A simple semi-automated plaque method for the detection of antibody-forming cell clones in microcultures

J o u r n a l o f I m m u n o l o g i c a I M e t h o d s , 52 (1982) 25--37

25

Elsevier/Biomedical Press

A SIMPLE SEMI-AUTOMATED PLAQUE METHOD FOR THE D E T E C T I O N OF A N T I B O D Y - F O R M I N G CELL CLONES IN MICROCULTURES

BEVERLEY L. PIKE, GAEL JENNINGS and KEN SHORTMAN The Walter a n d Eliza Hall I n s t i t u t e o f M e d i c a l Research, P o s t Office, R o y a l M e l b o u r n e Hospital, Victoria 3 0 5 0 , A u s t r a l i a

(Received 10 September 1981, accepted 18 December 1981)

A simple semi-automated method for the assay of large numbers of replicate microcultures for the presence of antibody-forming cell clones is described. The supernatant medium is removed from microcultures by a single sharp flick on inverting the tray. The cultured cells are mixed with 0.05 ml of a plaque-revealing mix containing indicator erythrocytes and complement and then transferred to new flat-bottomed 96-well microculture trays, using a multichannel pipette or 96-channel replicator. The tray is centrifuged, the indicator erythrocytes and cultured cells forming an even monolayer on the bottom surface of the well. Trays are held at 37°C for 1--1½ h to allow plaque development. Using a dissecting microscope, the number of plaques in each well is counted, or in the case of limiting dilution analysis, each well is simply scored as positive or negative. This assay procedure provides a simple, rapid and inexpensive means of assaying large numbers of microculture trays for the detection and enumeration of antibody-forming cell clones. There is no loss in sensitivity compared with the standard hemolytic plaque assay methods. The method is particularly useful for limiting dilution analysis which necessitates the assay of large numbers of replicate cultures for either the presence or absence of a clone of antibody-forming cells. Key words: a n t i b o d y - f o r m i n g c l o n e s - - assay f o r A F C - - a u t o m a t e d c l o n e assay INTRODUCTION T h e e n u m e r a t i o n o f the f r e q u e n c y w i t h i n a given cell p o p u l a t i o n o f prec u r s o r cells w h i c h are capable o f r e s p o n d i n g t o antigenic o r m i t o g e n i c stimulus b y t h e g e n e r a t i o n o f a c l o n e o f a n t i b o d y - f o r m i n g cells ( A F C ) in vitro can be statistically d e t e r m i n e d b y limiting d i l u t i o n analysis ( L e f k o v i t s , 1 9 7 2 ; Nossal and Pike, 1 9 7 6 ) . This t y p e o f a p p r o a c h allows analysis o f t h e antib o d y response at clonal level o f a v a r i e t y o f cell p o p u l a t i o n s , b o t h to T - d e p e n d e n t ( L e f k o v i t s , 1 9 7 2 ; L e f k o v i t s a n d Waldman, 1 9 7 9 ) a n d t o T - i n d e p e n d e n t (Nossal and Pike, 1 9 7 6 ; Q u i n t a n s a n d C o z e n a , 1 9 7 6 ) antigens. H o w e v e r , as limiting d i l u t i o n analysis necessitates the e v e n t u a l assaying o f large n u m b e r s o f replicate cultures f o r e i t h e r t h e p r e s e n c e or t h e a b s e n c e o f an A F C clone, t h e assay p r o c e d u r e is b o t h t i m e c o n s u m i n g a n d l a b o r 0022-1759/82/0000--0000/$02.75 © 1982 Elsevier Biomedical Press

