requires the availability of a convenient and sensitive assay m e t h o d of antibody-antigen binding. We were, therefore, interested in the m e t h o d for the rapid quantitation of membrane antigens using 12SI-labeled protein A, derived from S t a p h y l o c o c c u s aureus, described by Welsh et al. (1975). The assay they described offered a complement-independent radioimmunoassay applicable to a wide range of mammalian membrane antigens. Their assay has a sensitivity similar to that of the trypan blue exclusion and SlCr assays but they believed it to be more linear and thus more precise than these two tests. A major disadvantage of their assay, however, was that it depended upon the availability of fresh target cells, which could vary considerably from one experiment to another. The recent description of glutaraldehyde fixation of mouse l y m p h o m a (EL4BU and P388) cells onto concanavalin A-coated Sephadex beads by Sela and Edelman (1977), for the purpose of isolation of immunoglobulins specific for cell surface carbohydrates by cell column chromatography, led us to explore the development of a ~2SI-labeled protein A assay utilizing glutaraldehyde-fixed target cells. The purpose of this communication is to describe this assay which has the properties of sensitivity, convenience, rapidity and reproducibility. MATERIALS AND METHODS A n tisera
Pooled normal rabbit serum (NRS) was prepared from 5 normal, adult New Zealand white rabbits. Antisera against h u m a n lung cancer extract were prepared by the intramuscular injection of the extract (approximately 3 mg protein), prepared in 1.2 ml of complete Freund's adjuvant (Difco Lab., Detroit, MI), into adult New Zealand white rabbits. Antisera against cultured h u m a n lung cancer cells were prepared by the injection of 5--10 million Chago cells (Rabson et al., 1973), emulsified in complete Freund's adjuvant. Blood was taken from the ear veins beginning approximately 1 m o n t h after injection. Sera were centrifuged at 30,000 × g for 30 min in a refrigerated centrifuge to remove small particular matter before use, and stored at --20°C. In most experiments, 1 : 80 dilutions of antisera and NRS were used. This dilution was selected after it was observed to be the lowest dilution which could be consistently discriminated from background. Use of this
371 end point dilution facilitated the complete adsorption of antibody with a moderate number of cells.
Target cells Cultured cells were obtained by scraping the culture flasks, followed by washing 2 times in cold (4°C) PBS with vigorous vortexing between washes to separate the cell clumps. After each wash, the cells were centrifuged at 500 × g for 10 min. The cells to be fixed in glutaraldehyde after the final wash and centrifugation were vigorously vortexed, poured through a double layer of c o t t o n gauze into a new centrifuge tube with 10 ml of PBS, and then enough 3% glutaraldehyde (Practical grade, Eastman Kodak Co., Rochester, NY) was added to make a total volume of 50 ml. The cells in the glutaraldehyde solution were then continuously and gently mixed with an electric rotator (6 rev/min) at room temperature for 30 min, after which they were centrifuged for 10 min at 500 × g, washed 2 times with PBS and filtered through a double layer of gauze into a fresh tube, to be stored at 4 ° C until needed. Identical assay results were obtained with Chago target cells which had been fixed in glutaraldehyde for intervals ranging from 30 min to 48 h, when tested with an antiserum against human lung cancer extract (R-149) and with NRS. For storage purposes, 0.02% sodium azide solution (in PBS) was used to retard bacterial growth. The cell lines used in the experiments included Chago cultured human lung cancer, K-562 cultured myeloid leukemia cells (Lozzio and Lozzio, 1975) and MBL-2 mouse l y m p h o m a cells (Ng et al., 1977). Preparation o f [~2SI]protein A Purified, lyophilized protein A from Staphylococcus aureus Cowan strain I (Pharmacia, Uppsala, Sweden) was radioiodinated by a modification o f the m e t h o d of Bolton and Hunter (1973). Briefly, protein A (100 pg) in 0.1 ml of 0.01 M phosphate-buffered saline (PBS), pH 7.2, was added to 0.5 mCi of dried Bolton-Hunter reagent (New England Nuclear Corp., Bostot~, MA) and the reaction mixture was vortexed every 2 min for 15 min at 2 5 ° ( ', The reaction was stopped by addition of 1 ml of 0.2 M glycine in PBS to react with the unchanged Bolton-Hunter reagent for 5 min at 25 ° C. The 12sIlabeled protein A was separated from the other products and the reagent by gel filtration on Sephadex G-25 (PD-10, Pharmacia, Uppsala, Sweden) equilibrated with PBS containing 0.25% (w/v) gelatin. The first peak of radioactivity eluted from the Sephadex G-25 column in the void volume of the column was pooled, aliquoted and stored at --20°C. Specific activity of [12SI]protein A, assuming 100% recovery of protein A, was approximately 2 pCi/pg and subsequent preparations of [a2SI]protein A were adjusted to achieve this specific activity. Precipitability of [~2SI]protein A with 10% TCA was at least 85%.
