Proc. Natl. Acad. Sci. USA Vol. 87, pp. 9848-9852, December 1990 Biochemistry

Molecular epitope identification by limited proteolysis of an immobilized antigen-antibody complex and mass spectrometric peptide mapping (epitope mapping/molecular weight determination/plasma desorption mass spectrometry/complement component C3a/ monoclonal antibody)

DETLEV SUCKAU*, JORG KOHLt, GABRIELE KARWATHt, KLAUS SCHNEIDER*, MONIKA CASARETTOt, DIETER BITTER-SUERMANNt, AND MICHAEL PRZYBYLSKI*§ *Fakultat fur Chemie, Universitat Konstanz, D-7750 Konstanz, Federal Republic of Germany; tInstitut fur Medizinische Mikrobiologie, Medizinische Hochschule Hannover, D-3000 Hannover, Federal Republic of Germany; and tDeutsches Wollforschungsinstitut Aachen, D-5100 Aachen, Federal Republic of Germany

Communicated by Fred W. McLafferty, September 10, 1990 (received for review July 9, 1990)

ABSTRACT Sequences of antigenic determinants were identified by limited proteolysis of peptide antigens bound to an immobilized monoclonal antibody and direct molecular weight determination of the monoclonal antibody-bound peptide fragments by 252Cf plasma desorption mass spectrometry. The epitope peptides to the monoclonal antibody h453 [Burger, R., Zilow, G., Bader, A., Friedlein, A. & Naser, W. (1988) J. Immunol. 141, 553-558] were isolated from immobilized antigen-antibody complexes by partial trypsin digestion. A synthetic eicosapeptide comprised of the C-terminal sequence of the human complement component polypeptide des-Arg77-C3a as well as guinea pig des-Arg78-C3a was used as an antigen. Conditions were developed under which trypsin specifically degraded the antigens without inactivation of the immobilized antibody. After proteolysis, epitope peptides were dissociated from the antibody with 4 M MgCl2. The antigenic peptides were purified by HPLC and identified by 252Cf plasma desorption mass spectro! retry. The epitope recognized by h453 resides on the C-terminal tryptic peptides of human (residues 70-76) and guinea pig (residues 70-77) C3a. As an estimation of accuracy this method is able to provide, trypsin digestion of immune complexes caused cleavage of the antigen within a distance of two amino acid residues upstream from the epitope.

aration of complex digest mixtures followed by amino acid analysis or peptide sequencing may not enable unambiguous epitope identification due to unresolved peptides. The feasibility of fast atom bombardment mass spectrometry (FABMS) and 252Cf plasma desorption mass spectrometry (PDMS) for accurate molecular weight determination of polypeptides has been established in several bioanalytical applications (8). Particularly, abundant molecular ions of polypeptides up to small proteins have been obtained with high sensitivity by PDMS (9). A promising approach in recent work has been the application to multicomponent peptide mixtures, such as proteolytic digests (peptide mapping; ref. 8). The high molecular specificity provided by mass spectrometric peptide mapping has been successfully used in protein structural studies, such as the characterization of cDNA-derived sequences, identification of posttranslational modifications, and differentiation of isoenzyme structures (10-12). In this study the combination of limited enzymatic proteolysis and PDMS has been applied to the molecular epitope analysis of complement component C3a, which is recognized as a potent mediator of inflammation (13). Amino acid sequences (14, 15) and tertiary structure (16, 17) of C3a from several species including human (77-amino acid residues) and guinea pig (78 residues) have been reported. The threeresidue C-terminal sequence of C3a represents the essential receptor-binding site (18, 19). However, biological activity is lost by C-terminal desargination (13), concomitant with the exposure of a neoantigenic determinant that is recognized by mAb h453 (20). h453 coupled to tresyl-activated Sepharose (h453-TAS) was used for the preparation of immune complexes with (i) an eicosapeptide comprising human (h) C-terminal C3a, and (ii) guinea pig (gp) des-Arg78-C3a (des-Arg78gpC3a). For both antigens, mass spectrometric peptide mapping of tryptic peptides dissociated from the truncated immune complex by MgCl2 directly established the epitope, whereas the nonepitope peptides were identified after separation from the immune complex, as schematically illustrated in Fig. 1. Moreover, the synthetic peptide (hC21) comprising the sequence [Gln 65,Arg66]hC3a-(57-77) was designed to provide an estimation of the steric requirements for the ternary trypsin-antigen-antibody complex, by introducing three equidistant tryptic cleavage sites.

