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Cellular enzyme-linked immunocircle assay
Journal of Immunological Methods, 153 (1992) 141-149
141
© 1992 Elsevier SciencePublishers B.V. All rights reserved l)022-1759/92/$()5.00
JIM 06397
Cellular enzyme-linked immunocircle assay A rapid assay of hybridomas produced against cell surface antigens P6ter Balogh, Zsuzsa Beb~k and P6ter N 6 m e t h Immunological and Biotechnological Laboratory of the Unicersity Medical School of Pdcs, P~cs, Hungary
(Received 13 November1991, revisedreceived29 January 1992, accepted 3 April 1992)
A novel method has been developed for the initial screening of hybridomas produced against cell surface antigens. Giutaraldehyde-fixed cells were immobilized as targets on the lid of a 96-well tissue culture plate which had been precoated with po!y-L-lysine. Antibody binding was determined by an immunoenzymatic method in an arrangement permitting both macro- and microscopic examination. After optimization with mouse thymus cells using existing rat monoclonal antibodies, new rat-mouse hybridoma cell lines against mouse thymocytes and bone marrow cells were screened. The antibodies could be characterized immediately both by the localization of the immune reaction (surface or intracellular) or as estimated by the frequency of positive cells recognized by the antibody in the sample. Key words: Hybridomascreening; Circle assay; ELISA, cellular; lmmunocytochemistry
Introduction Hybridoma screening assays must be both specific and sensitive at the same time (Eshhar, 1985). in contrast to soluble antigens, where the detection of specific antibody is relatively easy using ELISA (Engvall, 1980), techniques for the initial screening of hybridomas against certain cell surface antigens have to balance speed of
Correspondence to: P. Balogh, Immunologicaland Biotechnological Laboratory of the University Medical School of P~cs, Szigeti u. 12. H-7643, Hungary. Abbreviations: AEC, 3-amin6-9-ethylcarbazole; DMSO, dimethyl-sulpho~de; ELISA, enzyme-linked immunosorbent assay; HBSS, Hanks' balanced salt solution; PBS, phosphatebuffered saline containing0.2% sodium azide; PBS-0.1% BSA, PBS containing 0.1% bovine serum albumin; PBS-5% FBS, PBS containing 5% fetal bovine serum; SABC, Streptavidinbiotinylated horseradisch peroxidase complex.
preparation and examination against the amount of information gained about each positive (Table l). For example, both radioisotopic and immunofluorescent detection, though sensitive and reliable (Johnstone and Thorpe, 1987), need special instrumentation and conditions. Conventional immunohistochemical methods are difficult to scale up to deal with, say, 800-1000 samples simultaneously. In contrast, large numbers of samples are easily screened by cellular ELISA systems (Douillard et al., 1980; Cobbold and Waldmann, 1981) but these methods do not reveal either the density of the antigen, or its localization (surface or cytoplasmic), or the frequency of positive cells among the whole population. Moreover, the reliability of the cellular ELISA procedure has been questioned (Drover and .Marshall, 1986) because of false positive reactions especially involving lgM class antibodies.
Here we report a simple immunoenzymatic hybridoma screening system performed on glutaraldehyde-fixed target cells. This assay can be carried out conveniently on large number of samples (at least 1000 in a day's assay) and it permits direct light microscopic observation of the test reaction. The approximate frequency of cells expressing the particular antigen can also be immediately estimated. Cells are linked by poly-L-lysine to 8 x 12 arrays of depressions ('circles') moulded to the lids of 96-well tissue culture plates (instead of to the wells themselves) and then fixed with glutaraldehyde. Conventional immunoperoxidase methods reveal samples macroscopically and these can then immediately be examined microscopically.
Materials and methods
Animals Inbred Wistar rats and inbred BALB/c, C57BL/6, CBA/J and AKR/J mice were used between 6 and 16 weeks of age. The experimental animals were maintained under conventional conditions in the breeding unit of our animal facility. Cell lines and antibodies "l~he Sp2/0-Agl4 murine myeloma cell line (Flow Labs, UK) is maintained in the Biotechnological Facility's cell repository. Tile !4.8 antiB220 rat-mouse hybridoma cell line (Kincade et al., 1981) was produced and generously provided by Dr. Paul W. Kincade of the Oklahoma Medi-
cal Research Foundation, Oklahoma, USA. Cells were cultured in RPMI 1640 medium (Flow Labs, UK) supplemented with 10% FBS (Sebak, Germany) and gentamicin (Chinoin, Hungary) at 50 /zg/ml concentration. Rat anti-mouse monoclonal antibodies (Serva, Heidelberg, Germany) were: anti-Ly2 (CD8) (clone: YTS 169.4 lgG2b, K), anti-L3T4 (CD4) (clone: YTS 191.1.1, lgG2b, K) (Cobbold et al., 1984). The anti-B220 (IgG2b, K) antibody was used as undiluted supernatant. Sheep anti-rat IgG serum was produced by the Mez6hegyes State Farm, Hungary, and the rat lgG-reactive antibodies were affinity purified on cyanogen bromide activated Sepharose 4B (Pharmacia LKB, Sweden) coupled with rat IgG, previously purified on a protein G (Pharmacia) column according to the manufacturer's instructions. The bound sheep antibodies were recovered by sequential elution with 0.1 M glycine-HCI (pH 2.5) and 0.1 M triethylamine (pH 11.5) (Harlow and Lane, 1988). The eluted material was pooled and dialyzed against PBS overnight at 4°C. After repeated absorption of mouse IgG-reactive sheep antibodies on a mouse IgG-coupled affinity column until no bound material was demonstrable, the remaining antibody fraction was labelled with either biotin-N-hydroxysuccinimide ester (Calbiochem, USA) or fluoresceinisothiocyanate (Sigma Chemical Co., USA) as previously described (Mason et al., 1987). The biotinylated anti-rat IgG antibodies were tested in ELISA with immobilized mouse or rat IgG as antigen followed by SABC as developing reagent,
TABLE ! COMPARISON BETWEENTHE MAIN TECHNICALASPECTSOF THE MOSTFREQUENTLYUSED HYBRIDOMA SCREENINGMETHODS Method CellularELISA Radioimmunobindingassay Flowcytometer (FACS) Immunofluorescence (suspension) lmmunohistochemistry (smearsor tissue)
Specialequipment No
Scale Large
Information Quantitative
Yes Yes
Large Limited
Quantitative Quantitative/ qualitative Qualitative topographical Qualitative topographical
Yes
Limited
No
Verylimited
143 and the reactivity of the conjugate against mouse IgG was observed to be weak (not shown). The FITC/protein molar ratio was determined photometrically and calculated as 2.65.