372 Assay p r o c e d u r e using glutaraldehyde-fixed target cells The target cells were taken from storage and observed under the microscope after vortexing. If clumping had occurred, additional washing and filtration as described above were p e r f o r m e d until single cells predominated. The cells were then c o u n t e d in a h e m o c y t o m e t e r . Centrifuge tubes containing known numbers o f cells were centrifuged at 500 X g for 10 min, then supernatant fluid withdrawn and the cells resuspended in 1% bovine serum albumin (BSA) (Reheis Chem. Co., Phoenix, AZ) in PBS at 5 X 10 ~ cells/ml. Into 12 mm X 75 m m glass culture tubes (Coming Glass Works, Coming, NY) were placed 100 pl of antiserum, NRS or PBS (as a background control) and then 100 pl of the cell suspension were added. Random i zat i on consisted o f adding coded antisera and cells and then treating all tubes alike, w i t h o u t reference to sequence. The tubes containing the antiserum, NRS or PBS and the cells in BSA were v o r t e x e d and incubated at r o o m t e m p e r a t u r e for 90 min, vortexing each t ube every 30 min. Then, 1 ml of cold PBS was added to each tube, the tubes were vor t e xe d, an additional 1 ml was added to each tube, v o r t e x e d again, and the tubes were centrifuged at 1800 X g for 10 min. T h e tubes were decanted, bl ot t ed and the washing procedure was repeated with PBS. After the second wash, 100 pl of [12SI]protein A (adjusted so t hat each aliquot contained 10 s c ount s / m i n) was added to each t ube and incubated for 90 min, vortexing initially and then every 30 min during the incubation. At the end o f the incubation, 1 ml of 1% BSA in P B S was added to each tube and, with vortexing, 1 ml of PBS was added and the tubes were centrifuged at 1800 X g for 10 min, decanted and blotted. The washing, centrifugation, decanting and blotting were repeated, after which the cont ent s of each tube were transferred to a fresh tube by adding 1 ml of 1% BSA, vortexing, pipetting, and then rinsing with 2 ml o f 1% BSA and pipetting, for a total volume o f 3 ml o f transfer solution. A final centrifugation, decantation and blotting were p e r f o r m e d as above and the pellets were c o u n t e d for 1 min. Trypan blue exclusion assay The t r y p a n blue exclusion c y t o t o x i c i t y assay, used to compare with the assay m e t h o d being described in this report, is essentially the same as t hat described previously (Takahashi et al., 1971; Princler and McIntire, 1975). After an incubation of the cultured Chago cells with the sera for 45 rain at 37 ° C, 0.4% t r y p a n blue in normal saline (Grand Island Biological Co., Grand Island, NY) was added. Fresh-frozen normal guinea pig serum (NGPS, prepared in o u r labor at or y) was diluted 1 : 4 as a c o m p l e m e n t source. Control tubes contained cells and diluent (Medium-199, Grand Island Biological Co.), cells and NGPS, and cells and antiserum. s 1Cr release cy to toxic i ty assay The StCr release assay used was described previously by Herberman (1972) and modified as follows. Fifty microliters of SlCr-labeled target cells, con-