A variety of methods have been applied to the study of monoclonal antibody (mAb)-antigen interactions and the characterization of their respective epitopes. Two major approaches that have been widely employed for epitope characterization are competitive binding analysis using synthetic peptides and fine specificity studies with panels of evolutionary variant or recombinant proteins (1). Although well established, these methods have major limitations; e.g., discontinuous or conformationally defined epitopes may not be detectable by using peptide probes (2). Site-directed mutagenesis experiments in epitope studies could be greatly facilitated if some information about the putative epitope is available in advance. A direct approach of epitope mapping, which seems promising in this respect, has been more recently introduced based on the finding that (i) mAbs exhibit remarkable resistance towards proteolytic enzymes, (ii) in immune complexes, antigenic determinants can be protected from proteolytic degradation, and (iii) proteolysis does not lead to dissociation of immune complexes (3-6). Limited proteolytic cleavage of immune complexes has been used for epitope characterization by means of PAGE (7) and by HPLC (6) of the respective peptide digests. However, HPLC sep-

Abbreviations: E;S, enzyme-to-substrate ratio (wt/wt); gpC3a, guinea pig C3a; hC3a, human C3a; h453-TAS, mAb h453 coupled to tresyl-activated Sepharose; hC21, [Gln65,Arg66]hC3a-(57-77); hC21dR, des-Arg21-hC21; mAb, monoclonal antibody; PDMS, 252Cf plasma desorption mass spectrometry; FABMS, fast atom bombard-

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ment mass spectrometry. §To whom reprint requests should be addressed.

9848

Biochemistry: Suckau et al. Free Antigen

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Immune-Complex

NO

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Mass Spectrometric Peptide Mapping Antigen Non-Epitope Epitope Peptide 4%h Peptides

m/z

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~.

Proc. Nati. Acad. Sci. USA 87 (1990)

0.2 ml of TS. Pooled supernatant and remaining immune complexes after dissociation were subjected to HPLC purification on a 0.4 x 25 cm Nucleosil 300-7-C18 column (Macherey & Nagel). Elution was carried out with a linear gradient of 0-52% acetonitrile in water containing 0.04% trifluoroacetic acid over 25 min at a flow rate of 1 ml/min. Peptide fractions were detected with an M490 multiwavelength detector (Waters), lyophilized, and redissolved in 5 ,ul of 0.1% trifluoroacetic acid for mass spectrometric analysis. Isolation of Epitope Peptides from des-Arg78-gpC3a. Sixty micrograms of des-Arg78-gpC3a was allowed to bind to 500 ,g of immobilized h453 for 1 hr at 20'C. The gel was rinsed with TS, and samples equivalent to 6.4 gg of bound antigen were incubated for 30 min at 370C with 50 gl of TS containing 0, 5, or 45 /ig of trypsin, After removal of supernatant and treatment three times with 1 ml of TS, dissociation and HPLC analysis were performed as described above. Mass Spectrometry. Nitrocellulose surfaces for sample adsorption in PDMS were prepared as described (24). Peptide solutions were allowed to adsorb for 2-3 min followed by spin drying (25). Spectra were obtained on a time-of-flight spectrometer (Bio-Ion 20 K, Uppsala, Sweden) at a 15-kV accelerating voltage. FABMS was performed on a Finnigan (San Jose, CA) MAT-312/AMD-5000 double-focusing spectrometer, with a 20-kV cesium primary ion source (AMD, Beckeln, F.R.G.); glycerol was used as a matrix for the sample.

m/z

FIG. 1. Scheme of the mass spectrometric epitope mapping method of an antigen-antibody complex as compared to peptide mapping of the free antigen. The use of an immobilized antibody (mAb) allows the separation of nonepitope and epitope peptides after limited proteolysis of the immune complex. Molecular ions of nonepitope and epitope proteolytic peptides are illustrated by solid and open bars, respectively.