Cellular immunocircle assay 96-well polystyrene tissue culture plate lids (Greiner, Germany, cat no. 656101)with 96 circular areas corresponding to the plate's wells were used. These provided 8 mm diameter wells with roughly 1 mm high rims moulded in an 8 x 12 array. They were rinsed with distilled water and precoated with 20 /zl poly-L-lysine (MW 10,000, Serva, Germany) dissolved in PBS at 1 m g / m l , and incubated in a humidified chamber at room temperature for 30 min. After rinsing with PBS, 20 t~! of a 2 × 107 cells/ml washed cell suspension in PBS were added per circle using a 12channel microdispensor, and allowed to sediment and to bind to the bottom for 30 min at room temperature. The cells were fixed by 20 ml 0.125% glutaraldehyde (Fluka, Germany) in PBS prechilled to 4°C (Carroll et al., 1990) carefully poured onto the periphery of the lid, in order to avoid disturbing the target cell layer. After 5 min fixation, the fixative was removed and the lids were thoroughly washed three times with PBS. The remaining active groups of glutaraldehyde were blocked with 5% FBS in PBS for 2 h at room temperature. The lids could be stored filled with 1% BSA-PBS containing 0.2% azide at 4°C for up to at least 1 month preserving the original (freshly prepared) antigenic activity. On the day of use, non-specific binding was blocked with normal sheep serum at 1/20 dilution for 20 min at room temperature. Then, after the removal of the serum, the circled areas were filled with 20 tzl of the first antibody at appropriate dilution. Positive and negative controls included immune and pre-immune sera at 1/100 dilution in 0.1% BSA-PBS. The lids were incubated at room temperature for 40 min, then washed three times by rinsing with PBS for 5 min. Each circle received 20 /~1 of biotinylated secondary antibody (sheep anti-rat IgG at 15 p.g/ml) and the reaction was allowed to proceed at room temperature for 40 min. The lids were washed as previously, and streptavidin-biotinylated horseradish peroxidase complex (SABC) (Amersham
International, Aylesbury, UK) was added and the plate incubated for a further 40 min at room temperature. All incubation steps were performed in a humid chamber to prevent drying out of the samples. After washing, the reaction was monitored using 2 0 / x l / w e l l AEC (Sigma, USA) as chromogen and H 2 0 2 as substrate in 0.1 M acetate buffer (pH 5.2) as previously described (Graham et al., 1965). Positive wells were easily recognized by the naked eye as red dots within the circular areas. The membrane-bound reaction was confirmed by subsequent microscopic examination comparing the results of an individual well with negative or positive control wells.
Cellular ELISA It was performed according to Carrol et ai. (1990) with the modification that the immune reaction was developed using biotinylated sheep anti-rat lgG antibodies and SABC. The colour reagent was 1.2-phenylenediamine dihydrochloride (Fluka, Germany) dissolved in 0.1 M citrate buffer, pH 5.2, and the absorption was recorded at a wavelength of 492 nm. Rat-mouse hybridoma production 8-week-old female Wistar rats were immunized intraperitoneaily with 107 spleen cells, derived from 4-week-old C 5 7 B L / 6 mice and boosted 3 and 5 weeks later. 1 week after the last immunization, the rats were injected intravenously with 107 spleen cells. 3 days later the spleen cells from the best responder rat were fused with Sp2/0-Agl4 cells as described previously (KiShler and Milstein, 1975), distributed in 96-well fiat bottomed plates (Greiner, Germany) and selected on H A T (Littlefield, 1964). The hybrid clones were tested for specific antibody production with the above cellular immunocircle assay using B A L B / c derived thymus cells or bone marrow ceils as targets. 20 ttl of supernatant were added and incubated at room temperature for 40 min, followed by extensive washing and further incubation with biotinylated sheep anti-rat IgG and SABC as described above. The positive results were confirmed by indirect immunofluorescence performed on unfixed cells according to previous methods (Harlow and Lane, 1988). The positive wells were cloned by limiting dilution,
and the clones were re-checked as above. The positive monoelones were expanded in 75 ml Costar tissue culture flasks.
lmmunofluorescence and immunohistochemistry In immunofluorescence, the target cell suspension in PBS-0.1% BSA (Sigma Chemical Co., USA) was incubated with the first antibody at 4"C for 30 min in a U bottomed 96-well microtiter plate (Greiner), washed three times with PBS and incubated for a further 30 rain at 4°C with FITC-labelled sheep anti-rat IgG (produced in our laboratory) diluted in 0.1% BSA-PBS. After washing with PBS, the cells were fixed with 1% buffered formaldehyde and stored at 4°C until examination. Monoclonal antibodies were further characterized on frozen and acetone-fixed sections from different mouse organs using the biotin-streptavidin-HRPO method as described previously (Hsu, 1985). After addition of the first antibody, the specimens were incubated with biotinylated sheep anti-rat IgG antibody followed by repeated washing. SABC was added and after a 40 rain incubation at room temperature the plate was thoroughly washed. The reaction was visualized with AEC, and the specimens were counterstained with Mayer's haematoxylin before mounting in gelatin.
Sepharose 4B (Sigma Chemical Co., USA) procoated with nonimmune rat IgG. The supernatant was incubated with hybridoma supernatant, and the immune complexes were collected with protein G-Sepharose 4B previously washed in lysis buffer. The unbound material was removed by repeated centrifugation and washing. The absorbed proteins were released by boiling for 10 rain in a sample buffer containing 0.125 M Tris-HCI, 2% SDS, 10% glycerol with or without 5% 2-mercaptoethanol, pH 6.8. SDS-PAGE was performed on Laemmli 10% polyacrylamide gels using the Mini-Protean I1 apparatus (Bio-Rad Laboratories, USA). After the electrophoresis, the gel was equilibrated for 30 min in a transfer buffer suitable for semi-dry transfer (48 mM Tris, 39 mM glycine, 0.037% SDS, 20% methanol). Proteins were transferred to a Hybond C nitrocellulose membrane (Amersham International, Aylesbury, UK) by blotting for 30 rain at 150 mA current using Sartoblot II-S (Sartorius, Germany). The membrane was soaked in blotting buffer (PBS containing 3% BSA, 1% FCS and 0.1% Tween 20) overnight at 4°C. After thorough washing in PBS containing 0.1% Tween-20, SABC at 1/i000 dilution in PBS-Tween was added for 2 h with constant agitation at room temperature. After extensive washing, the reaction was developed using AEC and 0.003% H20 2 in 0.1 M sodiumacetate buffer, pH 5.2.