373 taining 1.0--2.5 × 104 cells, were a d d e d to 25 pl o f serial, 2-fold dilutions o f serum. A f t e r i n c u b a t i o n at 37 ° C f o r 45 min, 50 pl o f I : 3 dilution o f r a b b i t serum ( G r a n d Island Biological Co.), which had b e e n p r e s c r e e n e d for lack o f c y t o t o x i c i t y , were a d d e d as the s o u r c e o f c o m p l e m e n t . T h e i n c u b a t i o n was t h e n c o n t i n u e d f o r 90 min at r o o m t e m p e r a t u r e . T h e r e a c t i o n was termin a t e d b y adding 2 ml o f Roswell Park Memorial I n s t i t u t e M e d i u m 1 6 4 0 (RPMI) c o n t a i n i n g 10% fetal bovine serum (FBS) ( G r a n d Island Biological Co.) at 4 °C. T h e cells were s e d i m e n t e d b y c e n t r i f u g a t i o n and the supernat a n t fluid was c o u n t e d f o r released r a d i o a c t i v i t y . C y t o t o x i c activities o f the antisera were calculated as follows: % c y t o t o x i c i t y = [ ( ~~Cr released f r o m a n t i s e r u m - t r e a t e d cells -- ~' Cr released f r o m c o m p l e m e n t c o n t r o l ) / ( T o t a l SlCr i n c o r p o r a t e d b y target cells)] × 100. RESULTS
Optimization o f assay procedures Selection o f optimum number o f cells per assay tube. Table 1 lists t h e c o u n t s / m i n ( c p m ) o b t a i n e d using Chago c u l t u r e d h u m a n lung c a n c e r cells as t h e target cells, varying the n u m b e r o f cells f r o m 104 to 107 cells per assay tube. T h e a n t i s e r u m t e s t e d was R-149, d e v e l o p e d in rabbits against fresh h u m a n lung c a n c e r e x t r a c t . As a c o n t r o l f o r non-specific binding o f i m m u noglobulin, NRS was used and, as a b a c k g r o u n d c o n t r o l , PBS was tested. As can be seen in Table 1, the c p m increased c o n t i n u o u s l y with increasing n u m b e r s o f cells for t h e a n t i s e r u m , the N R S and f o r t h e PBS. H o w e v e r , the ability to discriminate the a n t i s e r u m f r o m the N R S was d e t e r m i n e d b y a 'specific binding i n d e x ' , d e t e r m i n e d b y the f o r m u l a : ( a n t i s e r u m -- PBS) -- (NRS -- PBS) ( a n t i s e r u m -- PBS) This 'specific binding i n d e x ' expresses the p r o p o r t i o n o f t h e t o t a l i m m u n o globulin in the R-149 a n t i s e r u m b o u n d to t h e cells which is in excess o f t h e non-specific binding o f i m m u n o g l o b u l i n in NRS, s u b t r a c t i n g the noni m m u n o l o g i c a l a d h e r e n c e o f r a d i o a c t i v i t y as d e t e r m i n e d b y the i n c u b a t i o n o f cells with PBS. T h e i n d e x increased u p t o 5 × l 0 S Chago cells per assay t u b e , a f t e r which n o c o n s i s t e n t increase was o b s e r v e d with increasing n u m bers o f target cells. T h u s 5 × 10 s cells per assay t u b e a p p e a r t o r e p r e s e n t the smallest n u m b e r o f cells for o p t i m a l discrimination o f a n t i b o d y and nonspecific i m m u n o g l o b u l i n binding t o t h e Chago cells. Selection o f o p t i m u m incubation time o f cells with sera In the [~2sI]p r o t e i n A assay d e s c r i b e d b y B r o w n et al. ( 1 9 7 7 ) t h e first and s e c o n d incubations were p e r f o r m e d f o r 60 min. T o d e t e r m i n e the o p t i m u m i n c u b a t i o n t i m e for the a n t i s e r u m or c o n t r o l serum with t h e cells f o r t h e A b G f C - S p A
374 TABLE 1 EFFECT OF NUMBER T A I N E D IN A S S A Y N u m b e r o f Chago cells in assay t u b e
104 5 X 104 10 s 5 X l0 s 106 5 X 106 107
OF CHAGO
CELLS IN A S S A Y
TUBE
Counts/min
ON R E S U L T S
OB-
1 : 8 0 dilution R--149
1 : 8 0 dilution NRS
PBS
Specific binding i n d e x (AS--PBS)--(N RS--PBS ) (AS--PBS)
2 3 3 5 6 18 24
1458 1765 2021 2451 3087 6263 9387
645 711 783 1151 1379 1530 1941
0.44 0.55 0.50 0.68 0.66 0,73 0.68
083 037 248 154 409 748 946
