- Email: [email protected]
[28] Methods for binding cells to plastic: Application to solid phase immunoassays for cell-surface antigens
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sence of a compound; for instance, in the selection for metabolite overproducing plants or cell clones from microbial or plant cell cultures and the selection of plants devoid of an undesirable chemical character, such as a toxic or bitter principle. The assay is very precise (coefficients of variation are about twice as high as compared to the corresponding RIAs using the same reagents) and may therefore be used to obtain quantitative results as well, either by radioactivity determination in the supernatant fraction or by densitometry of the films. This latter mode is to be preferred in that it does not include the need for gamma-counting facilities and is not restricted by counting time. Acknowledgments Financial support for this investigation by the B u n d e s m i n i s t e r fiir F o r s c h u n g und Technologie, Bonn, is greatly appreciated. We would also like to thank Pablo S. Jourdan, Tampa, Florida, for critically reading the manuscript.
[28] M e t h o d s for B i n d i n g Cells to P l a s t i c : A p p l i c a t i o n to Solid P h a s e I m m u n o a s s a y s for C e l l - S u r f a c e A n t i g e n s
By C. H. HEUSSER, J. W. STOCKER, and R. H. GISLER Recent developments in cell fusion techniques allow the production of monoclonal antibodies specific for cell-surface antigens 1 and highlight the need for rapid and reproducible large-scale screening test systems to detect such antibodies in cell culture supernatants. Assays in which the binding of antibody is measured have an advantage over the test systems that rely on effector function of bound antibody (hemagglutination, cytotoxicity). Binding assays detect antibodies of all immunoglobulin (Ig) classes, and they can be applied to detect surface antigens of diverse cell types. A rather time-consuming part of conventional antibody-binding immunoassays is the centrifugation and resuspension of cells during the washing procedures. Stocker and Heusser2 as well as Kennett3 and
G. K6hler and C. Milstein, Nature (London) 265, 495 (1975). 2 j. W. Stocker and C. H. H e u s s e r , J. Immunol. Methods 26, 87 (1979). a R. H. Kennett, in " M o n o c l o n a l Antibodies. Hybridoma: A N e w Dimension in Biological A s s a y s " (R. H. K e n n e n , T. J. M c K e a r n , and K. B. Bechtol, eds.), p. 376. Plenum, N e w York, 1980.
METHODS IN ENZYMOLOGY,VOL. 73
Copyright © I981 by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181973-6
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Lansdorp et a l . 4 have described methods to attach cells to the bottom of microtiter plates forming stable cell monolayers suitable to detect antibodies against cell-surface antigens. Washing the cells simply involves dipping the plates into buffer and flicking the buffer into a sink. Compared with tests performed with cells in suspension, assays using attached cells have several advantages. First, the washing procedure is efficient and rapid. The 96 samples of a microtiter plate are washed four times within 20 sec. This fact alone enables one person to perform one thousand assays per day easily. Second, since cells do not need to be resuspended, mechanical disturbance is circumvented. This may explain the significantly better reproducibility with the solid-phase cell layer technique as compared to the suspension method. Moreover, improved reproducibility allows detection of low affinity and/or low quantities of antibodies that may be directed against a minor population of surface antigens. To attach cells to the plates, different methods can be used. Plates are either pretreated with a heterologous population of antibodies directed against cell-surface components or with lectins, such as phytohemagglutinin (PHA). Attached cells may be subsequently fixed with glutaraldehyde. On the other hand, fixation of cells with glutaraldehyde alone or in conjunction with poly-L-lysine coating of plates can be used further to prepare cell monolayers for solid-phase assays. Under all these conditions, cells remain firmly attached, and even if fixed by glutaraldehyde retain their specific surface antigen phenotype. Cell layers prepared in this way can be used for both radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA)P They yield specific and reproducible results. Finally, plates containing glutaraldehyde-fixed cells can be stored for several weeks without loss of cell membrane antigens, a significant advantage if cell types are to be studied that require time-consuming preparation procedures. Materials A s s a y Plates
Flexible polyvinyl chloride (PVC) fiat-bottom microtiter plates (M 29) from Cooke Engineering (distributed by Dynatech)
4 L. M. Lansdorp, F. Oosterhof, and M. Janssen,Protides Biol. Fluids, Proc. Colloq. 27, paper No. 52 (1980). 5 j. W. Stocker, F. Malavasi, and M. Trucco, in "ImmunologicalMethods" (I. Lefkovits and B. Pernis, eds.), Vol. 2, Chapter 18, p. 299. AcademicPress, New York, 1981.
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Buffer Solutions Phosphate-buffered saline (PBS): 8 g of NaC1, 1.44 g of NaH~PO4 • 2 H20, 0.2 g of KC1, 0.2 g of KH~PO4 dissolved in l liter of doubledistilled water. The solution was 0.15 M, and had a pH of 7.2. Radioimmunoassay buffer (RIA buffer): PBS, containing 1% bovine serum albumin (Pentex) and 0.2% sodium azide. For certain experiments, 1% normal rabbit serum was added to further quench protein-reactive sites on plates and to reduce nonspecific binding of antibodies to cells. This rabbit serum was absorbed on normal mouse Ig-Sepharose 4B columns prior to its use in order to eliminate possible contamination with anti-mouse Ig antibodies. Substrate buffer for the reaction with alkaline phosphatase: 20 mg of p-nitrophenyl phosphate (Merck, No. 6850) dissolved in 50 ml of 10% diethanolamine (Merck, No. 803116), pH 9.8, containing 2.5 mg of MgCI2 (Merck, No. 5833). Buffer is always freshly prepared just before use.
Reagents for Cell Attachment Glutaraldehyde (Sigma, No. G 5882), 25%, stored at 4° and diluted with cold PBS to obtain a 0.25% solution just prior to use Poly-L-lysine (PLL; Sigma, No. P 1886), dissolved in PBS to give a solution of 10/zg/ml Phytohemagglutinin-P (PHA) (Difco Laboratories), used at a concentration of 20/xg/ml in PBS
Antisera Rabbit anti-mouse lymphocyte globulin (ALG). This antiserum was made by injecting rabbits with thymocytes from outbred mice by the method of Levey and Medawar. n The serum was absorbed by passage through mouse IgG-Sepharose, 7 then, IgG was separated from the antiserum using protein A-Sepharose (Pharmacia, Uppsala). Rabbit antiserum to mouse immunoglobulins (RaMIg). Outbred rabbits were immunized with mouse IgG prepared from mouse serum by acid elution from staphylococcal protein A-coupled Sepharose. s The rabbits each received 0.5 mg of mouse IgG in Freund's complete adjuvant at multiple subcutaneous sites and were boosted 4 6 R. H. L e v e y and P. B. Medawar, Proc. Natl. Acad. Sci. U.S.A. 56, 1130 (1966). 7 J.-C. Jaton, D. C. Brandt, and P. Vassali, in " I m m u n o l o g i c a l M e t h o d s " (I. Lefkovits and B. Pernis, eds.), Vol. 1, p. 54. A c a d e m i c Press, N e w York, 1979. 8 j. W. Goding, J. lmmunol. Methods 13, 215 (1976).
