Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: Demonstration with region 56–62 (antigenic site 2) of myoglobin

This work was carried out in order to study the effects of substitutions outside antigenic site 2 of sperm whale myoglobin (SpMb) on the reactivity of protein variants with antisite 2 monoclonal antibodies (mAbs). A synthetic peptide corresponding to region 56–62 (site 2) of SpMb was used as an immunogen in mice in its free form (i.e., without coupling to any carrier) to prepare a panel of mAbs whose predetermined specificity is directed, by design, against this region. The binding of three of these mAbs to eight Mbs from different species was studied. Myoglobins of Pacific common dolphin, finback whale, and horse, which have no substitutions within region 56–62 relative to SpMb, showed remarkable differences in their cross-reactivities and relative affinities with each of the mAbs. Myoglobins of badger, chicken, and dog, although they have an identical substitution within the site (Ala-57 to Gly), exhibited cross-reactivities with a given mAb that were affected differently. Echidna Mb, which has one replacement (Glu-59 to Ala) within region 56–62, displayed greatly reduced cross-reactivities and relative binding affinities. The results, especially those from Mbs that have no substitutions within the boundaries of site 2, clearly indicate that substitutions outside site 2 of Mb can exert drastic effects on the binding of the Mb variants with mAbs whose specificity was predesigned to be against the site. These indirect effects and their impact on site reactivity will completely explain previous findings on cross-reactivities of Mb variants with mAbs of unknown specificity and will rule out the postulations of discontinuous sites in Mb, which were based on the assumption that every substitution affecting reactivity is directly involved in binding to antibody.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 15-22 (antigenic site 1) of myoglobin.

Monoclonal antibodies (mAbs) of predetermined specificity were prepared by immunizing with a free (i.e., not conjugated to any carrier) synthetic peptide representing region 15–22 (site 1) of sperm whale myoglobin (SpMb). The cross-reactions of Mb variants with three mAbs were studied in order to determine whether such interactions are influenced by substitutions outsde the site. Finback whale Mb, which has no substitutions within region 15–22, showed lower cross-reactivity and relative binding affinity than the reference antigen, SpMb. Bottle-nose Atlantic dolphin myoglobin (BdMb) and badger myoglobin (BgMb), although they have identical substitutions within region 15–22 (Ala-15 to Gly and Val-21 to Leu), showed very different binding properties. The cross-reaction of BdMb was quite comparable to that of SpMb, while that of BgMb was much lower. Since the two proteins have identical structures in regions 15–22, the differences in their cross-reactivities are readily attributed to the effects of substitutions outside this region. Another pair of myoglobins, horse myoglobins (HsMb) and chicken myoglobin (ChMb), also have two identical substitutions (Ala-15 to Gly and Val-21 to Ile) within region 15–22, but possessed different cross-reactivity. The results indicate that the reaction of mAbs, whose specificity is precisely known and predetermined by the immunizing free peptide, can be markedly affected by substitutions outside the indicated binding region on the protein.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 94-100 (antigenic site 3) of myoglobin.

Amino acid substitutions outside protein antigenic sites are very frequently assumed to exert no effect on binding to antiprotein antibodies, especially if these are monoclonal antibodies (mAbs). In fact, a very popular method for localization of residues in protein antigenic sites is based on the interpretation that whenever a replacement causes a change in binding to antibody, then that residue will be located in the antigenic site. To test this assumption, mAbs of predetermined specificity were prepared by immunization with a free (i.e., without coupling to any carrier) synthetic peptide representing region 94–100 of sperm whale myoglobin (Mb). The cross-reactivities and relative affinities of three mAbs with eight Mb variants were studied. Five Mb variants which had no substitutions within the boundaries of the designed antigenic site exhibited remarkable, and in two cases almost complete, loss in cross-reactivity relative to the reference antigen, sperm whale Mb. Two myoglobins, each of which had one substitution within region 94–100, showed little or no reactivity with the three mAbs. It is concluded that substitutions outside an antigenic site can exert drastic effects on the reactivity of a protein with mAbs against the site and that caution should be exercised in interpreting cross-reactivity data of proteins to implicate residues directly in an antigenic site.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: Demonstration with region 145–151 (antigenic site 5) of myoglobin

