Journal o f Immunological Methods, 15 (1977) 57--66 © Elsevier/North-Holland Biomedical Press
57
A MICROASSAY FOR ANTIBODY BINDING TO TUMOR CELL S U R F A C E A N T I G E N S U S I N G 12SI-LABELLED P R O T E I N A F R O M STAPHYLOCOCCUS A UREUS 1
JOSEPH P. BROWN, JACK M. KLITZMAN and KARL ERIK HELLSTROM Division o f Immunology,'Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington 98104, U.S.A. and Departments o f Pathology and Microbiology/Immunology, University o f Washignton Medical School, Seattle, Washington 98195, U.S.A.
(Received 8 September 1976, accepted 11 October 1976)
A sensitive assay for antibodies bound to the surface antigens of adherent tumor cells using protein A from Staphylococcus aureus is described. Cells, as monolayers in microtest wells, are incubated first with immune or control serum, and then with 12SI-labelled protein A (IPA), which binds to IgG antibodies bound to cell surface antigens. The IPA assay is shown to be more sensitive than the conventional isotopic antiglobulin (IAG) assay, because IPA binds to a lesser extent to IgG bound non-specifically to cells.
INTRODUCTION Cell surface antigens are c o m m o n l y d e m o n s t r a t e d b y m e a s u r i n g the binding t o the cells o f a n t i b o d i e s f r o m specific antisera. T e c h n i q u e s in w h i c h t h e a n t i b o d i e s b o u n d are d e t e c t e d b y an isotopically labelled a n t i - i m m u n o globulin (Ig) reagent, have been widely used f o r this p u r p o s e . In t h e i s o t o p i c a n t i g l o b u l i n ( I A G ) assay described b y H a r d e r and M c K h a n n ( 1 9 6 8 ) 12sIlabelled g o a t a n t i - m o u s e Ig was used. Cells were i n c u b a t e d first with i m m u n e or c o n t r o l serum, a n d t h e n with t h e I A G reagent. T h e a m o u n t o f I A G b o u n d t o cells was a m e a s u r e o f t h e a m o u n t o f Ig b o u n d in the first i n c u b a t i o n . H o w e v e r , this assay a n d s u b s e q u e n t m o d i f i c a t i o n s (Sparks et al., 1 9 6 9 ; Burdick et al., 1 9 7 3 ; G o l d s t e i n et al., 1 9 7 3 ; H u a n g et al., 1 9 7 5 ) suffer f r o m t h e d i s a d v a n t a g e t h a t , in a d d i t i o n t o a n t i b o d i e s b o u n d specifically to cell surface antigens, Ig b o u n d n o n - s p e c i f i c a l l y t o the cell surface is also d e t e c t e d , as s h o w n b y the relatively high b i n d i n g o f I A G t o t u m o r cells i n c u b a t e d with c o n t r o l sera. P r o t e i n A f r o m S. a u r e u s ( V e r w e y , 1 9 4 0 ) , w h i c h has a high a f f i n i t y f o r i This work was supported by grants CA 19148 and CA 19149 from the National Institutes of Health, by grant IM-43 F from the American Cancer Society, by contract NO1 CB 64018 from the National Cancer Institute and by contract NO1 CP 53570 within the Virus Cancer Program of the National Cancer Institute.