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weeks later with 0.5 mg of mouse IgG in F r e u n d ' s incomplete adjuvant. Serum was taken 1 week after this boost and was heatinactivated at 56 ° for 30 min. lZSI-Labeled rabbit anti-mouse Ig (['25I]RaMIg) was p r e p a r e d according to H e r z e n b e r g and H e r z e n b e r g 9 while bound to m o u s e Igcoated Sepharose 4B beads in order to protect the combining sites. ['2~I]RaMIg molecules were then eluted with 0.2 M glycine-HCl buffer at p H 2.3, which contained carrier protein, and were neutralized with borate buffer at p H 8.6. Alternatively, R a M I g was purified by affinity c h r o m a t o g r a p h y and radioiodinated in solution according to the method described by Hunter. '° Alkaline phosphatase-coupled RaMIg. Alkaline phosphatase was conjugated to R a M I g according to K e a r n e y et al." using 4.5 mg of alkaline phosphatase (Boehringer, 5 mg/ml solution), 1.5 mg of purified rabbit IgG anti-mouse Ig, and glutaraldehyde as a crosslinking reagent. The IgG antibody preparation was either purified by affinity c h r o m a t o g r a p h y on mouse Ig-coated Sepharose r or represented the isolated IgG fraction from a protein A-Sepharose column (Pharmacia, Uppsala). Technique of Solid-Phase I m m u n o a s s a y for Cell Surface Antigens
Methods for Immobilizing Cells Pretreatment possibilities for attaching cells to plastic surfaces are s u m m a r i z e d and evaluated in Table I. Pretreatment of Plates. The principle of coating plates with various substances is based on the property of plastic surfaces to absorb in a nonspecific w a y proteins and other reagents (Table I). One hundred microliters of coating reagent diluted in PBS are added to each well, and the plates are incubated for 1 hr at 37 °. The following concentrations result in optimal coating: poly-L-lysine, 10 /zg/ml; antilymphocyte globulin, 65 /zg/ml; phytohemagglutinin-P, 20/xg/ml. In order to wash the plates, they are i m m e r s e d into PBS, and the liquid is then r e m o v e d by flicking them. Preparation of Cell Monolayers. Cells are washed three times in PBS, and leukocytes are resuspended at a concentration of 107 cells/ml. Cul9 L. A. Herzenberg and L. A. Herzenberg, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), 3rd ed., Vol. 1, Chapter 12. Blackwell, Oxford, 1978. ,0 W. M. Hunter, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), 3rd ed., Vol. 1, Chapter 14.1. Blackwell, Oxford, 1978. 1, j. F. Kearney, A. Radbruch, B. Liesegang, and K. Rajewsky, J. lmmunol. 123, 1548 (1979).
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ANTIGEN
AND
ANTIBODY
DETECTION
BY IMMUNOASSAY
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TABLE 1 METHODS FOR IMMOBILIZING CELLS ON PLASTIC PLATES
Pretreatment of plates None a Poly-L-lysine Antibody b Antibody 0 Lectin c Lectin c
F i x a t i o n o f cell m o n o l a y e r by glutaraldehyde + + + + -
Stability of attachment + + + + + +
+ + + + + +
+/+ a + + + + +
Possibility of storage + + + + -
Not suitable for large cells or cells derived from tissue culture. b Rabbit anti-mouse lymphocyte globulin (ALG). c Phytohemagglutinin.
tured cell lines are diluted to 1 to 5 × 106 cells/ml (according to the size of the cells). Erythrocytes are used as a 0.1% suspension. One hundred microliters of the cell suspension are added per well using Titertek multichannel pipettes (Dynatech). Control wells without cells provide a measure for nonspecific binding of antibodies and/or developing probes to plates. The plates are centrifuged at 100g for 3 min. When glutaraldehyde fixation is required, the plates are then gently immersed in a l-liter glass beaker containing freshly prepared 0.25% glutaraldehyde (Merck, Darmstadt, G.F.R.) solution in PBS at 4° for 5 rain. The immersion has to be performed carefully to prevent disturbance of the cell layer and air trapping in the wells. Plates are then repeatedly washed by gently flicking them over a sink and reimmersion into PBS. Saturation of Protein-Reactive Sites. The plates are dipped into a beaker containing PBS with 1% bovine serum albumin and 0.2% sodium azide (RIA buffer). They are kept in this solution for 1 hr at room temperature to saturate protein-binding sites on the plastic. Under some circum:stances (see Discussion), 1% normal rabbit serum, 100 mM glycine, or 0.05% Tween 20 is added to the RIA buffer. After this incubation, the plates are either used immediately or stored at 4° without changing the buffer. In the case of PHA-coated plates, care should be taken to block the remaining PHA reactive sites. This is best achieved by adding buffer containing egg white (final concentration corresponds to OD280 of 14). Egg white buffer is prepared by stirring chicken egg white in PBS for 1 hr in the presence of 0.1% sodium azide. This is followed by a centrifugation at 10,000 g for 15 rain.
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Immltnoassays Binding of Antibody to Cells. Plates are washed once with PBS just before the addition of 50/zl of test antiserum or cell culture supernatant containing monoclonal antibodies. All dilutions of test samples are made in RIA buffer. The reaction is allowed to proceed for 1 hr at room temperature. The plates are then washed three times as described before. Development of Cell-Bound Antibody with 12~l-Labeled Anti-~. Fifty microliters of appropriately diluted [125I]RaMIg in RIA buffer are added to each well, and the plates are kept at room temperature for 1 hr. Unbound radioactive antibody is removed by washing as described above, except that the bulk of radioactivity is first removed. Three drops of PBS are added to each well, and the solution is sucked away or absorbed by paper towels backed by aluminum foil. After four wash cycles, the trays are allowed to dry and then are cut with a heated wire. During cutting, the trays are supported by a rigid, polystyrene Microtiter plate (Cooke Engineering Co., California, No. 220M-25 AR) with its border trimmed to hold the assay tray. This subsequently acts as a holder for the individual cut wells, which are then transferred to tubes for counting radioactivity. A "plate-cutter-distributor" device, which cuts and fills wells into counter racks, will soon be available from Biotec (Basel, Switzerland). Development of Cell-Bound Antibody with Enzyme-Labeled Anti-lg (ELISA). Fifty microliters of appropriately diluted alkaline phosphataselinked RaMIg in RIA buffer are added per well, and the plates are kept for 1 hr at room temperature. After three consecutive washings with PBS, 100 /zl of substrate buffer (containing p-nitrophenyl phosphate) are added to each well, and the colorimetric reaction is determined 30 min later, after incubation in the dark, either visually or in a vertical light path photometer (Titertek Multiscan, Flow Laboratories Ltd.). If delayed reading is desired, the reaction may be stopped by adding 50/~1 of 3 M NaOH.
Examples and Comments
Production of a Confluent Cell Monolayer. Optimal signals of antibody binding are obtained if cells form a confluent monolayer. The number of cells required depends obviously on the size of the cells used and can easily be checked under the microscope. Addition of 1 × 106 mouse spleen cells per well is optimal to establish monolayers as determined microscopically and through binding of [12~I]RaMIg.2 If lower cell numbers are used, the signals of label-bound antibodies will be proportionally smaller. In contrast, increasing the number of cells in each well leads to a decrease of
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T A B L E II COMPARISON OF BINDING ASSAY USING CELLS ATTACHED TO PLATES VERSUS CELLS IN SUSPENSION a-c Type o f a s s a y ~
T h y m o c y t e strain
Plate fixed (cpm)
Susp. fixed (cpm)
Susp. fresh (cpm)
DBA/2 C57BL/6 N o cells
3544 -+ 353 581 - 62 265 -+ 43
1264 _+ 98 380 -+ 111 333 -+ 38
1510 -+ 394 838 --- 196 299 - 69
a F r o m Stocker and Heusser. 2 b In the absence o f antiserum, the three types of assay gave the following nonspecific uptake: plate fixed, 160 -+ 31; suspension fixed, 117 -+ 11 ; suspension fresh, 526 --- 376. T h e s e values were similar for the two t h y m o c y t e donor strains. c Binding of anti-DBA/2 (1 : 100) to DBA/2 or C57BL/6 t h y m o c y t e s was m e a s u r e d in three different a s s a y s y s t e m s . U p t a k e o f anti-DBA/2 was determined by [~25I]RaMIg. n T h e three a s s a y s y s t e m s were: cell layer attached to the plate using glutaraldehyde method (Plate fixed); glutaraldehyde-fixed cells in suspension (Susp. fixed); and unfixed cells in suspension (Susp. fresh). E a c h value is the m e a n of 4 - 8 replicate a s s a y s _+SEM.
reproducibility, presumably because cells not directly bound to plates are lost during the washing procedure. Specificity of Signals with Respect to Solid-Phase versus Liquid-Phase Immunoassay. The binding of antibody to cells attached to plates was compared with the binding obtained with cells in suspension. Cells in suspension were either fresh or fixed by glutaraldehyde in suspension, 9'1z and attachment of cells was done by glutaraldehyde alone. The binding of anti-DBA/2 alloantisera to DBA/2 and C57B 1/6 thymocytes was investigated by development with [12'~I]RaMIg. Thymocytes were chosen in order to provide a cell population with few surface Ig-bearing cells so that direct binding of [125I]RaMIg to the cells would be minimized. As demonstrated in Table II, the plastic-bound cells exhibited a higher binding signal than did fixed or unfixed cells in suspension. As the same number of cells ( 10~) was used for each type of assay, this result suggested that the method using attached cells allows retention of more cells than assays done in suspension. The specific signal as judged by the difference between binding to relevant and irrelevant target cells was most pronounced for cells attached to the plate. Furthermore, cells attached to the plate by glutaraldehyde and stored at 4° in RIA buffer for 7 weeks before 12 M. Letarte-Muirhead, R. Acton, and A. F. Williams, Biochem. J. 143, 51 (1974).
