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Development of a rapid microELISA assay for screening hybridoma supernatants for murine monoclonal antibodies
Journal of Immunological Methods, 54 (1982) 291-296
291
Elsevier Biomedical Press
Development of a Rapid MicroELISA Assay for Screening Hybridoma Supernatants for Murine Monoclonal Antibodies H a r o l d H. H a n d l e y *" **' 1, M a r k C. G l a s s y *" * *, P a t r i c k H. C l e v e l a n d ** a n d Ivor R o y s t o n *'** • Division of Hematology/Oncology, Department of Medicine, University of California, San Diego and • * Veterans Administration Medical Center, San Diego, CA 92161, U.S.A.
(Received 10 February 1982, accepted 7 April 1982)
A micro enzyme-linked immunosorbent assay (ELISA) utilizing a filtration method has been developed which allows the rapid, simple, and sensitive detection of monoclonal antibodies that recognize either soluble or cell surface antigens. This assay involves the immobilization of target cells (or soluble antigen) onto glass fiber filter discs followed by an incubation with the test hybridoma supernatant and subsequent analysis by ELISA. A specially designed 96-well filtration device is employed which serves both as an incubation chamber and as a filtration manifold. This microELISA requires small volumes of antiserum, few target cells, and can be completed in less than 2 h. This assay is well suited for the rapid screening of murine hybridoma supernatants and can be adapted to detect monoclonal antibodies from other species.
Key words: monoclonal antibodies - - surface antigens - - ELISA
Introduction S i n c e t h e i n t r o d u c t i o n o f s o m a t i c cell h y b r i d i z a t i o n to p r o d u c e m o n o c l o n a l a n t i b o d i e s ( M o A b s ) p i o n e e r e d b y K 6 h l e r a n d M i l s t e i n (1975), g r e a t s t r i d e s h a v e b e e n m a d e i n t h e i d e n t i f i c a t i o n a n d c h a r a c t e r i z a t i o n o f a n t i g e n s ( M e l c h e r s et al., 1978; K e n n e r t et al., 1980). O n e o f t h e p r o b l e m s a s s o c i a t e d w i t h g e n e r a t i n g M o A b s entails the screening of a large number of hybridoma supernatants while searching
Address correspondence to: Harold Handley, University of California, San Diego, Hematology/Oncology, V-151, VA Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161, U.S.A. Abbreviations: MoAb, monoclonal antibody; lg, immunoglobulin; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; FBS buffer, fetal bovine serum buffer; OPD, o-phenylenediamine; HRP, horseradish peroxidase; PVC, poly-vinyl chloride; RIA, radioimmunoassay. 0022-1759/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
292 for antibody specificity. Therefore, immunoassays developed for these purposes must emphasize speed and simplicity. Recently, investigators have employed enzymed-linked immunosorbent assays (ELISA) in preference to radioimmunoassays (RIA) with equivalent or better sensitivity, improved assay time, longer shelf life of reagents, and lowered costs (Suter et al., 1980; Buchanan et al., 1981). In this report, we describe a simple 2 h microELISA for the detection of MoAbs to cell surface antigens. Our technique utilizes a highly versatile filtration manifold which serves as both an incubation chamber and filtration/washing device. A titration of a panel of MoAbs to a corresponding panel of known target antigens compares the sensitivity of polyvinyl chloride (PVC) plates with our filtration manifold method. This filtration device is well suited for the identification of both particulate antigens (Cleveland et al., 1979; Richman et al., 1981) and soluble antigens (Cleveland et al., 1981; Ryan et al., 1982).
Materials and Methods
Cell lines The following human cell lines were used in this report: CEM, MOLT-4, 8402, 8392 (the autologous Epstein-Barr virus transformed B cell counterpart to 8402, a T cell line), and WIL-2. All cells were grown in RPMI 1640 medium supplemented with 10% calf serum (Irvine Scientific) and glutamine. Cell preparation for ELISA Log-phase cells were washed 2 X (500 × g/wash) in phosphate-buffered saline (PBS; pH 7.4; N a H 2 P O 4, N a 2 H P O 4 0.9% saline) and resuspended to 2 × 107/ml in 10% fetal bovine serum (Irvine Scientific, Iruine, CA), 1% bovine serum albumin (Miles Laboratories, Elkhart, IN) and 0.3% gelatin (swine skin type I, Sigma, St. Louis, MO) in PBS with 0.01% NaN 3 (FBS buffer). Assay plates Filtration manifold plates (V and P Enterprises, San Diego, CA) have been previously described (Cleveland et al., 1979). Briefly, 96-well microtiter plates, which serve as a filter manifold, are loaded with glass-fiber filter discs and immediately prior to use moistened with FBS buffer. The plate is placed on a vacuum manifold to provide a source of vacuum to all 96 filters at once. Upon application of 380 m m / H g of vacuum 0.3 ml of fluid can be drawn through each of the 96 filters within 5 sec, thus efficiently removing soluble material and retaining particulate matter on the filter discs. Polyvinyl chloride plates (Dynatech, Alexandria, VA) were used for comparative analysis of ELISA plates. The PVC plates were precoated with FBS buffer overnight prior to use while the filter plates were not.