58 IgG antibodies (Kronvall et al., 1970b) has been used to measure IgG antibodies bound to surface antigens of lymphoid cells (Dorval et al., 1974a, b, 1976; Welsh et al., 1975). In this paper, we describe an assay which uses t2sIlabelled protein A (IPA) to measure antibody binding to surface antigens of cells from solid tumors, which are tested as adherent monlayers in microtest wells. The IPA assay is compared with a conventional IAG assay for the measurement of alloantigens and t u m o r antigens on murine MCA-induced fibrosarcomas. MATERIAL AND METHODS Mice
BALB/c and C57BL/6 mice bred in this laboratory, were maintained on a standard pellet diet and given water ad libitum. These mice accepted intrastrain skin grafts. Mice used in this study were approximately two months old. Tumors
Tumors 1315 and 1415 are fibrosarcomas induced in BALB/c mice and t u m o r 1412 is a fibrosarcoma induced in a C57BL/6 mouse, by intramuscular injection of 3-methylcholanthrene (MCA) as described previously (Hellstr6m et al., 1970). They were serially transplanted in syngeneic mice by subcutaneous inoculation of 5 × 106 cells to the flank. Tumors were palpable 7--10 days after transplantation, grew to 15 mm in diameter in 15--25 days, and killed their host in 30--50 days. Cells
Tumor cells were prepared for tissue culture by mincing and trypsinizing the tumor. The cells were filtered through cheese cloth, pelleted by centrifugation at 250 g, resuspended, and seeded into 75 cm 2 flasks (Falcon Plastics, Oxnard, California). Dulbecco's culture medium was used, supplemented with penicillin G, 100 units/ml (Squibb, Princeton, New Jersey), streptomycin, 100 pg/ml (GIBCO, Grand Island, New York), L-glutamine, 290 pg/ml (GIBCO), sodium pyruvate, 1 mM (Microbiological Associates, Bethesda, Maryland), non-essential amino acids, 10 ml/1 (GIBCO), and fetal calf serum (FCS), 200 ml/1 (Microbiological Associates), buffered with sodium bicarbonate (GIBCO). This medium is referred to as Dulbecco's FCS (DFCS). Cells were detached for passage to new culture flasks or to microtest wells with EDTA (0.2 g/1 in Tris-buffered saline) and were then diluted to the required concentration with DFCS.
59 Sera Mice were bled from the retro-orbital sinus. Sera were stored at --80°C in small volumes, so that samples were never frozen and thawed more than once.
Alloantisera designated C57BL/6 anti-BALB/c were obtained from C57BL/6 mice injected intraperitoneally with 5 × 107 BALB/c spleen cells, rechallenged at least twice at two week intervals, and bled two weeks later. Alloantisera designated BALB/c anti-C57BL/6 were obtained from BALB/c mice immunized similarly, but with C57BL/6 spleen cells. Tumor-bearer sera were obtained from BALB/c mice at various times after inoculation with 5 × 106 live 1315 t u m o r cells to the flank. Mice were never inoculated with cells which had been in tissue culture medium, so that there was no possibility of immunization against the proteins in FCS. Normal sera from age-matched untreated mice were used as controls. Proteins
Protein A, isolated from a m u t a n t strain of S. aureus, was purchased from Pharmacia (Uppsala, Sweden). Bovine serum albumin (BSA) was obtained from Sigma (St. Louis, Missouri). Specific antibody was purified from goat anti-mouse IgG1 serum (supplied by Dr. J. Clagett) by immunoadsorption on a column of a m m o n i u m sulphate purified mouse Ig (Herbert, 1974) coupled to Sepharose (Axen et al., 1967), followed by elution with NaSCN (3 M in PBS), and gel filtration into PBS on a column of Sephadex G-25. Protein concentrations were determined by spectrophotometry, using an A1% of 1.65 for protein A (SjSquist et al., 1972) and of 14.5 for IgG 280nm (Binaghi Benacerraf, 1964). Radioactivity measurements
A Packard Auto-Gamma scintillation counter was used for 12sI determination. Data are presented in counts per minute (cpm), as the mean of two replicates and the standard error of the mean (S.E.). Protein iodination
Protein A (30 pg) was labelled with Na '2sI (1 mCi, Amersham-Searle, Arlington Heights, Illinois) and chloramine-T (10 pg) in 0.5 ml of PBS, in a modification of the m e t h o d of McConahey and Dixon (1966). The reaction was carried out for 20 rain at 0°C, and stopped by addition of sodium metabisulphite (10 pg). The '2SI-labelled protein was separated from excess reagents by gel filtration on Sephadex G-25 in PBS and stored at --80 ° C. IPA had a specific radioactivity of 2 × 107 cpm/pg. Goat anti-mouse Ig antibody
60 (400 pg) was labelled similarly, the reaction being carried out for 5 min, which gave a specific acitivity of 1 × 106 cpm/pg.