26 intensive. Consequently, alternative, potentially semi-automated, assay procedures which provide equivalent sensitivity to the present widely used liquid monolayer slide method (Cunningham and Szenberg, 1968) or slide modifications of the Jerne assay m e t h o d (Jerne and Nordin, 1963; Mishell and Dutton, 1967) are essential to make this approach more practical. A semi-automated hemolysis-in-gel spot test (Lefkovits, 1972; Lefkovits and Kamber, 1972) has been successfully used for the detection of antisheep e r y t h r o c y t e (SRC) antibody in supernatants of cultures of spleen cells stimulated with SRC, thus enabling culture wells containing anti-SRC AFC clones to be both identified and enumerated. However, we have found this m e t h o d to be unreliable for detecting culture wells containing clones of antihapten AFC stimulated by T-independent antigens. The assay m e t h o d described in this paper is a modification of the 'in situ' microplate hemolytic plaque assay m e t h o d described by Kappler (1974) for revealing anti-SP~C AFC generated by either in vivo or in vitro immunization. We found that direct application of Kappler's m e t h o d to assay for in vitro generated anti-hapten AFC was unsuccessful due to poor plaque clarity, false plaques created by cell debris and dead cells, and to some undefined inhibitory factor present in the culture wells. The modified m e t h o d provides an inexpensive, simple, rapid and reproducible assay procedure for assaying large numbers of microculture trays for the detection of anti-hapten AFC clones without any loss in sensitivity compared with methods previously used. Furthermore, we have been able to extensively automate the procedure. MATERIALS AND METHODS

Mice Inbred, specific pathogen-free CBA/CaHWehi mice aged 8--10 weeks, were used as spleen donors in all experiments. CBA or (CBA X BALB/c)F1 hybrids aged 4--6 weeks, were used as t h y m u s donors. Antigens Unless indicated otherwise, antibody formation in vitro was induced by fluorescein (FLU) coupled polymerized flagellin (POL), the procedure for preparation of which has been published elsewhere (Nossal et al., 1978). The FLU-POL used had a substitution ratio of 1 mole of F L U per mole of monomeric flagellin. In some experiments antibody formation was induced by 4-hydroxy-3-iodo-5-nitrophenol (NIP) conjugated POL (Pike, 1975) or E. coli lipopolysaccharide (LPS) (Batch 0111:B4, Difco Laboratories, Detroit, MI) as specified. Preparation o f cell suspensions Spleen and t h y m u s cell suspensions were prepared in HEPES-buffered Eagle's medium {HEM) as previously described (Pike, 1975; Nossal and Pike,

27 1976). T h y m u s cells were treated with anti-Ia k antisera and c o m p l e m e n t in order to eliminate the majority of contaminating B cells prior to their use as 'filler' cells in the culture system (Nossal and Pike, 1978).

Culture procedure for antibody formation in vitro The microculture system used for the induction and generation of antibody-forming cell (AFC) clones in vitro has been previously described in detail (Pike, 1975; Nossal and Pike, 1976, 1978). Briefly, small numbers of unfractionated spleen cells were cultured with 2 × 106 anti-Ia k serum and c o m p l e m e n t pre-treated t h y m u s 'filler' cells in 96-well flat-bottomed microtest II tissue culture trays (Linbro cat. no. 76-003-05, Newhaven, CT) in 0.2 ml of culture medium containing either 0.1 t~g/ml of FLU-POL (or other hapten-POL conjugates, e.g., NIP-POL) or 20 t~g/ml of LPS. Where specified 96-well V-bottomed trays were used (Linbro no. 76-023-05). Cultures were held for 3 days at 37°C in a humidified atmosphere of 10% CO2 in air. Each culture well was then individually assayed for the presence of anti-FLU (or anti-NIP AFC) clones either by the standard Cunningham-Szenberg liquid monolayer method (Cunningham and Szenberg, 1968) or by the modified tray assay m e t h o d of Kappler (1974) as described in this paper. Preparation of hapten-coated target ery throcy tes The procedure for preparation of hapten-SRC for the plaque assay has been previously described (Pike, 1975; Nossal et al., 1978). Briefly, (Fab')2 fragments of rabbit anti-SRC antibody were prepared and conjugated with hapten by the standard methods. Haptenated (Fab')2 was attached to the SRC b y the m e t h o d of Strausbach et al. (1970) using a (Fab')~ to SRC ratio previously titrated to give optimal plaque quality. FLU-SRC and NIP-SRC were used as 20% (v/v) suspensions in human tonicity phosphate-buffered saline, pH 7.3. Complement Fresh guinea pig serum (Commonwealth Serum Laboratories, Melbourne) was absorbed twice with packed SRC prior to storage at --70°C in 1 ml aliquots until use. Complement was only thawed once prior to use and each batch was titrated to determine the concentration required to allow optimal plaque development without background (non-specific) lysis occurring. With most c o m p l e m e n t batches, a volume of 1--1.5 ml of c o m p l e m e n t per 10 ml o f plaque-revealing mix was optimal. Recipes for plaque-revealing mix Plaque-revealing mix was freshly prepared just prior to addition to the culture wells at the c o m m e n c e m e n t of the assay procedure and 0.05 ml was added to each well as described below. The standard recipes for 10 ml quantities of plaque-revealing mix (sufficient for 2 × 96-well culture trays) were as follows: (a) slide assay: 20% hapten-SRC, 1.5 ml; undiluted complement,