Initial characterization of the antigen detected by 3Hl l-A1 Mab Thymus cells were labelled with N-hydroxysuccinimidobiotin (NHS-biotin, Sigma Chemical Co., USA) essentially as described by Ingalls et al. (1986) with slight modifications. Briefly, the cells at 5 × 107/ml concentration in HBSS were washed in 0.15 M NaCI (pH 8.5 adjusted with 0A M sodium-bicarbonate/carbonate buffer pH 9) instead of alkaline HBSS used by others (Miyake et al., 1990). After incubation with NHS-biotin dissolved in DMSO at 50 mg/ml (2 mg/ml final concentration) on ice for 40 min, the cells were repeatedly washed with chilled PBS. The washed cells were extracted by lysis buffer containing 10 mM Tris-HCl, 0.15 M NaCI, 1% NP-40, 1 mM PMSF and 0.1% sodium-azide pH 7.5. After the removal of nuclei by centrifugation, the lysates were precleared twice with protein G-coated
Results
,
Preliminary experiments showed that rat antimouse CD4, CD8 (positive) and B220 (CD45) (negative) gave equivalent results to those from a parallel immunofluorescence assay on mouse thymocyte targets in the immunocircle assay. The precipitated red end-product of the reaction was readily discriminated from the negative colourless circles. Holding the lid at a shallow angle relative to the line of vision improved the visibility. The sensitivity of the assay was checked on mouse thymocyte targets by titrating pre-fusion sera from rats immunized with mouse splenocytes and comparing the results with the ELISA titre using the same cell numbers. Reactions were detectable using a dilution of immune sera of
145 2 w e e k s after fusion o f i m m u n e rat s p l e e n cells with S p 2 / 0 - A g I 4 cells, h y b r i d o m a s u p e r n a t a n t s were s c r e e n e d o n immobilized t h y m u s or b o n e m a r r o w cells. A b r o a d variation in t h e colour intensity w a s observed, w h i c h correlated well with t h e microscopic a p p e a r a n c e o f t h e s a m p l e s . A m o n g positive circles, t h e majority o f t h e cells was intensely labelled. However, in s o m e c a s e s t h e antibody in t h e s u p e r n a t a n t recognized only a s u b p o p u l a t i o n within t h e t h y m u s cells (Figs. 2 a 2d). In t h e case o f b o n e m a r r o w ceils we f o u n d only two positive s u p e r n a t a n t s , b o t h o f which r e a c t e d with t h e majority o f t h e cells. The hybridoma supernatants which were strongly positive o n t h y m o c y t e s in t h e i m m u n o c i r cle assay were also positive in t h e i m m u n o f l u o r e s c e n t assay u s i n g live cells w h e r e a s m o s t o f t h e weakly positive s u p e r n a t a n t s were negative by i m m u n o f l u o r e s c e n c e (Table II). T h e m o n o c l o n a l a n t i b o d i e s s h o w n o n T a b l e 111 were o b t a i n e d f r o m t h e two fusions.
D
Fig. 1. Titration of sera from mouse spleen cell immunized rats using fixed thymocytes as target. The assay was carried out as described in the materials and methods section. In the first antibody layer, immune (A - B) and preimmune (C = D) sera obtained from two different rats were added at different dilutions (indicated at the top).
1 / 1 0 0 0 (Fig. 1). Microscopic inspection t h e n revealed t h e cell s u r f a c e localization o f t h e i m m u n e reaction (see below). 1
2
3
4
5
6
?
~ , ~ ~ . ~ ~
8
9
I0 II 12
~..~, -~
A
.~-.--.. B.
C.
- .. :~.:.~,~ D.
Fig. 2. Macroscopic and microscopic appearance of the circle assay. BALB/c derived thymocytes were bound to the plastic surface and the circle assay was carried out using hybridoma supernatants. A: macroscopic appearance, where at bottom right (HI2 well) rat immune serum was added at !/100 dilution. B: microscopic picture of the G8 well. C: EIO well. D: GI2 well (negative control with normal pre-immune rat serum diluted 1/50 adde,]). Magnification: 380x.
TABLE !1 EFFICIENCY OF CIRCLE ASSAY IN THE SCREENING OF HYBRIDOMAS AGAINST MURINE CELL SURFACE MARKERS Fusion no." Target antigen
Frequencyof positive wells/their immunofluorescent control Strong b
R2 R3
Thymocytes I/ I Thymocytes 3/3 Bone marrow cells I / I
Weak ¢ 2/0 4/1 1/0
a Sp2/0-Agl4 cells were fused to Wistar rat spleen cells immunized with murine spleen cells and distributed in 96-well tissue culture plates. h Both macroscopically and microscopically detectable reaction. c Only microscopically detectable, ambiguous reaction.
Fig. 3. Reactivity of 3HII-AI monoclonal antibody on frozen section from BALB/c spleen. The first-layer monoclonal antibody was added as neat tissue culture supernatant. The overwhelming majority of cells surrounding the arteriole (indicated by *) are labelled. The reaction was developed using the streptavidin-biotin system. Magnification: 280×.
Ecaluation of the assay with low target cell frequency As the preliminary data showed, it was possible to isolate a monoclonal antibody which app e a r e d to recognize a T cell antigen (Figs. 3 and 4 and Table liD. E x p e r i m e n t s were p e r f o r m e d to d e t e r m i n e the lowest frequency of cells which were distinguishable by this assay. T h y m u s cells obtained from B A L B / c mice and Wistar rats were mixed t o g e t h e r at different ratios, immobi-
lized on the culture plate lids, and i m m u n e reactions p e r f o r m e d using a positive ( 3 H l l - A 1 ) and a negative (3H6-G7) hybridoma s u p e r n a t a n t . T h e sensitivity and selectivity of this assay were comp a r e d with those of the cellular E L I S A and these results are s u m m a r i z e d in Table IV. At low cell frequency (equal to or less t h a n 10%) the immunocircle assay, in contrast to the cellular
TABLE I!! LIST OF MONOCLONAL ANTIBODIES ISOLATED USING THE CIRCLE ASSAY FROM TWO INDEPENDENT FUSIONS Clone
Target antigen for screening
Specificity~
3HI I-AI
Thymocytes
3H6-HI0 3G8-A6 2Ai 1-Ai I 3H6-G7 d
Thymocytes Thymocytes Thymocytes Bone marrow cells
Thymus cells, splenic and lymph node T cells preferentially of non-AKR strains; neural cells, fibroblasts (probably a polymorphicdeterminant on Thy-l.2) b.c Thymus cells and T cells with no strain discrimination As for 3H6-HI0 As for 3H6-H10 Splenic red pulp cells, erythrocytes, large part of bone marrow cells ~
~' Assessed by immunohistochemistryon frozen sections b Wadsworth et al. (1989) c Morse et al. (1987) a From the original well (3H6); therefore, two distinct monoclones could be isolated with very. different specificities Evaluated by fluorescence activated cell sorter
147 E L I S A , w as still able to d e t e c t d i f f e r e n c e s bet w e e n t h e positive a n d n e g a t i v e s amp l es .