assay, an antiserum (R-170) which had been developed against the Chago cell line was tested, with NRS serving as the control serum, using 5 × 10 s Chago cells as target cells in each assay tube. As is shown in Fig. 1, a plateau of binding was reached for the antiserum and the NRS within 30 rain. Selection of optimum incubation time of cells with 12SI-labeled protein A. Having shown that 90 min was an adequate incubation time for cells with sera, we then determined the time of incubation of the cells with the 12sIlabeled protein A needed for maximal binding. As shown in Fig. 2, 90--120 KINETICS OF 1251-PROTEIN A INCUBATION
KINETICS OF ANTIBODY INCUBATION
4
4
/
o O
x E~
3
o
2
/
o R-170
x
•
#
NRS
/
o
1
3
o R 170 • NRS
2
1
I
I
1
l
I
I
30
60
90
120
150
180
INCUBATION TIME (rain.)
30
60
90
120
150
180
INCUBATION TIME (min.)
Fig. 1. D e t e r m i n a t i o n o f o p t i m u m i n c u b a t i o n t i m e for cells w i t h a n t i b o d y at a dilution o f 1 : 80. Chago cells at 5 × l 0 s cells per tube were i n c u b a t e d at 2 5 ° C w i t h R - 1 7 0 and N R S . I n c u b a t i o n w i t h [ 1 2 s I ] S p A was 90 rain at 25°C. Fig. 2. D e t e r m i n a t i o n o f o p t i m u m i n c u b a t i o n time for cells w i t h [ 1 2 s I ] S p A , after 90 min i n c u b a t i o n w i t h antisera R - 1 7 0 and N R S . The same cells and c o n d i t i o n s were used as in Fig. 1.
375
min appeared to be optimal for this second incubation. Incubation for less than 60 min seemed to give lower values for both antiserum and NRS, while incubation for 180 min resulted in a d i s p r o p o r t i o n a t e decrease in antiserum binding. Selection o f a m o u n t o f ['2SI]protein A to be added to each assay tube. In the [12SI]protein A assays described by Welsh et al. (1975) and by Brown et al. (1977), 10 s cpm per assay t ube was used. We, therefore, began our studies with this a m o u n t o f radioactivity present. After exploring the variables described above, we d e t e r m i n e d that l 0 s cpm was usually needed to adequately discriminate between the binding o f the antisera and NRS. We, t h er ef o r e, selected 10 s cpm/assay tube, based upon our own experience and t h at o f o t h e r investigators.
Determination o f effect o f glutaraldehyde fixation o f target cells on use o f assay. The applicability o f glutaraldehyde-fixed target cells, relative to the use o f the same n u m b e r of fresh cells, was studied by comparing the reactivity o f glutaraldehyde-fixed and unfixed cultured human lung cancer cells (Chago), using antisera developed against Chago cells (R-170) or against h u man lung cancer cells derived from a cancer pat i ent (R-149). The results o f this comparison are shown in Fig. 3. The immunoglobulin binding due to NRS was subtracted f r om the data presented for both antisera. In Fig. 3, it can be n o t e d t ha t the glutaraldehyde-fixed cells give greater or equivalent a n t i b o d y binding with R-170 through 1 : 640 dilution and with R-149 to a dilution of 1 : 160. Above a dilution o f 1 : 160 o f R-149 the unfixed cells appear to bind a n t i b o d y s om e w hat bet t er than the fixed cells. Specificity o f assay Results after adsorption o f immunoglobulin by glutaraldehyde-fixed cells. The application of the assay to quantitate the a m o u n t of specific a n t i b o d y ANTIBODY BINDING WITH FRESH AND FIXED CHAGO TARGET CELLS
3
A Fixed Ceils
8
\
6
~
Fixed Cells • Fresh Cells o
4
1,80
1 /160
1 ' 320
1 '640
ANTISERUM DILUTION R 170
1:80
1, 160
1,320
1 '640
ANTISERUM DILUTION R 149
Fig. 3. C o m p a r i s o n o f a n t i b o d y b i n d i n g to fresh and g l u t a r a l d e h y d e - f i x e d cells. Chago cells, 5 × 10 5 cells per t u b e , were r e a c t e d w i t h t w o d i f f e r e n t anti-lung t u m o r antisera. The c p m b o u n d by NRS at each d i l u t i o n have b e e n s u b t r a c t e d f r o m the total c p m b o u n d , for b o t h antisera.