28 1.0 ml (or as determined); HEM added to give a final volume of 10 ml; (b) tray assay: undiluted complement, 1.0 ml (or as determined); HEM (to 10 ml), 9.0 ml; 20% hapten SRC, 0.4 ml. For fully automated assay procedure add 1 drop of 0.5% antifoam solution (Sigma Antifoam Emulsion A5758).

Procedures for assay for AFC clones (a) Slide assay. The procedure for assay of microcultures for AFC clones by the Cunningham-Szenberg liquid monolayer (slide) m e t h o d has been previously described in detail (Pike, 1975; Nossal and Pike, 1976). Briefly, culture supernatants were removed by gentle suction and immediately replaced with 0.05 ml of plaque-revealing mix containing 3% FLUSRC or NIP-SRC, and complement. With a Pasteur pipette, the contents of each well were individually transferred to a slide chamber. The edges of the chamber were sealed with wax and it was incubated in the horizontal position at 37°C for 1 h before scoring for AFC clones under a dissecting microscope at X 15 magnification. (b) Modified assay. All assay procedures were performed at room temperature. Culture supernatants were removed by a single sharp shake on inverting the culture tray and the tray surface blotted. If a washing step was desired 0.2 ml of BSS was immediately added to each well using a multichannel pipettor (Titertek, Flow Laboratories, Finland). After thorough mixing with a mechanical plate shaker (Titertek, Flow Laboratories, Finland) the contents were transferred to a new 96-well flat-bott o m e d microtest tray (Disposable Products, no. STC1L, Australia or Falcon Plastics, No. 3040, U.S.A.) with a multichannel pipettor. The tray was centrifuged at 400 X g for 5 min. The supernatant medium was again removed with a single sharp shake on inverting the tray and immediately replaced with 0.05 ml of plaque-revealing mix containing 0.8% FLU-SRC (or NIP-SRC) and complement using an Oxford Microdoser pipette (Oxford Laboratories, Oxford) or multichannel pipettor. The contents of the tray were mixed on a mechanical shaker, and the tray was again centrifuged so that the cultured cells and the SRC formed an even layer on the b o t t o m of the well. After centrifugation, trays were covered and incubated for 1--11/2 h at 37°C prior to scoring. The number of wells containing antihapten AFC and/or the number of AFC per positive well were scored under a dissecting microscope at X20 magnification, with the aid of a special indexing stage (see Appendix, Darling et al., 1982). The initial washing and centrifugation procedure was found to be unnecessary when assaying for anti-hapten AFC clones amongst spleen cells cultured under limiting dilution conditions. A simpler and faster procedure was to add 0.05 ml of plaque-revealing mix directly to the original culture wells after removal of the supernatant, mix, and then transfer the cultured cells and plaque-revealing mix to a fresh tray. In this way only a single addition-transfer step and only one centrifugation step was necessary.

29

This basic procedure was readily adapted to automation, so tilat many trays could be assayed rapidly, as shown diagrammatically in Fig. 1. The washing step was eliminated to simplify the process. Antifoam was added to the plaque-revealing mix (see above) to prevent bubbles forming on transfer. The initial cultures were set up using a 96-channel replicator (0.2 ml/channel) and the addition of plaque-revealing mix and transfer to fresh

AUTOMATED ASSAYS FOR AFC AND AFC-PRECURSORS

Set up limit dilution cultures. Use 96 c h a n n e l r e p l i c a t o r

Culture 3days.

/

~'

Remove s u p e r n a t a n t . Flick. Blot.