IPA assay Cells were tested as monolayers in flat b o t t o m e d microtest wells {MicroTest II, Falcon) as in the IAG assay o f Burdick et al. (1973). Monolayers were prepared by adding l 0 s cells, suspended in 200 pl of DFCS, to each well and incubating t hem at 37°C in a humid i ncubat or with 5% CO2 in air. The assay was done the next day, by which time the cells had formed a confluent mo n o lay er . Cell viability, d e m o n s t r a t e d by t r y p a n blue exclusion, was at least 95%. At the start of the assay the DFCS was removed from the cells by aspiration, and 25 pl o f i m m une or control serum, usually at a 1/25 dilution in DFCS, was added to each well. Each serum was tested in duplicate. After incubation for 1 h at 37°C, the serum was removed, and the cells were washed twice with 200 pl of Dulbecco's medium containing the supplements m e n t i o n e d previously but with BSA (10 g/l) instead of FCS. This medium is referred to as Dulbecco's-BSA (DBSA). IPA (10 s cpm) was then added in 25 pl of DBSA. After incubation for I h at 37°C, the reagent was removed, and the cells were washed twice with 200 pl of DBSA. The cells were then dissolved in 200 pl of sodium h y d r o x i d e (2 M) and transferred to p o l y p r o p y l e n e vials (Bio-Vial, Beckman, Palo Alto, California) for 12sI determination. RESULTS
Demonstration of alloantigens The IPA assay was evaluated using t u m o r cells from MCA-induced fibrosarcomas. First, we showed that very little IPA, only 110 -+ 20 cpm (9 experiments), b o u n d to cells which had been incubated with DFCS alone in the first incubation. In all subsequent experiments we included control wells, in which cells were incubated with culture m edi um alone in the first incubation, the a m o u n t of IPA b o u n d to the cells in these control wells being subtracted from th at bound after incubation with the test sera. In this way, we c o r r ected for the small a m o u n t of IPA b o u n d directly to the cells, the remaining IPA being that b o u n d to IgG on the cells. Cells from tu m or s 1315 and 1415, induced by MCA in BALB/c mice, incubated with a C57BL/6 anti-BALB/c alloantiserum b o u n d up to several h u n d r e d times more IPA than cells incubated with a control normal serum fr o m C57BL/6 mice (table 1), demonstrating the high senstivity o f the IPA assay. Immunological specificity was shown in a criss-cross e x p e r i m e n t using cells f r o m t u m o r 1315, described above, and from t u m o r 1412, induced by
61 TABLE 1 Binding of IPA to BALB/c tumor cells incubated with specific alloantiserum or control serum. Serum *
C57BL/6 normal C57BL/6 anti-BALB/c
IPA bound (cpm ± S.E.) Tumor 1315
Tumor 1415
60 ± 10 12070 ± 140
180 ± 70 7530 ± 850
* Sera tested at a dilution of 1/25.
M C A i n a C 5 7 B L / 6 m o u s e . L i t t l e I P A b o u n d t o cells f r o m e i t h e r t u m o r a f t e r i n c u b a t i o n w i t h c o n t r o l n o r m a l s e r u m f r o m e i t h e r s t r a i n . H o w e v e r , cells f r o m each t u m o r i n c u b a t e d with the specific a l l o a n t i s e r u m b o u n d m o r e t h a n 7 0 0 0 c p m o f I P A ( t a b l e 2), w h e r e a s cells i n c u b a t e d w i t h t h e c o n t r o l a l l o a n t i s e r u m b o u n d a b o u t f o r t y t i m e s less I P A .
Demonstration of tumor antigens We a t t e m p t e d t o d e t e c t t u m o r - a s s o c i a t e d a n t i g e n s p r e s e n t o n cells f r o m tumor 1315, using serum from BALB/c mice bearing that tumor. Such t u m o r - b e a r e r sera h a v e b e e n r e p o r t e d t o c o n t a i n a n t i b o d i e s t o t u m o r a n t i g e n s d e t e c t a b l e b y b o t h t h e 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 assay a n d t h e I A G a s s a y ( F r i t z e et al., 1 9 7 5 ) . T h e I P A a s s a y gave r e s u l t s i n a g r e e m e n t w i t h t h i s r e p o r t ( t a b l e 3 ) ; t u m o r cells i n c u b a t e d w i t h t u m o r - b e a r e r s e r u m b o u n d a b o u t o n e h u n d r e d t i m e s m o r e I P A t h a n cells i n c u b a t e d w i t h c o n t r o l normal serum. We u s e d t h e I P A a s s a y t o m e a s u r e t h e d e v e l o p m e n t o f h u m o r a l i m m u n i t y in mice b e a r i n g a growing s y n g e n e i c t u m o r . Sera o b t a i n e d f r o m B A L B / c TABLE 2 Binding of IPA to tumor cells incubated with specific or control alloantisera or normal sera. Serum *
BALB/c normal C57BL/6 normal BALB/c anti-C57BL/6 C57BL/6 anti-BALB/c
IPA bound (cpm ± S.E.) BALB/c tumor **
C57BL/6 tumor ***
20 60 230 7150
70 50 9140 220
± ± ± ±
* Sera tested at a dilution of 1/25. ** Tumor 1315. *** Tumor 1412.