293
Antibody reagents A f f i n i t y purified I g G g o a t a n t i - m o u s e I g G c o n j u g a t e d to h o r s e r a d i s h p e r o x i d a s e ( H R P ; T A G O , Burlingame, C A ) was used at a 1 / 3 0 0 0 dilution in F B S buffer (without N a N 3 ) with 0.01% T h i m e r s o l (Sigma). o - P h e n y l e n e d i a m i n e ( O P D ; C a l b i o c h e m , La Jolla, C A ) was used at 4 0 0 / ~ g / m l in 0.05 M citrate buffer, p H 5.0, with 60 n g / m l p e r o x i d e ( M a l l i n k r o d t , Paris, K Y ) as s u b s t r a t e for the p e r o x i d a s e enzyme. P r o d u c t i o n a n d c h a r a c t e r i z a t i o n of m u r i n e M o A b s T101, T305.1, a n d L22, which react with T cells ( R o y s t o n et al., 1980), T cells a n d m o n o c y t e s ( F o x et al., 1981) a n d an a c t i v a t i o n antigen ( D i l l m a n et al., 1982) respectively, have been described; B522 recognizes a h u m a n Ia-like antigen (Yu et al., 1982); Leu 4a, a p a n T-cell M o A b was o b t a i n e d c o m m e r c i a l l y (Becton Dickson, Palo Alto, CA).
Assay procedure A 50 ~1 suspension of 4 X 10 6 target c e l l s / m l in F B S buffer was a d d e d to d u p l i c a t e wells in each test plate. Cells were i m m o b i l i z e d to the glass fiber filter discs b y a p p l i c a t i o n of a v a c u u m to each plate. F i f t y microliters of each test M o A b were then a d d e d a n d allowed to i n c u b a t e 30 rain, after which each well was washed 3 X with 0.3% gelatin in PBS ( g e l a t i n - P B S ) b y the s a m e v a c u u m filtration m e t h o d . PVC plates were w a s h e d 3 × (5 m i n / w a s h ) by c e n t r i f u g a t i o n at 500 X g. A f t e r washing, each well received 50 ~tl of the H R P c o n j u g a t e d goat a n t i - m o u s e I g G a n d allowed to i n c u b a t e a n a d d i t i o n a l 30 min when all wells were again w a s h e d 3 X with g e l a t i n PBS. A l l p r o c e d u r e s a n d i n c u b a t i o n s were carried out at r o o m t e m p e r a t u r e . The wells are sealed a n d 250/~1 of the O P D s u b s t r a t e are then a d d e d to each well a n d i n c u b a t e d in the d a r k for 30 rain. The r e a c t i o n was s t o p p e d b y a d d i n g 50 ~1 of 2.5 M H 2SO4 a n d the plates were r e a d visually at 490 nm using a D y n a t e c h m i c r o E L I S A reader, m o d e l M R 580.
TABLE I A TITRATION OF TI01 BINDING TO FILTER BOUND T(8402) AND B(8392) CELLS TI01 (ng/ml)
3 200 800 200 100 50 25 12.5 6.2 3.2
O1)49o readings a 8402 T cells
8392 B cells
Filter only
0.698 ± 0.049 0.631 + 0.051 0.616 ± 0.045 0.534±0.030 0.379±0.008 0.306±0.001 0.184±0.001 0.123 ± 0.004 0.076 ± 0.002
0.057 ± 0.024 0.043 ± 0.020 0.030 ± 0.008 0.037±0.013 0.012±0.004 0.035±0.001 0.030--+0.018 0.042 ± 0.010 0.052 ± 0.008
0.021 ± 0.021 0.027 ± 0.004 0.024 ± 0.003 0.031 ±0.013 0.018±0.012 0.018±0.005 0.053--+0.012 0.053 ± 0.035 0.074 ± 0.015
a OD49° readings were obtained on a microELISA reader. Numbers represent the average of 6 separate experiments, +S.D.