MATERIALS AND METHODS Preparation of Antigens and Immobilized Antibody. The peptide hC21, which has the sequence [Gln65,Arg661hC3a(57-77) was prepared by solid-phase synthesis (21) and contained a Cys'-S-acetamidomethyl protecting group. gpC3a was isolated and purified as described (22). The desarginated forms, des-Arg21-hC21 (hC2ldR) and des-Arg78-gpC3a, were prepared by carboxy-peptidase B treatment (23) and were purified by HPLC, which yielded -95% purity for des-ArgC3a. mAb h453 (20) was affinity purified using protein A-Sepharose (Pharmacia) and was coupled to tresylactivated Sepharose (Pharmacia) according to the supplier's procedures. The immobilized mAb (h453-TAS) was stored in phosphate-buffered saline containing 0.03% sodium azide at a concentration of 800 ,ug of bound mAb per ml of gel

RESULTS AND DISCUSSION Proteolytic Characterization of Free Antigen and the Antibody. The structure and purity of hC21dR was verified by FABMS of the intact peptide and by direct analysis of the mixture of tryptic peptides, which yielded abundant protonated molecular ions [M + H]+ for peptides T1, T3, and T4 (Fig. 2). Relative cleavage rates with trypsin were determined at conditions of limited proteolysis and showed rapid hydrolysis of peptide bonds at Arg'0 and Arg13 and a somewhat slower cleavage at Arg8 (data not shown). However, this reaction scheme is significantly different upon binding of the antibody as described below. In contrast, the antibody revealed a remarkable resistance towards proteolytic degradation. No cleavage of mAb h453 I

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Preparation and Dissociation of Immune Complexes. h453TAS (1 ml) was equilibrated with TS buffer (50 mM Tris HCI, pH 7.5/150 mM NaCI), and 50 ,g of hC21dR in 0.5 ml of TS was allowed to bind for 2 hr at 20°C. Free antigen was removed by three washes each with 1 ml of TS, and the gel volume was adjusted to 1 ml. Aliquots were used either for proteolysis or for direct dissociation by addition of 0.4 ml of 4 M MgCl2. Dissociation was allowed to proceed for 30 min at 37°C. The gel was then washed twice with 0.15 ml of 4 M MgCI2, and the pooled supernatant was subjected to HPLC analysis. Proteolytic Digestion and Purification of hC21dR Fragments. The immune complex of hC21dR and h453-TAS was digested at 37°C with trypsin (Sigma; see Table 1). The gel was chilled on ice, separated from supernatant by centrifugation (2000 x g, 4°C, 3 min), and washed three times with

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800

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FIG. 2. Amino acid sequence and FABMS analysis of tryptic peptides of the synthetic antigen hC21dR. Labeled peaks denote [M + H]' ions of peptide fragments T1, T3, and T4. The sequence shown represents [GIn65, Arg66]hC3a-(57-76). The stippled partial sequence represents the synthetic octapeptide used for production of the mAb h453. AAM, S-acetamidomethyl.

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Biochemistry: Suckau et al.

