Indirect 125I-labeled protein A assay for monoclonal antibodies to cell surface antigens

Journal o f Immunological Methods, 31 (1979) 201--209

201

© Elsevier/North-Holland Biomedical Press

INDIRECT I:SI-LABELED PROTEIN A ASSAY FOR MONOCLONAL ANTIBODIES TO CELL SURFACE ANTIGENS 1

JOSEPH P. BROWN, JOHN D. TAMERIUS 2 and INGEGERD HELLSTROM Division o f Tumor Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98104 and Departments o f Pathology and Microbiology~Immunology, University o f Washington Medical School, Seattle, WA 98195, U.S.A.

(Received 14 May 1979, accepted 3 August 1979)

An assay for detection of monoclonal hybridoma antibodies against cell surface antigens is described. Samples of spent medium from the hybridoma cultures are incubated in microtest wells with cells, either as adherent monolayers or in suspension. Antibodies bound to surface antigens are detected by successive incubations with rabbit anti-immunoglobulin serum and 12SI-labeled protein A from Staphylococcus aureus, followed by autoradiography of the microtest plate or scintillation counting of the individual wells. Particular advantages of this assay for screening hybridomas are: (1) commercially available reagents are used, (2) antibodies of any species and of any immunoglobulin class or subclass can be detected, and (3) large numbers of samples can be screened rapidly and inexpensively. We have used the assay to select hybridomas producing monoclonal antibodies to surface antigens of human melanomas and mouse sarcomas. INTRODUCTION H y b r i d o m a s , s o m a t i c cell h y b r i d s b e t w e e n m y e l o m a cells a n d spleen cells f r o m i m m u n i z e d mice (KShler and Milstein, 1 9 7 5 ) or rats (Galfre et al., 1 9 7 9 ) , w h i c h p r o d u c e large a m o u n t s o f specific m o n o c l o n a l a n t i b o d y , promise to r e v o l u t i o n i z e serological analysis o f cell surface antigens ( H e r z e n b e r g et al., 1 9 7 7 ; Melchers et al., 1 9 7 8 ) . D u r i n g the selection a n d c l o n i n g o f h y b r i d o m a s , m a n y h u n d r e d s o f samples m u s t be tested for a n t i b o d y to cell surface antigens. This requires rapid, sensitive serological tests t h a t are capable o f d e t e c t i n g b i n d i n g o f m o u s e or rat a n t i b o d i e s to a variety o f cell types. In this p a p e r we describe an assay, based on the ~:SI-labeled p r o t e i n A (IPA) assay ( B r o w n ' e t al., 1 9 7 7 ) , t h a t m e e t s these d e m a n d s . P r o t e i n A, a c o m p o n e n t o f the cell wall o f certain strains o f S t a p h y l o c o c c u s a u r e u s , binds with' high a f f i n i t y to t h e Fc region o f IgG a n t i b o d i e s o f m a n y m a m m a l s (Kronvall et al., 1 9 7 0 a ) . H o w e v e r , o n l y certain subclasses o f 1 This work was supported by Contract N01 CP53570 within the Virus Cancer Program of the National Cancer Institute, and by Grants CA14135 and CA25558 from the Na. tional Institutes of Health. 2 Present address: Department of Molecular Immunology, Scrip'ps Clinic and Research Foundation, 10666 North Torrey Pines Rd., La Jolla, CA 91037,,U,S.A. :