Mr ( 10 a )
R
N Discussion
66
--
45 34
"--
24 18 14
----
Fig. 4. lmmunoprecipitation by 3HII-AI monoclonal antibody. BALB/c thymus cells were biotinylated and the lysis buffer extract was precleared with protein G-coated beads preincubated with normal rat IgG. The supernatant was incubated with 3HI I-Ai hybridoma supernatant, and the immune complexes were collected onto protein G-coated beads. After boiling, the bound material was resolved by electrophoresis on 10% SDS-PAGE under both reducing (lane R) and non-reducing (lane N) conditions. The gel was blotted onto nitrocellulose, and the biotinylated antigen was revealed by incubation with SABC and development.
The present paper describes a simple alternative to o t h e r m e t h o d s f r e q u e n t l y u s ed for t e s t i n g h y b r i d o m a s p r o d u c i n g a n t i b o d i e s t o cell-surface an tig en s. O u r assay c o m b i n e s in a s i m p l e t es t s u i t a b l e for p r i m a r y s c r e e n i n g a rapid, a s em i q u a n t i t a t i v e a p p r o a c h (which co u l d if d e s i r e d b e m e a s u r e d by d e n s i t o m e t e r ) w i t h f u r t h e r microscopic i n f o r m a t i o n . T h e use o f c u l t u r e p l a t e lids p e r m i t s th e l o c a l i z a t i o n of a n t i g e n (i.e., m e m b r a n e - b o u n d o r i n t r a c e l l u l a r ) to b e e x a m i n e d , i n a d d i t i o n th e in ten sity of th e i m m u n e r e a c t i o n a n d also t h e a p p r o x i m a t e f r e q u e n c y o f t h e positive cells w i t h i n a mix ed t a r g e t cell p o p u l a t i o n can b e e v a l u a t e d . In mo s t p r o t o c o l s u s i n g c o n v e n t i o n a l c e l l u l a r E L I S A p r o c e d u r e s a f u r t h e r s t e p involving flow c y t o m e t r y o r m i c r o s c o p y is p e r f o r m e d . In o u r p r o c e d u r e , light m i c r o s c o p i c c o n f i r m a tion o f th e m a c r o s c o p i c a i l y d e t e c t e d r e s u l t s t a k e s no longer than 5 - 1 0 r a i n / 9 6 samples and needs
TABLE IV SELECTIVITY AND SENSITIVITY OF THE CIRCLE ASSAY (IC) COMPARED TO CELLULAR ELISA Clone
% of positive target ceils ~
Assay IC(M) b
IC(m) c
ELISA d
3HII-A!
100 50 25 10 5 0
+++ + +/ -
+ + + + + -
0.875+0.017 0.392_+0.021 0.147+0.026 0.092_+0.014 0.074 _+0.019 0.067 _+0.011
3H6-G7
100 50 25
+/+/ -
+/-
10
-
5 0
-
++ ++ + +
" BALB/c (positive) and Wistar rat (negative) thymocytes were mixed at differing ratios. Macroscopic observation. c Microscopic observation. d Average + SD of OD49z of three parallel samples.
0.137_+0.021 0.097_+0.01 I 0.081 _+0.016 0.069 _+0.009 0.065_+0.018 0.067 _+0.010
no specific instrumentation. After rinsing with distilled water, plates may be stored permanently, and rechecked if necessary after a short rehydration. In certain dot-methods previously developed, nitrocellulose paper is used as a solid support (Stya et al., 1984; Liebert et al., 1987) in order to retain the cells in the microwells, or in the radioisotopic version, to immobilize the cells. This setup, however, is not compatible with immediate microscopic inspection. In contrast, our assay is performed on plastic instead of nitrocellulose, and offers the possibility of immediate visual confirmation. Some previous publications using glutaraldehyde-fixed cells have claimed that there may be artifacts due to glutaraldehyde fixation (Posner et al., 1982; Letarte, 1984; Drover and Marshall, 1986). However, we found no differences when hybridoma supernatants strongly positive by the circle assay were retested by immunofluorescence on live cells in three separate fusions. In some cases of weak positivity, however, there were probably artifacts as the immunofluorescent control proved negative. 5 min fixation in 0.125% freshly prepared glutaraldehyde at 4°C has previously been found optimal for target preservation in cellular ELISAs (Klinman and Steinberg, 1987; Carroll et al., 1990) and has proved to be satisfactory in our assay also. Nevertheless, this does not rule out the possibility that cross-linking fixatives such as glutaraldehyde can be deleterious for some antigenic determinants (Hariow and Lane, 1988). Test lids can be prepared well in advance and stored for at least one month with no apparent loss of antigenic activity. The risk of cross-contamination between firstlayer antibodies in samples separated only by a narrow space was minimized by using only 20 pA of sample volume and relying on the raised rim of each circle. The rare macroscopically false positive, when it occurred, was visible in adjacent dots as a crescent or semicircle. The surface tension of the liquid in the circles contributed to the prevention of spillage. This assay may be a valuable alternative to previous methods for screening hybridoma cell lines against cell membrane antigens. Its simplicity and the important morphological information
it can provide offer useful advantages over other primary screening procedures. In principle it should be possible to develop a two-colour modification in which a sub-population is labelled with an alternative immunoenzyme reaction, thus immediately identifying the subset-labelling pattern of an unknown antibody.
Acknowledgements The authors wish to express their thanks to Dr. Simon V. Hunt from the Sir William D u n n School of Pathology, Oxford, England, lot his helpful critical comments on the manuscript. Also we are very grateful to Dr. Paul Kincade from the Oklahoma Medical Research Foundation, Oklahoma, USA, for the donation of 14.8 hybridoma line, and to Ms. Zsuzsa Cseri for photography.