294
Results A n a l y s i s o f T 1 0 1 b i n d i n g t o 8392 a n d 8402, t h e a u t o l o g o u s B a n d T cell lines, r e s p e c t i v e l y , is s h o w n i n T a b l e I. T h r o u g h o u t t h e e n t i r e c o n c e n t r a t i o n o f T I 0 1 u s e d , 3.2 m g / m l t o 3.2 n g / m l , t h e M o A b o n l y r e a c t e d w i t h 8402 T cells a n d n o t 8392 B cells. U s i n g 2 X 10 5 t a r g e t c e l l s / w e l l t h i s a s s a y c a n a c c u r a t e l y d e t e c t as little as 10
i:iL 0.8,
0.7
0.5 0.4 0.3 0.2 0.1 0.0 ABCDEI
,,
ABCDE
Ill,
ABCDE
Ill
ABCDE
ABCDE
T101 Leu 4 T305 BA4 L22 Fig. 1. ELISA determination of the reactivity of murine MoAbs to human cell lines. Each well of the immunofiltration plates contained 2 × 105 cells. OD490 units were obtained with a microELISA automated reader (see Materials and Methods). Cell lines used are 8402 (A), MOLT-4 (B), CEM (C), WI-L2 (D), and 8392 (El. Each peak represents the average of 3 separate determinations.
0.3
0.2
OlI~
0.0
1ODD 400
,
100 40 [T101] ng/ml
10
4
1
Fig. 2. ELISA comparison of immunofiltration plates [] (V and P Enterprises), with polyvinyl chloride plates A (Dynatech). Each well of the respective plates contained 2× 105 8402 cells. OD490 units were obtained with a microELISA automated reader (see Materials and Methods). ~ , immunofiltration plate controls in which no cells were added to the wells; O, PVC plate controls, no cells added.
295
n g / m l of MoAb, We have been able to effectively use as few as 5 × 10 4 target cells/well resulting in similar observations (data not shown). Shown in Fig. 1 are the results of the reactivity of a series of murine MoAbs with a panel of B and T cell lines. Using 2 × 105 target cells/well, this microELISA assay can detect differences in surface density of the antigens recognized by the MoAbs. At equivalent T101 concentrations, reactivity of T101 with 8402 results in higher OD490 readings (0.65 U) than either MOLT-4 (0.54 U) or CEM (0.38 U), all of which are T cell lines. Similar results can be seen with the anti-T cell MoAbs Leu4 and T305, in that Leu4 binding to MOLT-4 cells is significantly higher than to either 8402 or C E M and T305 binding is greatest with 8402 cells. Since m a n y immunoassays are performed using PVC plates we compared these plates and the immunofiltration plates used in our ELISA system. The results of this comparison are shown in Fig. 2. Here, significantly positive OD490 readings ( > 0.150 U) can be obtained with 8-fold less T101 bound to cells using the immunofiltration plates as opposed to the PVC plates. As little as 12 n g / m l of T101 were detected with the filter plates compared to 100 n g / m l TI01 with the PVC plates.