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IMMUNOASSAY
FOR CELL ANTIGENS
413
T A B L E III STABILITY OF CELL MONOI.AYERS: EFFECT OF WASHING PROCEDURES REQUIRED IN IMMUNOASSAYS ~25I-Labeled c e l l s a t t a c h e d a (cpm) Washing cycles *
Plate coating b ALG PHA PLL
1
3
4
9717_+ 118 10142 +- 45 9882 _+ 92
10238 +_ 61 10641 _+ 84 9713 +_ 114
9184 _+ 126 10060 _+ 43 9041 _+ 80
106 mouse t25I-labeled thymocytes were added on pretreated plates and subsequently fixed by glutaraldehyde. Values are mean of four replicate assays -+SEM. b Plates were coated with anti-lymphocyte globulin (ALG), phytohemagglutinin (PHA), or poly-L-lysine(PLL). c Cell monolayers were washed four times per cycle.
assay showed similar binding capacity to that of plates prepared on the day o f assay (data not shown). Stability of Cell Monolayers. Stability of the cell monolayers, produced by various means of attachment, was directly followed by using radiolabeled cells.12~I-Labeled thymocytes were centrifuged on ALG-, PHA-, or PLL-pretreated plates, respectively, and fixed by glutaraldehyde. After fixation, plates were passed through various washing cycles, each corresponding to four washing steps. One cycle would correspond to the cell layer at the beginning of an immunoassay, and three cycles would correspond to the end of a normal immunoassay in which a bound antibody is determined by a labeled second antibody. As can be seen in Table III, the cell attachment procedures result in firmly stable cell monolayers with a high degree of reproducibility.
Detection of Monoclonal Antibodies in Culture Supernatants Using Attached Cells. Culture supernatants from hybridomas producing monoclonal mouse antibodies were tested for binding to cells attached by glutaraldehyde alone. These antibodies, of known Ig subclass, have specificity for defined cell-surface antigens. Cell layers of human peripheral blood lymphocytes (PBL), and SRBC were used. Fifty microliters of supernatant media from hybrid cell cultures were added, and binding to the two cell types was assayed. Table IV shows that the antibodies could be detected on the appropriate target cells. In the case of N 5/1, an antibody directed against a human B-cell surface antigen, the binding was lower by a factor of 5 than binding of anti-fl2-microglobulin. This corresponds to the prevalence of B cells among PBL.
Comparison of Antibody Binding to Cells Attached by Different Means.
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TABLE IV SPECIFIC BINDING OF MONOCLONAL ANTIBODY TO CELL MONOLAYERS a'b Monoclonal antibody Designation N 34 N 26/114 N 5/I Sp 2/HL Hy 2-19 X63 Buffer
Binding to cell monolayer c
Specificity
H u m a n PBL
H L A complex Human /32microglobulin human B cell antigen SRBC TNP Unknown
SRBC
No cells
4279 ± 164 5416 ± 593
201 ± 166 ±
34 12
285 ± 21 356 ± 13
1150 ±
133 ±
17
182 ± 13
3596 ± 526 383 ± 33 129 ± 8 1 5 0 ± 46
264 ___ 22 672 ± 35 142___ 8 124± 9
323 465 264 180
± ± ± ±
8 12 53 14 8
" From Stocker and Heusser. 2 b Monoclonal mouse antibodies of various specificities were assayed for binding to cell layers of human peripheral lymphocytes (PBL) or sheep erythrocytes (SRBC) attached to plates using glutaraldehyde. Binding Of the monoclonal antibodies was detected by [l~5I]RaMIg. c Values as means of four replicate assays ± S E M .
TABLE V SPECIFICITY OF BINDING OF AN ANTI-H-2 MONOCLONAL A N T I B O D Y e TO CELLS IMMOBILIZED BY DIFFERENT MEANS Method of attachment b Cells
Attached"
AJJ IBALB/c AKR B 10A C57BL ]3W 5147 EL-4 No cell
Spleen Spleen Spleen Spleen Spleen ----
ALG + GLU 619 ± 588 ± 595 ±
555 14329 592 15207 688
± ± ± ± ±
186 11 13 62 508 102 1021 25
GLU 787 914 692 707 15987 595 15075 666
± ± ± ± --± ± ±
ALG 81 55 38 5 188 57 540 26
549 498 560 540 18486 789 16923 880
± 3 ± 5 ± 29 ± 20 ± 1146 ± 88 ± 1031 ± 26
H-2K b + + -
a 8 × 105 spleen cells or 2.5 × 105 cells from in vitro propagated cell lines were added per well and centrifuged onto the plastic surface. b Cells were centrifuged onto plates and allowed to react with anti-lymphocyte globulincoated wells for 5 min (ALG) and/or fixed with 0.25% glutaraldehyde for 5 rain at 4 ° (GLU). After washing, wells were quenched with RIA buffer as indicated. c B-8, a monoclonal IgG1 anti-H-2K b antibody was isolated and iodinated; 130,000 cpm were added per well and allowed to react for 1 hr at 22 °. d Values ± maximum deviations from the mean (cpm); N = 2.
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415
The binding of an isolated and radioiodinated monoclonal antibody directed against H-2K b~3 to cells attached by different means was compared. Spleen cells from different mouse strains and cells from two in vitro cell lines were immobilized on plates by glutaraldehyde fixation, by pretreating the plates with ALG, or by a combination of both. The experiment demonstrates (Table V) that a high specific signal can be obtained with either method of cell attachment. Since there is no development step involved in this experiment, the signal-to-noise ratio is a direct measure of specific binding. It is evident from the data that cells lacking H-2K b surface components show no significant binding compared to control wells without cells. This demonstrates that nonspecific binding is minimal. Since signals obtained with the three types of attachment procedures are similar, we conclude furthermore that glutaraldehyde treatment does not alter the epitope recognized by the anti-H-2K b antibody. Detection o f Monoclonal Antibody Binding to Cells Immobilized on PHA-Pretreated Plates and Using an Enzyme-Labeled Probe for the Immunoassay. In this example, taken from Stocker et al., ~ cells from two human cell lines were attached on PHA-coated plates and subsequently fixed by glutaraldehyde. Monolayers were incubated with various culture supernatants containing monoclonal antibody of indicated specificity (Table VI). The binding of antibodies was then revealed by alkaline phosphatase-labeled RaMIg followed by the enzyme-induced color reaction. As demonstrated, significant binding was obtained only where specific interaction of antibody and corresponding antigenic determinants was expected. The example demonstrates, furthermore, the use of an enzymelinked development step in an antibody-binding assay for cells immobilized on a plastic surface. Discussion In this paper, we described different possibilities for attaching cells to plastic so that they can be used in a solid-phase radioimmunoassay for cell-surface antigens. Advantage is taken of the property of plastic to bind protein by nonspecific absorption. This property is used to form anchors for cell attachment or to coat wells with reagents that actively bind to cells, thus functioning as cross-linking molecules. Glutaraldehyde treatment of cells spun onto untreated plates will remain attached on the plastic surface. la G. K6hler, F. Fischer-Lindahl,and C. H. Heusser,in "The Immune System" (C. Steinberg and I. Lefkovits,eds.). Karger, Basel (in press).
