[42] The spaghetti overlay procedure

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THE SPAGHETTI OVERLAY PROCEDURE

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we describe utilizes 3H-labeled anti-Ig probes. These probes exhibit longterm stability with little or no denaturation after labeling. Thus, the use of these probes can eliminate the hazards and inconvenience associated with frequent 125I labeling or enzyme conjugation procedures. The high sensitivity of these probes allows the assay to detect the presence of at least 510 APC.

[42] The Spaghetti Overlay Procedure

By W. STEVENADAIR Several procedures have been developed over the past few years for the identification of specific members of complex mixtures of polypeptides or peptides resolved by one -t or two-dimensional 2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Many probes have been employed, including antibodies, 3-8 lectins, 4,7,9-12 hormones, 13-16 ligands/7,18 and living cells. 19 In many cases it is useful to screen resolved components with several different probes simultaneously. The use of monoi U. K. Laemmli, Nature (London) 227, 680 (1970). 2 p. Z. O'Farrell, H. M. Goodman, and P. H. O'Farrell, Cell 12, 1133 (1977). 3 W. E. Stumph, S. C. R. Elgin, and L. Hood, J. lmmunol. 113, 1752 (1974). 4 K. Burridge, Proc. Natl. Acad. Sci. U.S.A. 73, 4457 (1976). 5 K. Olden and K. M. Yamada, Anal. Biochem. 78, 483 (1977). 6 W. S. Adair, D. Jurivich, and U. W. Goodenough, J. Cell Biol. 79, 281 (1978). 7 K. Burridge, this series, Vol. 50, p. 54. s W. S. Adair, Anal. Biochem. 125, 299 (1982). 9 j. W. Gurd and W. H. Evans, Can. J. Biochem. 54, 477 (1976). 10 N. A. Guzman, R. A. Berg, and D. J. Prokop, Biochem. Biophys. Res. Commun. 73, 279 (1976). 11 j. A. P. Rostas, P. T. Kelly, and C. W. Cotman, Anal. Biochem. 80, 366 (1977). 12 B. Monk, W. S. Adair, R. Cohen, and U. W. Goodenough, Planta 158, 517 (1983). ~3j. Massagne, B. J. Guillette, M. P. Czech, C. J. Morgan, and R. A. Bradshaw, J. Biol. Chem. 256, 9419 (1981). 14 S. Paglin and J. D. Jamieson, Proc. Natl. Acad. Sci. U.S.A. 79, 3739 (1982). 15 p. F. Pilch and M. P. Czech, J. Biol. Chem. 255, 1722 0980). 16 j. p. Yip, C. W. T. Yeung, and M. L. Moule, Biochemistry 19, 70 (1980). 17 j. R. Glenney and K. Weber, J. Biol. Chem. 225, 10551 (1980). t8 R. K. Carlin, D. J. Grab, and P. Siekevitz, J. Cell Biol. 89, 449 (1981). ~9 E. G. Hayman, E. Engvall, E. A'Hearn, D. Barnes, M. Pierschbacher, and E. Ruoslahti, J. Cell Biol. 95, 20 (1982).

METHODS IN ENZYMOLOGY, VOL. 121

Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

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clonal antibody libraries for epitope mapping, 2°-22 for example, is a powerful approach to dissecting the structural organization of polypeptides. For such analyses, a preparative gel (or transfer) is usually sliced into lanes which are reacted individually with each probe, a process which can be cumbersome and make precise alignment of closely migrating species problematic. An alternative approach, the spaghetti overlay, 8 has been developed to simplify the screening of multiple probes without the necessity of slicing gels. With this procedure, probes are dissolved in low melting temperature agarose and drawn into capillary tubes. After setting, the agarose strips are placed on the surface of a prepared gel. Lanes are created by the probes themselves. Antigen-antibody complexes are visualized by autoradiography as discrete spots which can be easily aligned with stained side strips. In this chapter, the spaghetti overlay technique is described in detail for use in immunoautoradiographic screening procedures employing polyclonal or monoclonal antibodies. Methods for performing multiple overlays on microgels and nitrocellulose transfers are presented together with a general outline of the relative merits of each approach and limitations of the overlay procedure itself. While the methods detailed here have been developed specifically for antibody overlay, they can obviously be adapted for use with other probes as well.