Discussion
Since m a n y laboratories are currently using MoAb technology it is of paramount interest to employ simple, rapid assays which can screen large numbers of hybridoma supernatants, especially since many investigators are searching for rare antigens, and consequently must screen thousands of hybridoma supernatants. To this end, we have developed a microELISA for MoAbs in which large numbers of hybridoma supernatants can be rapidly screened for the presence and specificity of antibody. The assay described in this report, when read visually, takes no more than 2 h to complete. It has several distinct advantages over other systems (e,g., RIA). Filter plate assays are more sensitive than equivalent PVC plate assays. There is no requirement for overnight pretreatment of the assay plate in order to reduce non-specific adsorption of antibody. Washes can be completed in less than 1 rain without centrifugation, poly-L-lysine, or glutaraldehyde fixation. Cells are not lost in centrifugation-wash steps nor is the antigenic structure of the target cell lost or reduced during the immobilization process. The assay plates may be used to immobilize soluble antigens such as keyhole limpet hemocyanin (Ryan et al., 1982), immunoglobulin G (Cleveland et al., 1981; Ryan et al., 1982) and for particulate antigens, such as cellular debris and whole cells infected with herpes simplex virus, varicella zoster virus and cytomegalovirus (Cleveland et al., 1979, 1982; Richman et al., 1981). In addition, this assay does not alter the density of cell surface antigens since our microELISA results are in reasonable agreement with a quantitative cytofluorographic analysis of T101 binding to a variety of T cell types (Wormsley et al., 1981). The versatility of this microELISA assay method should be emphasized. This assay is not restricted to some of the Ig subclasses as are other assays (Surer et al.,
296 1980; B u c h a n a n et al., 1981), b u t may be used to determine the subclass identity a n d c o n c e n t r a t i o n by use of affinity purified a n t i - m o u s e Ig class specific (e.g., IgM, IgGza, etc.) antisera. F u r t h e r m o r e , this assay can be a d a p t e d to detect h u m a n Igs, especially h u m a n h y b r i d o m a s secreting h u m a n M o A b s (Glassy et al., 1981; H a n d l e y a n d Royston, 1982). It is i m p o r t a n t to note that affinity purified a n t i b o d y reagents e l i m i n a t e d m a n y crossreactive problems seen by ourselves a n d others (Douillard et al., 1980) with less purified reagents. E n d o g e n o u s peroxidase activity was n o t observed by us with any of our cell lines. N o significant e n d o g e n o u s activity was present when c o m p a r i n g methanol-treated, s o d i u m azide-treated, or u n t r e a t e d cells, n o r did these treatments affect T101 b i n d i n g to each cell line tested ( u n p u b l i s h e d observations). I n summary, we have successfully developed a simple 2 h m i c r o E L I S A assay capable of detecting a n d q u a n t i f y i n g M o A b s to cell surface antigens. The assay utilizes an i m m u n o f i l t r a t i o n plate which provides both a solid support for immobilizing the a n t i g e n a n d facilitates rapid washing, a t i m e - c o n s u m i n g procedure in most c o n v e n t i o n a l assays.
References Buchanan, D., M. Kamarck and N.H. Ruddle, 1981, J. lmmunoi. Methods 42, 179. Cleveland, P.H., D.D. Richman, M.N. Oxman, M.G. Wickham, P.S. Binder and D.M. Worthen, 1979, J. Immunol. Methods 29, 369. Cleveland, P.H., G. Wickham, M. Goldbaum, A. Ryan and D.M. Worthen, 1981, J. Immunoassay 2, 117. Cleveland, P.H., D.D. Richman, M.N. Oxman and D.M. Worthen, 1982, J. Clin. Microbiol. 15, 402. Dillman, R.O., D.M. Frisman, C.F. Hockman, G.L. Yamamoto, M.C. Glassy and I. Royston, 1982, submitted. Douillard, J.Y., T. Hoffman and R.B. Herberman, 1980, J. lmmunol. Methods 39, 309. Fox, R.I., L. Thompson and J.R. Huddleston, 1981, J. Immunol. 126, 2062. Glassy, M.C., H. Handley, J.E. Seegmillerand I. Royston, 1981, Fed. Proc. 40, 996. Handley, H.H. and I. Royston, 1982, in: Hybridomas in the Diagnosis and Treatment of Cancer, eds. M. Mitchel and H. Oettgen (Raven Press, New York) p. 125. Kennert, R.H., T.J. McKearn and K.B. Bechtol (eds.), 1980, Monoclonal Antibodies (Plenum, New York). K6hler, G. and C. Milstein, 1975, Nature (London) 256, 495. Melchers, F., M. Potter and N.L. Warner (eds.), 1978, Curr. Top. Microbiol. lmmunol., Vol. 81, Lymphocyte Hybridomas (Springer-Verlag,New York). Richman, D.D., P.H. Cleveland, M.N. Oxman and J.A. Zaia, 1981, J. Inf. Dis. 143, 693. Royston, I., J.A. Majda, S.M. Baird, B.L. Meserve and J.C. Griffiths, 1980, J. Immunol. 125, 725. Ryan, A.F., P.H. Cleveland and A. Catanzaro, 1982, Ann. Otol. Rhinol. Laryngol. 91, 70. Suter, L., J. Bruggen and C. Sorg, 1980, J. Immunol. Methods 39, 407. Wormsley, S.B., M.L. Collins and I. Royston, 1981, Blood 57, 657. Yu, A.L., I. Royston, K.L. Leung, F.H. Kung, G.A. Hartman, A,L. Lightsey, J. Vangrove and R.E. Sobol, 1982, Hybridoma 2, 91.