Monoclonal antibodies of predetermined specificity were prepared by immunization with a free (i.e., without coupling to any protein carrier) synthetic peptide representing region 145–151 of sperm whale myoglobin (SpMb) and their cross-reactions with eight Mb variants were determined. Five Mbs—bottle-nose dolphin myoglobin (BdMb), pacific common dolphin myoglobin (PdMb), horse myoglobin (HsMb), dog myoglobin (DgMb), and badger myoglobin (BgMb)—have an identical sequence in that region. Nevertheless, these Mbs exhibited very different cross-reactivities. BdMb and PdMb exhibited cross-activities which were comparable to that of the reference antigen, SpMb; while the reactivity of HsMb was remarkedly decreased, DgMb and BgMb showed almost no cross-reactions with these mAbs. Since the region 145–151 has an identical sequence in all the five Mbs, it is concluded that the differences in their antigenic reactivities with anti-region 145–151 mAbs are due to the effects of amino acid substitutions outside the region 145–151. Another pair of myoglobins, echidna myoglobin (EdMb) and chicken myoglobin (ChMb), have the same sequence in that region, but reacted very differently with anti-region 145–151 mAbs. The reactivity and affinity of EdMb were substantially decreased while those of ChMb were almost completely absent, relative to SpMb. It is concluded, contrary to popular assumptions, that when an amino acid substitution influences the binding of a protein variant to a mAb, it is not necessary for that substitution to be an actual contact residue (i.e., a residue within the antigenic site where the mAb binds). Such effects, which are often very drastic, could be due to indirect influences of the substitution on the chemical and binding properties of the site residues. Furthermore, residues which had been postulated, on the basis of these assumptions, to constitute discontinuous antigenic sites in SpMb, were found [from the present studies and those recently reported with mAbs against the other four antigenic site of Mb (regions 15–22, 56–62, 94–100, and 113–120 of SpMb)] to merely be exerting indirect effects on the known five antigenic sites of Mb. The effects of substitutions, which can happen even in the absence of conformational changes, are determined by many factors, such as the chemical nature of the substitution, its environment, its distance from the site, and the nature of the site residue(s) being affected.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: Demonstration with region 113–120 (antigenic site 4) of myoglobin

Immunochemical cross-reactivity of protein variants has been very frequently used to map protein antigenic sites. The approach is based on the assumption that amino acid substitutions affecting the binding of a protein to its antibody, particularly when monoclonal antibodies (mAbs) are used, must be part of the antigenic site and not far from it. The assumption was investigated in this study by determining the effects of amino acid substitutions outside the antigenic site on the reactivity of six myglobin (Mb) variants with three mAbs of predetermined specificity prepared by immunization with a free synthetic peptide representing region 113–120 (antigenic site 4) of Mb. Two of the Mb variants used had no substitutions within residues 113–120 (the region to which the specificity of the mAbs is directed) and yet exhibited markedly decreased cross-reactions and binding affinities, relative to the reference antigen, sperm-whale Mb. The other three Mb variants possessed substitutions within, as well as outside, region 113–120 and showed very little cross-reactivities. The results of this study, particularly with the Mbs that have no substitutions within the indicated antigenic site, clearly show that substitutions outside the site, and which by design are not part of the site, can influence very markedly the reactivity of the protein variant with the anti-site mAbs. The approach can, therefore, lead to serious errors if used to identify residues of protein antigenic sites.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: Demonstration with region 15–22 (antigenic site 1) of myoglobin

Monoclonal antibodies (mAbs) of predetermined specificity were prepared by immunizing with a free (i.e., not conjugated to any carrier) synthetic peptide representing region 15–22 (site 1) of sperm whale myoglobin (SpMb). The cross-reactions of Mb variants with three mAbs were studied in order to determine whether such interactions are influenced by substitutions outsde the site. Finback whale Mb, which has no substitutions within region 15–22, showed lower cross-reactivity and relative binding affinity than the reference antigen, SpMb. Bottle-nose Atlantic dolphin myoglobin (BdMb) and badger myoglobin (BgMb), although they have identical substitutions within region 15–22 (Ala-15 to Gly and Val-21 to Leu), showed very different binding properties. The cross-reaction of BdMb was quite comparable to that of SpMb, while that of BgMb was much lower. Since the two proteins have identical structures in regions 15–22, the differences in their cross-reactivities are readily attributed to the effects of substitutions outside this region. Another pair of myoglobins, horse myoglobins (HsMb) and chicken myoglobin (ChMb), also have two identical substitutions (Ala-15 to Gly and Val-21 to Ile) within region 15–22, but possessed different cross-reactivity. The results indicate that the reaction of mAbs, whose specificity is precisely known and predetermined by the immunizing free peptide, can be markedly affected by substitutions outside the indicated binding region on the protein.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: Demonstration with region 94–100 (antigenic site 3) of myoglobin

