Detection of Antigen-Antibody Interactions by Surface Plasmon Resonance Application to Epitope Mapping Lars G. Fagerstam,* h a Frostell, Robert Karlsson, Mari Kullman, Anita L a w n , Magnus Malmqvist and Helena Butt? Pharmacia Biosensor AB and tPharmacia Diagnostics AB, S-751 82 Uppsala, Sweden

Surface plasmon resonance (SPR) detection requires no labelling of antigen or antibodies and allows quantification of two or more interacting molecular species. The automated S P R instrument used here consists of an optical detection unit, an integrated liquid handling unit, and an autosampler. A first molecule is immobilized to the dextran modified surface of the sensor chip. By sequential introduction, the stepwise formation of multimolecular complexes can then be monitored. A two-site binding assay which allows characterization of MoAb epitope specificities is described. A polyclonal rabbit anti-mouse IgGl (RAMGl) immobilized to the dextran surface is used to capture the first MoAb from unprocessed hybridoma culture supernatants. After introducing the antigen, the ability of a second MoAb to bind to the antigen is tested. The analysis cycle which is fully automated can be performed more than 100 times using the same RAMGl surface. Since the detection principle allows monitoring of each reactant in the consecutive formation of a multimolecular complex, multi-site binding experiments can be performed. Five MoAbs recognizing different epitopes on an antigen were shown to bind sequentially, forming a hexamolecular complex. MoAbs were further characterized by inhibition analysis using synthetic peptides derived from the primary structure of their antigen. As a model system MoAbs against recombinant HIV-1 core protein p24 were used in all experiments.

INTRODUCTION The detector of the instrument used to develop the methods presented here is based on surface plasmon resonance (SPR), which senses changes in the refractive index close to a metal surface. I t has previously been employed to study thin films deposited on such surfaces (Raether, 1977) and more recently for concentration analysis of biomolecules (Liedberg et al., 1983) and to monitor antibody-antigen interactions (Cullen et al., 1987; Mayo and Hallock, 1989). To fully exploit the evanescent field emanating from the surface and to allow immobilization of biomolecules under mild conditions a dextran matrix coupled to the sensor surface has been developed (Lofis and Johnsson, 1990). Monoclonal antibodies (MoAbs) are powerful tools for examining the structure and function of biological molecules (Goding, 1983; Van Regenmortel, 1989). Knowledge of the distribution and structural features of the epitopes defined by MoAbs can be utilized to identify regions involved in biological activities (Alonson-Whipple et al., 1988; Van Leuven et al., 1988). The term ‘epitope mapping’ is used by different authors to denote different levels of characterization of the epitope specificities of a panel of MoAbs. Sometimes it is used to denote procedures where the binding pattern, i.e., the mutual influence on binding among a panel of MoAbs is elucidated. In * Author to whom correspondence should be addressed.

other cases ‘epitope mapping’ means the precise determination of the structures to which the MoAbs bind. A number of methods to elucidate the binding pattern of MoAbs have been developed. After testing all possible pairs of MoAbs in a two-site or double antibody binding assay, each MoAb can be assigned a specific reaction pattern relative to the other MoAbs. All MoAbs showing the same pattern are then assigned to the same epitope. The resolution in the epitope map is dependent on the number of MoAbs available, since each additional MoAb might reveal a new pattern from those already tested. Gel filtration high performance liquid chromatography (Crawford et al., 1982; Wilson and Smith, 1984; Mazza and Retegui, 1989), gel electrophoresis (Wilson and Smith, 1984), and quasi-elastic light scattering spectroscopy (Yarmush et al., 1987) have been used to determine, from the size of the immune complexes formed, the ability of paired MoAbs to bind to the antigen. Most commonly though, RIA or ELISA methods have been employed. These require labelling of the MoAbs to be investigated or the use of a labelled detection antibody directed towards the MoAbs. For control experiments it might also be necessary to label the antigen. Methods used to localize protein epitopes involve cross reactivity studies with protein fragments (Atassi, 1984) or synthetic peptides (Geysen et al., 1988), comparison of reactivity to closely related proteins (Benjamin et al., 1984) or to intact proteins chemically modified at single residues (Burnens et af.,1987). Attempts have also been made to predict the location of continuous epitopes in proteins from their primary structure (Van Regenmortel and de Marcillac, 1988).