Add h a p t e n - SRC, complement, a n t i f o a m cocktail. Use g6 channel replicator

I

Q

I

Mix on shaker to suspend AFC

Transfer AFC in p l a q u e c o c k t a i l to f r e s h t r a y Use 96 channel r e p l i c a t o r

Centrifuge tray to form monolayer

Inlcubate 37 ° l h r

Count plaques or s c o r e + -

R

Use scanning microscope

[3

with indexing stage

Fig. 1. Diagrammatic illustration of fully automated procedure for the detection and enumeration of anti-hapten AFC clones generated in vitro.

30 trays by means of another 96-channel replicator (0.05 ml/channel). The replicators were made in the Hall Institute workshops, using the Byrd modification of a Lefkovits/Kamber design, as described elsewhere (Shortman and Wilson, 1981). With both the slide and tray assay systems the frequency of anti-FLU AFC precursors was statistically determined by Poisson analysis as previously described (Lefkovits, 1972; Nossal and Pike, 1976; Stocker, 1976; Lefkovits and Waldman, 1979). RESULTS

'In situ' plaque development in microculture wells In our hands, the m e t h o d described by Kappler (1974) for development of anti-SRC AFC in flat-bottomed microtrays worked well when used for detecting anti-hapten AFC amongst immunized spleen cells derived from intact mice. However, in situ assay of anti-hapten AFC amongst in vitro immunized spleen cells, using the same tray for both the culture and assay procedures, was a failure, particularly where spleen cells were cultured at limiting dilution with filler cells. Often no plaques could be seen. Where some plaques were visible in the wells, their quality was suboptimal and in addition, cell clumps and debris gave rise to false plaques making enumeration both difficult and highly inaccurate. Simultaneous assay of cultures using the conventional slide m e t h o d always revealed significantly higher numbers of AFC than the 'in situ' m e t h o d . The overall general impression was gained that the failure of 'in situ' plaque development was due to the presence of too m a n y cells and cell debris etc., in the culture wells. Effect o f reduction o f filler cell number and of cell debris on plaque development 'in situ' In an a t t e m p t to improve in situ plaque development, we reduced the number of filler cells added to the microcultures, with the knowledge that this would only be a partial solution as such action results in a lowering of the efficiency of the culture system (Nossal and Pike, 1976; Stocker, 1976). Cultures were set up containing spleen cells with fewer t h y m u s filler cells and assayed either by the slide m e t h o d or by the 'in situ' tray method. The results (Table 1) showed that apart from the anticipated reduction in responsiveness, the reduction in t h y m u s filler cell number resulted in very little, if any, improvement in the results obtained with 'in situ' assay. Another approach was to remove dead cells and debris from the culture wells by enzymic digestion at the end of the culture period just before to the assay procedure. This was achieved by the addition of 0.2% trypsin and 0.05 mg/ml DNA-ase for 1 h at 37°C at the end of the culture period, just prior to assay. Under these conditions, which effectively removed 80--85% of the dead cells w i t h o u t affecting the number of AFC

31 TABLE 1 Effect of decreasing filller cell number in microcultures on subsequent in situ plaque development. Number of thymus filler cells per culture

AFC/culture a using Cunningham slide assay b

AFC/culture using 'in situ' tray assay c

2X106 lX106 6x10 s 3X10 s

21±3 13±3 7±1 4±2

1.8±1.2 5.8±2.0 3.8±1.0 1.0±0.7

a Anti-FLU AFC generated by 3 x 104 CBA spleen cells cultured for 3 days in flat-bottomed microculture trays in the presence of the indicated number of thymus filler cells and 0.1 pg/ml of FLU-POL. b Cultures assayed for anti-FLU AFC by the conventional Cunningham slide method. c Cultures assayed for anti-FLU AFC 'in situ' as described by Kappler (1974). These plaques were in general of substandard quality.

s u b s e q u e n t l y d e t e c t e d (as d e t e r m i n e d b y t h e slide assay), 'in s i t u ' p l a q u e d e v e l o p m e n t was still highly u n s a t i s f a c t o r y .