291
Elsevier Biomedical Press
Development of a Rapid MicroELISA Assay for Screening Hybridoma Supernatants for Murine Monoclonal Antibodies H a r o l d H. H a n d l e y *" **' 1, M a r k C. G l a s s y *" * *, P a t r i c k H. C l e v e l a n d ** a n d Ivor R o y s t o n *'** • Division of Hematology/Oncology, Department of Medicine, University of California, San Diego and • * Veterans Administration Medical Center, San Diego, CA 92161, U.S.A.
(Received 10 February 1982, accepted 7 April 1982)
A micro enzyme-linked immunosorbent assay (ELISA) utilizing a filtration method has been developed which allows the rapid, simple, and sensitive detection of monoclonal antibodies that recognize either soluble or cell surface antigens. This assay involves the immobilization of target cells (or soluble antigen) onto glass fiber filter discs followed by an incubation with the test hybridoma supernatant and subsequent analysis by ELISA. A specially designed 96-well filtration device is employed which serves both as an incubation chamber and as a filtration manifold. This microELISA requires small volumes of antiserum, few target cells, and can be completed in less than 2 h. This assay is well suited for the rapid screening of murine hybridoma supernatants and can be adapted to detect monoclonal antibodies from other species.
Key words: monoclonal antibodies - - surface antigens - - ELISA
Introduction S i n c e t h e i n t r o d u c t i o n o f s o m a t i c cell h y b r i d i z a t i o n to p r o d u c e m o n o c l o n a l a n t i b o d i e s ( M o A b s ) p i o n e e r e d b y K 6 h l e r a n d M i l s t e i n (1975), g r e a t s t r i d e s h a v e b e e n m a d e i n t h e i d e n t i f i c a t i o n a n d c h a r a c t e r i z a t i o n o f a n t i g e n s ( M e l c h e r s et al., 1978; K e n n e r t et al., 1980). O n e o f t h e p r o b l e m s a s s o c i a t e d w i t h g e n e r a t i n g M o A b s entails the screening of a large number of hybridoma supernatants while searching
Address correspondence to: Harold Handley, University of California, San Diego, Hematology/Oncology, V-151, VA Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161, U.S.A. Abbreviations: MoAb, monoclonal antibody; lg, immunoglobulin; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; FBS buffer, fetal bovine serum buffer; OPD, o-phenylenediamine; HRP, horseradish peroxidase; PVC, poly-vinyl chloride; RIA, radioimmunoassay. 0022-1759/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
292 for antibody specificity. Therefore, immunoassays developed for these purposes must emphasize speed and simplicity. Recently, investigators have employed enzymed-linked immunosorbent assays (ELISA) in preference to radioimmunoassays (RIA) with equivalent or better sensitivity, improved assay time, longer shelf life of reagents, and lowered costs (Suter et al., 1980; Buchanan et al., 1981). In this report, we describe a simple 2 h microELISA for the detection of MoAbs to cell surface antigens. Our technique utilizes a highly versatile filtration manifold which serves as both an incubation chamber and filtration/washing device. A titration of a panel of MoAbs to a corresponding panel of known target antigens compares the sensitivity of polyvinyl chloride (PVC) plates with our filtration manifold method. This filtration device is well suited for the identification of both particulate antigens (Cleveland et al., 1979; Richman et al., 1981) and soluble antigens (Cleveland et al., 1981; Ryan et al., 1982).
Materials and Methods
Cell lines The following human cell lines were used in this report: CEM, MOLT-4, 8402, 8392 (the autologous Epstein-Barr virus transformed B cell counterpart to 8402, a T cell line), and WIL-2. All cells were grown in RPMI 1640 medium supplemented with 10% calf serum (Irvine Scientific) and glutamine. Cell preparation for ELISA Log-phase cells were washed 2 X (500 × g/wash) in phosphate-buffered saline (PBS; pH 7.4; N a H 2 P O 4, N a 2 H P O 4 0.9% saline) and resuspended to 2 × 107/ml in 10% fetal bovine serum (Irvine Scientific, Iruine, CA), 1% bovine serum albumin (Miles Laboratories, Elkhart, IN) and 0.3% gelatin (swine skin type I, Sigma, St. Louis, MO) in PBS with 0.01% NaN 3 (FBS buffer). Assay plates Filtration manifold plates (V and P Enterprises, San Diego, CA) have been previously described (Cleveland et al., 1979). Briefly, 96-well microtiter plates, which serve as a filter manifold, are loaded with glass-fiber filter discs and immediately prior to use moistened with FBS buffer. The plate is placed on a vacuum manifold to provide a source of vacuum to all 96 filters at once. Upon application of 380 m m / H g of vacuum 0.3 ml of fluid can be drawn through each of the 96 filters within 5 sec, thus efficiently removing soluble material and retaining particulate matter on the filter discs. Polyvinyl chloride plates (Dynatech, Alexandria, VA) were used for comparative analysis of ELISA plates. The PVC plates were precoated with FBS buffer overnight prior to use while the filter plates were not.