Proc. Natl. Acad. Sci. USA 87 (1990)

Table 1. Quantification of tryptic peptides isolated from h453-TAS-bound hC21dR at different proteolysis conditions mAb-bound

Trypsin,

hC21dR,

:S (wt/wt) hC21dR* h453-TAS 1:33 1:4400 1:440 1:3.3 3:1 1:44 6:1 1:18 15:1 1:7

Reaction time, min 20 20 20 10 120

% tryptic peptide

rycpepe

,ug Tlt T4 +T3/T4§ 1.67 80 ND 1.67 90 68 5 1.67 90 68 20 3.25 97 65 -11 50 -11 3.25 ND, not determined. *E:S for mAb-bound antigen. tExpressed as the molar percentage of mAb-bound-hC21dR as determined by HPLC. tPeptide T1 from supernatant. §Sum of peptides T4 and T3/T4 from MgCI2 dissociation. 1Estimated by ratio of [M + H]+ ion abundances in PDMS. "Not determined due to extensive digestion of mAb.

lug 0.05 0.05

was detectable by SDS/PAGE at conditions that provided complete proteolysis of the antigen [enzyme-to-substrate ratio (E:S) = 1:100 (wt/wt)]. The native mAb was not degraded, even at high protease concentrations (E:S = 1:2), whereas heat denaturation led to rapid proteolytic digestion. A comparable stability of the mAb was found towards a-chymotrypsin and Staphylococcus aureus V8 protease, as previously reported for different mAbs (6, 7), suggesting a similar utility of these enzymes for peptide mapping analysis of immune complexes. Molecular weight determination of intact gpC3a and desArg78-gpC3a by PDMS and mass spectrometric analysis of tryptic peptide mixtures provided structural information concerning the entire gpC3a sequence (26). Particularly, [M + H]+ ions of the N-terminal tryptic peptides and peptides containing residues 66-70 or residues 71-77 from the C terminus of des-Arg78-gpC3a were identified in high abundances, indicating complete cleavage at Arg65 and Arg70, which is in contrast to the epitope mapping discussed below (see Fig. 5). At high E:S (1:20), additional molecular ions of large polypeptide fragments could be assigned to partial digestion in the central part of the C3a sequence (data not shown). Formation and Dissociation of Immune Complexes. The binding capacity and specificity of h453-TAS, the dissociation procedure, and recovery ofintact antigen were evaluated with the aid of HPLC and mass spectral analysis. In a typical experiment, from 4 ,g of hC21dR bound to h453-TAS containing 75 ,g of mAb, 1.4 ,ug of antigen was recovered with TS buffer, and no free hC21dR was detectable by HPLC in the supernatant after additional TS treatment. Dissociation of the immune complex with MgCI2 led to the release of -1 jig of antigen (i.e., 40% of the theoretical binding capacity of the mAb). After HPLC purification, the antigen was identified as intact hC21dR by the [M + H]+ ion (m/z = 2382) and the doubly charged ion in the plasma desorption mass spectrum (see Fig. 4c). In a control experiment with Sepharose containing tresyl groups blocked by treatment with 0.1 M Tris-HCl/0.5 M NaCI, 2.4 ,ug of hC21dR was recovered in the TS supernatant, and no peptide was detectable in the MgCl2 eluate. In addition, the specificity of h453-TAS was tested with a mixture of partial tryptic peptides of hC21dR and exclusively yielded peptides in the MgCl2 eluate that contained the antigenic C terminus (data not shown). The binding and dissociation procedure could be performed repeatedly with the same batch of h453-TAS without loss of binding capacity, indicating the feasibility of the lyotropic agent MgCI2 (27) for efficient dissociation of the immobilized immune complex without affecting the mAb's function. Identification of Tryptic Epitope Peptides from mAb-Bound hC21dR. Samples of the purified immune complex of hC21dR

