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The ELISA-plaque assay for the detection and enumeration of antibody-secreting cells
Journal oflmmunologicalMethods, 87 (1986) 37-44 Elsevier
37
JIM03739
The ELISA-plaque assay for the detection and enumeration of antibody-secreting cells An overview J o n a t h o n D. Sedgwick* a n d Patrick G. H o l t Clinical Immunology Research Unit, Princess Margaret Hospital, Thomas Street, Subiaco, 6008, Western A ustralia, Australia
(Accepted 10 September 1985)
A solid-phase immunoenzymatic technique has been developed which allows the ready detection and enumeration of total- and antigen-specific immunoglobulin-secreting cells (ISC). The procedure involves the addition of putative ISC to plastic wells pre-coated with specific antigen or antisera. During incubation, the product of a single cell is immobilized by the solid phase at the point of release providing an immunological 'finger print' of the ISC which is subsequently developed by the application of appropriate enzyme-anti-Ig conjugates and an enzyme substrate which yields an insoluble product after incubation. Blue spots or 'ELISA plaques' are thus produced and can be counted macroscopically. This technique has been employed in rat, mouse and human systems and in each case appears to be of equivalent or greater sensitivity to existing haemolytic plaque techniques. The assay is particularly suited to the enumeration of antigen-specific ISC in which the antigen is difficult to couple to red cells or where a high degree of discriminating power is necessary as is required for example in the enumeration of IgE-ISC. Key words: lmmunoglobulin-secreting cells," ELISA-plaque assay," Solid phase," Single cell
Introduction
The haemolytic plaque-forming cell (PFC) assay in its original (Jerne and Nordin, 1963) and
* To whom correspondence should be addressed at the Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K. Abbreviations: ISC, immunoglobulin-secreting cells; PFC, plaque-forming cells; Ig, immunoglobulin; DNP, dinitrophenyl; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; FCS, foetal calf serum; AP, alkaline phosphatase; 5-BCIP, 5-bromo-4-chloro-3-indolyl phosphate; OVA, ovalbumin; ASC, Ascaris suum extract; PBL, peripheral blood leukocytes; HRP, horseradish peroxidase; LN, lymph nodes.
modified (Cunningham and Szenberg, 1968) forms has been used extensively to delineate the processes involved in antibody formation. While the impact of this technique on progress in the general area of humoral immunity is a matter of record, there remain however, a number of inherent disadvantages, many of which are related to the obligatory use of lysable erythrocyte targets as the indicator for antibody secretion (reviewed in Jerne et al., 1974). Limitations include the variation in susceptibility of target erythrocytes to lysis, and the difficulties associated with conjugating antigens to red blood cells - - the latter problem being one that often places constraints upon the choice of antigens to be used experimentally.
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
38 Additionally, the nature of the antibody isotype(s) which contribute to direct plaque formation cannot always be assumed (Wortis et al., 1969) and these may, furthermore, complicate the enumeration of developed or indirect plaques particularly where such indirect plaques are relatively few in number as may occur when attempting to enumerate IgE-ISC (for example see Sedgwick and Holt, 1983a). These limitations have prompted a number of investigators to pursue alternative haemolytic PFC methods for detecting plasma cells employing variations including the binding of erythrocyte indicator cells to a solid phase via poly-L-lysine (Kennedy and Axelrad, 1971) and the use of antigenbound latex particles to enhance red cell lysis (Bagasra and Damjanov, 1982). Most of these latter methods, however, are generally fairly tedious and in general do not have the sensitivity of the haemolysis-in-liquid assay of Cunningham and Szenberg (Richter et al., 1981). Other assays for detection of ISC which are not dependent on complement mediated lysis of target erythrocytes have also been developed. These include the formation of foci of intact erythrocytes around the ISC, held in situ by specific secreted antibody (Moore and Calkins, 1983) or the autoradiographic visualization of antibody secreted by an active cell which is precipitated by iodinated anti-immunoglobulin (Ig) (Pick and Feldman, 1967; Klinman and Taylor, 1969); alternatively, enzyme-labelled anti-Ig can be similarly employed (Cleveland and Erlanger, 1978). These methods also suffer a number of disadvantages, the former associated with binding of antigen to the fociforming erythrocytes while in the latter, lengthy washing (or electrophoretic) procedures are required to remove excess antisera from the agarose gel in which cells in these assays are incubated. A simple alternative to the above was devised by Mason (1976) and involved the construction of demountable Cunningham-like chambers consisting of glass slides coated with dinitrophenyl (DNP)-conjugated gelatin. Cells were allowed to sediment onto these antigen-covered slides and the subsequent area of bound anti-DNP antibody (a product of one ISC) was revealed by a second incubation using iodinated anti-Ig followed by autoradiography.
This method was extremely sensitive but was possibly limited in the range of antigens which could be employed. Additionally, the assay chambers were relatively time consuming to prepare and the use of radiolabelled antisera, both in terms of safety and stability, was disadvantageous. The success of the latter technique, however, underlined the usefulness of a solid-phase matrix for detection of antibody-secreting cells and subsequently we devised a simple procedure for the enumeration of ISC employing the well established principles of the enzyme-linked immunosorbent assay (ELISA) system (Sedgwick and Holt, 1983b). Since that time, a number of reports from our own laboratory and from others (Czerkinsky et al., 1983; see also Results) have attested to the versatility of this technique, employing it to enumerate both total- and antigen-specific ISC in human and rodent systems. It is the purpose of this report to bring together, in a condensed form, the observations made since the first description of the ELISA-plaque technique and in particular to outline certain features of the technique which have altered since the initial report and which appear important in obtaining maximum sensitivity.
Materials and methods
The general scheme for the ELISA-plaque technique is outlined in Fig. 1 and described in detail below. Important features from each step are discussed in the results and discussion section and summarized in Table I. Step 1 - - Coating the plates
Twenty-four-well tissue culture plates (Nunc, Denmark) were precoated by the addition of 0.5 ml of antigen or antisera dissolved in 0.05 M carbonate-bicarbonate buffer, pH 9.6 (Voller et al., 1976) and incubated overnight at 4°C. As the correct concentration and molecular form of the coating antigen was found to be crucial to the success of this technique, where required, antigens were polymerized with glutaraldehyde before coating (Holt et al., 1984a; see also the results and discussion section).
39 1,
AN T I G E N # C O N J U G A T E D TO P L A T E
Australia) in coating buffer was then added to each well and the plates incubated for 1 h at 37°C.
(["-t)
WASH
Step 3 - - Addition o f cells
2, B L O C K I N G P R O T E I N A D D E D
(|)
WASH
3- ADD C E L L S U S P E N S I O N ; I N C U B A T E
2 - - 4 HR.