293
Antibody reagents A f f i n i t y purified I g G g o a t a n t i - m o u s e I g G c o n j u g a t e d to h o r s e r a d i s h p e r o x i d a s e ( H R P ; T A G O , Burlingame, C A ) was used at a 1 / 3 0 0 0 dilution in F B S buffer (without N a N 3 ) with 0.01% T h i m e r s o l (Sigma). o - P h e n y l e n e d i a m i n e ( O P D ; C a l b i o c h e m , La Jolla, C A ) was used at 4 0 0 / ~ g / m l in 0.05 M citrate buffer, p H 5.0, with 60 n g / m l p e r o x i d e ( M a l l i n k r o d t , Paris, K Y ) as s u b s t r a t e for the p e r o x i d a s e enzyme. P r o d u c t i o n a n d c h a r a c t e r i z a t i o n of m u r i n e M o A b s T101, T305.1, a n d L22, which react with T cells ( R o y s t o n et al., 1980), T cells a n d m o n o c y t e s ( F o x et al., 1981) a n d an a c t i v a t i o n antigen ( D i l l m a n et al., 1982) respectively, have been described; B522 recognizes a h u m a n Ia-like antigen (Yu et al., 1982); Leu 4a, a p a n T-cell M o A b was o b t a i n e d c o m m e r c i a l l y (Becton Dickson, Palo Alto, CA).
Assay procedure A 50 ~1 suspension of 4 X 10 6 target c e l l s / m l in F B S buffer was a d d e d to d u p l i c a t e wells in each test plate. Cells were i m m o b i l i z e d to the glass fiber filter discs b y a p p l i c a t i o n of a v a c u u m to each plate. F i f t y microliters of each test M o A b were then a d d e d a n d allowed to i n c u b a t e 30 rain, after which each well was washed 3 X with 0.3% gelatin in PBS ( g e l a t i n - P B S ) b y the s a m e v a c u u m filtration m e t h o d . PVC plates were w a s h e d 3 × (5 m i n / w a s h ) by c e n t r i f u g a t i o n at 500 X g. A f t e r washing, each well received 50 ~tl of the H R P c o n j u g a t e d goat a n t i - m o u s e I g G a n d allowed to i n c u b a t e a n a d d i t i o n a l 30 min when all wells were again w a s h e d 3 X with g e l a t i n PBS. A l l p r o c e d u r e s a n d i n c u b a t i o n s were carried out at r o o m t e m p e r a t u r e . The wells are sealed a n d 250/~1 of the O P D s u b s t r a t e are then a d d e d to each well a n d i n c u b a t e d in the d a r k for 30 rain. The r e a c t i o n was s t o p p e d b y a d d i n g 50 ~1 of 2.5 M H 2SO4 a n d the plates were r e a d visually at 490 nm using a D y n a t e c h m i c r o E L I S A reader, m o d e l M R 580.
TABLE I A TITRATION OF TI01 BINDING TO FILTER BOUND T(8402) AND B(8392) CELLS TI01 (ng/ml)
3 200 800 200 100 50 25 12.5 6.2 3.2
O1)49o readings a 8402 T cells
8392 B cells
Filter only
0.698 ± 0.049 0.631 + 0.051 0.616 ± 0.045 0.534±0.030 0.379±0.008 0.306±0.001 0.184±0.001 0.123 ± 0.004 0.076 ± 0.002
0.057 ± 0.024 0.043 ± 0.020 0.030 ± 0.008 0.037±0.013 0.012±0.004 0.035±0.001 0.030--+0.018 0.042 ± 0.010 0.052 ± 0.008
0.021 ± 0.021 0.027 ± 0.004 0.024 ± 0.003 0.031 ±0.013 0.018±0.012 0.018±0.005 0.053--+0.012 0.053 ± 0.035 0.074 ± 0.015
a OD49° readings were obtained on a microELISA reader. Numbers represent the average of 6 separate experiments, +S.D.