376 p r e s e n t in an a n t i s e r u m to a p a r t i c u l a r h u m a n c a n c e r was t e s t e d b y a d s o r b i n g t w o p r e v i o u s l y d e s c r i b e d a n t i s e r a against h u m a n lung c a n c e r ( R - 1 7 0 a n d R - 1 4 9 ) w i t h m y e l o i d l e u k e m i a ( K - 5 6 2 ) tissue c u l t u r e cells. T h e a d s o r p t i o n s o f t h e s e a n t i s e r a a n d o f N R S were p e r f o r m e d using 1 : 80 d i l u t i o n s o f all sera, a d s o r b i n g w i t h 4 × 106 o f g l u t a r a l d e h y d e - f i x e d K - 5 6 2 cells/ml o f s e r u m . T h e i n c u b a t i o n t i m e o f t h e K - 5 6 2 cells w i t h t h e sera was 16 h, p e r f o r m e d w i t h gentle r o t a t i o n o f t h e sera a n d cells at 4°C o n an electric r o t a t o r at 6 rev/ m i n . T h e a d s o r b e d a n d u n a d s o r b e d sera w e r e t h e n a s s a y e d w i t h K - 5 6 2 a n d w i t h Chago t a r g e t cells, using t h e m e t h o d d e s c r i b e d in this p a p e r with t w o e x c e p t i o n s . T o e c o n o m i z e on t h e a m o u n t o f a d s o r b e d s e r u m , o n l y 25 pl o f s e r u m w e r e used in e a c h assay t u b e instead o f t h e 100 pl s t a n d a r d u s e d in o u r assay. T h e results o f this e x p e r i m e n t are s h o w n in Fig. 4. A d s o r p t i o n o f b o t h a n t i s e r a gave similar results w i t h t h e t w o antisera ( R - 1 7 0 a n d R - 1 4 9 ) . B e f o r e a d s o r p t i o n , b o t h a n t i s e r a r e a c t e d s t r o n g l y against K - 5 6 2 , w h e r e a s t h e a d s o r b e d sera gave similar results as N R S . On t h e o t h e r h a n d , m u c h o f t h e r e a c t i v i t y o f t h e a n t i s e r u m against C h a g o lung c a n c e r r e m a i n e d a f t e r a d s o r p t i o n . T h e results o f this e x p e r i m e n t i n d i c a t e t h a t t h e g l u t a r a l d e h y d e - f i x e d cells are c a p a b l e o f b i n d i n g t h e a n t i b o d y in t h e antis e r u m w h i c h is r e a c t i v e w i t h t h e antigens o f t h e a d s o r b i n g cell a n d t h a t selec-
EFFECT OF ADSORPTION ON ANTIBODY BINDING
34~ F 32~
K 562
CHAGO TARGET CELLS
Fig. 4. Effect o t adsorption of two anti-lung tumor antisera and NRS with cultured myeloid leukocytes, K-562, on antibody binding activity with the adsorbing cells and a lung tumor cell line. The horizontal lines indicate the cpm before adsorption and the downward pointing arrows show decrease in binding after adsorption. Bar graphs show post-adsorption data. Note retention of activity by antisera against the lung tumor cells, when compared with NRS, while activity of the adsorbed antisera against K-562 approximates the NRS control.
377 rive r e a c t i v i t y w i t h a s e c o n d c a n c e r cell can b e i d e n t i f i e d a f t e r such a d s o r p tion.