202 mouse IgG (Kronvall et al., 1970b) are recognized, and rat antibodies bind protein A poorly, if at all (Langone, 1978). We have found that the sensitivity of the IPA assay is increased by incubating the cells with a rabbit antiimmunoglobulin serum before the incubation with IPA, to allow detection of antibodies that do not bind IPA directly. A further modification is the use of autoradiography to detect positive reactions; for screening large numbers of samples this is simpler and cheaper than scintillation counting. The indirect IPA assay described here uses commercially available reagents to detect antibodies of most species irrespective of their immunoglobulin class or subclass, can be applied to both adherent and non-adherent cells, and is sensitive and accurate. We have used the indirect IPA assay in the isolation and cloning of hybridomas producing antibodies to cell surface antigens of human melanomas (Yeh et al., 1979) and mouse sarcomas (J. Brown, manuscript in preparation). MATERIALS AND METHODS Mice The BALB/c mice used for these studies were bred at FHCRC and maintained on a standard pellet diet and given water ad libitum. T h y m o c y t e s used to form feeder layers for hybridoma cultures were obtained from 2--4-weekold mice. Mice used for immunization were 8--16 weeks of age. Cells Sarcomas 1315 and 1460 were induced in BALB/c mice by intramuscular injection of 3-methylcholanthrene, and tissue culture lines were established. MC5-5, an MCA-transformed BALB/3T3 line, was obtained from Dr. Arup Sen (National Cancer Institute, Bethesda, MD). The cells were grown in 5% CO2 in air, in bicarbonate-buffered RPMI 1640 containing heat-inactivated fetal calf serum (80 ml/1) and supplemented with penicillin (100 U/ml), streptomycin (100 mg/1), non-essential amino acids (10 ml/1), sodium pyruvate (1 mM) and L-glutamine (2 mM). Serum and other supplements were obtained from GIBCO (Grand Island, NY), except sodium pyruvate, which was obtained from Microbiological Associates (Bethesda, MD). P3-NS1/1-Ag4-1 (NS-1) is an azaguanine-resistant BALB/c myeloma, which was provided by Dr. C. Milstein (MRC Laboratory of Molecular Biology, Cambridge, U.K.). NS-1 cells were grown in 7% CO2 in air, in bicarbonate-buffered RPMI 1640 containing heat-inactivated fetal calf serum (150 ml/1), supplemented as above, except that non-essential amino acids were not added; we refer to this as NS-1 medium. Immunization and cell fusion Two- to 4-month-old BALB/c mice were immunized with 2 intraperitoneal injections of sarcoma 1460 cells 1 week apart. Sarcomas used as immunogen were excised, minced with scissors, pressed through a stainless

203 steel screen, suspended in RPMI, and irradiated (15,000 rad). A volume of 0.2 ml, containing 50 mg of t u m o r tissue, was injected into each mouse. Four days after the second immunization, the mice were sacrificed, their spleens removed, and the spleen cells suspended in serum-free NS-1 medium. Erythrocytes were lysed by hypotonic shock. The spleen cells were mixed with NS-1 cells in a 4 : 1 ratio and pelleted in a 50 ml centrifuge tube. The cells were placed in a 37°C water bath, resuspended in serum-free NS-1 medium containing 50% (w/w) polyethylene glycol 1540 (Baker, Philipsburg, NJ) and stirred gently for 2 min. Ten volumes of NS-1 medium were added gradually and the cells spun down and resuspended gently in NS-1 medium to a concentration of 107 cells/ml. The cell suspension was dispensed in 0.1 ml aliquots into microtest wells {Falcon Plastics Co., Oxnard, CA, cat. no. 3040), and the plates were placed in a humidified 37°C incubator with 7% CO2 in air. Growth o f antibody-producing hybrids

One day after the fusion, 0.1 ml of NS-1 medium containing hypoxanthine (13.6 mg/1), aminopterin (0.176 mg/1) and thymidine (3.88 mg/1) HAT medium) was added to each well. For 2 weeks the cultures were fed at 2--3day intervals by replacing 0.1 ml of medium with fresh HAT medium. For the 3rd week NS-1 medium containing hypoxanthine and thymidine but not aminopterin was used. Subsequently we used NS-1 medium with none of these additives. A b o u t 3 weeks after the fusion, wells confluent with hybridoma cells were tested for production of specific antibody as described below. Hybridomas from selected wells were cloned by plating at 1 cell per microtest well with 6 × 10 s t h y m o c y t e s as a feeder layer. One week later the wells were examined microscopically for single clones, which were retested for antibody production. Antibody-producing clones were expanded to a b o u t 107 cells, frozen in RPMI containing heat-inactivated fetal calf serum (300 ml/1) and dimethylsulfoxide (100 ml/1) and stored in liquid nitrogen. A n tise ra