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141
© 1992 Elsevier SciencePublishers B.V. All rights reserved l)022-1759/92/$()5.00
JIM 06397
Cellular enzyme-linked immunocircle assay A rapid assay of hybridomas produced against cell surface antigens P6ter Balogh, Zsuzsa Beb~k and P6ter N 6 m e t h Immunological and Biotechnological Laboratory of the Unicersity Medical School of Pdcs, P~cs, Hungary
(Received 13 November1991, revisedreceived29 January 1992, accepted 3 April 1992)
A novel method has been developed for the initial screening of hybridomas produced against cell surface antigens. Giutaraldehyde-fixed cells were immobilized as targets on the lid of a 96-well tissue culture plate which had been precoated with po!y-L-lysine. Antibody binding was determined by an immunoenzymatic method in an arrangement permitting both macro- and microscopic examination. After optimization with mouse thymus cells using existing rat monoclonal antibodies, new rat-mouse hybridoma cell lines against mouse thymocytes and bone marrow cells were screened. The antibodies could be characterized immediately both by the localization of the immune reaction (surface or intracellular) or as estimated by the frequency of positive cells recognized by the antibody in the sample. Key words: Hybridomascreening; Circle assay; ELISA, cellular; lmmunocytochemistry
Introduction Hybridoma screening assays must be both specific and sensitive at the same time (Eshhar, 1985). in contrast to soluble antigens, where the detection of specific antibody is relatively easy using ELISA (Engvall, 1980), techniques for the initial screening of hybridomas against certain cell surface antigens have to balance speed of
Correspondence to: P. Balogh, Immunologicaland Biotechnological Laboratory of the University Medical School of P~cs, Szigeti u. 12. H-7643, Hungary. Abbreviations: AEC, 3-amin6-9-ethylcarbazole; DMSO, dimethyl-sulpho~de; ELISA, enzyme-linked immunosorbent assay; HBSS, Hanks' balanced salt solution; PBS, phosphatebuffered saline containing0.2% sodium azide; PBS-0.1% BSA, PBS containing 0.1% bovine serum albumin; PBS-5% FBS, PBS containing 5% fetal bovine serum; SABC, Streptavidinbiotinylated horseradisch peroxidase complex.
preparation and examination against the amount of information gained about each positive (Table l). For example, both radioisotopic and immunofluorescent detection, though sensitive and reliable (Johnstone and Thorpe, 1987), need special instrumentation and conditions. Conventional immunohistochemical methods are difficult to scale up to deal with, say, 800-1000 samples simultaneously. In contrast, large numbers of samples are easily screened by cellular ELISA systems (Douillard et al., 1980; Cobbold and Waldmann, 1981) but these methods do not reveal either the density of the antigen, or its localization (surface or cytoplasmic), or the frequency of positive cells among the whole population. Moreover, the reliability of the cellular ELISA procedure has been questioned (Drover and .Marshall, 1986) because of false positive reactions especially involving lgM class antibodies.
Here we report a simple immunoenzymatic hybridoma screening system performed on glutaraldehyde-fixed target cells. This assay can be carried out conveniently on large number of samples (at least 1000 in a day's assay) and it permits direct light microscopic observation of the test reaction. The approximate frequency of cells expressing the particular antigen can also be immediately estimated. Cells are linked by poly-L-lysine to 8 x 12 arrays of depressions ('circles') moulded to the lids of 96-well tissue culture plates (instead of to the wells themselves) and then fixed with glutaraldehyde. Conventional immunoperoxidase methods reveal samples macroscopically and these can then immediately be examined microscopically.
Materials and methods
Animals Inbred Wistar rats and inbred BALB/c, C57BL/6, CBA/J and AKR/J mice were used between 6 and 16 weeks of age. The experimental animals were maintained under conventional conditions in the breeding unit of our animal facility. Cell lines and antibodies "l~he Sp2/0-Agl4 murine myeloma cell line (Flow Labs, UK) is maintained in the Biotechnological Facility's cell repository. Tile !4.8 antiB220 rat-mouse hybridoma cell line (Kincade et al., 1981) was produced and generously provided by Dr. Paul W. Kincade of the Oklahoma Medi-
cal Research Foundation, Oklahoma, USA. Cells were cultured in RPMI 1640 medium (Flow Labs, UK) supplemented with 10% FBS (Sebak, Germany) and gentamicin (Chinoin, Hungary) at 50 /zg/ml concentration. Rat anti-mouse monoclonal antibodies (Serva, Heidelberg, Germany) were: anti-Ly2 (CD8) (clone: YTS 169.4 lgG2b, K), anti-L3T4 (CD4) (clone: YTS 191.1.1, lgG2b, K) (Cobbold et al., 1984). The anti-B220 (IgG2b, K) antibody was used as undiluted supernatant. Sheep anti-rat IgG serum was produced by the Mez6hegyes State Farm, Hungary, and the rat lgG-reactive antibodies were affinity purified on cyanogen bromide activated Sepharose 4B (Pharmacia LKB, Sweden) coupled with rat IgG, previously purified on a protein G (Pharmacia) column according to the manufacturer's instructions. The bound sheep antibodies were recovered by sequential elution with 0.1 M glycine-HCI (pH 2.5) and 0.1 M triethylamine (pH 11.5) (Harlow and Lane, 1988). The eluted material was pooled and dialyzed against PBS overnight at 4°C. After repeated absorption of mouse IgG-reactive sheep antibodies on a mouse IgG-coupled affinity column until no bound material was demonstrable, the remaining antibody fraction was labelled with either biotin-N-hydroxysuccinimide ester (Calbiochem, USA) or fluoresceinisothiocyanate (Sigma Chemical Co., USA) as previously described (Mason et al., 1987). The biotinylated anti-rat IgG antibodies were tested in ELISA with immobilized mouse or rat IgG as antigen followed by SABC as developing reagent,
TABLE ! COMPARISON BETWEENTHE MAIN TECHNICALASPECTSOF THE MOSTFREQUENTLYUSED HYBRIDOMA SCREENINGMETHODS Method CellularELISA Radioimmunobindingassay Flowcytometer (FACS) Immunofluorescence (suspension) lmmunohistochemistry (smearsor tissue)
Specialequipment No
Scale Large
Information Quantitative
Yes Yes
Large Limited
Quantitative Quantitative/ qualitative Qualitative topographical Qualitative topographical
Yes
Limited
No
Verylimited
143 and the reactivity of the conjugate against mouse IgG was observed to be weak (not shown). The FITC/protein molar ratio was determined photometrically and calculated as 2.65.