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DETECTION OF ANTIGEN-ANTIBODY INTERACTIONS BY SURFACE PLASMON RESONANCE ~~

EXPERIMENTAL Equipment and reagents The BIAcoreQ* system, Sensor Chip CM5, Surfactant P20, and Amine Coupling Kit containing N-hydroxysuccinimide (NHS), (N-ethyl-N'-(3-diethylaminopropyl)carbodiimide (EDC), and ethanolamine-hydrochloride were from Pharmacia Biosensor AB, Uppsala, Sweden. lmmunosorbent purified rabbit anti-mouse IgGI (RAMG 1 ), unprocessed hybridoma culture supernatants containing murine monoclonal antibodies (all IgG 1 ) against recombinant HIV-1 core protein p24, and a nonp24 specific MoAb (anti-human alfa-fetoprotein (antiAFP), subclass G 1) were from Pharmacia Diagnostics AB, Uppsala. Sweden, Recombinant HIV-I core protein p24 was obtained from Pharmacia Genetic Engineering, San Diego, USA. Synthetic p24 derived peptides corresponding to the regions ( I ) 198-217: MQMLKETINE[-] EAAERDRVHP, (2) 222-240: PlAPGQMREPRGS[-] DIAGTT. (3) 283--300: LDIRQGPKEPFRDYVDRF, (4) 300--319: FYKTLRAEQASQEVKNWMTE, (5a) 329-352: DCKTILKALGPAATLEEMMTACQG, (5b) 332-349: TILKALGPAATLEEMMTA, and (6) 353363: VGGPGHKARVL were obtained from Dr Ake Engstrom, Department of Immunology, Biomedical Center, University of Uppsala, Sweden. The peptide sequences according to Ratner et al. (1985) are numbered according to the Database by Myers et ai. (1988).

natant containing the first MoAb, (2) a non-specific MoAb (50 pg/mL of anti-AFP antibody in HBS omitting NaCI) to block any remaining free RAMG 1, (3) the antigen p24 at 10 pg/mL in HBS, (4) the second MoAb in undiluted hydridoma supernatant, and (5) 100 mM HCI to regenerate the RAMGl sensor surface. After equilibrating the surface with HBS it was ready for the next cycle. All volumes injected were 4 pL at a flow of 5 pL/min with HBS as the transport buffer. The multi-site binding assay cycle Multi-site binding experiments were performed by injecting several secondary MoAbs in sequence before regeneration. To assure that each epitope corresponding to the secondary MoAbs was saturated, the volumes needed were tested by two-site binding. In the samples shown 30 pL were used. Peptide inhibition experiments Forty-five microlitres, of each hybridoma supernatant were mixed with 10 pL of a 330 pg/mL solution in HBS of each of the seven peptides, or with 10 pL of HBS as control. After incubation for at least 30 min, 4 pL of each mixture, starting with the peptide-free contro1,'were injected over a p24 sensor surface, prepared as described above. Between each injection the p24 sensor surface was regenerated with 20 pL of 100 mM HCI.

Preparation of sensor surfaces RESULTS AND DISCUSSION Immobilization of RAMG 1 to the sensor chip via primary amine groups was performed in the instrument in the following manner. Separate vials containing 200 pL of 0.01 M NHS, 0.4 M EDC, 1 M ethanolamine-hydrochloride adjusted to pH 8.5 with sodium hydroxide and 100 mM HCI, respectively, and a vial for mixing EDC and NHS were placed in the autosampler together with a 30 pg/mL solution of RAMGl in 10 mM sodium acetate buffer, pH 5.0. After conditioning of the sensor chip with HBS ( 1 0 m ~HEPES, 0.15 M NaCI, 3.4 mM EDTA, 0.05% Surfactant P20 adjusted to pH 7.4 with sodium hydroxide), the automated immobilization cycle was performed at a continuous HBS flow of 5 pL/min. EDC and NHS, 70 pL of each, were transferred to the mixing vial after which 30 pL were injected to activate the carboxymethylated dextran matrix; 30 pL of RAMG1 were then injected followed by 30 pL ethanolamine to deactivate any remaining activated carboxyl groups. After conditioning with 15 pL of 100 mM HCI the RAMGl surface was then ready for use. Recombinant HIV-1 core protein p24 was immobilized to the sensor chip activated as above, by injection of 30 pL of a 30 pg/mL solution of p24 in 10 mM sodium acetate buffer. pH 5.0. The two-site binding assay cycle Each two-site binding assay cycle was performed using the injection sequence: ( 1 ) undiluted hybridoma superci:, 1990 by John Wiley & Sons, Ltd.