Ratio of T4 to T3/T49

1:5 2:1 13:1

and h453-TAS were subjected to trypsin digestion using a wide range of enzyme-to-hC21dR ratios and reaction times (Table 1). Under various digestion conditions, HPLC analyses of supernatants and MgC12 eluates consistently yielded major peptide fractions with retention times of 26.2 and 25.1 min, respectively (Fig. 3). In addition, nonpeptide contaminant components were present in the chromatograms at 21, 23.3, and 24.1 min. PDMS analysis of the purified peptides contained in the supernatant (i.e., nonepitope fraction) yielded a predominant [M + H]+ ion (m/z = 1083) of fragment T1 due to cleavage at Arg8 (Fig. 4b). By contrast, the spectrum of the fraction after MgC12 dissociation (Fig. 4a) showed a most abundant [M + H]+ ion of the C-terminal peptide T4 (m/z, = 669), together with a small ion of the coeluting peptide T3/T4 (m/z, = 1033), which was unresolved by HPLC. An =90% release of T1 from mAb-bound hC21dR in the supernatant and 65% release of T4 and T3/T4 from the truncated immune complex were found with E:S as

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FIG. 3. HPLC analysis of supernatant (traces d-f) and MgCI2dissociated peptide fragments (traces a-c) after trypsin digestion of h453-TAS-bound hC21dR, at different protease concentrations and reaction times (see Table 1). HPLC conditions were as described in Materials and Methods. Trace a, 20 ,ug of trypsin for 10 min; traces b and d, 5 ,ug of trypsin for 20 min; traces c and e, 0.5 jg of trypsin for 20 min; trace f, 0.05 ,ug of trypsin for 20 min. Unlabeled peaks in the chromatograms are due to nonpeptide contamination.

Proc. Natl. Acad. Sci. USA 87 (1990)

Biochemistry: Suckau et al. r

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FIG. 5. PDMS analysis of tryptic epitope peptides dissociated from the truncated immune complex of des-Arg78-gpC3a with the mAb h453 (E S = 6.5:1). Numbers in parentheses denote partial sequences of gpC3a; the ion at m/z = 1057 (70-78) originates from traces of gpC3a present. The C-terminal sequence of des-Arg78gpC3a is shown. The C-terminal tryptic cleavage sites in the free antigen (Arg70, open arrowhead) and in the immune complex (solid arrowhead) are indicated.

M2+

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m/z

FIG. 4. PDMS analysis of HPLC-purified hC21dR and tryptic peptides from the immune complex with h453-TAS. (a) Epitope fraction at 25.1 min in trace a of Fig. 3. (b) Nonepitope peptide fraction at 26.2 min in trace d of Fig. 3c. (c) Intact hC21dR after MgCI2 dissociation from the immune complex. M2' denotes the doubly protonated molecular ion (m/z = 1191); the ion at m/z = 794 is due to a nonpeptide contaminant.

high as 6:1 (see Table 1). At very high protease concentrations, specific peptide fragments were no longer detectable because of progressive destruction of the mAb. Low E:S led to the formation of increasing amounts of T3/T4 relative to T4 as estimated by their [M + H]+ ion abundances, indicating a reduced tryptic cleavage rate at Arg'3 relative to Arg8 due to steric hindrance by the mAb bound to its epitope. However, at any proteolytic conditions amenable to PDMS peptide mapping analysis and irrespective of nonpeptide contaminations, the epitope peptides T4 and T3/T4 were the only peptide fragments in the MgCI2 eluate. In contrast, some contamination of the nonepitope fraction by epitope peptides was observed, due to release of fragments from hC2ldR nonspecifically bound to the polypropylene cup surface. Only the epitope fraction was, therefore, used for the epitope identification of gpC3a. Epitope Identification from gpC3a. Proteolytic procedures and PDMS analysis were applied in the same manner to epitope mapping of des-Arg-gpC3a bound to h453-TAS. On the basis of the results obtained with the immune complex of hC21dR with h453-TAS, digestion was performed at two different trypsin concentrations that did not degrade the mAb while fragmenting C3a (enzyme-to-C3a ratios, 6.5:1 and 1:1.5). In all experiments, HPLC analysis yielded a single major peptide fraction at 23.8 min upon MgCI2 dissociation. PDMS analysis (Fig. 5) showed a predominant [M + H]+ ion (m/z = 901) of the C-terminal fragment containing residues