Cellular immunocircle assay 96-well polystyrene tissue culture plate lids (Greiner, Germany, cat no. 656101)with 96 circular areas corresponding to the plate's wells were used. These provided 8 mm diameter wells with roughly 1 mm high rims moulded in an 8 x 12 array. They were rinsed with distilled water and precoated with 20 /zl poly-L-lysine (MW 10,000, Serva, Germany) dissolved in PBS at 1 m g / m l , and incubated in a humidified chamber at room temperature for 30 min. After rinsing with PBS, 20 t~! of a 2 × 107 cells/ml washed cell suspension in PBS were added per circle using a 12channel microdispensor, and allowed to sediment and to bind to the bottom for 30 min at room temperature. The cells were fixed by 20 ml 0.125% glutaraldehyde (Fluka, Germany) in PBS prechilled to 4°C (Carroll et al., 1990) carefully poured onto the periphery of the lid, in order to avoid disturbing the target cell layer. After 5 min fixation, the fixative was removed and the lids were thoroughly washed three times with PBS. The remaining active groups of glutaraldehyde were blocked with 5% FBS in PBS for 2 h at room temperature. The lids could be stored filled with 1% BSA-PBS containing 0.2% azide at 4°C for up to at least 1 month preserving the original (freshly prepared) antigenic activity. On the day of use, non-specific binding was blocked with normal sheep serum at 1/20 dilution for 20 min at room temperature. Then, after the removal of the serum, the circled areas were filled with 20 tzl of the first antibody at appropriate dilution. Positive and negative controls included immune and pre-immune sera at 1/100 dilution in 0.1% BSA-PBS. The lids were incubated at room temperature for 40 min, then washed three times by rinsing with PBS for 5 min. Each circle received 20 /~1 of biotinylated secondary antibody (sheep anti-rat IgG at 15 p.g/ml) and the reaction was allowed to proceed at room temperature for 40 min. The lids were washed as previously, and streptavidin-biotinylated horseradish peroxidase complex (SABC) (Amersham
International, Aylesbury, UK) was added and the plate incubated for a further 40 min at room temperature. All incubation steps were performed in a humid chamber to prevent drying out of the samples. After washing, the reaction was monitored using 2 0 / x l / w e l l AEC (Sigma, USA) as chromogen and H 2 0 2 as substrate in 0.1 M acetate buffer (pH 5.2) as previously described (Graham et al., 1965). Positive wells were easily recognized by the naked eye as red dots within the circular areas. The membrane-bound reaction was confirmed by subsequent microscopic examination comparing the results of an individual well with negative or positive control wells.
Cellular ELISA It was performed according to Carrol et ai. (1990) with the modification that the immune reaction was developed using biotinylated sheep anti-rat lgG antibodies and SABC. The colour reagent was 1.2-phenylenediamine dihydrochloride (Fluka, Germany) dissolved in 0.1 M citrate buffer, pH 5.2, and the absorption was recorded at a wavelength of 492 nm. Rat-mouse hybridoma production 8-week-old female Wistar rats were immunized intraperitoneaily with 107 spleen cells, derived from 4-week-old C 5 7 B L / 6 mice and boosted 3 and 5 weeks later. 1 week after the last immunization, the rats were injected intravenously with 107 spleen cells. 3 days later the spleen cells from the best responder rat were fused with Sp2/0-Agl4 cells as described previously (KiShler and Milstein, 1975), distributed in 96-well fiat bottomed plates (Greiner, Germany) and selected on H A T (Littlefield, 1964). The hybrid clones were tested for specific antibody production with the above cellular immunocircle assay using B A L B / c derived thymus cells or bone marrow ceils as targets. 20 ttl of supernatant were added and incubated at room temperature for 40 min, followed by extensive washing and further incubation with biotinylated sheep anti-rat IgG and SABC as described above. The positive results were confirmed by indirect immunofluorescence performed on unfixed cells according to previous methods (Harlow and Lane, 1988). The positive wells were cloned by limiting dilution,
and the clones were re-checked as above. The positive monoelones were expanded in 75 ml Costar tissue culture flasks.
lmmunofluorescence and immunohistochemistry In immunofluorescence, the target cell suspension in PBS-0.1% BSA (Sigma Chemical Co., USA) was incubated with the first antibody at 4"C for 30 min in a U bottomed 96-well microtiter plate (Greiner), washed three times with PBS and incubated for a further 30 rain at 4°C with FITC-labelled sheep anti-rat IgG (produced in our laboratory) diluted in 0.1% BSA-PBS. After washing with PBS, the cells were fixed with 1% buffered formaldehyde and stored at 4°C until examination. Monoclonal antibodies were further characterized on frozen and acetone-fixed sections from different mouse organs using the biotin-streptavidin-HRPO method as described previously (Hsu, 1985). After addition of the first antibody, the specimens were incubated with biotinylated sheep anti-rat IgG antibody followed by repeated washing. SABC was added and after a 40 rain incubation at room temperature the plate was thoroughly washed. The reaction was visualized with AEC, and the specimens were counterstained with Mayer's haematoxylin before mounting in gelatin.
Sepharose 4B (Sigma Chemical Co., USA) procoated with nonimmune rat IgG. The supernatant was incubated with hybridoma supernatant, and the immune complexes were collected with protein G-Sepharose 4B previously washed in lysis buffer. The unbound material was removed by repeated centrifugation and washing. The absorbed proteins were released by boiling for 10 rain in a sample buffer containing 0.125 M Tris-HCI, 2% SDS, 10% glycerol with or without 5% 2-mercaptoethanol, pH 6.8. SDS-PAGE was performed on Laemmli 10% polyacrylamide gels using the Mini-Protean I1 apparatus (Bio-Rad Laboratories, USA). After the electrophoresis, the gel was equilibrated for 30 min in a transfer buffer suitable for semi-dry transfer (48 mM Tris, 39 mM glycine, 0.037% SDS, 20% methanol). Proteins were transferred to a Hybond C nitrocellulose membrane (Amersham International, Aylesbury, UK) by blotting for 30 rain at 150 mA current using Sartoblot II-S (Sartorius, Germany). The membrane was soaked in blotting buffer (PBS containing 3% BSA, 1% FCS and 0.1% Tween 20) overnight at 4°C. After thorough washing in PBS containing 0.1% Tween-20, SABC at 1/i000 dilution in PBS-Tween was added for 2 h with constant agitation at room temperature. After extensive washing, the reaction was developed using AEC and 0.003% H20 2 in 0.1 M sodiumacetate buffer, pH 5.2.