SECRETED ANTIBODY{8)BINOS
TO A N T I G E N
WASH
4. ADD
Rb--ANTI--RATIg
1H'HF
(~L.);
INCUBATE OVERNIGHT
WASH
5. ADD S h - - A N T I - - R b I g G - - E N Z V M E E C O N J U G A T E (rL1); I N C U B A T E 5 - - 6 HR A T RT
1 FEI--
WASH
6. ADD S O L U B L E S U B S T R A T E I N
AGAROSE(~);
'~. 7*E.E~E~:"r
I N C U B A T E TO A L L O W F O R M A T I O N OF INSOLUBLEPRODUCT(~]
The antigen-coated and BSA blocked wells were washed twice with 1.0 ml volumes of PBS-Tween followed by a third wash with normal PBS. One ml of cell suspension in a suitably iso-osmotic PBS or growth medium containing 10% foetal calf serum (FCS) was then added to each well slowly from a graduated pipette or 1 ml Oxford sampler. The plates were then covered and placed in a vibration free incubator at 37°C for 1-2 h (IgG and IgM) or 32°C for 4 h (lgE). After the incubation was complete, the plates were removed and immediately emptied by flicking over a sink. PBSTween (1 ml/well) was quickly added, swirled, and the wells emptied again. Three washes with 2 ml volumes of PBS-Tween followed. Steps 4 and 5 - - Addition of antisera
All antisera were diluted in PBS-Tween containing 1.0% ( w / v ) BSA. 0.4 ml of an appropriate dilution of the desired rabbit anti-rat Ig was added to each well, and the plates incubated overnight at 4°C. After washing, 0.4 ml of sheep anti-rabbit IgG-alkaline phosphatase (AP) conjugate was added, and the plates incubated at room temperature for 5 - 6 h. Preparation of the AP conjugate was exactly as detailed in Voller et al. (1976). Step 6 - - Addition of substrate
ENUMERATE MACROSCOPIC BLUE SPOTS
rig. 1. The EL1SA-plaque assay for enumeration of total- or antigen-specific ISC. * Specific antisera may be substituted for measurement of total |SC. Step 2 - - Blocking protein
One hour before use, the antigen-coated wells were washed twice with 1.0 ml volumes of PBS0.05% ( v / v ) Tween 20 (Sigma Chemical Co., U.S.A). Each wash consisted of the addition of buffer, followed by a 3 min interval, before its removal by flicking over a sink. 0.4 ml of a 1.0 m g / m l solution of bovine serum albumin (BSA; Commonwealth Serum Laboratories, Melbourne,
After incubation with the enzyme conjugate, the wells were again washed 3 times with PBSTween and emptied. The plates were placed on a level surface, and 0.5 ml of warm (40°C) agarosesubstrate mixture (substrate at approx. 1 m g / m l in 0.6% w / v agarose) was added to each well. The preparation and the use of the AP substrate utilized here (5-bromo-4-chloro-3-indolyl phosphate; 5BCIP) which forms a blue, insoluble product following cleavage by AP, is detailed elsewhere (Sedgwick and Holt, 1983b). After about 1 min at room temperature, the mixture hardened allowing the plates to be handled. Macroscopic blue spots or 'plaques' became visible within 15-30 min at room temperature and were routinely counted after 1 - 2 h. In some cases, where plaques were faint or small in size, the
40 TABLE I THE ELISA-PLAQUE ASSAY Summary of important technical considerations. Step
Procedure
1,2
Coating of the solid phase (i) Selection of the plastic vessel (ii) Antigen/antisera coating
3
Cell incubation (i) Medium (ii) Blocking protein (iii) Temperature
Special considerations
Ref. :'
(i) (ii) (i) (ii) (iii)
l 2 1,3 4 5
Square wells optimal Small round wells adequate High concentrations of some antigens required Low molecular weight antigens may require polymerization Use of glutaraldehyde to increase coating efficiency
(i) Growth medium for longer incubation times (ii) 10-15% FCS or equivalent (iii) Most isotypes; 37°C, 1-2 h IgE; 32°C, 4 h
1 2
4
Addition of antisera
(i) Ensure specificity for minor class or subclass ISC (ii) Low temperature incubations to avoid 'edge effects'
2 6
5
Addition of enzyme-anti-lg
Either AP or HRP can be used
1.3
6
Addition of substrate
(i) Substrate product must be insoluble (ii) Long term stability of A P / 5-BC1P product advantageous (iii) Agarose may be omitted
This report This report
7
Enumeration of plaques
(i) Use of microscope to distinguish artefacts from ELISA plaques (ii) Variation in the size of ELISA plaques related to antibody affinity
5
4
'~ 1, Sedgwick and Holt, 1983b; 2, Sedgwick and Holt, 1983a; 3, Czerkinsky et al., 1983: 4, Holt et al., 1984a; 5, Holt et al., 1984b: 6, Oliver et al., 1981.
p l a t e s w e r e left o v e r n i g h t to a l l o w f u r t h e r c o l o u r d e v e l o p m e n t to t a k e place. 3 M N a O H was t h e n a d d e d to the p l a t e s to s t o p the A P r e a c t i o n if s t o r a g e was desired.
Step 7 - - Enumeration of I S C T h e p l a t e s w e r e i n v e r t e d o v e r a light s o u r c e ( s u c h as an X - r a y v i e w i n g box), a n d m a c r o s c o p i c b l u e s p o t s ( p u t a t i v e z o n e s of a n t i b o d y b i n d i n g ) s c o r e d by m a r k i n g the u n d e r s u r f a c e w i t h a finep o i n t e d felt pen. S i m i l a r c o u n t s w e r e a c h i e v e d with a projecting microscope. An inverted micros c o p e was used to d i s t i n g u i s h o c c a s i o n a l a r t e f a c t s f r o m z o n e s of p u t a t i v e a n t i b o d y .
Results and discussion A s d e t a i l e d results h a v e b e e n p u b l i s h e d e l s e w h e r e ( S e d g w i c k a n d H o l t , 1983b) o n l y a n a l y s e s of i m p o r t a n t t e c h n i c a l c o n s i d e r a t i o n s w h i c h h a v e b e c o m e e v i d e n t since the initial r e p o r t o f this t e c h n i q u e are given.