Amino acid substitutions outside protein antigenic sites are very frequently assumed to exert no effect on binding to antiprotein antibodies, especially if these are monoclonal antibodies (mAbs). In fact, a very popular method for localization of residues in protein antigenic sites is based on the interpretation that whenever a replacement causes a change in binding to antibody, then that residue will be located in the antigenic site. To test this assumption, mAbs of predetermined specificity were prepared by immunization with a free (i.e., without coupling to any carrier) synthetic peptide representing region 94–100 of sperm whale myoglobin (Mb). The cross-reactivities and relative affinities of three mAbs with eight Mb variants were studied. Five Mb variants which had no substitutions within the boundaries of the designed antigenic site exhibited remarkable, and in two cases almost complete, loss in cross-reactivity relative to the reference antigen, sperm whale Mb. Two myoglobins, each of which had one substitution within region 94–100, showed little or no reactivity with the three mAbs. It is concluded that substitutions outside an antigenic site can exert drastic effects on the reactivity of a protein with mAbs against the site and that caution should be exercised in interpreting cross-reactivity data of proteins to implicate residues directly in an antigenic site.     

Amino acid substitutions outside a preselected antigenic region in hemoglobin affect the binding to monoclonal antibodies obtained by immunization with the synthetic region

It is often assumed that amino acid substitutions outside a protein antigenic site have no effect on the reactivity of a protein variant with antibodies, especially monoclonal antibodies (mAbs). Substitutions that exert an effect on the reactivity of a protein variant with mAbs are frequently considered to be within the antigenic site of the mAb. To test this assumption, two mAbs [IgG_l(k) and IgG_2a (k)] were prepared by immunization with a synthetic peptide corresponding to region 63–78 of the chain of human hemoglobin (Hb). The peptide was used as an immunogen in its free form (i.e., without conjugation to a carrier), so that the results will not be made ambiguous by peptide modification nor by an immune response to sites spanning peptide and protein carrier. In addition to their reaction with human Hb, the mAbs were also studied with four primate Hbs which had no substitutions within region 63–78 and only a limited number of substitutions which occurred outside of, and at considerable distances in three-dimensional (3D) structure from, this region. Inhibition studies revealed substantial differences in the binding affinities of some of the primate Hbs, relative to human Hb. Some of the substitutions caused major decreases in binding, although they were at considerable distances in the 3D structure from the indicated site residues. It is concluded that substitutions in a protein, even when distant from an antigenic site, can exert major influences on the protein's reactivity with anti-site mAbs.     

1H-NMR comparative study of the active site in shark (Galeorhinus japonicus), horse, and sperm whale deoxy myoglobins.

1H-NMR spectra of deoxy myoglobins (Mbs) from shark (Galeorhinus japonicus), horse, and sperm whale have been studied to gain insights into their active site structure. It has been demonstrated for the first time that nuclear Overhauser effect (NOE) can be observed between heme peripheral side-chain proton resonances of these paramagnetic complexes. Val-E11 methyl and His-F8 C delta H proton resonances of these Mbs were also assigned from the characteristic shift and line width. The hyperfine shift of the former resonance was used to calculate the magnetic anisotropy of the protein. The shift analysis of the latter resonance, together with the previously assigned His-F8 N delta H proton resonance, revealed that the strain on the Fe-N epsilon bond is in the order horse Mb approximately whale Mb < shark Mb and that the hydrogen bond strength of the His-F8 N delta H proton to the main-chain carbonyl oxygen in the preceding turn of the F helix is in the order shark Mb < horse Mb < whale Mb. Weaker Feporphyrin interaction in shark Mb was manifested in a smaller shift of the heme methyl proton resonance and appears to result from distortion of the coordination geometry in this Mb. Larger strain on the Fe-N epsilon bond in shark Mb should be to some extent attributed to its lowered O2 affinity (P50 = 1.1 mmHg at 20 degrees C), compared to whale and horse Mbs.     

