196, 174-177 (1991)

Detecting lmmunocomplex Formation in Sucrose Gradients by Enzyme Immunoassay: Application in Determining Epitope Accessibility on Ribosomes Wan-Jr

Syu,l Brenda



of Physiological


Kahaq2 Chemistry,

and Lawrence University


of Wisconsin,




A sensitive method using enzyme immunoassay and sucrose gradient to analyze immunocomplexes of biological particles has been developed. The sensitivity and application of this method were demonstrated by that the in situ accessibility of ribosomal protein epitopes could be easily determined. We used sucrose gradients to separate the ribosome-bound and the free antibodies and traced the antibodies in the gradients by an enzyme-linked immunosorbent assay. Epitopes exposed in situ are bound by specific antibodies, which in turn are detected in sucrose gradients migrating with ribosomes. This method of detecting antibody migration is more sensitive than the conventional means of the antibody-mediated dimeriusing Afeenm to monitor zation of ribosomes. Furthermore, an epitope defined by a biotin-labeled monoclonal antibody can be analyzed in the presence of other unlabeled antibodies. Thus, the relationship of different accessible epitopes in situ can be readily examined. Versatility and sensitivity of this method should make it useful in analyzing a variety of immunocomplex systems. 0 1991 Academic Press,



Surface regions of biological particles are of general interest because they provide clues to the structural organization of the particles and they are likely to be functionally important. We, and others, have been studying immunological accessible epitopes of Eschmichiu coli ribosomes and using specific antibodies to analyze the structures and functions of particular ribosomal proteins in situ (l-3). However, because most of the anti1 Present address: Graduate nology, National Yang-Ming * Department of Zoology, 53706.

Institute of Microbiology and ImmuMedical College, Taipei, R.O.C. 11221. University of Wisconsin-Madison, WI

bodies used were derived from antisera which had heterogeneous antibody molecules directed toward an unknown number of epitopes, the information obtained only reveals the general arrangement of individual proteins in situ. To map the ribosomal proteins at the level of individual epitopes, specific monoclonal antibodies (MAbs)3 offer significant advantages. However, a premise for the usefulness of such MAbs is that the recognized epitopes must be available in situ and that the reaction with the ribosomes could be monitored. To determine the accessibility of epitopes of ribosomal proteins in situ, two methods have been often used. One method is to use electron microscopy for a direct visualization of antibody-ribosome complexes (2,3). Since this methods is tedious and is not feasible for general laboratory practice, it is usually reserved for topological studies of ribosomes using well-characterized antibodies. A second method is to detect antibody (IgG)-linked ribosome dimers, which are separated from free ribosomes by sucrose gradient centrifugation. The ribosomal particles separated in gradients can be conveniently monitored by 260-nm absorbance (4-6). Thus, it has been widely used in testing reactions with newly generated antibodies. However, the sensitivity of detecting immunocomplexes becomes poor when few stable dimers are generated by low affinity antibodies. To circumvent this insensitivity, we improved the detection by monitoring antibody migrations with ribosomes in the sucrose gradients. By doing so, antibodies forming complexes with ribosome monomers and/or dimers were easily detected. Furthermore, this method provides a feasible way to analyze mutual relationship of different epitopes in situ.

3 Abbreviations used: MAb, monoclonal antibody; anti-ribosomal proteins S3 and S13, respectively; linked immunosorbent assay.

174 All

Copyright 0 1991 rights of reproduction

AS3 and AS13, ELISA, enzyme-

0003-2697/91 $3.00 by Academic Press, Inc. in any form reserved.













Ribosomes and Antibodies Isolation of E. coli K12 (strain

PR-ClO) 30 S ribosomal subunits was performed as described by Held et al, (7). Generation and epitope mapping for anti-S3 (AS3) MAbs, anti-S13 (AS13) MAbs have been described elsewhere (8-10). All MAbs used for reaction with 30 S ribosomal subunits were prepared in 10 mM Tris-HCI, pH 7.4, 10 mM MgCl,, 200 mM NH&l. Biotinylation of MAbs was performed as previously described (9).




Sucrose Gradient Centrifugation


The formation of MAb-linked ribosome dimers was monitored as described (5) except that tubes were centrifuged for 14 min (at speed) in a Beckman VTi 65 or VTi 65.2 rotor at 60,000 rpm in an L5-75B centrifuge (Beckman Instruments, Palo Alto, CA).