10 10 30 610
• ± ± ±
90 20 170 10
62 TABLE 3 B i n d i n g o f I P A t o cells f r o m t u m o r Serum *
1315 incubated with tumor-bearer
o r c o n t r o l sera.
I P A b o u n d ( c p m -+ S . E . )
BALB/c normal 1315 tumor-bearer
2 0 ~: 3 0 4220 ± 180
**
* Sera tested at a dilution of 1/25. ** Bled 16 days after tumor inoculation.
mice at various times after inoculation with tumor 1315 were tested on cells from the same tumor (fig. 1). This showed that antibodies directed against tumor-associated antigens were clearly detectable at 10 days and increased steadily thereafter.
Effect of serum dilution T h e e f f e c t o f diluting t h e t e s t sera on t h e s u b s e q u e n t b i n d i n g o f I P A is s h o w n in fig. 2. Serial f o u r f o l d dilutions o f an a l l o a n t i s e r u m , a t u m o r - b e a r e r s e r u m a n d a c o n t r o l s e r u m w e r e t e s t e d o n t u m o r cells, starting w i t h a dilut i o n o f 1 / 2 5 . T h e a m o u n t o f I P A b o u n d d e c r e a s e d w i t h increasing dilution, 5000-
4000A Cl
o
3000-
r~ 2000
57
A MICROASSAY FOR ANTIBODY BINDING TO TUMOR CELL S U R F A C E A N T I G E N S U S I N G 12SI-LABELLED P R O T E I N A F R O M STAPHYLOCOCCUS A UREUS 1
JOSEPH P. BROWN, JACK M. KLITZMAN and KARL ERIK HELLSTROM Division o f Immunology,'Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington 98104, U.S.A. and Departments o f Pathology and Microbiology/Immunology, University o f Washignton Medical School, Seattle, Washington 98195, U.S.A.
(Received 8 September 1976, accepted 11 October 1976)
A sensitive assay for antibodies bound to the surface antigens of adherent tumor cells using protein A from Staphylococcus aureus is described. Cells, as monolayers in microtest wells, are incubated first with immune or control serum, and then with 12SI-labelled protein A (IPA), which binds to IgG antibodies bound to cell surface antigens. The IPA assay is shown to be more sensitive than the conventional isotopic antiglobulin (IAG) assay, because IPA binds to a lesser extent to IgG bound non-specifically to cells.