Preparation of the solid phase T h e ideal vessels for this assay are s q u a r e - w e l l (2 x 2 cm) n o n - w e t t a b l e plastic repli dishes (25 wells p e r plate) as these a p p e a r to r e d u c e the d e g r e e to w h i c h cells swirl d u r i n g t r a n s p o r t a n d also b i n d a n t i g e n e x t r e m e l y well ( S e d g w i c k and
41 Holt, 1983b; Moskophidis and Lehmann-Grube, 1984). As these were not always available, 24 round-well, Nunc tissue-culture plates were substituted (Sedgwick and Holt, 1983a) and found to be adequate. Others (Czerkinsky et al., 1983) in their description of this technique employed larger plastic petri dishes (5 cm diameter). In general we found these latter plates to be somewhat tedious to use and often noted that cells were distributed unevenly over the plate which may reflect swirling of the cell suspension during transport. In a previous report it was shown that commercial preparations of low molecular weight antigens such as ovalbumin (OVA) differ in their content of higher molecular weight polymers (Holt et ai., 1981) and it is this latter component which appears to be the important solid-phase antigen in the ELISA-plaque assay (Holt et al., 1984a). This may explain why relatively high concentrations of these antigens are required to coat the wells (Czerkinsky et al., 1983; Sedgwick and Holt, 1983b). In contrast, considerably lower concentrations are needed if larger multi-determinant antigens such as Ascaris suum extract (ASC), thyroglobulin or haptenated proteins are employed (Holt et al., 1984a) or when the solid-phase consists of anti-Ig for detection of total ISC (Sedgwick and Holt, 1983a). To alleviate these problems, lower molecular weight antigens are routinely polymerized with glutaraldehyde before use (Holt et al., 1984a). It should be noted that increased binding of anti-immunoglobulin to the solid phase can be achieved by pretreatment of the plastic wells with glutaraldehyde. In comparative experiments, this procedure resulted in approximately 50% more lg bound than was observed with coating buffer alone (unpublished data), a strategy which in turn considerably increased the number of ISC detected in experiments which examined the production of ISC by human peripheral blood leukocytes (PBL) (Holt et al., 1984b). The value or otherwise of a subsequent 'blocking' protein in solid-phase assays (step 2) has not been established; however, in our hands, this procedure tended to reduce background blueing which, in turn, marginally increased the number of faint ELISA plaques which could be detected (unpublished data). It is conceivable that this additional
blocking protein may be of greater importance in the present assay since additives such as non-ionic detergents (e.g., Tween 20) which are successfully used in standard ELISA procedures to reduce non-specific binding (Bullock and Walls, 1977) must, for obvious reasons, be excluded during cell incubation (step 3). Cell preparation and incubation
A variety of cell types have been employed in the ELISA-plaque assay (see Table II) and in most cases resuspension and incubation in PBS containing 10% FCS has been adequate. For extended incubation times (i.e, for longer than 4 or 5 h) it is probably necessary to incubate the cells in growth medium in a humidified CO 2 incubator. A sufficiently high concentration of FCS (10-15%) or an equivalent protein supplement remains an essential additive to the cell suspension, however, in order to prevent both cell clumping and nonspecific binding of secreted antibody (Sedgwick and Holt, 1983b). The optimal temperature for cell incubation may not always be 37°C. For isotypes other than IgE we have noted 37°C for 1-2 h to be suitable, and little increase is apparent in the number of ISC detected after this time. Similar results have been noted by others (Czerkinsky et al., 1983). The IgE isotype, however, was not readily detected under these conditions and we subsequently showed, employing the standard liquid ELISA, that a slightly lower temperature (32°C) was more suitable (unpublished data). Extended incubation times (4 h) at this lower temperature proved to be optimal for the detection of IgE-ISC (Sedgwick and Holt, 1983a) and these conditions have been employed since that time. We have no definite explanation for this observation but it may indicate that the IgE-antigen complex is particularly susceptible to dissociation at the higher temperature. Antisera
Antisera which are used in the standard ELISA can also be employed in the ELISA-plaque assay. It is important, however, to titre out all antisera as prozone effects can occur and high concentrations of expensive antisera may be used needlessly. In general, affinity purified antibodies are prob-
42 T A B L E II S U M M A R Y OF E X P E R I M E N T A L SYSTEMS IN W H I C H T H E ELISA-PLAQUE ASSAY OR ITS E Q U I V A L E N T HAS BEEN EMPLOYED Species
Strain
Cell source
ISC detected
Rat
BrownNorway
Spleen. lymph node (LN)
Anti-OVA igE, IgG and IgM-1SC
1,2
LOU/M
Mesenteric LN
Total IgE-ISC
1
BrownNorway
Spleen, LN, bone marrow
Long-lived, radio-resistant anti-OVA IgE and IgG-ISC
3
PVG/c
Spleen
Anti-thyroglobulin IgG-ISC
4
BrownNorway
Respiratory LN
Total IgE-ISC
5
BALB/c
Spleen, LN, bone marrow
Long-lived, radio-resistant anti-OVA IgE and IgG-ISC
3
BALB/c
Spleen
Anti-BSA, ASC and DNPIgG-ISC
4
CBA/J
Spleen
Anti-LCM h and VSV " IgG and IgM-ISC
6
C57/BL6
Spleen
Anti-OVA and K L H d-ISC
7
BALB/c
Spleen
Total ISC
8
MRL/1
Spleen
lgG-rheumatoid factor-lSC
9
-
U266 lgE myeloma
Total IgE-ISC
10
-
Pokeweed-mitogen stimulated PBL
Total IgG-ISC
10
Epstein-Barr virus stimulated PBL
Total ISC
Tetanus toxoid stimulated PBL
Anti-tetanus toxoid ISC
Mouse
Human
-
-
Ref. '
8
11
1, Sedgwick and Holt, 1983a; 2, Sedgwick and Holt, 1983b; 3, Holt et al., 1984c; 4, Holt et al., 1984a: 5. Sedgwick and Holt, 1985: 6, Moskophidis and Lehmann-Grube, 1984; 7, Czerkinsky et al., 1983; 8, Czerkinsky et al., 1984a; 9, Tarkowski et al., 1984; 10, Holt et al., 1984b; 11, Czerkinsky et al., 1984b. b Lymphocytic choriomeningitis virus. Vesicular stomatitis virus. d Keyhole limpet haemocyanin.