Buffers and Solutions Buffer A: 25 m M sodium phosphate, 0.15 M NaC1, 0.02% NAN3, pH 7.0 Buffer B: 125 m M sodium borate, 0.15 M NaC1, pH 8.2 Fix solutions: 25% isopropanol/10% acetic acid (fix 1); 2% glutaraldehyde in buffer A (fix 2) Wash buffer: buffer A + 0.1% Tween-20 Quench solution: I% bovine hemoglobin, 0.1% Tween-20, in buffer A SDS-Polyacrylarnide Gel Eleetrophoresis (SDS-PAGE). The use of micro slab gels 23 allows short run times, rapid fixation, equilibration, and washing, as well as conservation of sample. Gels (0.5 mm thick) are cast eight at a time in a Plexiglas chamber, using lantern glass slides (8.2 × 10.2 cm) for gel plates, the exact gel specifications (concentration, gradient, etc.) depending on the specific application. When several antibodies are reacted with a common antigen mixture, a stacking gel is omitted; lanes are created by the antibody overlays themselves. When several 20 M. Dziadek, H. Richter, M. Schachner, and R. Timpl, FEBS Lett. 155, 321 (1983). 2, D. A. Winkelmann, S. Lowey, and J. L. Press, Cell34, 295 0983). 22 D. P. Kiehart, D. A. Kaiser, and T. D. Pollard, J. CelIBiol. 99, 1002 (1984). 23 p. T. Matsudaira and D. R. Burgess, Anal. Biochem. 87, 386 (1978).

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antigen mixtures are probed on the same gel, a 3% stacker and comb are employed. Gels are run at a constant voltage of 150 V for approximately 1.5 hr using a discontinuous buffer system. 1Following fixation (45 min, fix 1) gels are equilibrated in buffer A, then incubated for 1 hr in the quench solution. For certain applications (e.g., electrodestaining), a second fixation (fix 2) precedes equilibration and quenching. For orientation, the bottom right corner of the gel is clipped off diagonally. Reference strips are cut from each side and stained for protein by silver stain z4 or for carbohydrate by the periodic acid-Schiff (PAS) method, z° Prior to overlay, excess solution is removed by Pasteur pipet from the surface of the gel, which is supported on the glass plate. Preparation of Labeled Probes

In Vitro Labeling (Iodination). Antibodies and protein A are labeled using the Iodogen procedure. 25 A 50-/xl aliquot of methylene chloride containing 2/,tg Iodogen (Pierce) is added to a glass test tube (12 × 75 ram) and the methylene chloride evaporated off in a 37° water bath with rotation of the tube. To the dried film is added 200/zl buffer B containing 3550/zg protein, 250/zCi carrier-flee Nal25I (Amersham), and 0.5 /zg KI. The reaction is allowed to proceed on ice for 5 rain with gentle agitation. The labeling solution is then removed and rapidly desalted over a bed of Biogel P6-DG z6 as follows. The bottom of a 1.5-ml microfuge tube is pierced with a 25-gauge needle and a small plug of glass wool placed in the bottom. A settled bed of 1.3 ml BioGel P6-DG (Bio-Rad) in buffer A is poured and allowed to drain dry. The tube is then suspended in a 12 × 75mm polypropylene tube and spun for 1 min in a clinical centrifuge at 200 g. The flow-through buffer is discarded and the labeling solution added to the dried bed. The tube is then respun for 1 min at the same speed. Labeled protein is recovered in the bottom of the polypropylene tube while unreacted iodine is retained in the BioGel matrix. Residual iodine is removed by dialysis against buffer A after addition of carrier protein (hemoglobin) to 1%. Aliquots are stored at - 7 0 °. In Vivo Labeling. Monoclonal antibodies are labeled in vivo with [35S]methionine following the procedure of Haas and Kennett. z7 A sterile 24 j. H. Morrissey, Anal. Biochem. 117, 307 (1981). 25 p. j. Fraker and J. C. Speck, Jr., Biochern. Biophys. Res. Commun. 80, 849 (1978). 26 G. P. Tuszynski, L. Knight, J. R. Piperno, and P. N. Walsh, Anal. Biochem. 106, 118 (1980). 27 j. B. Haas and R. H. Kennett, in "Monoclonal Antibodies" (R. H. Kennett, T. J. McKearn, and K. B. Bechtol, eds.L Plenum, New York, 1980.