Rabbit antiserum to mouse immunoglobulin (IgG, IgA, IgM, cat. no. 21574-901, lot no. 8802D), was obtained from Behring Diagnostics (Sommerville, NJ). Rabbit antisera to mouse IgA, IgGl, IgG2a, IgG2b, IgG3 and IgM (cat. nos. 8403-01, -03, -04, -06, and -09, lot nos. AI100, AI099, AH096, 231-69-1, A J 1 0 6 and AJ105, respectively) were obtained from Bionetics (Kensington, MD). Rabbit antiserum to rat IgG (cat. no. 52-127, lot no. 15143) was obtained from Microbiological Associates. Radioiodination

Thirty micrograms of protein A (Pharmacia, Uppsala, Sweden) was incubated with 1 mCi of [12SI]Na (Amersham, Arlington Heights, IL) and

204 10 pg o f chloramine-T in 0.5 ml o f phosphate-buffered saline for 20 min at 0°C (McConahey and Dixon, 1966). The reaction was st opped by addition of 10 pg o f sodium metabisulfite. The ~2SI-labeled protein A was separated from reagents by gel filtration on Sephadex G-25 and had a specific radioactivity o f 2 × l 0 T cpm/pg. It was stored at --80° C. I n d i r e c t I P A assay Ad h er en t cells were tested as monolayers in f l a t - b o t t o m e d microtest wells (Falcon cat. no. 3040). Monolayers were prepared by adding to each well between 104 and 10 s cells (depending upon their size) suspended in 0.2 ml of culture m e d i u m and incubating t he m at 37°C in a humidified incubator with 5% CO2 in air. The assay was done the n e x t day, by which time the cells had f o r m e d a c o n f l u e n t m onol a ye r . N on- adher e nt cells were tested in suspension i n V - b o t t o m e d microtest wells (Cooke L a b o r a t o r y Products, Alexandria, VA). After each incubation and wash, suspended cells were pelleted by centrifugation for 3 min at 250 × g to allow removal of the supernatant. Multichannel pipettes (Flow Laboratories, Rockville, MD) were used for additions to the microtest wells, and a multichannel aspirator for removing m e d iu m f r o m the wells. At the start of the assay, the culture m e di um was removed from the cells by aspiration, and 0.05 ml of test sample (usually spent m edi um from h y b r i d o m a cultures) was added to each well. After incubation for 45 min in a humidified incubator, the cells were washed twice with 0.2 ml of RPMI containing heat-inactivated fetal calf serum (150 ml/1). Next, the cells were incubated with rabbit anti-mouse (or rat) immunoglobulin serum diluted in RPMI-fetal calf serum for 45 min at 37°C. The optimal dilution, chosen to give high binding to a n t i b o d y - c o a t e d cells but low non-specific binding, was d e t e r m i n e d empirically for each antiserum; in m ost cases a dilution of 1 / 1 0 0 0 was f o u n d to be suitable. The cells were then washed twice with 0.2 ml of RPMI containing bovine serum albumin (20 g/l). The final incubation was with 0.05 ml of IPA at 106 c p m / m l in RPMI-bovine serum albumin for 45 min at 37°C, followed by 2 washes with 0.2 ml of RPMI-bovine serum albumin. The microtest plates were dried at 70°C and autoradiographed overnight with Kodak X-R2 film and a Rarex-B Mid Speed X-ray intensifying screen (both obtained f r om N or t hw e s t Chemical Services, Seattle, WA) at --70°C. To facilitate these low t e m p e r a t u r e exposures, we used a light-tight cassette designed to hold up to 10 microtest plates in c o n t a c t with two 20 cm × 25 cm intensifying screens. For quantitative det erm i nat i on of 125I, the cells were transferred to 12 m m × 75 m m test tubes in 0.2 ml of 2 M NaOH (adherent cells) or RPMI-bovine serum albumin (non-adherent cells) and c o u n t e d in a Packard A u t o G a m m a scintillation counter. In the case of some of the hum a n cells that we studied, the n u m b e r of cells available for the assay was a limiting factor. These cells were tested as m on o lay er s of a b o u t 103 cells in Falcon cat. no. 3034 microtest plates. The assay was p e r f o r m e d as described above, except that incubation volumes