A260 0.0:


Me~ur~ng MAbs in sucrose Gradients by EnzymeLinked lmmunosorbent Assay CELISAf

Gradients were directly collected into Titertek 96well microtitration plates (Flow Laboratories, McLean, VA), 1 drop of about 80 ~1 per well; each well received additional 120 ~1 of 0.1 M Tris-HCl, pH 9.0, containing 0.15 M NaCl. The plates were vigorously agitated on a Mini-Orbital shaker (Bellco Glass, Vineland, NJ) for 2 min and then incubated for 20 h at 4’C. After three washes with buffer A (0.05 M Tris-HCl, pH 7.4, containing 0.15 M NaCl, 1.0 InM MgCl,, 0.02 mM ZnCl,, and 4.6 rnM NaN,), each well received 150 ~1 of 0.1 &g/ml alkaline phosphatase-labeled goat anti-mouse IgG (heavy and light chain specific; Kirkegaard and Perry Laboratories, Gaithersburg, MD) diluted in buffer A containing 0.25% (w/v) gelatin and 1% (w/v) bovine serum albumin. The plates were incubated at room temperature for 3 h, washed 5 times with buffer A, and developed by adding 200 ~1 of 0.2 mg/ml4-methylumbelliferyl phosphate (Research Organics, Cleveland, OH) in 1. M 2amino-2-methyl-1-propanol, 1.0 mM MgCl,, 0.02 mM ZnCl,, pH 10.3. The fluorescent product of the hydroiysis reaction, 4-methylumbelliferone, was measured at appropriate times using a Microfluor reader (Dynatech Lab., Alexandria, VA). Biotinylated MAbs in sucrose gradients were similarly measured by using avidin-labeled alkaline phosphatase (Sigma Chemical Co., St. Louis, MO). RESULTS

To determine whether an epitope recognized by an MAb is accessible in situ, the MAb was incubated with 30 S ribosomal subunits. The mixture was examined for the formation of MAb-linked ribosome dimers by sucrose gradient centrifugation (5); the gradient absorbance profiles were aligned and a typical result is shown in Fig. 1. AS3-MAb 1 reacted with 30 S subunits and formed 46 S subunit dimers (Fig. 1C) as the control an-





AJL ( Sedimwtatiin


FIG. 1.

Sucrose gradient analysis of reaction of MAbs with 53 in situ. Ribosomal30 S subunits (36 pmol) were incubated with individual antibodies for 20 min on ice and sedimented through 5 ml of 15-30% sucrose gradient at 4°C for analysis of immunocomplex formation. Measurement of absorbance was performed as described (5). A, 30 S subunits (contaminated with a small amount of 50 S subunits); B, with 3 81 of an S7-specific, dimer-forming antiserum (Kahan, unpublished data); C, with 15 pg of AS3-MAb 1. The arrow indicates the position of the 46 S dimers of 30 S subunits.

tiserum did (Fig. 1B). In titration experiments with several AS3-MAb 1 to ribosome ratios, dimers were increasingly detected in the gradients until about 50% of 30 S subunits had been converted to dimers. Thereafter, dimer formation gradually decreased as more MAbs were added to the reaction, a result similar to that with the control antiserum (data not shown), On the other hand, when AS3-MAbs 2 and 3 were tested in the same assay at several MAb to subunit ratios, absorbance profiles (data not shown; also, see Fig. 3A) were indistinguishable to that (Fig. 1A) with ribosomes alone. Such a lack of dimer formation could result from the corresponding S3 epitopes inaccessible to these two MAbs or from a lack of stability of the MAb-linked dimers during centrifugation. In the later case, few ribosome dimers are detected by absorbance but one might expect MAb molecules to migrate with 30 S subunits




FIG, 2. Reactions of AS3-MAbs with S3 in situ as detected by sucrose gradient combined with ELISA. Sucrose gradients were performed as described in Fig. 1. Gradients were collected into 96-well plates and measured for MAbs by ELISA (see Materials and Methods). MAbs applied were: (V), 15 ng AS3-MAbl; (O), 15 wg AS3-MAb 2; (O), 15 pg AS3-MAb 3. Continuous lines indicate the use of E. coli 30 S ribosomal subunits. Dotted line shows the negative control of reaction with chicken 80 S ribosomes; all three MAbs gave similar results and only results with AS3-MAb 2 is shown here for the reason of clarity. Arrows a and b indicate the positions of subunit monomers and dimers, respectively.