ably best as these are easier to standardize. Of paramount importance is the use of adequately absorbed antisera particularly when assessing minor class or subclass ISC. Incubation times for antisera are largely dictated by convenience. However, longer, low temperature incubations are probably preferable to short high temperature incubations since the latter may be susceptible to 'edge effects' which can
result in relatively higher temperatures in outer wells (Oliver et al., 1981). The choice of enzyme and substrate In all investigations, AP together with the substrate 5-BCIP which produces a vivid blue insoluble product following enzyme cleavage has been employed, whilst others (Czerkinsky et al., 1983) have successfully used horseradish peroxidase
43 ( H R P ) together with paraphenylenediamine and H202. In preliminary comparative experiments (unpublished) we tended to favour the former, both for the ease with which AP could be conjugated to Ig and also for the stability of the reaction product. By stopping the reaction with 0.2 ml per well of 3 M N a O H and storing at 4°C we have been able to maintain ELISA-plaque plates in their original condition for over a year. In contrast, spontaneous darkening of the agarose gel occurred when using the latter enzyme/substrate combination which severely limited storage. In spite of this, it is apparent that either enzyme can be employed, since both systems appear to be of similar sensitivity when compared to existing haemolytic PFC assays (Czerkinsky et al., 1983; Sedgwick and Holt, 1983b; Holt et al., 1984b). A formal examination of the 2 enzymes in a single experimental system is, nevertheless, required to establish their respective limits of sensitivity. The use of an agarose-substrate mixture to retain the substrate product at its site of generation (i.e., where antibody secretion occurred) may not be essential. In some recent experiments (to be published) we demonstrated that 5-BCIP in substrate buffer alone produced an equivalent number of blue spots although the intensity of colour was marginally reduced. Experiments are currently in progress to optimize this latter procedure and to assess its applicability to the detection of ISC in a variety of systems.
Enumeration of ISC and detection of artefacts As detailed in the materials and methods section, plaques are most readily detected by placing the inverted plates over a light source and counting by eye. With practice it is quite easy to distinguish artefacts from sites of antibody secretion as the former are usually much smaller, denser spots. These artefacts appear either at the same plane as the EEISA plaques or above the agarose/solidphase interface. The latter appear due to the presence of contaminating particulate matter in the agarose or the substrate and hence it is important that all solutions (water for agarose preparation and substrate solutions) are filtered before use. It is not clear what the other false spots are, although it is conceivable that they represent highly adher-
ent (and possibly also AP positive) cells. Indeed, activated B lymphocytes may well fit both the former and the latter (Garcia-Rozas et al., 1982) criteria, although no firm evidence in support of this possibility has been obtained. Consequently, the use of an inverted microscope is sometimes w a r r a n t e d and u n d e r as little as 10 x magnification, the typically granular ELISA plaques are readily distinguished (for example see Sedgwick and Holt, 1983a). During the course of these studies we became aware of considerable variation in the size of plaques, a feature which also did not go unnoticed in the studies by Czerkinsky et al. (1983). Subsequently we demonstrated that the plaque diameter increased in secondary when compared to primary responses and furthermore that the diameter of the plaques correlated with the affinity of serum antibody from the cell donors (Holt et al., 1984b). While further studies are clearly required to confirm this association it does nevertheless suggest that differences in the levels of antigen attached to the plates are not responsible as these effects would be expected to occur randomly.
Conclusion Since the initial report of the ELISA-plaque assay there has been a gradual accumulation of data employing the technique and it is now apparent that this system is applicable to a wide variety of experimental models, from the enumeration of ISC specific for defined protein antigens, to those secreting antibody against viruses and antigens relevant to autoimmune disease (see Table II). While the basic methodology involved in each of the reports listed in Table II remains the same, each system requires refinement to some extent and details of different assay conditions are given in Table I. In particular, it is our experience that the solid phase is invariably the component which limits the success of any one particular investigation and hence it is recommended that attention is given to optimizing this aspect of the ELISAplaque system. Whilst it is evident that this technique is sufficiently versatile to be employed in a number of different experimental systems for the detection of ISC, it may also be feasible to extend it to the
44
enumeration of cells, including T lymphocytes, secreting other biologically active molecules. These possibilities, and others, await investigation.
Acknowledgement The authors would like to thank Mrs. Valerie Boasten for typing the manuscript.
References Bagasra, O. and I. Damjanov, 1982, J. Immunol. Methods 49, 283. Bullock, S.L. and K.W. Walls, 1977, J. Infect. Dis. 136 (suppl.), $279. Cleveland, W.L. and B.F. Erlanger, 1978, Cell. Immunol. 37. 229. Cunningham, A.J. and A. Szenberg, 1968, Immunology 14, 599. Czerkinsky, C.C., L.-A. Nilsson, H. Nygren, O. Ouchterlony and A. Tarkowski, 1983, J. lmmunol. Methods 65, 109. Czerkinsky, C.C., A. Tarkowski, L.-A. Nilsson, O. Ouchterlony, H. Nygren and C. Gretzer, 1984a, J. immunol. Methods 72, 489. Czerkinsky, C., L.-A. Nilsson, O. Ouchterlony, A. Tarkowski and C. Gretzer, 1984b, Scand. J. Immunol. 19~ 573. Garcia-Rozas, C., A. Plaza, F. Diaz-Espada, M. Kreisler and C. Martinez-Alonso, 1982, J. Immunol. 129, 52. Holt, P.G., A.H. Rose, J.E. Batty and K.J. Turner, 1981, Int. Arch. Allergy Appl. Immunol. 65, 42.
Holt, P.G., J.D. Sedgwick, G.A. Stewart, C. O'Leary and K. Krska, 1984a, J. Immunol. Methods 74, 1. Holt, P.G., K.J. Cameron, G.A. Stewart, J.D. Sedgwick and K.J. Turner, 1984b, Clin. Immunol. lmmunopathol. 30, 159. Holt, P.G., J.D. Sedgwick, C. O'Leary, K. Krska and S. Leivers, 1984c, Cell. immunol. 89, 281. Jerne, N.K. and A.A. Nordin, 1963, Science 140, 405. Jerne, N.K., C. Henry, A.A. Nordin, H. Jufi, A.M.C. Koros and I. Lefkovits, 1974, Transplant. Rev. 18, 130. Kennedy, J.C. and M.A. Axelrad, 1971, Immunology 20, 253. Klinman, N.R. and R.B. Taylor, 1969, Clin. Exp. Immunol. 4, 473. Mason, D.W., 1976, J. lmmunol. Methods 10, 301. Moore, J. and C. Calkins, 1983, J. Immunol. Methods 63, 377. Moskophidis, D. and F. Lehmann-Grube, 1984, J. lmmunol. 133, 3366. Oliver, D.G., A.H. Sanders, R.D. Hogg and J.W. Hellman, 1981, J. Immunol. Methods 42. 195. Pick, E. and J.D. Feldman, 1967, Science 156, 964. Richter, M., M. Berry and N. Kazaniwsky, 1981, J. hnmunol. Methods 46, 77. Sedgwick, J.D. and P.G. Holt, 1983a, J. Exp. Med. 157, 2178. Sedgwick, J.D. and P.G. Holt. 1983b, J. Immunol. Methods 57, 301. Sedgwick, J.D. and P.G. Holt, 1985, Cell. Immunol. 94, t82. Tarkowski, A., C. Czerkinsky, L.-A. Nilsson, H. Nygren and O. Ouchterlony, 1984, J. lmmunol. Methods 72, 451. Voller, A., D. Bidwell and A. Bartlett, 1976, in: Manual of Clinical Microbiology, eds. N.R. Rose and H. Friedman (American Society for Microbiology, Washington, DC) p. 506. Wortis, H.H., D.H. Dresser and H.R. Anderson, 1969, Immunology 17. 93.