70-77, associated with minor ions of the peptides containing residues 66-77 and residues 70-78 of gpC3a, due to incomplete cleavage at Arg69 and a small amount of nondesarginated gpC3a, respectively. These peptides clearly can be named epitope peptides since they all contain the sequence motif Leu-Gly-Leu-Ala, which is part of the epitope sequence (see Fig. 2 and the sequence in Fig. 5). In contrast to the cleavage at Arg70 found by tryptic digestion of free gpC3a (26), cleavage of the mAb-bound antigen occurred exclusively at Arg69. As noted above, this effect is in accordance with the steric requirements of trypsin binding (28) and with inaccessibility of Arg70 due to binding of the mAb to its epitope approximately three amino acid residues downstream.

Moreover, ELISA data yielded a 30-fold lower affinity of h453 for des-Arg78-gpC3a as compared to des-Arg77-hC3a (data not shown), which strongly indicates sequence variation within the epitope region. In addition to the sequence motif Leu-Gly-Leu-Ala, which is conserved in both species and is responsible for the immunological cross-reactivity of h453, the epitope on des-Arg77-hC3a must therefore contain one or both of the evolutionary variant residues, His72 and Ser71. On the other hand, the weak exopeptidase activity of trypsin and crystal structure data of trypsin inhibitor complexes (28) indicate binding approximately two residues downstream as a prerequisite to tryptic cleavage at Arg69 in the gpC3a sequence. Supported by these additional data, the mass spectrometric results provide evidence for His-Leu-Gly-Leu-Ala [i.e., hC3a-(72-76)] being the epitope recognized by h453, with Ser71 as a possible part of the epitope sequence. CONCLUSIONS

described in this study presents several advantages to the molecular characterization of epitopes by combining the selectivity of partial proteolysis of immune complexes with the molecular specificity of accurate mass The

approach

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Biochemistry: Suckau et al.

spectrometric molecular weight determination. Irreversible immobilization of the mAb and analysis of the bound peptides after proteolytic digestion provide the basis to name the selectively dissociated fragments epitope peptides. Important prerequisites for accurately defining epitope structures are (i) high affinity of the proteolytic peptides (i.e., truncated antigen) to the mAb and (ii) the availability and accessibility of suitable proteolytic cleavage sites. Despite some suppression by the mAb, tryptic cleavage was shown to occur approximately two amino acid residues away from the epitope. At least two other endoproteases, a-chymotrypsin and S. aureus V8 protease, appear to be well suited and could be supplemented by exopeptidases to further define epitope sequences. Epitope extraction and direct identification of peptide fragment mixtures by PDMS was shown to be applicable to the analysis of a sequential epitope and may enable the characterization of even conformationally defined and assembled topographic epitopes, which are not detectable by synthetic peptides as antigenic probes. In the course of an exact description of an epitope (e.g., using site-directed mutagenesis), its application will greatly reduce the number of possible antigenic residues to be tested. Thus, epitope extraction and mass spectrometric peptide mapping of a single immune complex represents a sensitive and rapid method of high molecular specificity in the analysis of protein

antigens. We thank R. Burger for the generous gift of the mAb h453 and R. Gerardy-Schahn for stimulating discussions. This work was supported by grants from the Deutsche Forschungsgemeinschaft, Bonn, F.R.G. (Pr 175/2), by the Bundesministerium fur Forschung und Technologie, Bonn (01VM89014), and by the University of Konstanz. 1. 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., SmithGill, S. J., Todd, P. E. & Wilson, A. C. (1984) Annu. Rev. Immunol. 2, 67-101. 2. Berzofsky, J. A. (1985) Science 229, 932-940. 3. Schwyzer, M., Weil, R., Frank, G. & Zuber, H. (1980) J. Biol. Chem. 255, 5627-5634. 4. Moelling, K., Scott, A., Dittmann, K. E. J. & Owada, M. (1980) J. Virol. 33, 680-688. 5. Eisenberg, R. J., Long, D., Pereira, L., Hampar, B., Zweig, M. & Cohen, G. H. (1982) J. Virol. 41, 478-488. 6. Jemmerson, D. & Paterson, Y. (1986) Science 232, 1001-1004.