The SPR detector in the instrument responds to changes in the refractive index close to the sensor surface. The refractive index increment for proteins is proportional to the mass present within the detected volume and varies little between protein species. The refractive index changes which are monitored continuously over time are registered as a sensorgran?. The abscissa of the sensorgram is denoted the resonance signal and is indicated in resonance units (RU); 1000 RU correspond to a 0.1" shift in the surface plasmon resonance angle and for the average protein this corresponds to a surface concentration change of approximately 1 ng/mm' (Stenberg cf a/., 1990). The SPR detector covers a range of 3" or 30 000 RU. The resonance signal at a certain point in time will be the sum of the contributions to the refractive index from the sensor surface, the captured molecules, and the buffer. During runs of the type shown here, the amount of captured molecules can be quantified by readings between the sample injections. where the transport buffer passes the sensor surface. Since immobilization of the first molecule is performed in the instrument and the SPR detector senses all changes in the refractive index, the course of the immobilization steps during preparation of a sensor surface can be followed as indicated in Fig. I . The amount of RAMG 1 immobilized can therefore be directly compared between all surfaces prepared. Figure 2 shows a sensorgram of the two-site binding assay cycle using the RAMGl sensor surface. After

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F i g u r e 1 Sensorgram of the immobilization of R A M G l to the sensor chip A continuous HBS buffer flow of 5 ItL/rnin is passing over the sensor surface At 280 s a 30 p L pulse ( A ) of EDC/NHS IS injected followed by 30 IIL of R A M G I at 700 s (B) Ethanolaminehydrochloride is then injected (C) to deactivate the surface The R A M G l surface is then conditioned with 1 5 pL of 100 mM HCI (D) The amount, 1 0 5 kRU of RAMG1 immobilized is indicated at E

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F i g u r e 2. Sensorgram of a two-site binding assay cycle. A t point 1 a resonance signal reading is registered as the continuous HBS buffer is passing over the R A M G l surface at a flow of 5 pL/rnin. The first M o A b (A), blocking M o A b (B), antigen p24 ( C ) , and the second M o A b (D) are then injected in sequence. A t the indicated times (2-5) successive resonance signal readings are registered. A t E the R A M G I surface is regenerated with 100 mM HCI and is then ready for the next cycle.

inJection of the first MoAb, remaining free RAMGl is blocked by injection of a non-p24 specific lgCl MoAb. The negative resonance signal during this injection is due to the lower ionic strength, and hence refractive index, of the solvent buffer a s compared to the transport buffer HBS. Since there is a finite number of binding sites on the RAMG 1 surface, the total uptake of the specific first MoAb and of the blocking MoAb used at a saturating concentration should be independent of the concentration of the first MoAb in the hybridoma supernatant. In experiments where the first MoAb was injected at five concentrations from zero to about 100 pg/mL, the total uptake deviated less than 3% from the mean value. To assure that the resonance signal obtained emanates from the interaction of the second MoAb with the antigen and not with the RAMG 1 surface, the efficiency of the blocking was tested. When reversing the order of injection, i.e., first the blocking MoAb then a purified p24 specific MoAb (200 pg/mL) and then $24, no resonance signal was obtained for either MoAb or p24. This also indicates that there is no detectable exchange between captured MoAb and free MoAb in solution. The non-specific background signal for each hybridoma supernatant was tested against four arbitrarily chosen first MoAbs in the