294
Results A n a l y s i s o f T 1 0 1 b i n d i n g t o 8392 a n d 8402, t h e a u t o l o g o u s B a n d T cell lines, r e s p e c t i v e l y , is s h o w n i n T a b l e I. T h r o u g h o u t t h e e n t i r e c o n c e n t r a t i o n o f T I 0 1 u s e d , 3.2 m g / m l t o 3.2 n g / m l , t h e M o A b o n l y r e a c t e d w i t h 8402 T cells a n d n o t 8392 B cells. U s i n g 2 X 10 5 t a r g e t c e l l s / w e l l t h i s a s s a y c a n a c c u r a t e l y d e t e c t as little as 10
i:iL 0.8,
0.7
0.5 0.4 0.3 0.2 0.1 0.0 ABCDEI
,,
ABCDE
Ill,
ABCDE
Ill
ABCDE
ABCDE
T101 Leu 4 T305 BA4 L22 Fig. 1. ELISA determination of the reactivity of murine MoAbs to human cell lines. Each well of the immunofiltration plates contained 2 × 105 cells. OD490 units were obtained with a microELISA automated reader (see Materials and Methods). Cell lines used are 8402 (A), MOLT-4 (B), CEM (C), WI-L2 (D), and 8392 (El. Each peak represents the average of 3 separate determinations.
0.3
0.2
OlI~
0.0
1ODD 400
,
100 40 [T101] ng/ml
10
4
1
Fig. 2. ELISA comparison of immunofiltration plates [] (V and P Enterprises), with polyvinyl chloride plates A (Dynatech). Each well of the respective plates contained 2× 105 8402 cells. OD490 units were obtained with a microELISA automated reader (see Materials and Methods). ~ , immunofiltration plate controls in which no cells were added to the wells; O, PVC plate controls, no cells added.
295
n g / m l of MoAb, We have been able to effectively use as few as 5 × 10 4 target cells/well resulting in similar observations (data not shown). Shown in Fig. 1 are the results of the reactivity of a series of murine MoAbs with a panel of B and T cell lines. Using 2 × 105 target cells/well, this microELISA assay can detect differences in surface density of the antigens recognized by the MoAbs. At equivalent T101 concentrations, reactivity of T101 with 8402 results in higher OD490 readings (0.65 U) than either MOLT-4 (0.54 U) or CEM (0.38 U), all of which are T cell lines. Similar results can be seen with the anti-T cell MoAbs Leu4 and T305, in that Leu4 binding to MOLT-4 cells is significantly higher than to either 8402 or C E M and T305 binding is greatest with 8402 cells. Since m a n y immunoassays are performed using PVC plates we compared these plates and the immunofiltration plates used in our ELISA system. The results of this comparison are shown in Fig. 2. Here, significantly positive OD490 readings ( > 0.150 U) can be obtained with 8-fold less T101 bound to cells using the immunofiltration plates as opposed to the PVC plates. As little as 12 n g / m l of T101 were detected with the filter plates compared to 100 n g / m l TI01 with the PVC plates.