Common structural features among monoclonal antibodies binding the same antigenic region of cytochrome c.

To examine if there are common physicochemical features among antibodies binding the same antigenic region of a protein, B cell hybridomas were prepared against the two major antigenic regions on mammalian cytochromes c, and the nucleotide sequences encoding the monoclonal antibody (mAb) heavy (H) and light (L) chains were determined and compared. Although the genetic elements used were somewhat diverse, similarities among mAbs to a given antigenic region were observed. In particular, mAbs binding in a region situated at a bend in the antigen around residues 44 and 47 had longer complementarity-determining regions (4-5 additional amino acid residues in L1 and 1-2 in H3) than mAbs binding the other region around residues 60 and 62 located on a relatively flat surface. These observations indicate that the topography of an antigenic site and the lengths of certain complementarity-determining regions are important physicochemical properties determining, at least in part, which antibodies (B cells) will participate in an immune response to a particular site on a protein antigen.     

Haloperidol binding to monoclonal antibodies. Predictions of three-dimensional combining site structure via computer modeling

The amino acid sequences of five monoclonal antibodies (designated mAbs A-E) which bind to the dopaminergic D-2 antagonist, haloperidol, with a variety of affinities (Kd = 4-810 nM), have been used to build theoretical, three-dimensional, computer models of the variable region combining sites. Physiocochemical interactions which have been previously determined from in vitro binding data have been used to orient the drug molecule within the combining site model. The results indicate that hydrophobic, aromatic, and ionic amino acids are involved in specific interactions with the antagonist molecule. For example, fluorescence quenching data suggests that a tryptophan residue is intimately involved in the binding of haloperidol by mAb A. Examination of the modeled structure reveals five tryptophans within the variable fragment, only one of which (H-50) is within the classical beta-barrel binding pocket and is readily accessible to the antigen. Haloperidol's relatively electron poor fluorophenyl ring system stacks with the electron-rich tryptophan ring system at a distance of 3.3 A and in so doing, places haloperidol's positively charged piperidinyl nitrogen atom within hydrogen bond distance of the negatively charged Glu-95 and Asp-100A residues of the H3 loop (Glu-H-95 and Asp-H-100A). This type of analysis for each antibody provides an interesting profile of changes in amino acid composition and hypervariable loop length which markedly effect binding affinity and specificity for a series of proteins which have similar combining site.     

The immunoglobulin heavy chain constant region affects kinetic and thermodynamic parameters of antibody variable region interactions with antigen

A central dogma in immunology is that antibody specificity is a function of the variable (V) region. However serological analysis of IgG(1), IgG(2a), and IgG(2b) switch variants of murine monoclonal antibody (mAb) 3E5 IgG(3) with identical V domains revealed apparent specificity differences for Cryptococcus neoformans glucuronoxylomannan (GXM). Kinetic and thermodynamic binding properties of mAbs 3E5 to a 12-mer peptide mimetic of GXM revealed differences in the affinity of these mAbs for a monovalent ligand, a result that implied that the constant (C) region affects the secondary structure of the antigen binding site, thus accounting for variations in specificity. Structural models of mAbs 3E5 suggested that isotype-related differences in binding resulted from amino acid sequence polymorphisms in the C region. This study implies that isotype switching is another mechanism for generating diversity in antigen binding and that isotype restriction of certain antibody responses may reflect structural constraints imposed by C region on V region binding. Furthermore, isotype affected the polyreactivity of V region identical antibodies, implying a role for C region in determining self-reactivity.     

Gonococcal outer-membrane protein PIB: comparative sequence analysis and localization of epitopes which are recognized by type-specific and cross-reacting monoclonal antibodies.