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suspension of 1 × 106 log phase hybridoma cells is washed once with methionine-deficient medium and resuspended in 2.5 ml medium containing 10% dialyzed horse serum, 5% dialyzed fetal bovine serum, 30 mg glutamine/ml, and 50-100/zCi [35S]methionine. Cells are incubated for 18-24 hr at 37 ° in a 5% CO2 incubator. The supernatant is removed, rapidly desalted as above, dialyzed against buffer A, and stored at - 7 0 ° in aliquots. Overlay Procedures Prior to the overlay, the gel and its glass support are placed on a reference plate which is constructed of transparent Plexiglas and has grooves spaced at 3.5-ram intervals. This entire ensemble rests on a light box. Grooves in the reference plate serve as guides for the application of spaghetti strips in evenly spaced lanes. Spaghetti strips are formed by mixing equal aliquots (35/zl) of either antibody (0.5-5 tzg in buffer A) or spent hybridoma medium with liquified low melting temperature agarose (2% in buffer A, 0.2% Tween-20), then drawing the mixture into a 50-tzl glass capillary tube (Corning), and allowing it to set (1-2 min). To facilitate removal of the agarose strips during overlay, the capillary tubes are precoated with 0.05% Photo-flo 200 (Eastman-Kodak). When a large number of antibodies are to be screened at one time, a Plexiglas plate containing labeled grooves is used to hold the capillary tubes in order horizontally until overlays are performed. After all spaghetti strips have set, each is gently applied to the surface of the gel by gentle blowing through a mouth adapter. When all strips are in place, excess agarose is trimmed from the top and bottom ends of each and the overlay placed in a humidified box for 12-24 hr at 4°. Following the primary incubation, spaghetti strips are washed from the surface of the gel with a gentle stream of buffer A, and excess antibody removed by washing (2-3 hr in wash buffer with several changes). The gel is then reacted with a radiolabeled second probe. The labeled probe, either a second antibody or protein A, is used at 5 × 105 dpm/ml (in quench buffer) and incubation of the gel in this solution carried out at 25 ° for 2-3 hr. When a second antibody is employed, lower backgrounds are usually obtained with F(ab')2 fragments. After the secondary incubation, the gel is washed extensively until counts in the wash solution reach background levels (usually 4-5 hr). The gel is then dried and placed in contact with preflashed XAR film (Kodak) and Cronex intensifying screens, for 12-48 hr at - 7 0 °. For orientation, radioactive ink spots are applied to a corner of the dried gel prior to autoradiography. The

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THE SPAGHETTI OVERLAY PROCEDURE

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developed autoradiogram is placed on the reference plate and compared with stained side strips of the gel. Variations of the Overlay

One-Step Overlay. Several steps can be eliminated by combining primary and secondary incubations. This can be accomplished by using either labeled primary antibody or a conjugate of secondary antibody with 125I-labeled protein A (2 : 1 molar ratio). Primary antibody can be labeled in vitro by iodination or in vivo metabolically. While more time-consuming, metabolic labeling is generally preferred for monoclonal antibodies, since iodination can affect the binding properties of antibodies even when performed under relatively gentle conditions. The use of antibody-protein A conjugates takes advantage of the fact that protein A binds to the Fc fragment of immunoglobulins, leaving the antigen-combining sites free. This approach is limited, however, to certain IgG antibodies (e.g., rabbit) which are bound by protein A with relatively high affinity. Mouse IgGs show a wide variability in this regard and are less suitable for this method. Electrodestain. This procedure can be used in conjunction with onestep overlays as an alternative to postincubation washing of the gel. Gels are fixed in glutaraldehyde (fix 2) prior to equilibration, quenching, and overlay. After the overlay, they are placed in contact with a sheet of wet DE-81 paper and electroblotted 24for 1 hr using a 12-V battery charger as a power source. Other (more expensive) power sources can obviously be substituted but a battery charger does a very adequate job. After blotting, unreacted probe is bound to the DE-81 paper for convenient disposal, while the gel is ready for drying and autoradiography. Although the antibodies examined to date using this electrodestain procedure have remained sufficiently complexed with antigen for detection, antibodies with low binding affinities could be lost using this method. Transfer Overlays. While original overlay procedures were performed on gels, most workers now routinely transfer electrophoresed components to a solid substrate, such as nitrocellulose or Zeta-bind, prior to incubation with probes (see Ref. 28 for an excellent recent review). Spaghetti overlays have been successfully performed on nitrocellulose transfers using the protocol outlined in Fig. 1.29 The major advantages and disadvantages of each approach are summarized in the table. 28 j. M. Gershoni and G. E. Palade, Anal. Biochem. 131, 1 (1983). 29 K. H a n c o c k and V. C. W. Tsang, Anal. Biochem. 133, 157 (1983).