Proc. Natl. Acad. Sci. USA 87 (1990) 7. Sheshberadaran, H. & Payne, L. G. (1988) Proc. Natl. Acad. Sci. USA 85, 1-5. 8. Biemann, K. & Martin, S. A. (1987) Mass Spectrom. Rev. 6, 1-75. 9. Cotter, R. J. (1988) Anal. Chem. 60, 781A-793A. 10. Suckau, D., Manz, I., Schneider, K., Przybylski, M., Thomas, H., Milbert, U., Klein, J., Post, K. & Oesch, F. (1989) Adv. Mass Spectrom. 11, 492-493. 11. Gauss, C., Klein, J., Post, K., Suckau, D., Schneider, K., Thomas, H., Oesch, F. & Przybylski, M. (1990) Environ. Health Perspect. 88, 57-62. 12. Svoboda, M., Przybylski, M., Schreurs, J., Miyajima, A., Hogeland, K. & Deinzer, M. (1990) J. Chromatogr., in press. 13. Hugh, T. E. (1986) Complement 3, 111-127. 14. Hugh, T. E. (1975) J. Biol. Chem. 250, 8293-8301. 15. Gerard, N. P., Lively, M. 0. & Gerard, C. (1988) Protein Sequences Data Anal. 1, 473-478. 16. Huber, R., Scholze, H., Paques, E. P. & Deisenhofer, J. (1980) Hoppe-Seyler Z. Physiol. Chem. 361, 1389-1399. 17. Chazin, W. J., Hugh, T. E. & Wright, P. E. (1988) Biochemistry 27, 9139-9148. 18. Gerardy-Schahn, R., Ambrosius, D., Saunders, D., Casaretto, M., Mittler, C., Karwarth, G., Gorgen, S. & Bitter-Suermann, D. (1989) Eur. J. Immunol. 19, 1095-1102. 19. Kohl, J., Casaretto, M., Gier, M., Karwath, G., Gietz, C., Bautsch, W., Saunders, D. & Bitter-Suermann, D. (1990) Eur. J. Immunol. 20, 1463-1468. 20. Burger, R., Zilow, G., Bader, A., Friedlein, A. & Naser, W. (1988) J. Immunol. 141, 553-558. 21. Ambrosius, D., Casaretto, M., Gerardy-Schahn, R., Saunders,

D., Brandenburg, D. & Zahn, H. (1989) Biol. Chem. HoppeSeyler 370, 217-227. 22. Hoffmann, T., Bottger, E. C., Baum, H. P., Messner, M., Hadding, U. & Bitter-Suermann, D. (1988) Clin. Exp. Immunol. 71, 487-492. 23. Gerardy-Schahn, R., Ambrosius, D., Casaretto, M., Grotzinger, J., Saunders, D., Wollmer, A., Brandenburg, D. & Bitter-Suermann, D. (1988) Biochem. J. 255, 209-216. 24. Jonsson, G. P., Hedin, A. B., Hakansson, P. L., Sundqvist, B. U. R., Save, B. G. S., Nielsen, P. F., Roepstorff, P., Johansson, K. E., Kamensky, 1. & Lindberg, M. S. L. (1986) J. Anal. Chem. 58, 1084-1087. 25. Nielsen, P. F., Klarskov, K., Hojrup, P. & Roepstorff, P. (1988) Biomed. Environ. Mass Spectrom. 17, 355-362. 26. Schneider, K., Manz, 1., Messner, M., Gerardy-Schahn, R., Bitter-Suermann, D. & Przybylski, M. (1988) Adv. Mass Spectrom. 11, 1338-1339. 27. Kessler, S. W. (1975) J. Immunol. 115, 1617-1624. 28. Walter, J. & Bode, W. (1983) Hoppe-Seyler Z. Physiol. Chem. 364, 949-959.