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ANTIGEN AND ANTIBODY DETECTION BY IMMUNOASSAY
[28]
TABLE VI ENZYME-LINKED IMMUNOSORBENT ASSAY USING CELLS
ATTACHEDTO PHA-CoATED PLATESa Cells a t t a c h e d Culture supernatant° Medium alone X 63 Anti-/32 m Anti-HLA ABC Anti-DR Anti-DR 3 + 5 + 6
c'a
Daudi
WT 18
No cells
0 0 0 0 5 5
0 1 6 5 7 2
0 1 1 1 1 1
" From Stocker e t al. 5 b The specificity of the antibodies produced by X 63 cells is unknown (negative control). c The Daudi cell line is a Burkitt lymphoma line that lacks chromosome 15, which is responsible for/32 m expression and as a consequence lacks HLA A, B, and C surface antigens as well. It still expresses D determinants. The WT 18 cell line is Epstein-Barr virus transformed (HLA l~omozygous donors). Daudi and WT 18 cells were centrifuged onto PHA-P pretreated plates and fixed by glutaraldehyde. Remaining reactive sites on plates, glutaraldehyde, and lectin were saturated with radioimmunoassay buffer containing egg white. a Reading was done with a vertical light path photometer (Titertek Multiskan, Flow Laboratories Ltd.). The machine was set to express the measured absorbance as numbers (from 1 to 10) corresponding to multiples of 0 . 3 0 D (matrix mode). H o w e v e r , if cells are c e n t r i f u g e d o n t o p o l y - L - l y s i n e - p r e t r e a t e d plates a n d g l u t a r a l d e h y d e - t r e a t e d t h e r e a f t e r , the r e s u l t i n g m o n o l a y e r s are m o r e stable. A l t e r n a t i v e l y , cells can be b o u n d to a c t i v a t e d s u r f a c e s b y c o a t i n g the plates with a n t i b o d i e s d i r e c t e d a g a i n s t c e l l - s u r f a c e c o m p o n e n t s or with lectins, s u c h as P H A , that r e a c t w i t h c e r t a i n c e l l - s u r f a c e c a r b o h y d r a t e s . U n d e r t h e s e c o n d i t i o n s , the i m m o b i l i z e d cells f o r m stable m o n o l a y e r s w i t h o u t s u b s e q u e n t g l u t a r a l d e h y d e fixation. S u c h cell l a y e r s m a y be u s e d to e x c l u d e p o s s i b l e m o d i f i c a t i o n o f the a n t i g e n s i n d u c e d b y g l u t a r a l d e h y d e fixation. It has to be s t r e s s e d , h o w e v e r , that we n e v e r o b s e r v e d a n y loss or c h a n g e s in specificity o f surface a n t i g e n s due to g l u t a r a l d e h y d e fixation. This w a s m o s t c l e a r l y d e m o n s t r a t e d in c a s e s o f m o n o c l o n a l a n t i b o d i e s that are e x p e c t e d to r e a c t with a single a n t i g e n i c d e t e r m i n a n t . So far, all a n t i b o d i e s that had b e e n o r i g i n a l l y s e l e c t e d for b i n d i n g to u n f i x e d cells, b o u n d to fixed cells as well.
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417
Some of the anchoring methods described have intrinsic disadvantages under some assay conditions. Coating the plate with antibodies requires the preparation of the IgG fraction of a specific antiserum, which may contain added specificities that interfere with the test. Furthermore, radioiodinated or enzyme-linked protein A cannot be used as a developing reagent because it binds not only to antibodies of the cell culture supernatant to be analyzed, but also to antibodies used to coat the plates. The coating by lectins, on the other hand, requires special quenching procedures to saturate the sugar-reactive sites of the lectin, which in the case of PHA was achieved by adding egg-white glycoproteins. Poly-Llysine as a coating reagent forms anchors for the subsequent glutaraldehyde fixation and allows for the preparation of stable monolayers suitable for the purpose of an immunoassay. This method has the advantage of simplicity and applicability to many different cell types, and it now represents a widely used method for cell immobilization. 3 Immobilization of cells by glutaraldehyde alone is a rapid and convenient method for preparing cell layers for many types of cells 2 and has been used successfully for parasites as well. TM It results in stable monolayers suitable for storage at 4°. Where cells from tissue culture are to be attached, one of the anchor methods should be chosen, and this may be combined with glutaraldehyde fixation as described. One problem of cell binding assays in general is the nonspecific binding of antibodies to reactive sites on the plate. The performance of the assay in 1% BSA eliminates most of the problem. The addition of 100 mM glycine will further help to neutralize remaining glutaraldehyde reactive sites. The addition of 1% heterologous serum or 0.05% Tween 20 prior to and during the incubation with the test sample leads to further reduction of nonspecific binding. For general purposes, we recommend the use of glutaraldehyde with the additional use of poly-L-lysine for cell types where this is found necessary. In any case, cell attachment should occasionally be checked microscopically at the end of an immunoassay procedure. Unsuccessful cell attachment will result in visible holes in the cell layer. The binding of antibodies to a cell layer can be detected either by radioactively labeled or by enzyme-labeled anti-immunoglobulins. Several examples have been presented in this paper. Labeled staphylococcal protein A may be used, either to replace the anti-immunoglobulin or as a second indirect label for maximal amplification. The use of radiolabeled probes involves time-consuming procedures such as cutting the plate and putting the wells into the gamma counter. This step can be automated with ~4E. Handman and J. S. Remington,Immunology 40, 597 (1980).
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[29]
a "plate-cutter-separator" device (Biotech, Basel). Alternatively, a semiquantitative assessment of positive wells is also possible by autoradiography of the whole plate. 15"16On the other hand, enzyme-linked detection methods offer the advantage of convenience, safety, and speed. T M Furthermore, enzyme-linked probes are more stable than radioiodinated probes and need no expensive equipment. It is shown that optical densities of the enzyme reaction can actually be determined with a vertical photometer across the cell monolayers. Alkaline phosphatases (Table V1), ~ and horseradish peroxidase 3"4have been used successfully in a cell binding assay with mouse lymphocyte monolayers. However, ELISA techniques are limited by the fact that certain cell types express enzymatic activities themselves. The method for detection of antibodies binding to cells immobilized on solid phase will be extremely helpful when large numbers of samples need to be rapidly screened. It has been successfully employed in extended hybridization experiments in which many culture supernatants had to be assayed for possible antibody activity against cell-surface antigens. The method offers speed, reproducibility, and the possibility of preparing plates in advance and storing them until use. ,5 j. p. Brown, J. D. Tamerius, and I. Hellstr6m, J. Immunol. Methods 31,201 (1979). i6 R. M. Parkhouse and G. Garnotta, Cur. Top. Microbiol. lmmunol. 81, 142 (1978). 17 E. Engvall and P. Perlmann, Immunochemistry 8, 871 (1971). is A. H. Schuurs and B. K. Van Weemen, Clin. Chim. Acta 81, I (1977).
[29] M i c r o p r o c e d u r e s f o r Q u a n t i t a t i v e I m m u n o c h e m i c a l Analysis of Antigenic Molecules and Antigenic Determinants 1
By KAZIMIERZ MALINOWSKI and WLADYSLAW MANSKI The quantitative determination of antigens and antibodies, first developed by Heidelberger and Kendall, TM is at the foundation of molecular immunology. The theoretical basis of the antigen-antibody reaction has been reviewed by Kabat 2 and by Day. 3 The sensitivity and accuracy of 1 This work was supported by U.S. Public Health Service Research Grants EY 00189 and EY 00198. la M. Heidelberger and F. E. Kendall, J. Exp. Med. 50, 808 (1929). 2 I. Kabat, "Structural Concept in Immunology and l m m u n o c h e m i s t r y . " Holt, N e w York, 1976. 3 E. D. Day, " A d v a n c e d I m m u n o c h e m i s t r y . " Williams & Wilkins, Baltimore, Maryland, 1972.