Reproducibility o f assay T a b l e 2 gives t h e assay results o b t a i n e d f r o m 5 i n d e p e n d e n t , c o n s e c u t i v e e x p e r i m e n t s p e r f o r m e d using 1 : 80 d i l u t i o n s o f a n t i s e r a R - 1 7 0 a n d R - 1 4 9 , N R S a n d PBS c o n t r o l , using t h e C h a g o cells as t a r g e t s in t h e assay. T h e s e e x p e r i m e n t s w e r e p e r f o r m e d w i t h n e w l y p r e s e r v e d t a r g e t cells a n d w i t h cells w h i c h h a d b e e n g l u t a r a l d e h y d e - f i x e d a n d s t o r e d f o r as l o n g as 3 m o n t h s . T h e results are p r e s e n t e d as t h e t o t a l r a d i o a c t i v i t y c o u n t s p e r m i n u t e ( c p m ) obt a i n e d f r o m t h e [12SI]protein A b o u n d t o t h e t a r g e t cell surface a n t i g e n s or p r e s e n t in t h e t u b e s as b a c k g r o u n d a n d , also, as t h e 'specific b i n d i n g i n d e x ' d e f i n e d earlier. As is s h o w n in T a b l e 2, a l t h o u g h t h e r e was general a g r e e m e n t in t h e values o b t a i n e d f o r t h e t w o a n t i s e r a in t h e five e x p e r i m e n t s , r a t h e r large s t a n d a r d d e v i a t i o n s o f t h e c p m w e r e o b s e r v e d . T h e v a r i a t i o n is m o s t likely d u e t o diff e r e n c e s in a c t u a l n u m b e r s o f t a r g e t cells p r e s e n t (cell c o u n t i n g error), e x p e r i m e n t a l e r r o r in a d d i t i o n o f r e a g e n t s , a n d a l t e r a t i o n s in t h e specific a c t i v i t y o f t h e 12SI-labeled p r o t e i n A. H o w e v e r , if o n e l o o k s at t h e indices, w h i c h w o u l d t e n d to c o r r e c t f o r t h e s e variations, closer r e p l i c a t i o n was o b t a i n e d f o r b o t h antisera.
Comparison with trypan blue exlusion assay T h e sensitivity a n d q u a n t i t a t i o n capabilities o f t h e A b G f C - S p A assay w e r e c o m p a r e d w i t h t h e t r y p a n b l u e e x c l u s i o n assay b y c o m p a r i n g t h e d i l u t i o n s at w h i c h t h e t w o tests gave d e t e c t a b l e d i f f e r e n c e s w h e n c o m p a r e d w i t h c o n t r o l
TABLE 2 REPRODUCIBILITY OF ASSAY RESULTS IN FIVE CONSECUTIVE, INDEPENDENT EXPERIMENTS WITH CHAGO TARGET CELLS Reagent tested
Counts/rain Experiment 1
R-170 R-149 NRS PBS
33 9 3 3
095 473 229 054
2
3
35 524 9 502 2 148 893
33 10 1 1
4
184 334 804 206
24 8 2 1
Mean
S.D.
29 397 6 722 1 443 606
31 8 2 1
4163 1383 669 958
0.97 0.86
0.98 0.91
5
888 503 054 591
218 907 136 470
Computed index R-170 R-149
0.99 0.97
0.96 0.85
0.98 0.93
0.98 0.93
0.02 0.10
378
TRYPAN BLUE EXCLUSION A S S A Y
Antisera
R-170: R-149:
IO0
~_
ANTIBODY BINDING A S S A Y
/k R-170 o R149 a NRS
Complement Control
o ~
• •
8o
o
8o
30
4O
20
2O
10
1/2
1 ,'4
1/8
1/64
1/256 1/128
DILUTION
1/1024
1/512
1/4096
1/2048
DILUTION
Fig. 5. C o m p a r i s o n o f a n t i b o d y binding with the trypan blue e x c l u s i o n assay o f c y t o t o x icity using R - 1 7 0 and R - 1 4 9 against Chago tissue culture cells.
conditions (e.g., the complement control in the trypan blue exclusion assay and the NRS control in the AbGfC-SpA assay). In Fig. 5 the comparison of antisera R-170 and R-149 with the Chago cultured cell being used as the target cell in each of the assays is shown.