INTRODUCTION Cell surface antigens are c o m m o n l y d e m o n s t r a t e d b y m e a s u r i n g the binding t o the cells o f a n t i b o d i e s f r o m specific antisera. T e c h n i q u e s in w h i c h t h e a n t i b o d i e s b o u n d are d e t e c t e d b y an isotopically labelled a n t i - i m m u n o globulin (Ig) reagent, have been widely used f o r this p u r p o s e . In t h e i s o t o p i c a n t i g l o b u l i n ( I A G ) assay described b y H a r d e r and M c K h a n n ( 1 9 6 8 ) 12sIlabelled g o a t a n t i - m o u s e Ig was used. Cells were i n c u b a t e d first with i m m u n e or c o n t r o l serum, a n d t h e n with t h e I A G reagent. T h e a m o u n t o f I A G b o u n d t o cells was a m e a s u r e o f t h e a m o u n t o f Ig b o u n d in the first i n c u b a t i o n . H o w e v e r , this assay a n d s u b s e q u e n t m o d i f i c a t i o n s (Sparks et al., 1 9 6 9 ; Burdick et al., 1 9 7 3 ; G o l d s t e i n et al., 1 9 7 3 ; H u a n g et al., 1 9 7 5 ) suffer f r o m t h e d i s a d v a n t a g e t h a t , in a d d i t i o n t o a n t i b o d i e s b o u n d specifically to cell surface antigens, Ig b o u n d n o n - s p e c i f i c a l l y t o the cell surface is also d e t e c t e d , as s h o w n b y the relatively high b i n d i n g o f I A G t o t u m o r cells i n c u b a t e d with c o n t r o l sera. P r o t e i n A f r o m S. a u r e u s ( V e r w e y , 1 9 4 0 ) , w h i c h has a high a f f i n i t y f o r i This work was supported by grants CA 19148 and CA 19149 from the National Institutes of Health, by grant IM-43 F from the American Cancer Society, by contract NO1 CB 64018 from the National Cancer Institute and by contract NO1 CP 53570 within the Virus Cancer Program of the National Cancer Institute.
58 IgG antibodies (Kronvall et al., 1970b) has been used to measure IgG antibodies bound to surface antigens of lymphoid cells (Dorval et al., 1974a, b, 1976; Welsh et al., 1975). In this paper, we describe an assay which uses t2sIlabelled protein A (IPA) to measure antibody binding to surface antigens of cells from solid tumors, which are tested as adherent monlayers in microtest wells. The IPA assay is compared with a conventional IAG assay for the measurement of alloantigens and t u m o r antigens on murine MCA-induced fibrosarcomas. MATERIAL AND METHODS Mice
BALB/c and C57BL/6 mice bred in this laboratory, were maintained on a standard pellet diet and given water ad libitum. These mice accepted intrastrain skin grafts. Mice used in this study were approximately two months old. Tumors
Tumors 1315 and 1415 are fibrosarcomas induced in BALB/c mice and t u m o r 1412 is a fibrosarcoma induced in a C57BL/6 mouse, by intramuscular injection of 3-methylcholanthrene (MCA) as described previously (Hellstr6m et al., 1970). They were serially transplanted in syngeneic mice by subcutaneous inoculation of 5 × 106 cells to the flank. Tumors were palpable 7--10 days after transplantation, grew to 15 mm in diameter in 15--25 days, and killed their host in 30--50 days. Cells
Tumor cells were prepared for tissue culture by mincing and trypsinizing the tumor. The cells were filtered through cheese cloth, pelleted by centrifugation at 250 g, resuspended, and seeded into 75 cm 2 flasks (Falcon Plastics, Oxnard, California). Dulbecco's culture medium was used, supplemented with penicillin G, 100 units/ml (Squibb, Princeton, New Jersey), streptomycin, 100 pg/ml (GIBCO, Grand Island, New York), L-glutamine, 290 pg/ml (GIBCO), sodium pyruvate, 1 mM (Microbiological Associates, Bethesda, Maryland), non-essential amino acids, 10 ml/1 (GIBCO), and fetal calf serum (FCS), 200 ml/1 (Microbiological Associates), buffered with sodium bicarbonate (GIBCO). This medium is referred to as Dulbecco's FCS (DFCS). Cells were detached for passage to new culture flasks or to microtest wells with EDTA (0.2 g/1 in Tris-buffered saline) and were then diluted to the required concentration with DFCS.
59 Sera Mice were bled from the retro-orbital sinus. Sera were stored at --80°C in small volumes, so that samples were never frozen and thawed more than once.