37
JIM03739
The ELISA-plaque assay for the detection and enumeration of antibody-secreting cells An overview J o n a t h o n D. Sedgwick* a n d Patrick G. H o l t Clinical Immunology Research Unit, Princess Margaret Hospital, Thomas Street, Subiaco, 6008, Western A ustralia, Australia
(Accepted 10 September 1985)
A solid-phase immunoenzymatic technique has been developed which allows the ready detection and enumeration of total- and antigen-specific immunoglobulin-secreting cells (ISC). The procedure involves the addition of putative ISC to plastic wells pre-coated with specific antigen or antisera. During incubation, the product of a single cell is immobilized by the solid phase at the point of release providing an immunological 'finger print' of the ISC which is subsequently developed by the application of appropriate enzyme-anti-Ig conjugates and an enzyme substrate which yields an insoluble product after incubation. Blue spots or 'ELISA plaques' are thus produced and can be counted macroscopically. This technique has been employed in rat, mouse and human systems and in each case appears to be of equivalent or greater sensitivity to existing haemolytic plaque techniques. The assay is particularly suited to the enumeration of antigen-specific ISC in which the antigen is difficult to couple to red cells or where a high degree of discriminating power is necessary as is required for example in the enumeration of IgE-ISC. Key words: lmmunoglobulin-secreting cells," ELISA-plaque assay," Solid phase," Single cell
Introduction
The haemolytic plaque-forming cell (PFC) assay in its original (Jerne and Nordin, 1963) and
* To whom correspondence should be addressed at the Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, U.K. Abbreviations: ISC, immunoglobulin-secreting cells; PFC, plaque-forming cells; Ig, immunoglobulin; DNP, dinitrophenyl; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; FCS, foetal calf serum; AP, alkaline phosphatase; 5-BCIP, 5-bromo-4-chloro-3-indolyl phosphate; OVA, ovalbumin; ASC, Ascaris suum extract; PBL, peripheral blood leukocytes; HRP, horseradish peroxidase; LN, lymph nodes.
modified (Cunningham and Szenberg, 1968) forms has been used extensively to delineate the processes involved in antibody formation. While the impact of this technique on progress in the general area of humoral immunity is a matter of record, there remain however, a number of inherent disadvantages, many of which are related to the obligatory use of lysable erythrocyte targets as the indicator for antibody secretion (reviewed in Jerne et al., 1974). Limitations include the variation in susceptibility of target erythrocytes to lysis, and the difficulties associated with conjugating antigens to red blood cells - - the latter problem being one that often places constraints upon the choice of antigens to be used experimentally.
0022-1759/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
38 Additionally, the nature of the antibody isotype(s) which contribute to direct plaque formation cannot always be assumed (Wortis et al., 1969) and these may, furthermore, complicate the enumeration of developed or indirect plaques particularly where such indirect plaques are relatively few in number as may occur when attempting to enumerate IgE-ISC (for example see Sedgwick and Holt, 1983a). These limitations have prompted a number of investigators to pursue alternative haemolytic PFC methods for detecting plasma cells employing variations including the binding of erythrocyte indicator cells to a solid phase via poly-L-lysine (Kennedy and Axelrad, 1971) and the use of antigenbound latex particles to enhance red cell lysis (Bagasra and Damjanov, 1982). Most of these latter methods, however, are generally fairly tedious and in general do not have the sensitivity of the haemolysis-in-liquid assay of Cunningham and Szenberg (Richter et al., 1981). Other assays for detection of ISC which are not dependent on complement mediated lysis of target erythrocytes have also been developed. These include the formation of foci of intact erythrocytes around the ISC, held in situ by specific secreted antibody (Moore and Calkins, 1983) or the autoradiographic visualization of antibody secreted by an active cell which is precipitated by iodinated anti-immunoglobulin (Ig) (Pick and Feldman, 1967; Klinman and Taylor, 1969); alternatively, enzyme-labelled anti-Ig can be similarly employed (Cleveland and Erlanger, 1978). These methods also suffer a number of disadvantages, the former associated with binding of antigen to the fociforming erythrocytes while in the latter, lengthy washing (or electrophoretic) procedures are required to remove excess antisera from the agarose gel in which cells in these assays are incubated. A simple alternative to the above was devised by Mason (1976) and involved the construction of demountable Cunningham-like chambers consisting of glass slides coated with dinitrophenyl (DNP)-conjugated gelatin. Cells were allowed to sediment onto these antigen-covered slides and the subsequent area of bound anti-DNP antibody (a product of one ISC) was revealed by a second incubation using iodinated anti-Ig followed by autoradiography.
This method was extremely sensitive but was possibly limited in the range of antigens which could be employed. Additionally, the assay chambers were relatively time consuming to prepare and the use of radiolabelled antisera, both in terms of safety and stability, was disadvantageous. The success of the latter technique, however, underlined the usefulness of a solid-phase matrix for detection of antibody-secreting cells and subsequently we devised a simple procedure for the enumeration of ISC employing the well established principles of the enzyme-linked immunosorbent assay (ELISA) system (Sedgwick and Holt, 1983b). Since that time, a number of reports from our own laboratory and from others (Czerkinsky et al., 1983; see also Results) have attested to the versatility of this technique, employing it to enumerate both total- and antigen-specific ISC in human and rodent systems. It is the purpose of this report to bring together, in a condensed form, the observations made since the first description of the ELISA-plaque technique and in particular to outline certain features of the technique which have altered since the initial report and which appear important in obtaining maximum sensitivity.
Materials and methods
The general scheme for the ELISA-plaque technique is outlined in Fig. 1 and described in detail below. Important features from each step are discussed in the results and discussion section and summarized in Table I. Step 1 - - Coating the plates
Twenty-four-well tissue culture plates (Nunc, Denmark) were precoated by the addition of 0.5 ml of antigen or antisera dissolved in 0.05 M carbonate-bicarbonate buffer, pH 9.6 (Voller et al., 1976) and incubated overnight at 4°C. As the correct concentration and molecular form of the coating antigen was found to be crucial to the success of this technique, where required, antigens were polymerized with glutaraldehyde before coating (Holt et al., 1984a; see also the results and discussion section).