205 were 0.01 ml, and the wells were washed by rinsing the plate with 5 ml of medium. RESULTS AND DISCUSSION Faced with the task of testing hundreds of samples of h y b r i d o m a culture m e d iu m for antibodies to cell surface antigens, we first considered using the 12SI-labeled protein A assay (Brown et al., 1977), in which cells are incubated first with a n t i b o d y and then with IPA, which binds to IgG antibodies b o u n d to cell surface antigens. The IPA assay had several features that made it seem attractive for screening hybridomas: (1) protein A is available commercially, (2) microtest plates are used for the assay, which greatly simplifies transferring and testing large numbers of samples, and (3) the IPA assay is accurate enough to make replicate determinations unnecessary. For the particular purpose of screening hybridomas, however, the IPA assay has a serious drawback: protein A binds p r e d o m i n a t e l y to mouse antibodies of certain subclasses (IgG: and IgG3) (Kronvall et al., 1970b), and with low affinity relative to its binding to rabbit antibodies (Langone, 1978). Binding of protein A to rat antibodies is r e p o r t e d to be very p o o r (Langone, 1978). This problem can be solved by using rabbit anti-mouse (or rat) immunoglobulin serum in an intermediate incubation; the rabbit antibodies bind to mouse antibodies of any subclass, and in turn themselves are b o u n d by the IPA. Although an alternative is to use an affinity-purified, radiolabeled antimouse immunoglobulin a n t i b o d y , the indirect IPA assay has the advantage of being more readily adapted to detecting a particular immunoglobulin class or subclass, or immunoglobulins of o t h e r species. The indirect IPA assay is a b o u t 5-fold m ore sensitive than the direct assay in detecting mouse antibodies; in one experi m ent , for example, BALB/c sarcoma cells incubated with a 1/100 dilution of a h y p e r i m m u n e C57BL/6 anti-BALB/c alloantiserum b o u n d 7760 cpm in the indirect assay but only 1540 cpm in the direct assay. For d e t e c t i o n of rat antibodies the indirect assay was at least 80-fold more sensitive than the direct assay, mouse sarcoma cells incubated with a 1 / 2 0 0 0 dilution of a rat anti-mouse sarcoma antiserum binding 4670 cpm in the indirect assay but only 60 cpm in the direct assay. We also tested several m o n o c l o n a l mouse antibodies in bot h direct and indirect IPA assays. Protein A did n o t bind directly to any of the IgM antibodies tested, or to two IgG1 antibodies, but it did bind to t w o IgG2 antibodies. However, one of the IgG2 antibodies b o u n d protein A poorl y, a 10-fold amplification being obtained in the indirect assay. These results show (1) that the indirect IPA assay detects more a n t i b o d y classes and subclasses than the direct IPA assay, and (2) that for antibodies that bind protein A directly the indirect assay can be m or e sensitive. Th e detailed p r o c e d u r e described in Material and Methods is the one t hat we have developed for r out i ne screening of hybridomas. Here we shall