51Cr RELEASE A S S A Y
A N T I B O D Y BINDING A S S A Y
o Anti-MBL-2
• NRS
(J
x 0 o
O Anti-MBL*2
20
• NRS
60
10
50
5 ×
~ 3o
(J
~,-r 20 m 10
1/8
1/32
1/128 DILUTION
1/512 1/2048
. . . . . . . . . . . . . . . . . . . . . I I I I I I I I I 1/8 1/32 1/128 1/512 1/2048 DILUTION
Fig. 6. C o m p a r i s o n o f the SlCr release assay ( A ) w i t h the A b G f C - S p A assay (B) using MBL-2 murine l y m p h o m a cells and anti-MBL-2 antiserum. The assays have similar detection sensitivity to a dilution o f at least 1 : 512. N o t e , h o w e v e r , the e x c e l l e n t doseresponse characteristics o f the A b G f C - S p A assay.
379 In the trypan blue exclusion assay, both antisera gave significant cytotoxicity above the complement controls at dilutions of 1 : 4 or less. In comparison, using the AbGfC-SpA assay, R-170 had a titer of greater than 1 : 4096 while R-149, although less reactive than R-170, had a titer of 1 : 4096, and the results at this dilution were still significantly above the NRS control. Comparison with S~Cr release assay A comparison of the sensitivity and quantitation capabilities of the AbGfC-SpA assay with the SlCr release assay was performed using the MBL-2 mouse l y m p h o m a cell system (Fig. 6). In the 5~Cr release assay, the antiMBL-2 antiserum had a titer of 1 : 512. This antiserum gave a similar titer in the AbGfC-SpA assay. However, note that the AbGfC-SpA assay provided a dose response curve of immunoglobulin binding whereas the 51Cr release m e t h o d gave a plateau until the 'end point' dilution.
DISCUSSION The identification and isolation of antibodies" specific for antigens of cancer cells require an antiserum containing such antibodies, an effective means of removing selectively these antibodies from the antiserum, and a means of identifying the nature and a m o u n t of reactive antibody. Existing assays did not fulfill our needs because t h e y were either too insensitive (i.e., the trypan blue exclusion method), were sensitive but semi-quantitative and variable on a daily basis (i.e., the SlCr release assay) or depended upon the ready availability of fresh cells (i.e., the standard 125I-labeled protein A assay). This latter disadvantage we found to be prohibitive when we began dealing with fresh human cancer cells obtained by mincing t u m o r and washing the cells. With existing tests, the cells either had to be used that same day, or a culture line of the t u m o r had to be started, with all of the inherent problems including a possible change in the antigenicity of the cells involved. Recently, Brown et al. (1977) described a monolayer microwell modification of the 12SI-labeled protein A assay using cultured fibrosarcoma cells induc, ed in BALB/c and C57BL/6 mice and alloantisera developed against these t u m o r cells and against spleen cells. They demonstrated that the ~2sIlabeled protein A assay was more sensitive than the conventional isotopic antiglobulin assay, ascribing the increased sensitivity to the lesser binding of the protein A to IgG bound non-specifically to cells. Zeltzer and Seeger (1977) described a microassay using radioiodinated protein A for antibodies bound to cell surface antigens of t u m o r cells which were adherent, thus being a variation of the m e t h o d of Welsh et al. (1975), who developed the radioiodinated protein A assay for suspensions of cells. Both assays depend upon the availability of viable cells, thus presenting a major obstacle to the elective use of the m e t h o d to assay for antibodies binding to the surfaces of h u m a n cancers on days other than when the tissue or cells al~ ~resifly available.