METHODS IN ENZYMOLOGY,VOL. 73
Copyright @ 1981 by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181973-6
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sence of a compound; for instance, in the selection for metabolite overproducing plants or cell clones from microbial or plant cell cultures and the selection of plants devoid of an undesirable chemical character, such as a toxic or bitter principle. The assay is very precise (coefficients of variation are about twice as high as compared to the corresponding RIAs using the same reagents) and may therefore be used to obtain quantitative results as well, either by radioactivity determination in the supernatant fraction or by densitometry of the films. This latter mode is to be preferred in that it does not include the need for gamma-counting facilities and is not restricted by counting time. Acknowledgments Financial support for this investigation by the B u n d e s m i n i s t e r fiir F o r s c h u n g und Technologie, Bonn, is greatly appreciated. We would also like to thank Pablo S. Jourdan, Tampa, Florida, for critically reading the manuscript.
[28] M e t h o d s for B i n d i n g Cells to P l a s t i c : A p p l i c a t i o n to Solid P h a s e I m m u n o a s s a y s for C e l l - S u r f a c e A n t i g e n s
By C. H. HEUSSER, J. W. STOCKER, and R. H. GISLER Recent developments in cell fusion techniques allow the production of monoclonal antibodies specific for cell-surface antigens 1 and highlight the need for rapid and reproducible large-scale screening test systems to detect such antibodies in cell culture supernatants. Assays in which the binding of antibody is measured have an advantage over the test systems that rely on effector function of bound antibody (hemagglutination, cytotoxicity). Binding assays detect antibodies of all immunoglobulin (Ig) classes, and they can be applied to detect surface antigens of diverse cell types. A rather time-consuming part of conventional antibody-binding immunoassays is the centrifugation and resuspension of cells during the washing procedures. Stocker and Heusser2 as well as Kennett3 and
G. K6hler and C. Milstein, Nature (London) 265, 495 (1975). 2 j. W. Stocker and C. H. H e u s s e r , J. Immunol. Methods 26, 87 (1979). a R. H. Kennett, in " M o n o c l o n a l Antibodies. Hybridoma: A N e w Dimension in Biological A s s a y s " (R. H. K e n n e n , T. J. M c K e a r n , and K. B. Bechtol, eds.), p. 376. Plenum, N e w York, 1980.
METHODS IN ENZYMOLOGY,VOL. 73
Copyright © I981 by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181973-6
[28]
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407
Lansdorp et a l . 4 have described methods to attach cells to the bottom of microtiter plates forming stable cell monolayers suitable to detect antibodies against cell-surface antigens. Washing the cells simply involves dipping the plates into buffer and flicking the buffer into a sink. Compared with tests performed with cells in suspension, assays using attached cells have several advantages. First, the washing procedure is efficient and rapid. The 96 samples of a microtiter plate are washed four times within 20 sec. This fact alone enables one person to perform one thousand assays per day easily. Second, since cells do not need to be resuspended, mechanical disturbance is circumvented. This may explain the significantly better reproducibility with the solid-phase cell layer technique as compared to the suspension method. Moreover, improved reproducibility allows detection of low affinity and/or low quantities of antibodies that may be directed against a minor population of surface antigens. To attach cells to the plates, different methods can be used. Plates are either pretreated with a heterologous population of antibodies directed against cell-surface components or with lectins, such as phytohemagglutinin (PHA). Attached cells may be subsequently fixed with glutaraldehyde. On the other hand, fixation of cells with glutaraldehyde alone or in conjunction with poly-L-lysine coating of plates can be used further to prepare cell monolayers for solid-phase assays. Under all these conditions, cells remain firmly attached, and even if fixed by glutaraldehyde retain their specific surface antigen phenotype. Cell layers prepared in this way can be used for both radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA)P They yield specific and reproducible results. Finally, plates containing glutaraldehyde-fixed cells can be stored for several weeks without loss of cell membrane antigens, a significant advantage if cell types are to be studied that require time-consuming preparation procedures. Materials A s s a y Plates
Flexible polyvinyl chloride (PVC) fiat-bottom microtiter plates (M 29) from Cooke Engineering (distributed by Dynatech)
4 L. M. Lansdorp, F. Oosterhof, and M. Janssen,Protides Biol. Fluids, Proc. Colloq. 27, paper No. 52 (1980). 5 j. W. Stocker, F. Malavasi, and M. Trucco, in "ImmunologicalMethods" (I. Lefkovits and B. Pernis, eds.), Vol. 2, Chapter 18, p. 299. AcademicPress, New York, 1981.
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Buffer Solutions Phosphate-buffered saline (PBS): 8 g of NaC1, 1.44 g of NaH~PO4 • 2 H20, 0.2 g of KC1, 0.2 g of KH~PO4 dissolved in l liter of doubledistilled water. The solution was 0.15 M, and had a pH of 7.2. Radioimmunoassay buffer (RIA buffer): PBS, containing 1% bovine serum albumin (Pentex) and 0.2% sodium azide. For certain experiments, 1% normal rabbit serum was added to further quench protein-reactive sites on plates and to reduce nonspecific binding of antibodies to cells. This rabbit serum was absorbed on normal mouse Ig-Sepharose 4B columns prior to its use in order to eliminate possible contamination with anti-mouse Ig antibodies. Substrate buffer for the reaction with alkaline phosphatase: 20 mg of p-nitrophenyl phosphate (Merck, No. 6850) dissolved in 50 ml of 10% diethanolamine (Merck, No. 803116), pH 9.8, containing 2.5 mg of MgCI2 (Merck, No. 5833). Buffer is always freshly prepared just before use.
Reagents for Cell Attachment Glutaraldehyde (Sigma, No. G 5882), 25%, stored at 4° and diluted with cold PBS to obtain a 0.25% solution just prior to use Poly-L-lysine (PLL; Sigma, No. P 1886), dissolved in PBS to give a solution of 10/zg/ml Phytohemagglutinin-P (PHA) (Difco Laboratories), used at a concentration of 20/xg/ml in PBS
Antisera Rabbit anti-mouse lymphocyte globulin (ALG). This antiserum was made by injecting rabbits with thymocytes from outbred mice by the method of Levey and Medawar. n The serum was absorbed by passage through mouse IgG-Sepharose, 7 then, IgG was separated from the antiserum using protein A-Sepharose (Pharmacia, Uppsala). Rabbit antiserum to mouse immunoglobulins (RaMIg). Outbred rabbits were immunized with mouse IgG prepared from mouse serum by acid elution from staphylococcal protein A-coupled Sepharose. s The rabbits each received 0.5 mg of mouse IgG in Freund's complete adjuvant at multiple subcutaneous sites and were boosted 4 6 R. H. L e v e y and P. B. Medawar, Proc. Natl. Acad. Sci. U.S.A. 56, 1130 (1966). 7 J.-C. Jaton, D. C. Brandt, and P. Vassali, in " I m m u n o l o g i c a l M e t h o d s " (I. Lefkovits and B. Pernis, eds.), Vol. 1, p. 54. A c a d e m i c Press, N e w York, 1979. 8 j. W. Goding, J. lmmunol. Methods 13, 215 (1976).