Transfer of cultures to new microculture tray for assay procedure In a n o t h e r a p p r o a c h u n d e r t a k e n to r e d u c e t h e t h y m u s filler cell n u m b e r w i t h o u t significantly a f f e c t i n g t h e e f f i c i e n c y o f t h e c u l t u r e s y s t e m , spleen cells w e r e c u l t u r e d at limiting d i l u t i o n in e i t h e r V- or U - b o t t o m e d m i c r o c u l t u r e t r a y s , t h e s e r e q u i r i n g o n l y 3 - - 6 X l 0 s t h y m o c y t e s f o r o p t i m a l stimu l a t i o n (Pike, u n p u b l i s h e d d a t a ) . This n e c e s s i t a t e d t h e t r a n s f e r o f cult u r e s to f l a t - b o t t o m e d m i c r o c u l t u r e t r a y s at t h e e n d o f t h e c u l t u r e p e r i o d f o r assay f o r A F C . This p r o c e d u r e r e s u l t e d in e x c e l l e n t p l a q u e d e v e l o p m e n t a n d v i r t u a l l y i d e n t i c a l results w e r e o b t a i n e d w h e n c u l t u r e s w e r e a s s a y e d f o r A F C clones b y t h e slide m e t h o d or b y t h e t r a n s f e r t o a f l a t - b o t t o m e d m i c r o c u l t u r e t r a y ( T a b l e 2). F u r t h e r i n v e s t i g a t i o n s h o w e d t h a t this d r a m a t i c i m p r o v e m e n t in p l a q u e d e v e l o p m e n t in t r a y s was u n r e l a t e d t o t h e r e d u c e d filler cell n u m b e r . Cultures set u p in f l a t - b o t t o m e d wells w i t h t h e o p t i m a l n u m b e r o f t h y m u s filler cells, a n d t r a n s f e r r e d to a fresh t r a y j u s t b e f o r e assay s h o w e d e x c e l l e n t p l a q u e d e v e l o p m e n t w i t h results similar t o t h o s e o b t a i n e d f r o m c u l t u r e s a s s a y e d s i m u l t a n e o u s l y b y t h e slide m e t h o d (Tables 3 and 4). This i n d i c a t e d t h a t t h e difficulties e n c o u n t e r e d with 'in s i t u ' p l a q u e r e v e l a t i o n w e r e d u e t o s o m e c h a n g e in t h e original c u l t u r e well d u r i n g t h e 3 - d a y c u l t u r e p e r i o d a n d t h a t t r a n s f e r o f c u l t u r e s to a n e w t r a y f o r assay c o u l d e l i m i n a t e t h e p r o b l e m . As a c o m p a r i s o n , T a b l e 3 s h o w s results o b t a i n e d w h e n c u l t u r e s o f spleen cells set u p in f l a t - b o t t o m e d well m i c r o t r a y s at a c o n c e n t r a t i o n w h e r e e a c h well w o u l d c o n t a i n at least o n e A F C c l o n e (i.e., n o t at l i m i t i n g d i l u t i o n ) w e r e a s s a y e d b y t h e slide m e t h o d , b y d i r e c t a p p l i c a t i o n o f t h e K a p p l e r

32 TABLE 2 C o m p a r a t i v e results o b t a i n e d w h e n spleen cell cultures were t r a n s f e r r e d t o n e w flat-bott o m e d m i c r o c u l t u r e plate for assay a Experiment

P e r c e n t o f culture wells positive b

N u m b e r o f A F C per positive well c

Slide assay

Tray assay

Slide assay

Tray assay

83 94 63 63

83 94 88 46

5.0 5.6 4.1 3.2

6.5 5.0 4.1 7.8

no.

1 2 3 4

a All cultures were in 0.1 ml o f m e d i u m in V - b o t t o m e d c u l t u r e wells. In e x p e r i m e n t s 1 and 2, 104 spleen cells were c u l t u r e d with 4 X l 0 s t h y m u s filler cells and 20 p g / m l o f LPS a n d in e x p e r i m e n t s 3 and 4, 5000 spleen cells were c u l t u r e d with 4 X 10 s t h y m o c y t e s and 0.1 p g / m l o f F L U - P O L . I d e n t i c a l culture groups were assayed a f t e r 3 days e i t h e r by the C u n n i n g h a m slide m e t h o d or b y t r a n s f e r t o f l a t - b o t t o m e d m i c r o c u l t u r e trays as d e s c r i b e d in Materials and M e t h o d s . b The n u m b e r o f culture wells c o n t a i n i n g a n t i - F L U A F C e x p r e s s e d as a p e r c e n t a g e o f the total n u m b e r o f wells assayed w i t h i n each c u l t u r e group. c The average n u m b e r o f A F C d e t e c t e d per positive culture well.