206 discuss t h e individual steps o f t h e assay. In t h e first i n c u b a t i o n , we generally t e s t e d 0.05 ml o f s p e n t h y b r i d o m a c u l t u r e m e d i u m , since smaller v o l u m e s are barely e n o u g h to c o v e r a m o n o l a y e r o f cells in a f l a t - b o t t o m e d m i c r o t e s t well. F o r testing cells in s u s p e n s i o n in V - b o t t o m e d wells, h o w e v e r , as little as 0.01 ml m a y be used. I n c u b a t i o n t i m e s o f 45 m i n are a d e q u a t e a n d allow t h e assay t o be c o m p l e t e d w i t h i n a f e w hours. In s o m e s i t u a t i o n s it was desirable to t e s t several d i l u t i o n s o f h y b r i d o m a m e d i u m to o b t a i n b e t t e r q u a n t i t a t i o n . T w o washes w i t h 0.2 ml o f m e d i u m a f t e r each i n c u b a t i o n c o m p l e t e l y r e m o v e u n b o u n d reagents. D u r i n g the washes, cells t e s t e d in s u s p e n s i o n are p e l l e t e d b y b r i e f c e n t r i f u g a t i o n a n d r e s u s p e n d e d b y f o r c e f u l a d d i t i o n o f t h e m e d i u m i n t o t h e c e n t e r o f t h e well, or, if t h e pellet is particularly cohesive, b y a s p i r a t i n g it into a p i p e t t e tip a n d expelling t h e cells b a c k i n t o t h e well. In t h e s e c o n d i n c u b a t i o n we used 0.05 ml o f r a b b i t a n t i - i m m u n o g l o b u l i n s e r u m . An a n t i s e r u m t h a t is e f f e c t i v e at a d i l u t i o n a b o v e 1 / 1 0 0 0 s h o u l d be selected; at l o w e r d i l u t i o n s n o n - s p e c i f i c b i n d i n g o f r a b b i t a n t i b o d i e s to the cells m a y be significant. G o a t a n t i s e r u m is n o t suitable, b e c a u s e g o a t antib o d i e s b i n d p r o t e i n A p o o r l y ( L a n g o n e , 1978). In place o f a b r o a d l y reactive a n t i - i m m u n o g l o b u l i n s e r u m , a n t i s e r a o f m o r e r e s t r i c t e d s p e c i f i c i t y , e.g. anti-

Fig. 1. Autoradiograph of a microtest plate in which spent media from 96 primary hybridoma cultures were tested by the indirect IPA assay as described in Materials and Methods. Sarcoma 1460 cells were used as targets; they were tested in suspension in V-bottomed microtest wells. The autoradiograph was exposed at --70°C with preflashed film and an intensifying screen for 17 h.

207 IgG1, m a y be used if o n e wishes to c h a r a c t e r i z e t h e b o u n d a n t i b o d y (see below). F o r the t h i r d i n c u b a t i o n , p r o t e i n A labeled with 125I to a specific radioactivity o f a b o u t 2 × 10 ~ cpm//~g was used. A c o n c e n t r a t i o n o f 106 c p m / m l gives up to 2 0 , 0 0 0 c p m for a s t r o n g positive r e a c t i o n a n d less t h a n a few h u n d r e d c p m in negative wells. F o r the i n c u b a t i o n with IPA, and f o r the washes b e f o r e a n d after it, m e d i u m c o n t a i n i n g bovine s e r u m a l b u m i n is used, since fetal calf s e r u m appears to inhibit the binding o f I P A (data n o t s h o w n ) . F o r the d e t e c t i o n o f b o u n d r a d i o a c t i v i t y , either scintillation c o u n t i n g or a u t o r a d i o g r a p h y ( P a r k h o u s e and G u a r n o t t a , 1 9 7 8 ) was used. We f o u n d the a u t o r a d i o g r a p h i c p r o c e d u r e t o be m o r e c o n v e n i e n t w h e n m o r e t h a n one o r t w o m i c r o t e s t plates are being tested. By using preflashed film a n d an intensifying screen at a t e m p e r a t u r e o f - - 7 0 ° C to p r e v e n t l a t e n t image fading (Lask e y and Mills, 1 9 7 7 ; S w a n s t r o m and S h a n k , 1 9 7 8 ) , one can d e t e c t as little as 100 c p m in an o v e r n i g h t e x p o s u r e , while 5 0 0 0 c p m is seen as an intense d a r k spot. T o d e m o n s t r a t e the use o f the indirect I P A assay, an a u t o r a d i o g r a p h o f a

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Fig. 2. Indirect IPA assays of hybridoma culture media. Serial dilutions of spent culture medium from hybridomas 1.4 (A) and 1.5 (o) were tested on sarcoma 1315 cells in suspension. Hybridoma 1.5 no longer produces antibody and serves as a negative control. Results are given as IPA bound/10 s cells.