absence of antigen. The non-specific background signal varied between supernatants (typically 30- 100 RU). but was largely independent of the first MoAb. The nonspecific signal was subtracted from the corresponding second MoAb signal for each two-site binding pair. Theoretically 900 assay cycles would be necessary to obtain data on all possible combinations of 30 MoAbs in a two-site binding study. In practice however once a particular pair of MoAbs tested is found to bind simultaneously to the antigen, it is assumed that they bind to non-overlapping sites and it is not necessary t o test them in reverse order. Negative results however might be due to effects that depend on the order of binding and can only be interpreted after testing both orders. However, in the model system used here all o f the 92 pairs of MoAbs that could not bind to the antigen concurrently showed this behaviour independent of the binding order tested. Since SPR detection allows monitoring of all reactants, the reason for absence of ;I signal for the second MoAb can be looked into in detail. An example is behaviour of MoAbs 9, 10. 13, and 14. For sensorgrams (Fig. 3) where these four MoAbs have been used as the first antibody it can be seen that the absence of a resonance signal from thc secondary MoAbs is due to an unexpected absence of the ability of the first MoAbs to bind antigen when captured by RAMGI. We know of no obvious explanation for this behaviour. The part of the reactivity matrix representing the 4 x 4 possible pairs of these MoAbs must be approached by a different strategy, e.g., by amine coupling of the first MoAb directly to the sensor surface t o avoid the RAMGlLMoAb interaction. This requires purification of the MoAbs since they represent a minor fraction of the total protein in the hybridoina supernatants and was therefore not attempted here. The strength of the resonance signal obtained for the second MoAb will depend on: ( I ) the binding capacity of first MoAb, (2) the degree ~ f s a t u r a t i o nof the first MoAb with antigen, (3) the concentration of the second MoAb, and (4) its affinity and association rate when reacting with the captured antigen. Since all of these parameters vary between MoAbs from different hybridoma supernatants, so d o the resonance signals obtained. Yet, the majority of the signal data offer no difficulty in interpretation as positive (typically 30(.&1000 RU for the second MoAb) or negative and can be directly transferred to the reactivity pattern matrix (Fig. 4). Four combinations of MoAbs (31/9, 4719, 52/53, and 53/52) out of the 544 combinations tested however showed values for the second MoAb slightly above background which might indicate binding.

Resonance Signal [kRU] 22

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F i g u r e 3. Sensorgram of a two-site binding assay cycle using M o A b 1 0 as first M o A b The cycle was performed as in Fig 2 Note the absence of antigen binding by the first M o A b (4)

210 JOURNAL OF MOLECULAR RECOGNITION, VOL 3, N o 5/6,1990

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DETECTION OF ANTIGEN-ANTlBODY INTERACTIONS BY SURFACE PLASMON RESONANCE First MoAb

Second M o A b 5 6 7 9 1 0 1 3 1 4 15 1 7 1 8 1 9 23 26 3 0 31 3 2 33 3 4 3 6 41 4 3 4 4 45 46 47 48 51 52 5 3 5 4

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(These pairs of M o A b s bind simultaneously in the reverse cornbination ) Pairs of MoAbs that interfere i n binding in either combination

Figure 4. The reactivity pattern matrix obtained by two-site binding assays Question marks indicate uncertain interpretatlon or, for MoAbs 9-14, assays that could not be performed (see results)

Further investigation, e.g., by saturation experiments, is needed to classify them into either category and was not attempted here. By grouping MoAbs that showed the same reaction pattern 17 groups were obtained, as shown in Fig. 5. Transformation of the pattern into a two-dimensional diagram (Fig. 6) gives a surface-like representation of the binding interferences between different MoAb groups. The diagram should not be interpreted as physical locations of the epitopes on the surface of the antigen since influences such as allosteric conformational changes in the antigen or electrostatic forces between MoAbs might distort the pattern. In the case presented here there are however no results that contradict such a presentation. To further probe the relation between MoAbs from different reactivity pattern groups, their reactivity towards synthetic peptides corresponding to seven regions of the antigen was tested (Table I ) . The selection of peptides to be synthesized was done using the prediction method Of HoPP and Woods (1981). Peptides 5a and b were included due to the disulfide loop structure, and thereby inherent

8 1990 by John Wiley & Sons, Ltd.