Alloantisera designated C57BL/6 anti-BALB/c were obtained from C57BL/6 mice injected intraperitoneally with 5 × 107 BALB/c spleen cells, rechallenged at least twice at two week intervals, and bled two weeks later. Alloantisera designated BALB/c anti-C57BL/6 were obtained from BALB/c mice immunized similarly, but with C57BL/6 spleen cells. Tumor-bearer sera were obtained from BALB/c mice at various times after inoculation with 5 × 106 live 1315 t u m o r cells to the flank. Mice were never inoculated with cells which had been in tissue culture medium, so that there was no possibility of immunization against the proteins in FCS. Normal sera from age-matched untreated mice were used as controls. Proteins
Protein A, isolated from a m u t a n t strain of S. aureus, was purchased from Pharmacia (Uppsala, Sweden). Bovine serum albumin (BSA) was obtained from Sigma (St. Louis, Missouri). Specific antibody was purified from goat anti-mouse IgG1 serum (supplied by Dr. J. Clagett) by immunoadsorption on a column of a m m o n i u m sulphate purified mouse Ig (Herbert, 1974) coupled to Sepharose (Axen et al., 1967), followed by elution with NaSCN (3 M in PBS), and gel filtration into PBS on a column of Sephadex G-25. Protein concentrations were determined by spectrophotometry, using an A1% of 1.65 for protein A (SjSquist et al., 1972) and of 14.5 for IgG 280nm (Binaghi Benacerraf, 1964). Radioactivity measurements
A Packard Auto-Gamma scintillation counter was used for 12sI determination. Data are presented in counts per minute (cpm), as the mean of two replicates and the standard error of the mean (S.E.). Protein iodination
Protein A (30 pg) was labelled with Na '2sI (1 mCi, Amersham-Searle, Arlington Heights, Illinois) and chloramine-T (10 pg) in 0.5 ml of PBS, in a modification of the m e t h o d of McConahey and Dixon (1966). The reaction was carried out for 20 rain at 0°C, and stopped by addition of sodium metabisulphite (10 pg). The '2SI-labelled protein was separated from excess reagents by gel filtration on Sephadex G-25 in PBS and stored at --80 ° C. IPA had a specific radioactivity of 2 × 107 cpm/pg. Goat anti-mouse Ig antibody
60 (400 pg) was labelled similarly, the reaction being carried out for 5 min, which gave a specific acitivity of 1 × 106 cpm/pg.
IPA assay Cells were tested as monolayers in flat b o t t o m e d microtest wells {MicroTest II, Falcon) as in the IAG assay o f Burdick et al. (1973). Monolayers were prepared by adding l 0 s cells, suspended in 200 pl of DFCS, to each well and incubating t hem at 37°C in a humid i ncubat or with 5% CO2 in air. The assay was done the next day, by which time the cells had formed a confluent mo n o lay er . Cell viability, d e m o n s t r a t e d by t r y p a n blue exclusion, was at least 95%. At the start of the assay the DFCS was removed from the cells by aspiration, and 25 pl o f i m m une or control serum, usually at a 1/25 dilution in DFCS, was added to each well. Each serum was tested in duplicate. After incubation for 1 h at 37°C, the serum was removed, and the cells were washed twice with 200 pl of Dulbecco's medium containing the supplements m e n t i o n e d previously but with BSA (10 g/l) instead of FCS. This medium is referred to as Dulbecco's-BSA (DBSA). IPA (10 s cpm) was then added in 25 pl of DBSA. After incubation for I h at 37°C, the reagent was removed, and the cells were washed twice with 200 pl of DBSA. The cells were then dissolved in 200 pl of sodium h y d r o x i d e (2 M) and transferred to p o l y p r o p y l e n e vials (Bio-Vial, Beckman, Palo Alto, California) for 12sI determination. RESULTS
Demonstration of alloantigens The IPA assay was evaluated using t u m o r cells from MCA-induced fibrosarcomas. First, we showed that very little IPA, only 110 -+ 20 cpm (9 experiments), b o u n d to cells which had been incubated with DFCS alone in the first incubation. In all subsequent experiments we included control wells, in which cells were incubated with culture m edi um alone in the first incubation, the a m o u n t of IPA b o u n d to the cells in these control wells being subtracted from th at bound after incubation with the test sera. In this way, we c o r r ected for the small a m o u n t of IPA b o u n d directly to the cells, the remaining IPA being that b o u n d to IgG on the cells. Cells from tu m or s 1315 and 1415, induced by MCA in BALB/c mice, incubated with a C57BL/6 anti-BALB/c alloantiserum b o u n d up to several h u n d r e d times more IPA than cells incubated with a control normal serum fr o m C57BL/6 mice (table 1), demonstrating the high senstivity o f the IPA assay. Immunological specificity was shown in a criss-cross e x p e r i m e n t using cells f r o m t u m o r 1315, described above, and from t u m o r 1412, induced by
61 TABLE 1 Binding of IPA to BALB/c tumor cells incubated with specific alloantiserum or control serum. Serum *
C57BL/6 normal C57BL/6 anti-BALB/c
IPA bound (cpm ± S.E.) Tumor 1315
Tumor 1415
60 ± 10 12070 ± 140
180 ± 70 7530 ± 850
* Sera tested at a dilution of 1/25.