TABLE 3 N u m b e r s o f A F C f r o m in vitro s t i m u l a t e d spleen cells d e t e c t e d b y various assay procedures. Assay m e t h o d a Slide

' I n s i t u ' tray 36 32 37 42 14

24 8 45 28 15

30 22 26 5

5 + + +

T r a n s f e r r e d tray

AFC detected per c u l t u r e well b

16 7 27 1 45

+

32 16 31 28

32 2 44 12

29 15 37 2

Mean -+ S.E. c

25.1 -+ 3.7

17.6 -+ 5.3 c

23.3 -+ 3.9

Comments

All wells positive, excellent plaque quality

Only 5/9 wells s c o r e d d u e to p o o r plaque quality

All wells positive, e x c e l l e n t plaque quality

a Cultures were assayed b y t h e m e t h o d s i n d i c a t e d as d e s c r i b e d in Materials and M e t h o d s . b N u m b e r o f a n t i - F L U SRC p l a q u e s g e n e r a t e d b y 104 CBA spleen cells c u l t u r e d in flatb o t t o m e d wells w i t h F L U - P O L and LPS in t h e p r e s e n c e o f 106 t h y m u s filler cells for 3 days p r i o r t o assay. Cultures were assayed by o n e o f t h e t h r e e i n d i c a t e d m e t h o d s . c Mean -+ S.E. o f s c o r e d c u l t u r e s o n l y .

33 'TABLE 4 Comparative data obtained by assaying limiting dilution microcultures for AFC clones by either the conventional slide method or the modified tray method a. Percent positive culture wells b

Mean value

Average number of AFC per positive well

Slide

Tray

Slide

Tray

42 44 45 63 79 83 94

50 41 63 88 88 79 94

3.4 5.2 5.0 6.4 7.8 10.8 16.4

5.7 2.8 9.6 9.9 10.6 13.0 15.1

64 -+ 8

72 -+ 8

7.9 -+ 1.7

9.5 -+ 1.6

a CBA spleen cells were cultured at or near limiting dilution in flat-bottomed microculture trays with thymus fillers and FLU-POL for 3 days, then assayed for anti-FLU AFC clones using either the conventional slide or the modified tray assay method. Statistical analysis using Student's t-test showed there to be no significant difference between the results obtained with either assay method in terms of either percent positive wells or clone size. (P -<<0.2). b See footnote 2, Table 2.

'in s i t u ' m e t h o d or b y t h e m o d i f i e d t r a y m e t h o d as described in this paper. O n l y 5 o u t o f the 9 wells assayed c o u l d be s c o r e d with t h e 'in s i t u ' m e t h o d o w i n g t o p o o r p l a q u e q u a l i t y or lack o f visible plaques, whereas with b o t h t h e slide and m o d i f i e d t r a y m e t h o d s p l a q u e q u a l i t y was excellent and virt u a l l y e q u i v a l e n t n u m b e r s were d e t e c t e d .

Comparative results obtained with slide and optimized modified tray assay methods for limit dilution assay T o test t h e m o d i f i e d t r a y s y s t e m f o r r o u t i n e a p p l i c a t i o n t o limiting d i l u t i o n analysis, l o w n u m b e r s o f CBA spleen cells were c u l t u r e d with t h y m u s filler cells and F L U - P O L in f l a t - b o t t o m e d m i c r o c u l t u r e t r a y s as d e s c r i b e d in Materials and M e t h o d s f o r 3 d a y s p r i o r t o assay f o r a n t i - F L U A F C clones. C u l t u r e g r o u p s were divided so t h a t h a l f were assayed b y t h e c o n v e n t i o n a l C u n n i n g h a m slide m e t h o d a n d half b y the m o d i f i e d t r a y assay m e t h o d as described in Materials and M e t h o d s . Cultures were s c o r e d either as positive o r negative and the n u m b e r o f A F C in t h e positive wells c o u n t e d . T h e results o f 7 separate c o m p a r a t i v e e x p e r i m e n t s (Table 4) s h o w t h a t t h e r e was n o significant d i f f e r e n c e in either the p e r c e n t a g e o f wells c o n taining A F C clones or the n u m b e r o f A F C d e t e c t e d in the positive wells w h e n cultures were assayed b y either m e t h o d . In fact, these results indicate t h a t t h e t r a y assay m e t h o d m a y be slightly m o r e sensitive t h a n the slide m e t h o d . In general, the size o f plaques d e v e l o p e d in the t r a y s y s t e m was