through the gradients. To test this notion, we increased the detection sensitivity by monitoring the migration of MAbs in the sucrose gradients after centrifugation (Fig. 2) as described under Materials and Methods. In control experiments with E. coli 50 S ribosomal subunits (data not shown) or with 80 S ribosomal particles (Fig. 2) which do not contain E. coli S3, the free MAbs were found at the top of gradients and in gradually decreasing amounts toward the bottom. When the method was tested with the dimer forming AS3-MAb 1, the MAb migrated into the gradient and gave peaks at positions a and b (Fig. 2) which correspond to the locations of the monomers and the dimers of 30 S ribosomal subunits, respectively. AS3-MAbs 2 and 3 migrated with 30 S subunits through the gradients (Fig. 2), mainly to the monomer position and some to the dimer position. (The imprecise alignment of MAb migration patterns shown in Fig. 2 was mainly due to the difficulty of aligning sucrose gradients.) These results suggested that the two MAbs indeed reacted with S3 in situ and the resulting dimers may not be stable enough to give a peak detected by absorbance. The reactivity of AS3-MAbs 2 and 3 with 30 S subunits could be more clearly demonstrated by increasing association of MAbs with ribosomal dimers, providing that dimers could be generated by other means. One way of doing so was to pair AS13-MAb 2 and AS13MAb 21 to react with 30 S subunits, a reaction that shifted almost all of the 30 S subunits to dimers (1,9). Moreover, addition of AS3-MAb 3 did not affect the A 26,,nm gradient profile generated by these AS13-MAbs



(data not shown). Therefore, we used biotinylated AS3MAb 3 in conjunction with unlabeled AS13-MAbs 2 and 21 to react with ribosomes and monitored biotin-MAbs in the gradients (Fig. 3). Biotinylated ASS-MAb 3 alone reacted with ribosomes and migrated mainly to the monomer position as expected. When added together with the AS13-MAbs, the biotin-AS3 migrated accordingly further to dimer position (comparing the dotted line and the solid line in Fig. 3B); a result strongly supports the notion that antibodies like AS3-MAb 3 are reactive with ribosomes and that less dimers generated are probably due to the lack of stability of 30 S-MAb-30 S complexes. The usefulness of this method was further demonstrated in analyzing the relationship of different epitopes in situ. AS3-MAb 2 and AS3-MAb 3 were generated from different hybridoma fusions and epitopes have been mapped to an N-terminal about 20 residues of S3 (10). We reacted these two AS3 MAbs with ribosomes simultaneously to test whether their epitopes are mutual exclusive in situ. As shown in Fig. 4, binding of biotinylated AS3-MAb 3 was completely blocked by a prior incubation with AS3-MAb 2. Reciprocally, AS3MAb 3 blocked the binding of biotinylated AS3-MAb 2

FIG. 3. Binding of AS3-MAb 3 to the subunit dimers. Biotinylated AS3-MAb 3 (1 ag) was added together with AS13-MAb 2 (5 pg) and AS13-MAb 21 (4 ag) to react with 30 S subunits. Sucrose gradient analysis was done as described in Fig. 1. A, Are,, gradient scanning; B, biotinylated MAbs in the gradients in A measured by ELISA using avidin-conjugated alkaline phosphatase. (-) Biotinylated AS3-MAb 3 alone; (- - -) AS13-MAbs added. See legend to Fig. 2 for explanation of arrows a and b.


0-j 1






I 40



I 60



FIG. 4. Analysis of simultaneous binding of different AS3-MAbs to S3 in situ. A purified, unlabeled MAb (15 pg) was incubated with 36 pmol of 30 S subunits at 4°C for 5 min, followed by an additional ZO-min incubation after adding 0.3 pg of biotinylated MAbs 3. Sucrose gradient centrifugation and detection of biotinylated MAbs were performed as described in Fig. 3. (U) Biotinylated AS&MAb 3 only; (0) unlabeled AS3-MAb I added; (m) unlabeled AS3-MAb 2 added. See legend to Fig. 2 for explanation of arrows a and b.