Comparison of the inferred amino acid sequence of outer-membrane protein PIB from gonococcal strain P9 with those from other serovars reveals that sequence variations occur in two discrete regions of the molecule centred on residues 196 (Var1) and 237 (Var2). A series of peptides spanning the amino acid sequence of the protein were synthesized on solid-phase supports and reacted with a panel of monoclonal antibodies (mAbs) which recognize either type-specific or conserved antigenic determinants on PIB. Four type-specific mAbs reacted with overlapping peptides in Var1 between residues 192-198. Analysis of the effect of amino acid substitutions revealed that the mAb specificity is generated by differences in the effect of single amino acid changes on mAb binding, so that antigenic differences between strains are revealed by different patterns of reactivity within a panel of antibodies. The variable epitopes in Var1 recognized by the type-specific mAbs lie in a hydrophilic region of the protein exposed on the gonococcal surface, and are accessible to complement-mediated bactericidal lysis. In contrast, the epitope recognized by mAb SM198 is highly conserved but is not exposed in the native protein and the antibody is non-bactericidal. However, the conserved epitope recognized by mAb SM24 is centred on residues 198-199, close to Var1 , and is exposed for bactericidal killing.     

Altered target site specificity variants of the I-PpoI His-Cys box homing endonuclease

We used a yeast one-hybrid assay to isolate and characterize variants of the eukaryotic homing endonuclease I-PpoI that were able to bind a mutant, cleavage-resistant I-PpoI target or ‘homing’ site DNA in vivo. Native I-PpoI recognizes and cleaves a semi-palindromic 15-bp target site with high specificity in vivo and in vitro. This target site is present in the 28S or equivalent large subunit rDNA genes of all eukaryotes. I-PpoI variants able to bind mutant target site DNA had from 1 to 8 amino acid substitutions in the DNA–protein interface. Biochemical characterization of these proteins revealed a wide range of site–binding affinities and site discrimination. One-third of variants were able to cleave target site DNA, but there was no systematic relationship between site-binding affinity and site cleavage. Computational modeling of several variants provided mechanistic insight into how amino acid substitutions that contact, or are adjacent to, specific target site DNA base pairs determine I-PpoI site-binding affinity and site discrimination, and may affect cleavage efficiency.     

Monoclonal antibodies to Torpedo acetylcholine receptor. Characterisation of antigenic determinants within the cholinergic binding site

Thirteen monoclonal antibodies (mAb) to the acetylcholine receptor (AChR) from Torpedo marmorata showed high avidity for the receptor but none exhibited binding to muscle AChR solubilised from seven other animal species. Five mAb and Fab monomer fragments prepared from two of them, inhibited alpha-bungarotoxin (alpha BuTx) binding to receptor by a maximum of 50%. In the presence of excess mAb the 125I-alpha BuTx bound could be precipitated by anti-IgG indicating that the mAb bound to only one of the two alpha BuTx binding sites on each AChR monomer. This site appeared to have a lower affinity for d-tubocurarine and decamethonium than the non-mAb site. Binding of five anti-site mAb was mutually competitive and four of them (AS2-AS5) were inhibited by other cholinergic ligands and influenced by four non-toxin binding site antibodies. One (AS1) bound within the toxin binding site yet outside the main neurotransmitter binding region. It is concluded that these five mAb distinguish between the two alpha BuTx binding sites on the Torpedo AChR, and bind only to the site which displays lower affinity for d-tubocurarine and other competitive ligands.     

A neutralizing monoclonal antibody specific for the dimer interface region of IL-8

We have generated two mAbs, 6G4.2.5 and A5.12.14, that are similarly capable of neutralizing the biologic activity of wild-type IL-8. To characterize these antibodies further, their reactivity against a series of engineered IL-8 monomer and dimer variants was examined using a neutrophil degranulation assay. While 6G4.2.5 was found to block effectively the biologic activity of all variants regardless of their dimerization status, the results for A5.12.14 differed dramatically. A5.12.14 fully inhibited the agonist activity of one of the monomer variants, partially blocked the activity of another, and had no effect on the activity of two other variants. These results suggested that the binding epitope of A5.12.14 was being affected by the particular amino acid substitutions introduced into the dimer interface region of the variants to disfavor dimerization. If A5.12.14 indeed binds to the dimer interface region of IL-8, it could be predicted that this mAb would be unable to inhibit the activity of dimeric IL-8. This was confirmed in studies which showed that A5.12.14 had no demonstrable effect on the activity of a constitutively dimeric IL-8 variant. These studies represent the first example of a mAb specific for the dimerization status of IL-8.     