380 O u r assay appears t o o v e r c o m e all o f the disadvantages e n u m e r a t e d for the o t h e r t y p e s o f assays. F o r e x a m p l e , we have s t o c k s o f g l u t a r a l d e h y d e - f i x e d h u m a n lung c a n c e r cells, n o r m a l h u m a n cells o f various organs, and o f a n u m b e r o f c u l t u r e d h u m a n c a n c e r cells r e a d y for use. We have been able to screen 40 antisera d e v e l o p e d against h u m a n lung cancers for reactivity against cell surface antigens on h u m a n lung c a n c e r cells in o n e d a y and have identified t h o s e with high reactivity w h i c h m i g h t be p r o m i s i n g sources o f specific a n t i b o d y . Zeltzer and Seeger ( 1 9 7 7 ) suggested t h a t fetal calf serum (FCS) can act as cell surface antigens in a p r o t e i n A microassay. In this regard, a l t h o u g h the cells used b y us were g r o w n in FCS and s h o w e d reactivity, we also n o t e d a similarly high r e a c t i v i t y o f g l u t a r a l d e h y d e - f i x e d , fresh h u m a n lung c a n c e r cells, w h i c h had never been in c o n t a c t with FCS, with antisera against lung c a n c e r extract. This suggests t h a t the c u l t u r e c o n d i t i o n s are n o t the source o f the m a j o r p o r t i o n o f t h e a n t i g e n - a n t i b o d y i n t e r a c t i o n s being observed. The loss o f r e a c t i v i t y o f t h e antisera with K - 5 6 2 m y e l o i d l e u k e m i a cells a f t e r a d s o r p t i o n with K - 5 6 2 cells (Fig. 4) and the r e t e n t i o n o f activity o f these antisera against a cell line (Chago) derived f r o m lung c a n c e r indicate t h e applicability o f these g l u t a r a l d e h y d e - f i x e d cells t o a d s o r p t i o n steps w h i c h we are using t o m a k e antisera m o r e specific for the antigens o f certain c a n c e r cells. O u r studies t o date indicate the assay permits sensitive, c o n v e n i e n t and rapid q u a n t i t a t i o n o f a n t i b o d y b i n d i n g t o cellular antigens a n d t h e r e f o r e m a y p r o v i d e an i m p o r t a n t t e c h n i q u e in research in this area. ACKNOWLEDGEMENTS T h e a u t h o r s wish t o t h a n k Dr. Grace C a n n o n o f Bionetics, Inc. for her g e n e r o u s gift o f tissue c u l t u r e cell lines and Dr. Alan R a b s o n o f the National C a n c e r I n s t i t u t e f o r the C h a g o cell line and his helpful e n c o u r a g e m e n t . REFERENCES Bolton, A.E. and W.M. Hunter, 1973, Biochem. J. 133, 529. Brown, J.P., J.P. Klitzman and K.E. HellstrSm, 1977, J. Immunol. Methods 15, 57. Herberman, R.B., 1972, J. Natl. Cancer Inst. 48, 265. Lozzio, C.B. and B.B. Lozzio, 1975, Blood 45, 321. Ng, A., K.R. McIntire, J.A. Braatz and R.B. Herberman, 1977, in: Regulatory Mechanisms in Lymphocyte Activation: Proceedings of Eleventh Leukocyte Culture Conference, ed. D.O. Lucas (Academic Press, New York) p. 374. Princler, G.L. and K.R. McIntire, 1975, Cell. Immunol. 15, 197. Rabson, A.S., S.W. Rosen, A.H. Tashjian, Jr. and B.D. Weintraub, 1973, J. Natl. Cancer Inst. 50,669. Rosenberg, S.A., S. Schwarz, H. Anding, C. Hyatt and G.M. Williams, 1977, J. Immunol. Methods 17, 225. Sela, B. and G.M. Edelman, 1977, J. Exp. Med. 145,443. Takahashi, T., L.J. Old, K.R. McIntire and E.A. Boyse, 1971, J. Exp. Med. 134,815. Ting, C.C. and R.B. Herberman, 1976, in: International Review of Experimental Pathology, Vol. 15, eds. G.W. Richter and M.A. Eptein (Academic Press, New York) p. 94. Welsh, K.I., G. Dorval and H. Wigzell, 1975, Nature 254, 67. Zeltzer, P.M. and R.C. Seeger, 1977, J. Immunol. Methods 17, 163.