[28]
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409
weeks later with 0.5 mg of mouse IgG in F r e u n d ' s incomplete adjuvant. Serum was taken 1 week after this boost and was heatinactivated at 56 ° for 30 min. lZSI-Labeled rabbit anti-mouse Ig (['25I]RaMIg) was p r e p a r e d according to H e r z e n b e r g and H e r z e n b e r g 9 while bound to m o u s e Igcoated Sepharose 4B beads in order to protect the combining sites. ['2~I]RaMIg molecules were then eluted with 0.2 M glycine-HCl buffer at p H 2.3, which contained carrier protein, and were neutralized with borate buffer at p H 8.6. Alternatively, R a M I g was purified by affinity c h r o m a t o g r a p h y and radioiodinated in solution according to the method described by Hunter. '° Alkaline phosphatase-coupled RaMIg. Alkaline phosphatase was conjugated to R a M I g according to K e a r n e y et al." using 4.5 mg of alkaline phosphatase (Boehringer, 5 mg/ml solution), 1.5 mg of purified rabbit IgG anti-mouse Ig, and glutaraldehyde as a crosslinking reagent. The IgG antibody preparation was either purified by affinity c h r o m a t o g r a p h y on mouse Ig-coated Sepharose r or represented the isolated IgG fraction from a protein A-Sepharose column (Pharmacia, Uppsala). Technique of Solid-Phase I m m u n o a s s a y for Cell Surface Antigens
Methods for Immobilizing Cells Pretreatment possibilities for attaching cells to plastic surfaces are s u m m a r i z e d and evaluated in Table I. Pretreatment of Plates. The principle of coating plates with various substances is based on the property of plastic surfaces to absorb in a nonspecific w a y proteins and other reagents (Table I). One hundred microliters of coating reagent diluted in PBS are added to each well, and the plates are incubated for 1 hr at 37 °. The following concentrations result in optimal coating: poly-L-lysine, 10 /zg/ml; antilymphocyte globulin, 65 /zg/ml; phytohemagglutinin-P, 20/xg/ml. In order to wash the plates, they are i m m e r s e d into PBS, and the liquid is then r e m o v e d by flicking them. Preparation of Cell Monolayers. Cells are washed three times in PBS, and leukocytes are resuspended at a concentration of 107 cells/ml. Cul9 L. A. Herzenberg and L. A. Herzenberg, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), 3rd ed., Vol. 1, Chapter 12. Blackwell, Oxford, 1978. ,0 W. M. Hunter, in "Handbook of Experimental Immunology" (D. M. Weir, ed.), 3rd ed., Vol. 1, Chapter 14.1. Blackwell, Oxford, 1978. 1, j. F. Kearney, A. Radbruch, B. Liesegang, and K. Rajewsky, J. lmmunol. 123, 1548 (1979).
410
ANTIGEN
AND
ANTIBODY
DETECTION
BY IMMUNOASSAY
[28]
TABLE 1 METHODS FOR IMMOBILIZING CELLS ON PLASTIC PLATES
Pretreatment of plates None a Poly-L-lysine Antibody b Antibody 0 Lectin c Lectin c
F i x a t i o n o f cell m o n o l a y e r by glutaraldehyde + + + + -
Stability of attachment + + + + + +
+ + + + + +
+/+ a + + + + +
Possibility of storage + + + + -
Not suitable for large cells or cells derived from tissue culture. b Rabbit anti-mouse lymphocyte globulin (ALG). c Phytohemagglutinin.
tured cell lines are diluted to 1 to 5 × 106 cells/ml (according to the size of the cells). Erythrocytes are used as a 0.1% suspension. One hundred microliters of the cell suspension are added per well using Titertek multichannel pipettes (Dynatech). Control wells without cells provide a measure for nonspecific binding of antibodies and/or developing probes to plates. The plates are centrifuged at 100g for 3 min. When glutaraldehyde fixation is required, the plates are then gently immersed in a l-liter glass beaker containing freshly prepared 0.25% glutaraldehyde (Merck, Darmstadt, G.F.R.) solution in PBS at 4° for 5 rain. The immersion has to be performed carefully to prevent disturbance of the cell layer and air trapping in the wells. Plates are then repeatedly washed by gently flicking them over a sink and reimmersion into PBS. Saturation of Protein-Reactive Sites. The plates are dipped into a beaker containing PBS with 1% bovine serum albumin and 0.2% sodium azide (RIA buffer). They are kept in this solution for 1 hr at room temperature to saturate protein-binding sites on the plastic. Under some circum:stances (see Discussion), 1% normal rabbit serum, 100 mM glycine, or 0.05% Tween 20 is added to the RIA buffer. After this incubation, the plates are either used immediately or stored at 4° without changing the buffer. In the case of PHA-coated plates, care should be taken to block the remaining PHA reactive sites. This is best achieved by adding buffer containing egg white (final concentration corresponds to OD280 of 14). Egg white buffer is prepared by stirring chicken egg white in PBS for 1 hr in the presence of 0.1% sodium azide. This is followed by a centrifugation at 10,000 g for 15 rain.
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411
Immltnoassays Binding of Antibody to Cells. Plates are washed once with PBS just before the addition of 50/zl of test antiserum or cell culture supernatant containing monoclonal antibodies. All dilutions of test samples are made in RIA buffer. The reaction is allowed to proceed for 1 hr at room temperature. The plates are then washed three times as described before. Development of Cell-Bound Antibody with 12~l-Labeled Anti-~. Fifty microliters of appropriately diluted [125I]RaMIg in RIA buffer are added to each well, and the plates are kept at room temperature for 1 hr. Unbound radioactive antibody is removed by washing as described above, except that the bulk of radioactivity is first removed. Three drops of PBS are added to each well, and the solution is sucked away or absorbed by paper towels backed by aluminum foil. After four wash cycles, the trays are allowed to dry and then are cut with a heated wire. During cutting, the trays are supported by a rigid, polystyrene Microtiter plate (Cooke Engineering Co., California, No. 220M-25 AR) with its border trimmed to hold the assay tray. This subsequently acts as a holder for the individual cut wells, which are then transferred to tubes for counting radioactivity. A "plate-cutter-distributor" device, which cuts and fills wells into counter racks, will soon be available from Biotec (Basel, Switzerland). Development of Cell-Bound Antibody with Enzyme-Labeled Anti-lg (ELISA). Fifty microliters of appropriately diluted alkaline phosphataselinked RaMIg in RIA buffer are added per well, and the plates are kept for 1 hr at room temperature. After three consecutive washings with PBS, 100 /zl of substrate buffer (containing p-nitrophenyl phosphate) are added to each well, and the colorimetric reaction is determined 30 min later, after incubation in the dark, either visually or in a vertical light path photometer (Titertek Multiscan, Flow Laboratories Ltd.). If delayed reading is desired, the reaction may be stopped by adding 50/~1 of 3 M NaOH.
Examples and Comments
Production of a Confluent Cell Monolayer. Optimal signals of antibody binding are obtained if cells form a confluent monolayer. The number of cells required depends obviously on the size of the cells used and can easily be checked under the microscope. Addition of 1 × 106 mouse spleen cells per well is optimal to establish monolayers as determined microscopically and through binding of [12~I]RaMIg.2 If lower cell numbers are used, the signals of label-bound antibodies will be proportionally smaller. In contrast, increasing the number of cells in each well leads to a decrease of
412
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[28]
T A B L E II COMPARISON OF BINDING ASSAY USING CELLS ATTACHED TO PLATES VERSUS CELLS IN SUSPENSION a-c Type o f a s s a y ~
T h y m o c y t e strain
Plate fixed (cpm)
Susp. fixed (cpm)
Susp. fresh (cpm)
DBA/2 C57BL/6 N o cells
3544 -+ 353 581 - 62 265 -+ 43
1264 _+ 98 380 -+ 111 333 -+ 38
1510 -+ 394 838 --- 196 299 - 69
a F r o m Stocker and Heusser. 2 b In the absence o f antiserum, the three types of assay gave the following nonspecific uptake: plate fixed, 160 -+ 31; suspension fixed, 117 -+ 11 ; suspension fresh, 526 --- 376. T h e s e values were similar for the two t h y m o c y t e donor strains. c Binding of anti-DBA/2 (1 : 100) to DBA/2 or C57BL/6 t h y m o c y t e s was m e a s u r e d in three different a s s a y s y s t e m s . U p t a k e o f anti-DBA/2 was determined by [~25I]RaMIg. n T h e three a s s a y s y s t e m s were: cell layer attached to the plate using glutaraldehyde method (Plate fixed); glutaraldehyde-fixed cells in suspension (Susp. fixed); and unfixed cells in suspension (Susp. fresh). E a c h value is the m e a n of 4 - 8 replicate a s s a y s _+SEM.
reproducibility, presumably because cells not directly bound to plates are lost during the washing procedure. Specificity of Signals with Respect to Solid-Phase versus Liquid-Phase Immunoassay. The binding of antibody to cells attached to plates was compared with the binding obtained with cells in suspension. Cells in suspension were either fresh or fixed by glutaraldehyde in suspension, 9'1z and attachment of cells was done by glutaraldehyde alone. The binding of anti-DBA/2 alloantisera to DBA/2 and C57B 1/6 thymocytes was investigated by development with [12'~I]RaMIg. Thymocytes were chosen in order to provide a cell population with few surface Ig-bearing cells so that direct binding of [125I]RaMIg to the cells would be minimized. As demonstrated in Table II, the plastic-bound cells exhibited a higher binding signal than did fixed or unfixed cells in suspension. As the same number of cells ( 10~) was used for each type of assay, this result suggested that the method using attached cells allows retention of more cells than assays done in suspension. The specific signal as judged by the difference between binding to relevant and irrelevant target cells was most pronounced for cells attached to the plate. Furthermore, cells attached to the plate by glutaraldehyde and stored at 4° in RIA buffer for 7 weeks before 12 M. Letarte-Muirhead, R. Acton, and A. F. Williams, Biochem. J. 143, 51 (1974).