208 m i c r o t e s t plate, in w h i c h s p e n t m e d i u m f r o m 96 p r i m a r y h y b r i d o m a c u l t u r e s w e r e t e s t e d on m o u s e s a r c o m a cells, is s h o w n in Fig. 1. T i t r a t i o n experim e n t s have s h o w n t h a t the i n d i r e c t I P A assay Can d e t e c t a n t i b o d y to cell surface antigens in s p e n t h y b r i d o m a c u l t u r e m e d i a at d i l u t i o n s u p to 1 / 1 2 5 (Fig. 2), a n d u p to 1 / 1 0 0 , 0 0 0 in sera a n d ascites fluids f r o m h y b r i d o m a bearing m i c e ( d a t a n o t s h o w n ) . We have also used the assay to d e t e r m i n e the i m m u n o g l o b u l i n class o f h y b r i d o m a a n t i b o d i e s b y r e p l a c i n g t h e r a b b i t antim o u s e i m m u n o g l o b u l i n s e r u m w i t h subclass-specific antisera (Fig. 3). D u r i n g t h e s e studies we have used t h e i n d i r e c t I P A assay to test a considerable n u m b e r o f d i f f e r e n t cell t y p e s , including s a r c o m a s , m e l a n o m a s , c a r c i n o m a s , f i b r o b l a s t s , l y m p h o m a s , t h y m o c y t e s and e r y t h r o c y t e s . T h e assay has also b e e n used to test f o r a n t i b o d i e s to viral antigens, using as t a r g e t s virions a d s o r b e d t o m i c r o t e s t wells ( N o w i n s k i et al., 1979). T h e s e d a t a i n d i c a t e t h a t t h e i n d i r e c t I P A assay will be generally useful f o r screening h y b r i d o m a s f o r a n t i b o d i e s to cell surface antigens, a n d f o r p r e l i m i n a r y char-

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Fig. 3. Determination of Ig class of hybridoma antibodies. Spent culture media from hybridomas 1.4 (at a dilution of 1/25) and 2.10 (undiluted) were tested on sarcoma 1315 cells in Suspension. The indirect IPA assay was used as described in Materials and Methods except that rabbit antisera specific for different mouse Ig classes and subclasses were used (at a dilution of 1/1000) in the second incubation. Results are given as IPA bound/10 s cells.

209

acterization of the antibodies with respect to both immunoglobulin class and antigen specificity. REFERENCES Brown, J.P., J.M. Klitzman and K.E. HellstrSm, 1977, J. Immunol. Methods 15, 57. Galfre, G., C. Milstein and B. Wright, 1979, Nature 277, 133. Herzenberg, L.A., L.A. Herzenberg and C. Milstein, 1977, in: Handbook of Experimental Immunology, ed. D.M. Weir (Blackwell, Oxford) Ch. 25. KShler, G. and C. Milstein, 1975, Nature 2 5 6 , 4 9 7 . Kronvall, G., U.S. Seal, J. Finstad and R.C. Williams, Jr., 1970a, J. Immunol. 104, 140. Kronvall, G., H.M. Grey and R.C. Williams, Jr., 1970b, J. Immunol. 105, 116. Langone, J.J., 1978, J. Immunol. Methods 24, 269. Laskey, R.A. and A.D. Mills, 1977, FEBS Lett. 82,314. McConahey, P.J. and F.J. Dixon, 1966, Int. Arch. Allergy 29, 185. Melchers, F., M. Potter and N.L. Warner, ]978, Curr. Top. Microbiol. Immunol. 81, ix. Nowinski, R.C., M.E. Lostrom, M.R. Tam, M.R. Stone and W.N. Burnette, 1979, Virology 93, 111. Parkhouse, R.M.E. and G. Guarnotta, 1978, Curr. Top. Microbiol. Immunol. 81,142. Swanstrom, R. and P.R. Shank, 1978, Anal. Biochem. 86, 78. Yeh, M.Y., I. HellstrSm, J.P. Brown, G.A. Warner, J.A. Hansen and K.E. HellstrSm, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 2927.