in a similar way (data not shown). In contrast, AS3MAb 1, which is directing to a central portion of S3 molecule (10) and reacted with S3 in situ (Fig. l), did not interfere with the binding of biotinylated ASS-MAb 3. The gradient absorbance profile of reaction with AS3MAb 1 alone was indistinguishable to that of having biotin-AS3 MAb 3 added (data not shown). However, the pattern of biotin-MAb 3 migration apparently changed when AS3-MAb 1 was added first; more biotin-MAb shifted from the ribosome monomer position to the dimer position (Fig. 4). These results indicated that biotin-MAb 3 bound to the ribosomes even when the subunits were bound or dimerized by AS3-MAb 1. Thus, the epitope recognized by AS3-MAb 1 and that of AS3-MAb 3 must be simultaneously available in situ.





on detecting particles dimerized by antibody molecules often encounters the difficulty of detecting dimeric complexes, which are less stable than monomeric complexes due to the possibility of dissociation into monomeric complexes during the gradient centrifugation. Therefore, monitoring the later complexes becomes increasingly important. In case of monitoring monomeric ribosomes associated with a single MAb, it can hardly be achieved by the sucrose gradient absorbance profiles because that the monomeric immunocomplexes are not separable from free ribosomes by the mass difference. To circumvent this difficulty and to analyze immunocomplexes formed in gradients, we have developed a method, as described above, of detecting MAb migration with ribosomes. This new method is sensitive and specific since that the migrations could be well correlated with the moving of ribosomal monomers and dimers and that little migration was detected when unrelated ribosomes were used. Furthermore, this method provides an easy way to analyze whether different epitopes of ribosomes are simultaneously available in situ. Versatility and sensitivity of this method should be equally useful for analyzing epitopes of other biological systems. ACKNOWLEDGMENTS This work was supported by NIH ported in part by Award from Minist~

Grant 22150. of Education,

W.J.S. was supTaiwan, R.O.C.

REFERENCES W.-J., and Kahan, L. (1987) Fed. Proc. 46, 2219. G., and Stoffler-Meilicke, M. (1986) in Structure, Function and Genetics of Ribosomes (Hard&y, B., and Kramer, G., Eds.), pp. 28-46, Springer-Verlag, New York.

1. Syu,

2. Stoffler, 3. Oakes,

M., Henderson, A., Scheinman, A., Clark, M., and Lake, J. A. (1986) in Structure, Function and Genetics of Ribosomes (Hardesty, B., and Kramer, G., Eds.), pp. 47-67, Sponger-Verlag, New York.

4. Winkelmann,

D,, and Kahan, L. (1980) in Ribosome: Structure, Function, and Genetics (Chambliss, G., Craven, G. R., Davis, K., Kahan, L., and Nomura, M., Eds.), pp. 255-265, University Park Press, Baltimore, MD.

DlSCUSSION To probe epitopes in situ requires a way to preserve the integrity of the particles and a means to monitor the immunocomplex formation. Sommer et al. (11) has used an approach with coating ribosomes on plates and detecting antibody interactions by ELISA (11). This method provides a quick way to screen a library of antibodies for potential ones which are reactive in situ. However, limitations encountered are that the status of particles coated on plates is not clear and that the immunocomplexes formed are difficult to analyze. In these regards, sucrose gradient centrifugation appears to be the method of choice since particles are integrally maintained and different immunocomplexes of particles could be separated by gradients and accordingly monitored (2,3,12). However, the system mainly relying


5. Winkelmann, 443-455. 6. Breitenreuter, 7.

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G., Lotti, M., Stiiffler-Meilicke, G. (1984) Mol. Gen. Genet. 197,189-195. Held, W. A., Mizushima, S., and Nomura,

M., M.

and Stiiffler,




Chem. 248,5720-5730. 8. Syu, W.-J.,

and Kahan,

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9. Syu, W.-J.,


B., and Kahan,

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Protein Chem. 8, Chem. 9,

11. Sommer, A., Etchison, J. R., Gavino, G., Zecherle, N., Casiano, C., and Traut, R. R. (1985) J. Biol. Chem. 260,6522-652?. 12. Conti-Tronconi, B. M., Tzartos, S. J., and Lindstrom, J. M. (1981) Bioc~rn~t~ 20,2181-2191.

Detecting immunocomplex formation in sucrose gradients by enzyme immunoassay: application in determining epitope accessibility on ribosomes.

A sensitive method using enzyme immunoassay and sucrose gradient to analyze immunocomplexes of biological particles has been developed. The sensitivit...
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