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SDS-PAGE

I

Transfer Overlays

I

Gel Overlays

I

Electro-transfer to Nitrocellulose

Fix, I h

I I I I

I

Quench, i h

Equilibrate in Buffer A, I h, 25*

I I

Primary Overlay, 2-8 h, 25 °

Quench, 1 h, 25 °

Wash, 1 h

Primary Overlay, 12-24 h, 4 ° ,

Secondary Overlay, l h , 25 °

I

Wash~ I h

I

1-Step Overlay

2-Step Overlay

I

I

Electro-Destain~ I h, or Wash, 2-3 h

I

Wash, 4-5 h

I

Secondary Overlay 4-5 h, 25* Wash, 2-3 h

Autoradiography,

Autoradiography,

2-24 h

12-48 h

I DiY Autoradlography, 12-48 h

FIG. 1. Steps for spaghetti overlay on gels and nitrocellulose transfers. Incubation, wash, and autoradiographic exposure times are approximate. Actual times will vary in practice with the concentration of primary antibody and the specific activities of labeled probes. For orientation, gel slices are stained as described in the text; strips of nitrocellulose are stained with india ink. 29

Concluding R e m a r k s The procedures outlined a b o v e have been used successfully for confirming the specificity o f polyclonal and m o n o c l o n a l antibodies, for screening o f hybridomas supernatants prior to cloning, and for domainmapping procedures employing peptide digests. Other potential applications include lectin and ligand screening, probing for receptors (e.g., horm o n e or growth factors), and other protein-protein interactions. The primary limitation is the c o n s e r v a t i o n of binding functions following electrophoresis under denaturing conditions. In practice, h o w e v e r , this has not turned out to be as much a problem as one might expect.

[43]

RIA AND EIA AVIDIN--BIOTINIMMUNOASSAYS

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COMPARISON OF G E L AND T R A N S F E R OVERLAYS

Advantages Gel overlays

Transfer overlays

Polypeptides are probed in an unbiased fashion; particularly important for very high and very low molecular weight components Conformationally dependent epitopes often retained to a greater degree More sensitive; less material required Faster Multiple reactions possible

Disadvantages More time-consuming; longer incubation and washing times Lower sensitivity Gels are more fragile than paper Gels cannot be reused for multiple reactions Polypeptides often not sampled equally. High molecular weight polypeptides transfer inefficiently or not at all, while low molecular weight components can be lost. Also, some polypeptides bind weakly or not at all to nitrocellulose Reactivities to some antibodies can be lost due to the denaturing affects of adsorption

Acknowledgments The application of the spaghetti overlay technique to nitrocellulose transfers was developed in collaboration with Dr. Jerry Bryant, Washington University Biology Department. This work was supported by NIH Grants GM-26117 and GM-26150, awarded to Dr. Ursula Goodenough.

[43] Solution-Phase RIA and Solid-Phase EIA Using Avidin-Biotin Systems for Analysis of Monoclonal Antibody Epitopes and Affinity Constants

By

J O H N E . SHIVELY, CHRISTOPH WAGENER, a n d BRIAN R. CLARK

A monoclonal antibody (MAb) should recognize a single epitope on the antigen used for immunization. Although this generalization can be complicated by the occurrence of multiple identical or cross-reactive epitopes, or the presence of overlapping epitopes, it is a good starting point for epitope analysis. One of the initial problems in selecting a suitable MAb for antigen screening is a determination of which epitope(s) is recogMETHODS IN ENZYMOLOGY, VOL. 121

Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.