M C A i n a C 5 7 B L / 6 m o u s e . L i t t l e I P A b o u n d t o cells f r o m e i t h e r t u m o r a f t e r i n c u b a t i o n w i t h c o n t r o l n o r m a l s e r u m f r o m e i t h e r s t r a i n . H o w e v e r , cells f r o m each t u m o r i n c u b a t e d with the specific a l l o a n t i s e r u m b o u n d m o r e t h a n 7 0 0 0 c p m o f I P A ( t a b l e 2), w h e r e a s cells i n c u b a t e d w i t h t h e c o n t r o l a l l o a n t i s e r u m b o u n d a b o u t f o r t y t i m e s less I P A .
Demonstration of tumor antigens We a t t e m p t e d t o d e t e c t t u m o r - a s s o c i a t e d a n t i g e n s p r e s e n t o n cells f r o m tumor 1315, using serum from BALB/c mice bearing that tumor. Such t u m o r - b e a r e r sera h a v e b e e n r e p o r t e d t o c o n t a i n a n t i b o d i e s t o t u m o r a n t i g e n s d e t e c t a b l e b y b o t h t h e 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 assay a n d t h e I A G a s s a y ( F r i t z e et al., 1 9 7 5 ) . T h e I P A a s s a y gave r e s u l t s i n a g r e e m e n t w i t h t h i s r e p o r t ( t a b l e 3 ) ; t u m o r cells i n c u b a t e d w i t h t u m o r - b e a r e r s e r u m b o u n d a b o u t o n e h u n d r e d t i m e s m o r e I P A t h a n cells i n c u b a t e d w i t h c o n t r o l normal serum. We u s e d t h e I P A a s s a y t o m e a s u r e t h e d e v e l o p m e n t o f h u m o r a l i m m u n i t y in mice b e a r i n g a growing s y n g e n e i c t u m o r . Sera o b t a i n e d f r o m B A L B / c TABLE 2 Binding of IPA to tumor cells incubated with specific or control alloantisera or normal sera. Serum *
BALB/c normal C57BL/6 normal BALB/c anti-C57BL/6 C57BL/6 anti-BALB/c
IPA bound (cpm ± S.E.) BALB/c tumor **
C57BL/6 tumor ***
20 60 230 7150
70 50 9140 220
± ± ± ±
* Sera tested at a dilution of 1/25. ** Tumor 1315. *** Tumor 1412.
10 10 30 610
• ± ± ±
90 20 170 10
62 TABLE 3 B i n d i n g o f I P A t o cells f r o m t u m o r Serum *
1315 incubated with tumor-bearer
o r c o n t r o l sera.
I P A b o u n d ( c p m -+ S . E . )
BALB/c normal 1315 tumor-bearer
2 0 ~: 3 0 4220 ± 180
**
* Sera tested at a dilution of 1/25. ** Bled 16 days after tumor inoculation.
mice at various times after inoculation with tumor 1315 were tested on cells from the same tumor (fig. 1). This showed that antibodies directed against tumor-associated antigens were clearly detectable at 10 days and increased steadily thereafter.
Effect of serum dilution T h e e f f e c t o f diluting t h e t e s t sera on t h e s u b s e q u e n t b i n d i n g o f I P A is s h o w n in fig. 2. Serial f o u r f o l d dilutions o f an a l l o a n t i s e r u m , a t u m o r - b e a r e r s e r u m a n d a c o n t r o l s e r u m w e r e t e s t e d o n t u m o r cells, starting w i t h a dilut i o n o f 1 / 2 5 . T h e a m o u n t o f I P A b o u n d d e c r e a s e d w i t h increasing dilution, 5000-
4000A Cl
o
3000-
r~ 2000