34 smaller than of those in the slide system. This, however, produced no difficulty in their identification or enumeration as no difference in plaque clarity occurred. Plaque clarity in the tray assays was excellent and allowed easy discrimination of plaque morphology into clear, mixed and cloudy (Short,man et al., 1979). A further advantage of the tray m e t h o d was that the contents of the assay well could be recovered for subsequent assay against another indicator SRC if necessary. An 87% recovery of AFC was achieved when the contents of wells scored as positive in the tray system were reassayed using the slide system.

Further technical aspects influencing modified tray assay system Further investigations revealed that the following technical points are crucial for successful reproducibility of good plaque quality and visualization in the modified tray assays. (1) Transfer of cultures to a new, flatb o t t o m e d microculture tray was essential. Washed, previously used trays were found to produce unsatisfactory results. Microculture trays with the most vertical sides to the wells should be chosen to obtain better optical conditions for counting AFC and to minimize peripheralization of SRC around the rim of the well. The brands of trays mentioned in Materials and Methods were found to be the most satisfactory. (2) The total volume of liquid in the wells should be kept to a 0.05 ml m a x i m u m as greater volumes created optical difficulties at the AFC readout stage. (3) All centrifugation procedures and media, etc., used in the assay procedure, should be at or around 25°C. This procedure was found to reduce markedly the chance of formation of tiny gas bubbles formed on the b o t t o m of the wells during the 37°C incubation step. These bubbles if formed are easily discriminated from plaques but can in fact at times create difficulties in scoring wells positive or negative. The addition of antifoam reagent, however, abrogated this problem. {4) The number of indicator-SRC added should be such that the b o t t o m of the well is just completely covered at the end of the incubation period. The presence of too m a n y SRC can result in smaller plaques. Interestingly, the concentration of SRC optimal for assay for anti-SRC plaques was found to be lower than for anti-hapten SRC. (5) The final centrifugation step should ideally be performed with the centrifuge brake inactivated to ensure that the indicator-SRC and cultured cells form an undisrupted even cell layer on the b o t t o m of the well. Care should be taken in the subsequent handling of the trays. (6) The enumeration of plaques became inaccurate where more than 40--50 plaques were present in any one well. In cases where high plaque numbers were anticipated, for example the assay of non-limiting dilution cultures, this problem was overcome by assaying only a measured portion of the original culture. Application to assay for protein A-SRC plaques The modified tray assay system as described in Materials and Methods has been successfully used for the protein A plaque m e t h o d (Gronowicz

35 et al., 1976). In this situation, the first washing step with transfer to assay plate was f o u n d to be essential to eliminate non-specific lysis of protein A-SRC by antibody carried over in the culture supernatant. The conditions for the assay and preparation of protein A-SRC were as previously described (Burns and Pike, 1981). Each batch of SRC absorbed guinea pig complement was carefully titrated against the developing antiserum (rabbit anti-mouse immunoglobulin) prior to use. Most complement batches were titrated to be used at a final concentration of 1 in 30 to 1 in 50 in the plaque-revealing mix. Our present developing antiserum is used at a final concentration of I in 200. Protein A-SRC were used at 0.4 ml per 10 ml of mix as for anti-hapten plaques. Trays were incubated for 4--6 h at 37°C prior to scoring. The plaque quality obtained was excellent and trays could be left overnight at room temperature (or 4°C) before scoring without noticeable deterioration.