Initial characterization of the antigen detected by 3Hl l-A1 Mab Thymus cells were labelled with N-hydroxysuccinimidobiotin (NHS-biotin, Sigma Chemical Co., USA) essentially as described by Ingalls et al. (1986) with slight modifications. Briefly, the cells at 5 × 107/ml concentration in HBSS were washed in 0.15 M NaCI (pH 8.5 adjusted with 0A M sodium-bicarbonate/carbonate buffer pH 9) instead of alkaline HBSS used by others (Miyake et al., 1990). After incubation with NHS-biotin dissolved in DMSO at 50 mg/ml (2 mg/ml final concentration) on ice for 40 min, the cells were repeatedly washed with chilled PBS. The washed cells were extracted by lysis buffer containing 10 mM Tris-HCl, 0.15 M NaCI, 1% NP-40, 1 mM PMSF and 0.1% sodium-azide pH 7.5. After the removal of nuclei by centrifugation, the lysates were precleared twice with protein G-coated
Results
,
Preliminary experiments showed that rat antimouse CD4, CD8 (positive) and B220 (CD45) (negative) gave equivalent results to those from a parallel immunofluorescence assay on mouse thymocyte targets in the immunocircle assay. The precipitated red end-product of the reaction was readily discriminated from the negative colourless circles. Holding the lid at a shallow angle relative to the line of vision improved the visibility. The sensitivity of the assay was checked on mouse thymocyte targets by titrating pre-fusion sera from rats immunized with mouse splenocytes and comparing the results with the ELISA titre using the same cell numbers. Reactions were detectable using a dilution of immune sera of
145 2 w e e k s after fusion o f i m m u n e rat s p l e e n cells with S p 2 / 0 - A g I 4 cells, h y b r i d o m a s u p e r n a t a n t s were s c r e e n e d o n immobilized t h y m u s or b o n e m a r r o w cells. A b r o a d variation in t h e colour intensity w a s observed, w h i c h correlated well with t h e microscopic a p p e a r a n c e o f t h e s a m p l e s . A m o n g positive circles, t h e majority o f t h e cells was intensely labelled. However, in s o m e c a s e s t h e antibody in t h e s u p e r n a t a n t recognized only a s u b p o p u l a t i o n within t h e t h y m u s cells (Figs. 2 a 2d). In t h e case o f b o n e m a r r o w ceils we f o u n d only two positive s u p e r n a t a n t s , b o t h o f which r e a c t e d with t h e majority o f t h e cells. The hybridoma supernatants which were strongly positive o n t h y m o c y t e s in t h e i m m u n o c i r cle assay were also positive in t h e i m m u n o f l u o r e s c e n t assay u s i n g live cells w h e r e a s m o s t o f t h e weakly positive s u p e r n a t a n t s were negative by i m m u n o f l u o r e s c e n c e (Table II). T h e m o n o c l o n a l a n t i b o d i e s s h o w n o n T a b l e 111 were o b t a i n e d f r o m t h e two fusions.
D
Fig. 1. Titration of sera from mouse spleen cell immunized rats using fixed thymocytes as target. The assay was carried out as described in the materials and methods section. In the first antibody layer, immune (A - B) and preimmune (C = D) sera obtained from two different rats were added at different dilutions (indicated at the top).
1 / 1 0 0 0 (Fig. 1). Microscopic inspection t h e n revealed t h e cell s u r f a c e localization o f t h e i m m u n e reaction (see below). 1
2
3
4
5
6
?
~ , ~ ~ . ~ ~
8
9
I0 II 12
~..~, -~
A
.~-.--.. B.
C.
- .. :~.:.~,~ D.
Fig. 2. Macroscopic and microscopic appearance of the circle assay. BALB/c derived thymocytes were bound to the plastic surface and the circle assay was carried out using hybridoma supernatants. A: macroscopic appearance, where at bottom right (HI2 well) rat immune serum was added at !/100 dilution. B: microscopic picture of the G8 well. C: EIO well. D: GI2 well (negative control with normal pre-immune rat serum diluted 1/50 adde,]). Magnification: 380x.
TABLE !1 EFFICIENCY OF CIRCLE ASSAY IN THE SCREENING OF HYBRIDOMAS AGAINST MURINE CELL SURFACE MARKERS Fusion no." Target antigen
Frequencyof positive wells/their immunofluorescent control Strong b
R2 R3
Thymocytes I/ I Thymocytes 3/3 Bone marrow cells I / I
Weak ¢ 2/0 4/1 1/0
a Sp2/0-Agl4 cells were fused to Wistar rat spleen cells immunized with murine spleen cells and distributed in 96-well tissue culture plates. h Both macroscopically and microscopically detectable reaction. c Only microscopically detectable, ambiguous reaction.
Fig. 3. Reactivity of 3HII-AI monoclonal antibody on frozen section from BALB/c spleen. The first-layer monoclonal antibody was added as neat tissue culture supernatant. The overwhelming majority of cells surrounding the arteriole (indicated by *) are labelled. The reaction was developed using the streptavidin-biotin system. Magnification: 280×.
Ecaluation of the assay with low target cell frequency As the preliminary data showed, it was possible to isolate a monoclonal antibody which app e a r e d to recognize a T cell antigen (Figs. 3 and 4 and Table liD. E x p e r i m e n t s were p e r f o r m e d to d e t e r m i n e the lowest frequency of cells which were distinguishable by this assay. T h y m u s cells obtained from B A L B / c mice and Wistar rats were mixed t o g e t h e r at different ratios, immobi-
lized on the culture plate lids, and i m m u n e reactions p e r f o r m e d using a positive ( 3 H l l - A 1 ) and a negative (3H6-G7) hybridoma s u p e r n a t a n t . T h e sensitivity and selectivity of this assay were comp a r e d with those of the cellular E L I S A and these results are s u m m a r i z e d in Table IV. At low cell frequency (equal to or less t h a n 10%) the immunocircle assay, in contrast to the cellular
TABLE I!! LIST OF MONOCLONAL ANTIBODIES ISOLATED USING THE CIRCLE ASSAY FROM TWO INDEPENDENT FUSIONS Clone
Target antigen for screening
Specificity~
3HI I-AI
Thymocytes
3H6-HI0 3G8-A6 2Ai 1-Ai I 3H6-G7 d
Thymocytes Thymocytes Thymocytes Bone marrow cells
Thymus cells, splenic and lymph node T cells preferentially of non-AKR strains; neural cells, fibroblasts (probably a polymorphicdeterminant on Thy-l.2) b.c Thymus cells and T cells with no strain discrimination As for 3H6-HI0 As for 3H6-H10 Splenic red pulp cells, erythrocytes, large part of bone marrow cells ~
~' Assessed by immunohistochemistryon frozen sections b Wadsworth et al. (1989) c Morse et al. (1987) a From the original well (3H6); therefore, two distinct monoclones could be isolated with very. different specificities Evaluated by fluorescence activated cell sorter
147 E L I S A , w as still able to d e t e c t d i f f e r e n c e s bet w e e n t h e positive a n d n e g a t i v e s amp l es .