Epitope MoAb

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47 33 5, 6, 7, 10, 13, 14, 15 17 23,26, 51, 54 34 _I

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Groups of MoAbs that can bind concurrently. Groups of MoAbs that interfere in binding.

Figure 5. By grouping MoAbs that show the same reaction pattern 17 groups were distinguished. The groups are shown sorted in descending order of binding interference.

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![I>

0 Figure 6. From the matrix in Fig. 5 a two-dimensional 'surface-like' map can be constructed. Overlapping circles denote MoAb groups that cannot bind concurrently.

Table 1. The synthetic p24 peptides used correspond to the regions: (1) 198-217: MQMLKETINEEAAERDRVHP, (2) 222-240: PIAPGQMREPRGSDIAGTT, (3) 283-300: LDI RQG PK E PFR DY VDRF, (4) 300-319: FYKTLRAEQASQEVKNWMTE, (Sa) 329-352: DCKTI LKALGPAATLEEMMTACQG, (Sb) 332-349: TILKALGPAATLEEMMTA, and (6) 353-363: VGGPGHKARVL. MoAb

5 6 7 9 10 13 14 15 17 18 19 23 26 30 31 32 33 34 41 43 44 45 46 47 48 51 52 53 54

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101 102 95 98 99 99 100 100 106 100 100 101 100 102 101 101 103 101 103 102 98 98 103 100 103 100 101 100 100

102 102 92 96 100 101 99 100 105 100 99 99 102 101 100 101 99 101 63 79 98 101 63 103 105 100 102 100 101

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102 102 94 91 99 100 99 99 110 100 97 101 100 104 99 102 98 98 107 102 99 99 99 103 105 99 102 101 101

Data in the table are given as percent response for each MoAbpeptide pair as compared to the peptide-free control.

conformational restraint. Here again a pattern evolves which resembles the grouping of MoAbs by two-site binding (Fig. 6). Peptide 2 inhibits MoAbs 41. 43, and 46 which all belong to the same reactivity pattern group and peptide 4 inhibits only MoAb 47 which also is the only representative of its group. A more complex pattern is obtained for the peptide pair 5a and 5b. The 24 amino acid residues long peptide 5a contains a disulfide bond bridging 19 residues forming a loop. This loop can be assumed to have restricted conformational mobility as compared to peptide 5b which corresponds to 18 of the residues within the loop. Peptide 5a inhibits MoAbs 5. 6, 7. 9, 10, 13. and 14 which by two-site binding show very similar reactivity patterns. However, MoAb 15 which also belongs to this group shows no reactivity at all. The linear peptide 5b inhibits most strongly MoAb 9 and to a lesser extent MoAb 7 but none of the other MoAbs reactive towards peptide 5a. For the MoAbs showing reactivity towards the peptides tested (except MoAb 15). these results indicate that the grouping deduced from the two-site binding patterns in fact represent groups of MoAbs recognizing neighbouring structural motifs in the antigen (Fig. 6). Hinkula ct a/. (1990) have identified five immunogenic regions in the primary sequence of p24 by testing 30 amino acid residues long peptides, overlapping by 10 residues. in direct binding and inhibition assays using solid phase ELISA. In order to obtain a more precise localization of the epitopes, 15 amino acid residue peptides were also used. Where applicable, due to the limited number of peptides used by us, the data obtained are in good agreement. However, in addition to some of the regions identified by Hinkula t't al., we also found MoAbs reactive to the region residues 222-240 (peptide 2). Since no labelling is needed when using SPR detection, multideterminant binding experiments can be performed Resonance Signal [kRU]

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Figure 7 . Sensorgram of a multideterminant binding experiment with eight hybridoma supernatants. Reagents were injected in the sequence indicated in the figure; 30 p L of each MoAb were injected to assure saturation of the binding sites.