Other applications of tray assay system The modified tray assay m e t h o d is used extensively in our laboratory as a means of assaying for antibody formation in cultures set up in trays o f different geometric shape up to 96-well microtrays. A particular example is the assay of cultures set up in 0.01 ml 60-well culture trays (Lux Scientific Corp., CA). Cultures are washed prior to assay by flooding the tray with HEM. After removing the HEM, the contents of each well are individually transferred in about 0.025 ml of medium to the corresponding well of a 96-well flat-bottomed tray. Fifty microliters of plaque revealing mix are added and the tray processed as described in Materials and Methods. It is also used for assaying 0.025 ml samples of pooled bulk cultures or immunized spleen cell suspensions from intact mice. Replicate 25 #1 samples of the cell suspension under test are placed in a 96-well flat-bott o m e d tray, 0.05 ml of plaque-revealing mix added and the tray processed as described in Materials and Methods. DISCUSSION This paper describes a microtray hemolytic plaque assay m e t h o d which is a modification of the 'in situ' m e t h o d of Kappler (1974). It provides a simple, rapid and inexpensive m e t h o d for assay of microcultures for the presence or absence of AFC clones with equivalent sensitivity to the liquid monolayer slide m e t h o d (Cunningham and Szenberg, 1968) (Tables 2 and 4). Furthermore, as shown in Fig. 1, it allows a fully a u t o m a t e d approach to limiting dilution analysis of antibody formation, from the initiation of cultures to assay for the presence or absence of AFC clones. This provides a considerable practical advantage, allowing processing of large numbers of replicate cultures within a single experiment. The major factor contributing to the success of the modified m e t h o d was the transfer of cultures to a new, previously unused, 96-well flat-bottomed

36 tray for the assay procedure. For reasons which are still n o t clear, we f o u n d the direct 'in situ' m e t h o d of Kappler highly unsatisfactory for detecting anti-hapten AFC clones (Tables 1 and 3). Removal o f dead cells and debris b y trypsin t r e a t m e n t p r o d u c e d no i m provem ent . Later studies revealed that preincubation of wells with m edi um and antigen alone resulted in inhibition o f plaque f o r m a t i o n by added immunized spleen cells from intact mice. One min o r disadvantage of the tray assay m e t h o d is t hat the wells do n o t have the same optical quality as slides. Satisfactory microscopic visualization o f plaque-forming cells can be obtained in wells using an inverted phase microscope b u t this is s om e w hat inferior when com pared to the slide system. However, for the majority of purposes, such refined analysis is n o t required. Th e tray assay system is now used in our l aborat ory as a general m e t h o d for the assay for a n t i b o d y f o r m a t i o n to a variety of haptens such as FLU, NIP, DNP (dinitrophenyl) and TNP (trinitrophenyl). It has also been successfully used for indirect plaque m e t h o d s such as the protein A-SRC plaque m e t h o d , which demonstrates its versatility. Under optimized conditions, the quality o f plaques obtained is excellent and allows morphological discrimination. One of the major advantages of this m e t h o d is its appriopriateness for limiting dilution analysis, providing a simple, speedy and efficient means of assaying large numbers of replicate microcultures for a n t i b o d y formation. ACKNOWLEDGEMENTS We wish to tha nk Professor G.J.V. Nossal for his helt)ful advice and suggestions. This wo r k was s uppor t ed by the National Health and Medical Research Council, Canberra, Australia; by Grant no. AI-03958 from the National Instit u t e o f Allergy and Infectious Diseases, U.S.A., Public Health Services; and by the generosity of a n u m b e r of private donors to The Walter and Eliza Hall Institute. REFERENCES Burns, G.F. and B.L. Pike, 1981, J. Immunol. Methods 41,269. Cunningham, A.J. and A. Szenberg, 1968, Immunology 14,599. Darling, W., O. Justin, B.L. Pike, G. Jennings and K. Shortman, 1982, J. Immunol. Methods 52, 39. Gronowicz, E., A. Coutinho and F. Melchers, 1976, Eur. J. Immunol. 6, 588. Jerne, N.K. and A.A. Nordin, 1963, Science 140,405. Kappler, J.W., 1974, J. Immunol. 112, 1271. Lefkovits, I., 1972, Eur. J. Immunol. 2, 360. Lefkovits, I. and O. Kamber, 1972, Eur. J. Immunol. 2,365. Lefkovits, I. and H. Waldman, 1979, Limiting Dilution Analysis of Cells in the Immune System (Cambridge University Press, Cambridge, MA). Mishetl, R.I. and R.W. Dutton, 1967, J. Exp. Med. 126,423. Nossal, G.J.V. and B.L. Pike, 1976, Immunology 30,189. Nossal, G.J.V. and B.L. Pike, 1978, J. Immunol. 120, 145.

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