[28]
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IMMUNOASSAY
FOR CELL ANTIGENS
413
T A B L E III STABILITY OF CELL MONOI.AYERS: EFFECT OF WASHING PROCEDURES REQUIRED IN IMMUNOASSAYS ~25I-Labeled c e l l s a t t a c h e d a (cpm) Washing cycles *
Plate coating b ALG PHA PLL
1
3
4
9717_+ 118 10142 +- 45 9882 _+ 92
10238 +_ 61 10641 _+ 84 9713 +_ 114
9184 _+ 126 10060 _+ 43 9041 _+ 80
106 mouse t25I-labeled thymocytes were added on pretreated plates and subsequently fixed by glutaraldehyde. Values are mean of four replicate assays -+SEM. b Plates were coated with anti-lymphocyte globulin (ALG), phytohemagglutinin (PHA), or poly-L-lysine(PLL). c Cell monolayers were washed four times per cycle.
assay showed similar binding capacity to that of plates prepared on the day o f assay (data not shown). Stability of Cell Monolayers. Stability of the cell monolayers, produced by various means of attachment, was directly followed by using radiolabeled cells.12~I-Labeled thymocytes were centrifuged on ALG-, PHA-, or PLL-pretreated plates, respectively, and fixed by glutaraldehyde. After fixation, plates were passed through various washing cycles, each corresponding to four washing steps. One cycle would correspond to the cell layer at the beginning of an immunoassay, and three cycles would correspond to the end of a normal immunoassay in which a bound antibody is determined by a labeled second antibody. As can be seen in Table III, the cell attachment procedures result in firmly stable cell monolayers with a high degree of reproducibility.
Detection of Monoclonal Antibodies in Culture Supernatants Using Attached Cells. Culture supernatants from hybridomas producing monoclonal mouse antibodies were tested for binding to cells attached by glutaraldehyde alone. These antibodies, of known Ig subclass, have specificity for defined cell-surface antigens. Cell layers of human peripheral blood lymphocytes (PBL), and SRBC were used. Fifty microliters of supernatant media from hybrid cell cultures were added, and binding to the two cell types was assayed. Table IV shows that the antibodies could be detected on the appropriate target cells. In the case of N 5/1, an antibody directed against a human B-cell surface antigen, the binding was lower by a factor of 5 than binding of anti-fl2-microglobulin. This corresponds to the prevalence of B cells among PBL.
Comparison of Antibody Binding to Cells Attached by Different Means.
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TABLE IV SPECIFIC BINDING OF MONOCLONAL ANTIBODY TO CELL MONOLAYERS a'b Monoclonal antibody Designation N 34 N 26/114 N 5/I Sp 2/HL Hy 2-19 X63 Buffer
Binding to cell monolayer c
Specificity
H u m a n PBL
H L A complex Human /32microglobulin human B cell antigen SRBC TNP Unknown
SRBC
No cells
4279 ± 164 5416 ± 593
201 ± 166 ±
34 12
285 ± 21 356 ± 13
1150 ±
133 ±
17
182 ± 13
3596 ± 526 383 ± 33 129 ± 8 1 5 0 ± 46
264 ___ 22 672 ± 35 142___ 8 124± 9
323 465 264 180
± ± ± ±
8 12 53 14 8
" From Stocker and Heusser. 2 b Monoclonal mouse antibodies of various specificities were assayed for binding to cell layers of human peripheral lymphocytes (PBL) or sheep erythrocytes (SRBC) attached to plates using glutaraldehyde. Binding Of the monoclonal antibodies was detected by [l~5I]RaMIg. c Values as means of four replicate assays ± S E M .
TABLE V SPECIFICITY OF BINDING OF AN ANTI-H-2 MONOCLONAL A N T I B O D Y e TO CELLS IMMOBILIZED BY DIFFERENT MEANS Method of attachment b Cells
Attached"
AJJ IBALB/c AKR B 10A C57BL ]3W 5147 EL-4 No cell
Spleen Spleen Spleen Spleen Spleen ----
ALG + GLU 619 ± 588 ± 595 ±
555 14329 592 15207 688
± ± ± ± ±
186 11 13 62 508 102 1021 25
GLU 787 914 692 707 15987 595 15075 666
± ± ± ± --± ± ±
ALG 81 55 38 5 188 57 540 26
549 498 560 540 18486 789 16923 880
± 3 ± 5 ± 29 ± 20 ± 1146 ± 88 ± 1031 ± 26
H-2K b + + -
a 8 × 105 spleen cells or 2.5 × 105 cells from in vitro propagated cell lines were added per well and centrifuged onto the plastic surface. b Cells were centrifuged onto plates and allowed to react with anti-lymphocyte globulincoated wells for 5 min (ALG) and/or fixed with 0.25% glutaraldehyde for 5 rain at 4 ° (GLU). After washing, wells were quenched with RIA buffer as indicated. c B-8, a monoclonal IgG1 anti-H-2K b antibody was isolated and iodinated; 130,000 cpm were added per well and allowed to react for 1 hr at 22 °. d Values ± maximum deviations from the mean (cpm); N = 2.
[28]
SOLID-PHASE 1MMUNOASSAY FOR CELL ANTIGENS
415
The binding of an isolated and radioiodinated monoclonal antibody directed against H-2K b~3 to cells attached by different means was compared. Spleen cells from different mouse strains and cells from two in vitro cell lines were immobilized on plates by glutaraldehyde fixation, by pretreating the plates with ALG, or by a combination of both. The experiment demonstrates (Table V) that a high specific signal can be obtained with either method of cell attachment. Since there is no development step involved in this experiment, the signal-to-noise ratio is a direct measure of specific binding. It is evident from the data that cells lacking H-2K b surface components show no significant binding compared to control wells without cells. This demonstrates that nonspecific binding is minimal. Since signals obtained with the three types of attachment procedures are similar, we conclude furthermore that glutaraldehyde treatment does not alter the epitope recognized by the anti-H-2K b antibody. Detection o f Monoclonal Antibody Binding to Cells Immobilized on PHA-Pretreated Plates and Using an Enzyme-Labeled Probe for the Immunoassay. In this example, taken from Stocker et al., ~ cells from two human cell lines were attached on PHA-coated plates and subsequently fixed by glutaraldehyde. Monolayers were incubated with various culture supernatants containing monoclonal antibody of indicated specificity (Table VI). The binding of antibodies was then revealed by alkaline phosphatase-labeled RaMIg followed by the enzyme-induced color reaction. As demonstrated, significant binding was obtained only where specific interaction of antibody and corresponding antigenic determinants was expected. The example demonstrates, furthermore, the use of an enzymelinked development step in an antibody-binding assay for cells immobilized on a plastic surface. Discussion In this paper, we described different possibilities for attaching cells to plastic so that they can be used in a solid-phase radioimmunoassay for cell-surface antigens. Advantage is taken of the property of plastic to bind protein by nonspecific absorption. This property is used to form anchors for cell attachment or to coat wells with reagents that actively bind to cells, thus functioning as cross-linking molecules. Glutaraldehyde treatment of cells spun onto untreated plates will remain attached on the plastic surface. la G. K6hler, F. Fischer-Lindahl,and C. H. Heusser,in "The Immune System" (C. Steinberg and I. Lefkovits,eds.). Karger, Basel (in press).