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simply by sequential injection of the hybridoma supernatants whilst monitoring the stepwise formation of the antigen-antibody complex. Here saturation of the secondary antibody binding sites is important if one wants to be able to infer effects on the binding that emanate from hindrance other than that seen in the two-site binding experiments. For example, the binding of an antibody to the antigen might be reduced by the presence of a combination of other antibodies, even though each one of them alone does not have this effect. Fig. 7 shows an example of the sequential injection of seven MoAbs that together with the first MoAb represent five reactivity pattern groups. As can be seen, MoAbs from all five groups can bind simultaneously, forming a hexamolecular complex.

GENERAL DISCUSSION When using surface plasmon resonance detection, molecular interactions can be monitored without any labelling. The signal obtained is with good correlation related to the protein mass in the detected volume. The behaviour of each reactant in the sequential formation of a multimolecular complex can thus be monitored. In assays requiring labelling usually only the last reactant added is detected and hence no information is obtained on the course of the previous steps of the experiment. The labelling procedure itself may also interfere with the ability of an antibody to bind its antigen and sometimes experiments cannot be performed at all due to low efficiency in the labelling procedure for certain antibodies. In the procedures shown here, all antibodies were of the IgG1 subclass and therefore a rabbit anti-mouse IgGl (RAMGI) surface was used to capture the first antibody from unprocessed hybridoma culture supernatants in two-site and multi-site binding experiments. The use of a RAMGI surface, through its selectivity eliminates the need for purification of the antibodies and provides constant experimental conditions since fresh first antibody is used in each cycle. In the peptide inhibition studies surface selectivity was obtained by immobilization of the antigen to the sensor surface.

The immobilization did not abolish the antigen binding ability of any of the MoAbs, indicating random orientation of the antigen. When using SPR detection, the amount of first antibody and its antigen binding ability is monitored in each cycle and deviations from the expected are easily identified. An example is the loss of the antigen binding ability of four of the MoAbs used in this study when captured by the RAMGl surface. Without this information, the absence of a signal for the second antibody in a two-site binding experiment might easily have been misinterpreted as hindrance in binding of the second antibody, rather than absence of antigen. Since hybridoma cultivation, antibody purification, and labelling procedures are tedious, epitope mapping is often performed with carefully selected antibodies at a late stage in the hybridoma production. Using SPR detection in combination with miniaturized liquid handling consumed less than 3 ml of each hybridoma supernatant for a panel of 30 MoAbs, including control experiments. Epitope mapping can thus, with little effort, be included in early screening procedures. Multideterminant binding experiments using labelling methods would require an elaborate experimental protocol. One approach which has obvious practical limitations is to use different labels for the different antibodies and to quantify them by discriminative detection. Another approach would be to monitor the stepwise buildup of the multimolecular complex to successively larger forms by detection of a labelled antibody added after preincubation with different combinations of unlabelled antibodies. Our results show that the use of SPR detection in a single experiment using unprocessed hybridoma supernatants allows mult-site binding experiments. In the example, antibodies from five different reactivity pattern groups were shown to bind in sequence to the antigen, forming a hexamolecular complex. To our knowledge no other technique allows such visualization of the stepwise formation of multimolecular complexes.

Acknowledgements The authors wish to thank professor Marc H. V. Van Regenmortel for many constructive discussions during the preparation of the manuscript.

REFERENCES Alonson-Whipple, C., Couet, M. L., Doss, R., Koziarz, J., Ogunro, E. A. and Crowley Jr., W. F. (1988). Epitope mapping of human luteinizing hormone using monoclonal antibodies. Endocrinology 123, 1854-1 860. Atassi, M. Z. (1984). Antigenic structures of proteins. fur. J. Biochem. 145.1-20. Benjamin, D. C.. Berzofsky, J. A,, East, I. J., Gurd, F. R . N., Hannum, C., Leach, S. J., Margoliash, E., Michael, J. G., Miller, A,, Prager, E. M.. Reichlin, M., Sercarz, E. E.. Smith-Gill, S. J., Todd, P. E., and Wilson, A. C. (1 984). The antigenic structure of proteins: a reappraisal. Annual. Rev. Immunol. 2, 67-1 01. Burnens. A.. Demotz, S., Corradin, G., Binz, H., and Bosshard, R. (1 987). Epitope mapping by chemical modification of free and antibody-bound protein antigen. Science 235, 78C783. Crawford, G. D.. Correa, L., and Salvaterra, P. M. (1982). Interaction of monoclonal antibodies with mammalian choline acetyltransferase. Proc. Natl. Acad. Sci. USA 79, 7031 -7035.