39 1,
AN T I G E N # C O N J U G A T E D TO P L A T E
Australia) in coating buffer was then added to each well and the plates incubated for 1 h at 37°C.
(["-t)
WASH
Step 3 - - Addition o f cells
2, B L O C K I N G P R O T E I N A D D E D
(|)
WASH
3- ADD C E L L S U S P E N S I O N ; I N C U B A T E
2 - - 4 HR.
SECRETED ANTIBODY{8)BINOS
TO A N T I G E N
WASH
4. ADD
Rb--ANTI--RATIg
1H'HF
(~L.);
INCUBATE OVERNIGHT
WASH
5. ADD S h - - A N T I - - R b I g G - - E N Z V M E E C O N J U G A T E (rL1); I N C U B A T E 5 - - 6 HR A T RT
1 FEI--
WASH
6. ADD S O L U B L E S U B S T R A T E I N
AGAROSE(~);
'~. 7*E.E~E~:"r
I N C U B A T E TO A L L O W F O R M A T I O N OF INSOLUBLEPRODUCT(~]
The antigen-coated and BSA blocked wells were washed twice with 1.0 ml volumes of PBS-Tween followed by a third wash with normal PBS. One ml of cell suspension in a suitably iso-osmotic PBS or growth medium containing 10% foetal calf serum (FCS) was then added to each well slowly from a graduated pipette or 1 ml Oxford sampler. The plates were then covered and placed in a vibration free incubator at 37°C for 1-2 h (IgG and IgM) or 32°C for 4 h (lgE). After the incubation was complete, the plates were removed and immediately emptied by flicking over a sink. PBSTween (1 ml/well) was quickly added, swirled, and the wells emptied again. Three washes with 2 ml volumes of PBS-Tween followed. Steps 4 and 5 - - Addition of antisera
All antisera were diluted in PBS-Tween containing 1.0% ( w / v ) BSA. 0.4 ml of an appropriate dilution of the desired rabbit anti-rat Ig was added to each well, and the plates incubated overnight at 4°C. After washing, 0.4 ml of sheep anti-rabbit IgG-alkaline phosphatase (AP) conjugate was added, and the plates incubated at room temperature for 5 - 6 h. Preparation of the AP conjugate was exactly as detailed in Voller et al. (1976). Step 6 - - Addition of substrate
ENUMERATE MACROSCOPIC BLUE SPOTS
rig. 1. The EL1SA-plaque assay for enumeration of total- or antigen-specific ISC. * Specific antisera may be substituted for measurement of total |SC. Step 2 - - Blocking protein
One hour before use, the antigen-coated wells were washed twice with 1.0 ml volumes of PBS0.05% ( v / v ) Tween 20 (Sigma Chemical Co., U.S.A). Each wash consisted of the addition of buffer, followed by a 3 min interval, before its removal by flicking over a sink. 0.4 ml of a 1.0 m g / m l solution of bovine serum albumin (BSA; Commonwealth Serum Laboratories, Melbourne,
After incubation with the enzyme conjugate, the wells were again washed 3 times with PBSTween and emptied. The plates were placed on a level surface, and 0.5 ml of warm (40°C) agarosesubstrate mixture (substrate at approx. 1 m g / m l in 0.6% w / v agarose) was added to each well. The preparation and the use of the AP substrate utilized here (5-bromo-4-chloro-3-indolyl phosphate; 5BCIP) which forms a blue, insoluble product following cleavage by AP, is detailed elsewhere (Sedgwick and Holt, 1983b). After about 1 min at room temperature, the mixture hardened allowing the plates to be handled. Macroscopic blue spots or 'plaques' became visible within 15-30 min at room temperature and were routinely counted after 1 - 2 h. In some cases, where plaques were faint or small in size, the
40 TABLE I THE ELISA-PLAQUE ASSAY Summary of important technical considerations. Step
Procedure
1,2
Coating of the solid phase (i) Selection of the plastic vessel (ii) Antigen/antisera coating
3
Cell incubation (i) Medium (ii) Blocking protein (iii) Temperature
Special considerations
Ref. :'
(i) (ii) (i) (ii) (iii)
l 2 1,3 4 5
Square wells optimal Small round wells adequate High concentrations of some antigens required Low molecular weight antigens may require polymerization Use of glutaraldehyde to increase coating efficiency
(i) Growth medium for longer incubation times (ii) 10-15% FCS or equivalent (iii) Most isotypes; 37°C, 1-2 h IgE; 32°C, 4 h
1 2
4
Addition of antisera
(i) Ensure specificity for minor class or subclass ISC (ii) Low temperature incubations to avoid 'edge effects'
2 6
5
Addition of enzyme-anti-lg
Either AP or HRP can be used
1.3
6
Addition of substrate
(i) Substrate product must be insoluble (ii) Long term stability of A P / 5-BC1P product advantageous (iii) Agarose may be omitted
This report This report
7
Enumeration of plaques
(i) Use of microscope to distinguish artefacts from ELISA plaques (ii) Variation in the size of ELISA plaques related to antibody affinity
5
4
'~ 1, Sedgwick and Holt, 1983b; 2, Sedgwick and Holt, 1983a; 3, Czerkinsky et al., 1983: 4, Holt et al., 1984a; 5, Holt et al., 1984b: 6, Oliver et al., 1981.
p l a t e s w e r e left o v e r n i g h t to a l l o w f u r t h e r c o l o u r d e v e l o p m e n t to t a k e place. 3 M N a O H was t h e n a d d e d to the p l a t e s to s t o p the A P r e a c t i o n if s t o r a g e was desired.
Step 7 - - Enumeration of I S C T h e p l a t e s w e r e i n v e r t e d o v e r a light s o u r c e ( s u c h as an X - r a y v i e w i n g box), a n d m a c r o s c o p i c b l u e s p o t s ( p u t a t i v e z o n e s of a n t i b o d y b i n d i n g ) s c o r e d by m a r k i n g the u n d e r s u r f a c e w i t h a finep o i n t e d felt pen. S i m i l a r c o u n t s w e r e a c h i e v e d with a projecting microscope. An inverted micros c o p e was used to d i s t i n g u i s h o c c a s i o n a l a r t e f a c t s f r o m z o n e s of p u t a t i v e a n t i b o d y .