Discussion
Since m a n y laboratories are currently using MoAb technology it is of paramount interest to employ simple, rapid assays which can screen large numbers of hybridoma supernatants, especially since many investigators are searching for rare antigens, and consequently must screen thousands of hybridoma supernatants. To this end, we have developed a microELISA for MoAbs in which large numbers of hybridoma supernatants can be rapidly screened for the presence and specificity of antibody. The assay described in this report, when read visually, takes no more than 2 h to complete. It has several distinct advantages over other systems (e,g., RIA). Filter plate assays are more sensitive than equivalent PVC plate assays. There is no requirement for overnight pretreatment of the assay plate in order to reduce non-specific adsorption of antibody. Washes can be completed in less than 1 rain without centrifugation, poly-L-lysine, or glutaraldehyde fixation. Cells are not lost in centrifugation-wash steps nor is the antigenic structure of the target cell lost or reduced during the immobilization process. The assay plates may be used to immobilize soluble antigens such as keyhole limpet hemocyanin (Ryan et al., 1982), immunoglobulin G (Cleveland et al., 1981; Ryan et al., 1982) and for particulate antigens, such as cellular debris and whole cells infected with herpes simplex virus, varicella zoster virus and cytomegalovirus (Cleveland et al., 1979, 1982; Richman et al., 1981). In addition, this assay does not alter the density of cell surface antigens since our microELISA results are in reasonable agreement with a quantitative cytofluorographic analysis of T101 binding to a variety of T cell types (Wormsley et al., 1981). The versatility of this microELISA assay method should be emphasized. This assay is not restricted to some of the Ig subclasses as are other assays (Surer et al.,
296 1980; B u c h a n a n et al., 1981), b u t may be used to determine the subclass identity a n d c o n c e n t r a t i o n by use of affinity purified a n t i - m o u s e Ig class specific (e.g., IgM, IgGza, etc.) antisera. F u r t h e r m o r e , this assay can be a d a p t e d to detect h u m a n Igs, especially h u m a n h y b r i d o m a s secreting h u m a n M o A b s (Glassy et al., 1981; H a n d l e y a n d Royston, 1982). It is i m p o r t a n t to note that affinity purified a n t i b o d y reagents e l i m i n a t e d m a n y crossreactive problems seen by ourselves a n d others (Douillard et al., 1980) with less purified reagents. E n d o g e n o u s peroxidase activity was n o t observed by us with any of our cell lines. N o significant e n d o g e n o u s activity was present when c o m p a r i n g methanol-treated, s o d i u m azide-treated, or u n t r e a t e d cells, n o r did these treatments affect T101 b i n d i n g to each cell line tested ( u n p u b l i s h e d observations). I n summary, we have successfully developed a simple 2 h m i c r o E L I S A assay capable of detecting a n d q u a n t i f y i n g M o A b s to cell surface antigens. The assay utilizes an i m m u n o f i l t r a t i o n plate which provides both a solid support for immobilizing the a n t i g e n a n d facilitates rapid washing, a t i m e - c o n s u m i n g procedure in most c o n v e n t i o n a l assays.
References Buchanan, D., M. Kamarck and N.H. Ruddle, 1981, J. lmmunoi. Methods 42, 179. Cleveland, P.H., D.D. Richman, M.N. Oxman, M.G. Wickham, P.S. Binder and D.M. Worthen, 1979, J. Immunol. Methods 29, 369. Cleveland, P.H., G. Wickham, M. Goldbaum, A. Ryan and D.M. Worthen, 1981, J. Immunoassay 2, 117. Cleveland, P.H., D.D. Richman, M.N. Oxman and D.M. Worthen, 1982, J. Clin. Microbiol. 15, 402. Dillman, R.O., D.M. Frisman, C.F. Hockman, G.L. Yamamoto, M.C. Glassy and I. Royston, 1982, submitted. Douillard, J.Y., T. Hoffman and R.B. Herberman, 1980, J. lmmunol. Methods 39, 309. Fox, R.I., L. Thompson and J.R. Huddleston, 1981, J. Immunol. 126, 2062. Glassy, M.C., H. Handley, J.E. Seegmillerand I. Royston, 1981, Fed. Proc. 40, 996. Handley, H.H. and I. Royston, 1982, in: Hybridomas in the Diagnosis and Treatment of Cancer, eds. M. Mitchel and H. Oettgen (Raven Press, New York) p. 125. Kennert, R.H., T.J. McKearn and K.B. Bechtol (eds.), 1980, Monoclonal Antibodies (Plenum, New York). K6hler, G. and C. Milstein, 1975, Nature (London) 256, 495. Melchers, F., M. Potter and N.L. Warner (eds.), 1978, Curr. Top. Microbiol. lmmunol., Vol. 81, Lymphocyte Hybridomas (Springer-Verlag,New York). Richman, D.D., P.H. Cleveland, M.N. Oxman and J.A. Zaia, 1981, J. Inf. Dis. 143, 693. Royston, I., J.A. Majda, S.M. Baird, B.L. Meserve and J.C. Griffiths, 1980, J. Immunol. 125, 725. Ryan, A.F., P.H. Cleveland and A. Catanzaro, 1982, Ann. Otol. Rhinol. Laryngol. 91, 70. Suter, L., J. Bruggen and C. Sorg, 1980, J. Immunol. Methods 39, 407. Wormsley, S.B., M.L. Collins and I. Royston, 1981, Blood 57, 657. Yu, A.L., I. Royston, K.L. Leung, F.H. Kung, G.A. Hartman, A,L. Lightsey, J. Vangrove and R.E. Sobol, 1982, Hybridoma 2, 91.