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Cullen, D. C., Brown, R. G. V., and Lowe, C. R. (1987). Detection of imrnuno-complex formation via surface plasmon resonance on gold-coated diffraction gratings. Biosensors 3, 21 1-225. Geysen, H. M., Mason, T. M., and Rodda, S. J. (1988). Cognitive features of continuous antigenic determinants. J. Mot. Recognition 1, 32-41. Goding, J. W. (1 983). Monoclonal Antibodies: Principles and Practice. Academic Press, London. Hinkula, J., Rosen. J., Sundqvist, V.-AA., Stigbrand, T., and Wahren, 6 . (1990). Epitope mapping of the HIV-1 gag region with monoclonal antibodies. Molecular Immunology 27, 3 9 5 4 0 3 . Hopp, T. P., and Woods, K. R . (1981). Prediction of protein antigenic determinants from amino acid sequences. Proc. Natl. Acad. Sci. USA 78,3824-3828. Liedberg, B., Nylander. C.. and Lundstrom, I. (1983). Surface plasmon resonance for gas detection and biosensing. Sensors and Actuators 4. 299-304.

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L. G . FAGERSTAM E T A L . LofBs. S., and Johnsson, B. (1990). A novel hydrogel matrix on gold surface in surface plasmon resonance sensors for fast and efficient covalent imrnunobilization of ligands. J. Chem. SOC., Chem. Commun., 1526-1 528. Mayo, C. S.. and Hallock, R. B. (1989). lrnmunoassay based on surface plasmon oscillations. J. Immunol. Methods 120, 1 0 5 114. Mazza. M. M.. and Retegui, L. A. (1989). The antigenic topography of human growth hormone. Molecular lmmunology 26, 231 240. Myers. G., Josephs, S., Rabson, A., Smith, T., and Wong-Staal, F. (Eds) (1988). Database Human Retroviruses and AIDS. Los Alamos National Laboratory, Los Alamos. Raether. H. (1977). In. G. Hass (Ed.), Surface Plasrnon Oscillations and Their Application in Physics of Thin Films. Vol. 9, Acad. Press, New York, p. 145. Ratner, L.. Haseltine, W., Partarca, R., Livak, K. J., Starcich, B., Josephs, S. F., Doran, E. R., Rafalski, J. A,, Whitehorn, E. A,, Baumeister, K., Ivanoff, L., Petteway, S. R., Jr., Pearson, M. L.. Lautenberg, J. A.. Papas, T. S., Ghrayeb, J., Chang, N. T., Gallo, R . C., and Wong-Staal, F. (1985). Complete nucleotide sequence of the AIDS virus, HTLV-Ill. Nature313, 277-284. Stenberg. E., Persson, 6..Roos, H., and Urbaniczky, C. (1990).

Quantitative determination of surface concentration of protein with surface plasmon resonance by using radiolabelled proteins (submitted). Van Leuven, F.. Maryen, P., Cassiman, J.-J., and Van den Berghe, H. (1988). Mapping of structure-function relationships in proteins with a panel of monoclonal antibodies. J. lmmunol. Methods 111,3949. Van Regenmortel, M. H. V., and de Marcillac, G. D. (1988). An assessment of prediction methods for locating continuous epitopes in proteins. Immunology Letters 17, 95-1 08. Van Regenmortel, M. H. V. (1989). Structural and functional approaches to the study of protein antigenicity. Immunology Today 10, 266-272. Wilson, J. E., and Smith, A. D. (1984). A gel electrophoresis method for epitope mapping studies with monoclonal antibodies. Analytical Biochem. 143. 179-1 87. Yarmush, D. M., Morel, G., and Yarmush, M. L. (1987). A new technique for mapping epitope specificities of monoclonal antibodies using quasi-elastic light scattering spectroscopy. J. Biochem. Biophys. Methods 14.279-289. Received 7 September 1990; accepted (revised) 6 November 1990.

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