Mr ( 10 a )
R
N Discussion
66
--
45 34
"--
24 18 14
----
Fig. 4. lmmunoprecipitation by 3HII-AI monoclonal antibody. BALB/c thymus cells were biotinylated and the lysis buffer extract was precleared with protein G-coated beads preincubated with normal rat IgG. The supernatant was incubated with 3HI I-Ai hybridoma supernatant, and the immune complexes were collected onto protein G-coated beads. After boiling, the bound material was resolved by electrophoresis on 10% SDS-PAGE under both reducing (lane R) and non-reducing (lane N) conditions. The gel was blotted onto nitrocellulose, and the biotinylated antigen was revealed by incubation with SABC and development.
The present paper describes a simple alternative to o t h e r m e t h o d s f r e q u e n t l y u s ed for t e s t i n g h y b r i d o m a s p r o d u c i n g a n t i b o d i e s t o cell-surface an tig en s. O u r assay c o m b i n e s in a s i m p l e t es t s u i t a b l e for p r i m a r y s c r e e n i n g a rapid, a s em i q u a n t i t a t i v e a p p r o a c h (which co u l d if d e s i r e d b e m e a s u r e d by d e n s i t o m e t e r ) w i t h f u r t h e r microscopic i n f o r m a t i o n . T h e use o f c u l t u r e p l a t e lids p e r m i t s th e l o c a l i z a t i o n of a n t i g e n (i.e., m e m b r a n e - b o u n d o r i n t r a c e l l u l a r ) to b e e x a m i n e d , i n a d d i t i o n th e in ten sity of th e i m m u n e r e a c t i o n a n d also t h e a p p r o x i m a t e f r e q u e n c y o f t h e positive cells w i t h i n a mix ed t a r g e t cell p o p u l a t i o n can b e e v a l u a t e d . In mo s t p r o t o c o l s u s i n g c o n v e n t i o n a l c e l l u l a r E L I S A p r o c e d u r e s a f u r t h e r s t e p involving flow c y t o m e t r y o r m i c r o s c o p y is p e r f o r m e d . In o u r p r o c e d u r e , light m i c r o s c o p i c c o n f i r m a tion o f th e m a c r o s c o p i c a i l y d e t e c t e d r e s u l t s t a k e s no longer than 5 - 1 0 r a i n / 9 6 samples and needs
TABLE IV SELECTIVITY AND SENSITIVITY OF THE CIRCLE ASSAY (IC) COMPARED TO CELLULAR ELISA Clone
% of positive target ceils ~
Assay IC(M) b
IC(m) c
ELISA d
3HII-A!
100 50 25 10 5 0
+++ + +/ -
+ + + + + -
0.875+0.017 0.392_+0.021 0.147+0.026 0.092_+0.014 0.074 _+0.019 0.067 _+0.011
3H6-G7
100 50 25
+/+/ -
+/-
10
-
5 0
-
++ ++ + +
" BALB/c (positive) and Wistar rat (negative) thymocytes were mixed at differing ratios. Macroscopic observation. c Microscopic observation. d Average + SD of OD49z of three parallel samples.
0.137_+0.021 0.097_+0.01 I 0.081 _+0.016 0.069 _+0.009 0.065_+0.018 0.067 _+0.010
no specific instrumentation. After rinsing with distilled water, plates may be stored permanently, and rechecked if necessary after a short rehydration. In certain dot-methods previously developed, nitrocellulose paper is used as a solid support (Stya et al., 1984; Liebert et al., 1987) in order to retain the cells in the microwells, or in the radioisotopic version, to immobilize the cells. This setup, however, is not compatible with immediate microscopic inspection. In contrast, our assay is performed on plastic instead of nitrocellulose, and offers the possibility of immediate visual confirmation. Some previous publications using glutaraldehyde-fixed cells have claimed that there may be artifacts due to glutaraldehyde fixation (Posner et al., 1982; Letarte, 1984; Drover and Marshall, 1986). However, we found no differences when hybridoma supernatants strongly positive by the circle assay were retested by immunofluorescence on live cells in three separate fusions. In some cases of weak positivity, however, there were probably artifacts as the immunofluorescent control proved negative. 5 min fixation in 0.125% freshly prepared glutaraldehyde at 4°C has previously been found optimal for target preservation in cellular ELISAs (Klinman and Steinberg, 1987; Carroll et al., 1990) and has proved to be satisfactory in our assay also. Nevertheless, this does not rule out the possibility that cross-linking fixatives such as glutaraldehyde can be deleterious for some antigenic determinants (Hariow and Lane, 1988). Test lids can be prepared well in advance and stored for at least one month with no apparent loss of antigenic activity. The risk of cross-contamination between firstlayer antibodies in samples separated only by a narrow space was minimized by using only 20 pA of sample volume and relying on the raised rim of each circle. The rare macroscopically false positive, when it occurred, was visible in adjacent dots as a crescent or semicircle. The surface tension of the liquid in the circles contributed to the prevention of spillage. This assay may be a valuable alternative to previous methods for screening hybridoma cell lines against cell membrane antigens. Its simplicity and the important morphological information
it can provide offer useful advantages over other primary screening procedures. In principle it should be possible to develop a two-colour modification in which a sub-population is labelled with an alternative immunoenzyme reaction, thus immediately identifying the subset-labelling pattern of an unknown antibody.
Acknowledgements The authors wish to express their thanks to Dr. Simon V. Hunt from the Sir William D u n n School of Pathology, Oxford, England, lot his helpful critical comments on the manuscript. Also we are very grateful to Dr. Paul Kincade from the Oklahoma Medical Research Foundation, Oklahoma, USA, for the donation of 14.8 hybridoma line, and to Ms. Zsuzsa Cseri for photography.
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