The tyrosine phosphorylation site of the acetylcholine receptor beta subunit is located in a highly immunogenic epitope implicated in channel function: antibody probes for beta subunit phosphorylation and function.

Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) seems to be involved in AChR desensitization and localization on the postsynaptic membrane. This study reveals a probable function of the single known beta subunit phosphorylation site (beta Tyr355) and provides suitable tools for its study. The epitopes for 15 monoclonal antibodies (mAbs) against the cytoplasmic side of the AChR beta subunit were precisely mapped using > 100 synthetic peptides attached on polyethylene rods. Eleven mAbs bound to a very immunogenic cytoplasmic epitope (VICE-beta) on Torpedo beta 352-359, which contains the beta Tyr355, and to the corresponding sequence of human AChR. The contribution of each VICE-beta residue to mAb binding was then studied by peptide analogues having single residue substitutions. Overall, each of the residues beta 354-359, including beta Tyr355, proved critical for mAb binding. Two of our four mAbs known to block the ion channel were found to bind at (mAb148) or close (mAb10) to VICE-beta. Tyrosine phosphorylation of Torpedo AChR by endogenous kinase(s) selectively reduced binding of some VICE-beta mAbs, including the channel blocking mAb148. We conclude that VICE-beta probably plays a key role in AChR function. Elucidation of this role should be facilitated by the identified mAb tools.     

Amino Acid Residues Within the Sequence Region α55–74 of Torpedo Nicotinic Acetylcholine Receptor Interacting with Antibodies to the Main Immunogenic Region and with Snake α-Neurotoxins

Abstract

The sequence region 55–74 of the α-subunit of the acetylcholine receptor (AChR) from Torpedo californica electroplax comprises the amino-terminal end of a sequence segment—residues α67–76—forming the main immunogenic region (MIR), which is most frequently recognized by anti-AChR autoantibodies in myasthenia gravis. The synthetic sequence α55–74 of Torpedo AChR binds α-bungarotoxin (αBTX), suggesting that amino acid residues within this sequence region may contribute to formation of an αBTX binding site.

Using single-residue substituted synthetic analogues of the sequence α55–74 of Torpedo AChR, in which each residue was sequentially substituted by either glycine or alanine, we sought identification of the amino acids involved in interaction with α-neurotoxins and with three different anti-MIR monoclonal antibodies (mAbs 6, 22, and 198). Substitution of Arg₅₅, Arg₅₇, Trp₆₀, Arg₆₄, Leu₆₅, Arg₆₆, Trp₆₇, or Asn₆₈ strongly inhibited α-toxin binding, whereas substitutions of Ile₆₁, Val₆₃, Pro₆₉, Ala₇₀, Asp₇₁, or Tyr₇₂ had marginal effects. Substitutions within the region α68–72 significantly diminished binding of anti-MIR mAbs, although residue preferences differed among mAbs. Further, substituting Trp₆₀ substantially reduced binding of mAb 198, and moderately affected binding of mAb 6, and substitution of Asp₆₂ slightly but consistently affected binding of mAbs 6 and 22.     

Mutational analysis of two stefin A epitopes.

Stefin A, an intracellular inhibitor of cysteine proteinases, is expressed most abundantly in epithelial cells and in cells of lymphatic origin. In order to study its role in normal and pathological conditions we have prepared and characterized monoclonal antibodies against recombinant stefin A. Two high affinity monoclonal antibodies (mAbs) (A22 and C52) were tested for binding to free and papain-complexed stefin A and to a chimeric inhibitor, consisting of 61 amino acid residues of stefin A and 37 carboxy-terminal residues of stefin B. mAb A22 recognized not only free stefin A but also stefin A in complex with papain. The mAbs were further tested for their cross-reactivity against stefin A and B isolated from different mammalian species. On the basis of sequence similarity and tertiary structure of human stefin A we have prepared three mutants - Glu33Lys, Asp61Gly and Asn62Tyr and their reactivity with the mAbs was tested. The binding affinities of mAb A22 for the Asp61Gly and Asn62Tyr mutants were significantly lower, indicating thatthe two amino acids are part of the stefin A epitope recognized by A22. The binding of both mAbs to the mutants Gly4Arg and Gly4Glu was comparable to wild-type stefin A.