Results and discussion A s d e t a i l e d results h a v e b e e n p u b l i s h e d e l s e w h e r e ( S e d g w i c k a n d H o l t , 1983b) o n l y a n a l y s e s of i m p o r t a n t t e c h n i c a l c o n s i d e r a t i o n s w h i c h h a v e b e c o m e e v i d e n t since the initial r e p o r t o f this t e c h n i q u e are given.
Preparation of the solid phase T h e ideal vessels for this assay are s q u a r e - w e l l (2 x 2 cm) n o n - w e t t a b l e plastic repli dishes (25 wells p e r plate) as these a p p e a r to r e d u c e the d e g r e e to w h i c h cells swirl d u r i n g t r a n s p o r t a n d also b i n d a n t i g e n e x t r e m e l y well ( S e d g w i c k and
41 Holt, 1983b; Moskophidis and Lehmann-Grube, 1984). As these were not always available, 24 round-well, Nunc tissue-culture plates were substituted (Sedgwick and Holt, 1983a) and found to be adequate. Others (Czerkinsky et al., 1983) in their description of this technique employed larger plastic petri dishes (5 cm diameter). In general we found these latter plates to be somewhat tedious to use and often noted that cells were distributed unevenly over the plate which may reflect swirling of the cell suspension during transport. In a previous report it was shown that commercial preparations of low molecular weight antigens such as ovalbumin (OVA) differ in their content of higher molecular weight polymers (Holt et ai., 1981) and it is this latter component which appears to be the important solid-phase antigen in the ELISA-plaque assay (Holt et al., 1984a). This may explain why relatively high concentrations of these antigens are required to coat the wells (Czerkinsky et al., 1983; Sedgwick and Holt, 1983b). In contrast, considerably lower concentrations are needed if larger multi-determinant antigens such as Ascaris suum extract (ASC), thyroglobulin or haptenated proteins are employed (Holt et al., 1984a) or when the solid-phase consists of anti-Ig for detection of total ISC (Sedgwick and Holt, 1983a). To alleviate these problems, lower molecular weight antigens are routinely polymerized with glutaraldehyde before use (Holt et al., 1984a). It should be noted that increased binding of anti-immunoglobulin to the solid phase can be achieved by pretreatment of the plastic wells with glutaraldehyde. In comparative experiments, this procedure resulted in approximately 50% more lg bound than was observed with coating buffer alone (unpublished data), a strategy which in turn considerably increased the number of ISC detected in experiments which examined the production of ISC by human peripheral blood leukocytes (PBL) (Holt et al., 1984b). The value or otherwise of a subsequent 'blocking' protein in solid-phase assays (step 2) has not been established; however, in our hands, this procedure tended to reduce background blueing which, in turn, marginally increased the number of faint ELISA plaques which could be detected (unpublished data). It is conceivable that this additional
blocking protein may be of greater importance in the present assay since additives such as non-ionic detergents (e.g., Tween 20) which are successfully used in standard ELISA procedures to reduce non-specific binding (Bullock and Walls, 1977) must, for obvious reasons, be excluded during cell incubation (step 3). Cell preparation and incubation
A variety of cell types have been employed in the ELISA-plaque assay (see Table II) and in most cases resuspension and incubation in PBS containing 10% FCS has been adequate. For extended incubation times (i.e, for longer than 4 or 5 h) it is probably necessary to incubate the cells in growth medium in a humidified CO 2 incubator. A sufficiently high concentration of FCS (10-15%) or an equivalent protein supplement remains an essential additive to the cell suspension, however, in order to prevent both cell clumping and nonspecific binding of secreted antibody (Sedgwick and Holt, 1983b). The optimal temperature for cell incubation may not always be 37°C. For isotypes other than IgE we have noted 37°C for 1-2 h to be suitable, and little increase is apparent in the number of ISC detected after this time. Similar results have been noted by others (Czerkinsky et al., 1983). The IgE isotype, however, was not readily detected under these conditions and we subsequently showed, employing the standard liquid ELISA, that a slightly lower temperature (32°C) was more suitable (unpublished data). Extended incubation times (4 h) at this lower temperature proved to be optimal for the detection of IgE-ISC (Sedgwick and Holt, 1983a) and these conditions have been employed since that time. We have no definite explanation for this observation but it may indicate that the IgE-antigen complex is particularly susceptible to dissociation at the higher temperature. Antisera
Antisera which are used in the standard ELISA can also be employed in the ELISA-plaque assay. It is important, however, to titre out all antisera as prozone effects can occur and high concentrations of expensive antisera may be used needlessly. In general, affinity purified antibodies are prob-
42 T A B L E II S U M M A R Y OF E X P E R I M E N T A L SYSTEMS IN W H I C H T H E ELISA-PLAQUE ASSAY OR ITS E Q U I V A L E N T HAS BEEN EMPLOYED Species
Strain
Cell source
ISC detected
Rat
BrownNorway
Spleen. lymph node (LN)
Anti-OVA igE, IgG and IgM-1SC
1,2
LOU/M
Mesenteric LN
Total IgE-ISC
1
BrownNorway
Spleen, LN, bone marrow
Long-lived, radio-resistant anti-OVA IgE and IgG-ISC
3
PVG/c
Spleen
Anti-thyroglobulin IgG-ISC
4
BrownNorway
Respiratory LN
Total IgE-ISC
5
BALB/c
Spleen, LN, bone marrow
Long-lived, radio-resistant anti-OVA IgE and IgG-ISC
3
BALB/c
Spleen
Anti-BSA, ASC and DNPIgG-ISC
4
CBA/J
Spleen
Anti-LCM h and VSV " IgG and IgM-ISC
6
C57/BL6
Spleen
Anti-OVA and K L H d-ISC
7
BALB/c
Spleen
Total ISC
8
MRL/1
Spleen
lgG-rheumatoid factor-lSC
9
-
U266 lgE myeloma
Total IgE-ISC
10
-
Pokeweed-mitogen stimulated PBL
Total IgG-ISC
10
Epstein-Barr virus stimulated PBL
Total ISC
Tetanus toxoid stimulated PBL
Anti-tetanus toxoid ISC
Mouse
Human
-
-
Ref. '
8
11
1, Sedgwick and Holt, 1983a; 2, Sedgwick and Holt, 1983b; 3, Holt et al., 1984c; 4, Holt et al., 1984a: 5. Sedgwick and Holt, 1985: 6, Moskophidis and Lehmann-Grube, 1984; 7, Czerkinsky et al., 1983; 8, Czerkinsky et al., 1984a; 9, Tarkowski et al., 1984; 10, Holt et al., 1984b; 11, Czerkinsky et al., 1984b. b Lymphocytic choriomeningitis virus. Vesicular stomatitis virus. d Keyhole limpet haemocyanin.