416
ANTIGEN AND ANTIBODY DETECTION BY IMMUNOASSAY
[28]
TABLE VI ENZYME-LINKED IMMUNOSORBENT ASSAY USING CELLS
ATTACHEDTO PHA-CoATED PLATESa Cells a t t a c h e d Culture supernatant° Medium alone X 63 Anti-/32 m Anti-HLA ABC Anti-DR Anti-DR 3 + 5 + 6
c'a
Daudi
WT 18
No cells
0 0 0 0 5 5
0 1 6 5 7 2
0 1 1 1 1 1
" From Stocker e t al. 5 b The specificity of the antibodies produced by X 63 cells is unknown (negative control). c The Daudi cell line is a Burkitt lymphoma line that lacks chromosome 15, which is responsible for/32 m expression and as a consequence lacks HLA A, B, and C surface antigens as well. It still expresses D determinants. The WT 18 cell line is Epstein-Barr virus transformed (HLA l~omozygous donors). Daudi and WT 18 cells were centrifuged onto PHA-P pretreated plates and fixed by glutaraldehyde. Remaining reactive sites on plates, glutaraldehyde, and lectin were saturated with radioimmunoassay buffer containing egg white. a Reading was done with a vertical light path photometer (Titertek Multiskan, Flow Laboratories Ltd.). The machine was set to express the measured absorbance as numbers (from 1 to 10) corresponding to multiples of 0 . 3 0 D (matrix mode). H o w e v e r , if cells are c e n t r i f u g e d o n t o p o l y - L - l y s i n e - p r e t r e a t e d plates a n d g l u t a r a l d e h y d e - t r e a t e d t h e r e a f t e r , the r e s u l t i n g m o n o l a y e r s are m o r e stable. A l t e r n a t i v e l y , cells can be b o u n d to a c t i v a t e d s u r f a c e s b y c o a t i n g the plates with a n t i b o d i e s d i r e c t e d a g a i n s t c e l l - s u r f a c e c o m p o n e n t s or with lectins, s u c h as P H A , that r e a c t w i t h c e r t a i n c e l l - s u r f a c e c a r b o h y d r a t e s . U n d e r t h e s e c o n d i t i o n s , the i m m o b i l i z e d cells f o r m stable m o n o l a y e r s w i t h o u t s u b s e q u e n t g l u t a r a l d e h y d e fixation. S u c h cell l a y e r s m a y be u s e d to e x c l u d e p o s s i b l e m o d i f i c a t i o n o f the a n t i g e n s i n d u c e d b y g l u t a r a l d e h y d e fixation. It has to be s t r e s s e d , h o w e v e r , that we n e v e r o b s e r v e d a n y loss or c h a n g e s in specificity o f surface a n t i g e n s due to g l u t a r a l d e h y d e fixation. This w a s m o s t c l e a r l y d e m o n s t r a t e d in c a s e s o f m o n o c l o n a l a n t i b o d i e s that are e x p e c t e d to r e a c t with a single a n t i g e n i c d e t e r m i n a n t . So far, all a n t i b o d i e s that had b e e n o r i g i n a l l y s e l e c t e d for b i n d i n g to u n f i x e d cells, b o u n d to fixed cells as well.
[28]
SOLID-PHASE IMMUNOASSAY FOR CELL ANTIGENS
417
Some of the anchoring methods described have intrinsic disadvantages under some assay conditions. Coating the plate with antibodies requires the preparation of the IgG fraction of a specific antiserum, which may contain added specificities that interfere with the test. Furthermore, radioiodinated or enzyme-linked protein A cannot be used as a developing reagent because it binds not only to antibodies of the cell culture supernatant to be analyzed, but also to antibodies used to coat the plates. The coating by lectins, on the other hand, requires special quenching procedures to saturate the sugar-reactive sites of the lectin, which in the case of PHA was achieved by adding egg-white glycoproteins. Poly-Llysine as a coating reagent forms anchors for the subsequent glutaraldehyde fixation and allows for the preparation of stable monolayers suitable for the purpose of an immunoassay. This method has the advantage of simplicity and applicability to many different cell types, and it now represents a widely used method for cell immobilization. 3 Immobilization of cells by glutaraldehyde alone is a rapid and convenient method for preparing cell layers for many types of cells 2 and has been used successfully for parasites as well. TM It results in stable monolayers suitable for storage at 4°. Where cells from tissue culture are to be attached, one of the anchor methods should be chosen, and this may be combined with glutaraldehyde fixation as described. One problem of cell binding assays in general is the nonspecific binding of antibodies to reactive sites on the plate. The performance of the assay in 1% BSA eliminates most of the problem. The addition of 100 mM glycine will further help to neutralize remaining glutaraldehyde reactive sites. The addition of 1% heterologous serum or 0.05% Tween 20 prior to and during the incubation with the test sample leads to further reduction of nonspecific binding. For general purposes, we recommend the use of glutaraldehyde with the additional use of poly-L-lysine for cell types where this is found necessary. In any case, cell attachment should occasionally be checked microscopically at the end of an immunoassay procedure. Unsuccessful cell attachment will result in visible holes in the cell layer. The binding of antibodies to a cell layer can be detected either by radioactively labeled or by enzyme-labeled anti-immunoglobulins. Several examples have been presented in this paper. Labeled staphylococcal protein A may be used, either to replace the anti-immunoglobulin or as a second indirect label for maximal amplification. The use of radiolabeled probes involves time-consuming procedures such as cutting the plate and putting the wells into the gamma counter. This step can be automated with ~4E. Handman and J. S. Remington,Immunology 40, 597 (1980).
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[29]
a "plate-cutter-separator" device (Biotech, Basel). Alternatively, a semiquantitative assessment of positive wells is also possible by autoradiography of the whole plate. 15"16On the other hand, enzyme-linked detection methods offer the advantage of convenience, safety, and speed. T M Furthermore, enzyme-linked probes are more stable than radioiodinated probes and need no expensive equipment. It is shown that optical densities of the enzyme reaction can actually be determined with a vertical photometer across the cell monolayers. Alkaline phosphatases (Table V1), ~ and horseradish peroxidase 3"4have been used successfully in a cell binding assay with mouse lymphocyte monolayers. However, ELISA techniques are limited by the fact that certain cell types express enzymatic activities themselves. The method for detection of antibodies binding to cells immobilized on solid phase will be extremely helpful when large numbers of samples need to be rapidly screened. It has been successfully employed in extended hybridization experiments in which many culture supernatants had to be assayed for possible antibody activity against cell-surface antigens. The method offers speed, reproducibility, and the possibility of preparing plates in advance and storing them until use. ,5 j. p. Brown, J. D. Tamerius, and I. Hellstr6m, J. Immunol. Methods 31,201 (1979). i6 R. M. Parkhouse and G. Garnotta, Cur. Top. Microbiol. lmmunol. 81, 142 (1978). 17 E. Engvall and P. Perlmann, Immunochemistry 8, 871 (1971). is A. H. Schuurs and B. K. Van Weemen, Clin. Chim. Acta 81, I (1977).
[29] M i c r o p r o c e d u r e s f o r Q u a n t i t a t i v e I m m u n o c h e m i c a l Analysis of Antigenic Molecules and Antigenic Determinants 1
By KAZIMIERZ MALINOWSKI and WLADYSLAW MANSKI The quantitative determination of antigens and antibodies, first developed by Heidelberger and Kendall, TM is at the foundation of molecular immunology. The theoretical basis of the antigen-antibody reaction has been reviewed by Kabat 2 and by Day. 3 The sensitivity and accuracy of 1 This work was supported by U.S. Public Health Service Research Grants EY 00189 and EY 00198. la M. Heidelberger and F. E. Kendall, J. Exp. Med. 50, 808 (1929). 2 I. Kabat, "Structural Concept in Immunology and l m m u n o c h e m i s t r y . " Holt, N e w York, 1976. 3 E. D. Day, " A d v a n c e d I m m u n o c h e m i s t r y . " Williams & Wilkins, Baltimore, Maryland, 1972.
METHODS IN ENZYMOLOGY,VOL. 73
Copyright @ 1981 by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181973-6