ably best as these are easier to standardize. Of paramount importance is the use of adequately absorbed antisera particularly when assessing minor class or subclass ISC. Incubation times for antisera are largely dictated by convenience. However, longer, low temperature incubations are probably preferable to short high temperature incubations since the latter may be susceptible to 'edge effects' which can
result in relatively higher temperatures in outer wells (Oliver et al., 1981). The choice of enzyme and substrate In all investigations, AP together with the substrate 5-BCIP which produces a vivid blue insoluble product following enzyme cleavage has been employed, whilst others (Czerkinsky et al., 1983) have successfully used horseradish peroxidase
43 ( H R P ) together with paraphenylenediamine and H202. In preliminary comparative experiments (unpublished) we tended to favour the former, both for the ease with which AP could be conjugated to Ig and also for the stability of the reaction product. By stopping the reaction with 0.2 ml per well of 3 M N a O H and storing at 4°C we have been able to maintain ELISA-plaque plates in their original condition for over a year. In contrast, spontaneous darkening of the agarose gel occurred when using the latter enzyme/substrate combination which severely limited storage. In spite of this, it is apparent that either enzyme can be employed, since both systems appear to be of similar sensitivity when compared to existing haemolytic PFC assays (Czerkinsky et al., 1983; Sedgwick and Holt, 1983b; Holt et al., 1984b). A formal examination of the 2 enzymes in a single experimental system is, nevertheless, required to establish their respective limits of sensitivity. The use of an agarose-substrate mixture to retain the substrate product at its site of generation (i.e., where antibody secretion occurred) may not be essential. In some recent experiments (to be published) we demonstrated that 5-BCIP in substrate buffer alone produced an equivalent number of blue spots although the intensity of colour was marginally reduced. Experiments are currently in progress to optimize this latter procedure and to assess its applicability to the detection of ISC in a variety of systems.
Enumeration of ISC and detection of artefacts As detailed in the materials and methods section, plaques are most readily detected by placing the inverted plates over a light source and counting by eye. With practice it is quite easy to distinguish artefacts from sites of antibody secretion as the former are usually much smaller, denser spots. These artefacts appear either at the same plane as the EEISA plaques or above the agarose/solidphase interface. The latter appear due to the presence of contaminating particulate matter in the agarose or the substrate and hence it is important that all solutions (water for agarose preparation and substrate solutions) are filtered before use. It is not clear what the other false spots are, although it is conceivable that they represent highly adher-
ent (and possibly also AP positive) cells. Indeed, activated B lymphocytes may well fit both the former and the latter (Garcia-Rozas et al., 1982) criteria, although no firm evidence in support of this possibility has been obtained. Consequently, the use of an inverted microscope is sometimes w a r r a n t e d and u n d e r as little as 10 x magnification, the typically granular ELISA plaques are readily distinguished (for example see Sedgwick and Holt, 1983a). During the course of these studies we became aware of considerable variation in the size of plaques, a feature which also did not go unnoticed in the studies by Czerkinsky et al. (1983). Subsequently we demonstrated that the plaque diameter increased in secondary when compared to primary responses and furthermore that the diameter of the plaques correlated with the affinity of serum antibody from the cell donors (Holt et al., 1984b). While further studies are clearly required to confirm this association it does nevertheless suggest that differences in the levels of antigen attached to the plates are not responsible as these effects would be expected to occur randomly.
Conclusion Since the initial report of the ELISA-plaque assay there has been a gradual accumulation of data employing the technique and it is now apparent that this system is applicable to a wide variety of experimental models, from the enumeration of ISC specific for defined protein antigens, to those secreting antibody against viruses and antigens relevant to autoimmune disease (see Table II). While the basic methodology involved in each of the reports listed in Table II remains the same, each system requires refinement to some extent and details of different assay conditions are given in Table I. In particular, it is our experience that the solid phase is invariably the component which limits the success of any one particular investigation and hence it is recommended that attention is given to optimizing this aspect of the ELISAplaque system. Whilst it is evident that this technique is sufficiently versatile to be employed in a number of different experimental systems for the detection of ISC, it may also be feasible to extend it to the
44
enumeration of cells, including T lymphocytes, secreting other biologically active molecules. These possibilities, and others, await investigation.
Acknowledgement The authors would like to thank Mrs. Valerie Boasten for typing the manuscript.
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Holt, P.G., J.D. Sedgwick, G.A. Stewart, C. O'Leary and K. Krska, 1984a, J. Immunol. Methods 74, 1. Holt, P.G., K.J. Cameron, G.A. Stewart, J.D. Sedgwick and K.J. Turner, 1984b, Clin. Immunol. lmmunopathol. 30, 159. Holt, P.G., J.D. Sedgwick, C. O'Leary, K. Krska and S. Leivers, 1984c, Cell. immunol. 89, 281. Jerne, N.K. and A.A. Nordin, 1963, Science 140, 405. Jerne, N.K., C. Henry, A.A. Nordin, H. Jufi, A.M.C. Koros and I. Lefkovits, 1974, Transplant. Rev. 18, 130. Kennedy, J.C. and M.A. Axelrad, 1971, Immunology 20, 253. Klinman, N.R. and R.B. Taylor, 1969, Clin. Exp. Immunol. 4, 473. Mason, D.W., 1976, J. lmmunol. Methods 10, 301. Moore, J. and C. Calkins, 1983, J. Immunol. Methods 63, 377. Moskophidis, D. and F. Lehmann-Grube, 1984, J. lmmunol. 133, 3366. Oliver, D.G., A.H. Sanders, R.D. Hogg and J.W. Hellman, 1981, J. Immunol. Methods 42. 195. Pick, E. and J.D. Feldman, 1967, Science 156, 964. Richter, M., M. Berry and N. Kazaniwsky, 1981, J. hnmunol. Methods 46, 77. Sedgwick, J.D. and P.G. Holt, 1983a, J. Exp. Med. 157, 2178. Sedgwick, J.D. and P.G. Holt. 1983b, J. Immunol. Methods 57, 301. Sedgwick, J.D. and P.G. Holt, 1985, Cell. Immunol. 94, t82. Tarkowski, A., C. Czerkinsky, L.-A. Nilsson, H. Nygren and O. Ouchterlony, 1984, J. lmmunol. Methods 72, 451. Voller, A., D. Bidwell and A. Bartlett, 1976, in: Manual of Clinical Microbiology, eds. N.R. Rose and H. Friedman (American Society for Microbiology, Washington, DC) p. 506. Wortis, H.H., D.H. Dresser and H.R. Anderson, 1969, Immunology 17. 93.