Studies on the allergenic significance and structure of rat serum albumin

This study evaluated the capacity of rat serum albumin and of its proteolytic fragments to activate human basophils for IgE-mediated histamine release. The leukocytes from 8 out of 33 patients allergic to rats released histamine with rat serum albumin. Two proteolytic fragments of rat serum albumin, each constituting half of the molecule, were used to study the IgE-reactive antigenic sites. These fragments released histamine with the cells of some of the donors, thus demonstrating the presence of at least 2 antigenic determinants on each fragment for a total minimum of 4 sites on the intact rat serum albumin molecule. Most of the allergenic activity, however, was not recovered in the 2 fragments (total recovery mean = 6.4%, range between 0.1 and 31%). This loss could be due to cleavage of the rat albumin molecule in the middle of the third domain with loss of antigenic sites and/or due to minor conformational changes in the fragments as compared with the intact molecule. There was up to a 500-fold difference in the percent of activity recovered in the fragments when tested on cells from different patients. Therefore, there is no single immunodominant site on the molecule equally important for all patients. The cells of all 8 patients also reacted with mouse serum albumin but only 2 with bovine serum albumin. At least 1 determinant on mouse and rat serum albumin is cross-reactive with IgE.     

Location of antigenic epitopes on antibody molecules

Using X-ray crystallographic co-ordinates of immunoglobulins, surface regions accessible to a large spherical probe, comparable in size to an antibody domain, were computed. Locations of these exposed regions were compared with those of experimentally determined antigenic sites, i.e. idiotypic, allotypic and isotypic serological markers. In all cases, an excellent agreement was found. The most prominent computed epitopes correspond to convex parts of antibody surface made by reverse turn segments of the polypeptide chain. The computed epitopes occur in homologous positions in all the immunoglobulin domains, and most of the beta-sheet surfaces on the domains are poorly antigenic. The CH2 domain (Fc fragment) has many more antigenic sites than the Fab fragments (antigen-binding fragments). Variable domain epitopes (idiotypes) involve both hypervariable and framework residues, and only about 25% of the hypervariable residues are strongly antigenic. The results indicate that, in a vertebrate body, each antibody molecule may be recognized, and its concentration regulated, by at least 40 complementary anti-immunoglobulin antibodies; therefore, a possibility of an "immune network" with much higher connectivity than is generally assumed should be seriously contemplated.     

A mouse plasma cell surface antigenic determinant (PLKB) recognized by xenoantiserum. Its relationship to the PC.1 antigenic determinant.

The specificities of the xenoantisera made against mouse myeloma cells have been compared to those recognized by alloantiserum by studying patterns of cytotoxicity on both normal and malignant plasma cells. Goat antiserum obtained by immunization with Balb/c mouse myeloma ADJ-PC-22A cells and purified by in vivo absorption could detect cell surface antigenic determinants present on plasma cells and on cells of liver, kidney, and brain (PLKB antigen), as we had previously reported for a similarly prepared rabbit antiserum. In spite of an apparent similarity between the tissue representation of the PLKB determinant and that of PC.1 antigenic determinants which were detected by DBA/2 anti-ADJ-PC-22A cell alloantiserum, the PLKB antigenic determinant is not identical with the PC.1 antigenic determinant, since the former is found on the tissues of PC.1-negative as well as PC.1-positive strains of mice. However, it was deduced that the PLKB antigenic determinant and the PC.1 antigenic determinant reside in close proximity on the cell surface or maybe even on the same molecule, since Fab fragments of antiserum against either PLKB or PC.1 blocked the cytotoxicity against both antigens. On the other hand, these Fab fragments did not inhibit the cytotoxicity of anti-H-2 antiserum, indicating that neither PLKB nor PC.1 antigenic determinants are in close proximity to H-2 antigens. Association of PLKB and PC.1 determinants was further supported by the finding that the loss of the PLKB determinant in a variant of myeloma MOPC-70A corresponds to the loss of PC.1 determinant on the same cells.     

Cleavage of bacterial flagellin with cyanogen bromide. Antigenic properties of the protein fragments

1. Four polypeptide fragments, obtained by cyanogen bromide treatment of the protein flagellin from Salmonella adelaide, were tested for their antigenic activity by using them as inhibitors in three different assays: bacterial immobilization, haemagglutination of sensitized erythrocytes and quantitative micro precipitation. Immunodiffusion studies were also performed on the protein fragments. 2. Cleavage of the flagellin molecule in this way gave no detectable loss of antigenic determinants. Fragment A (mol.wt. 18000), the largest of the polypeptides, contained all the antigenic specificities present on flagellin that were recognized by the antisera used. In one test, fragment B (mol.wt. 12000) also contained antigenic activity to an extent not easily explainable by contamination with fragment A. Fragments C (mol.wt. 5500) and D (mol.wt. 4500) appeared to be antigenically inactive.     

Structure of antigenic determinants in the N-terminal region of dermatosparactic sheep procollagen type I.

About half of the rabbit antisera raised against type-I procollagen, p alpha 1(I) chain or nonreduced procollagen peptides reacted in a radioimmunoassay with the reduced form of peptide Col 1, which comprises the whole non-collagenous region at the N-terminus of procollagen. Proteolytic fragments prepared from reduced peptide Col 1 were still effective inhibitors of the antibodies and allowed the localization of two antigenic determinants. The antigenically active regions have the sequences less than Glu-Glu-Glu-Gly-Gln-Gln-Glu and Gly-Asp-Thr-Gly-Pro-Arg, and are located at the N- and C-termini of the peptide respectively. Antibodies raised against reduced peptide Col 1 bind to a determinant localized in a different region of the peptide.     

Epitopes of herpes simplex virus type 1 glycoprotein gC are clustered in two distinct antigenic sites.

Epitopes of herpes simplex virus type 1 (HSV-1) strain KOS glycoprotein gC were identified by using a panel of gC-specific, virus-neutralizing monoclonal antibodies and a series of antigenic variants selected for resistance to neutralization with individual members of the antibody panel. Variants that were resistant to neutralization and expressed an antigenically altered form of gC were designated monoclonal antibody-resistant (mar) mutants. mar mutants were isolated at frequencies of 10(-3) to 10(-5), depending on the antibody used for selection. The epitopes on gC were operationally grouped into antigenic sites by evaluating the patterns of neutralization observed when a panel of 22 antibodies was tested against 22 mar mutants. A minimum of nine epitopes was identified by this process. Three epitopes were assigned to one antigenic site (I), and six were clustered in a second complex site (II) composed of three distinct subsites, IIa, IIb, and IIc. The two antigenic sites were shown to reside in physically distinct domains of the glycoprotein, by radioimmunoprecipitation of truncated forms of gC. These polypeptides lacked portions of the carboxy terminus and ranged in size from approximately one-half that of the wild-type molecule to nearly full size. Antibodies recognizing epitopes in site II immunoprecipitated the entire series of truncated polypeptides and thereby demonstrated that site II resided in the N-terminal half of gC. Antibodies reactive with site I, however, did not immunoprecipitate fragments smaller than at least two-thirds the size of the wild-type polypeptide, suggesting that site I was located in the C-terminal portion. Sites I and II were also shown to be spatially separate on the gC polypeptide by competition enzyme-linked immunosorbent assay with monoclonal antibodies representative of different site I and site II epitopes.     

Topographic antigenic determinants recognized by monoclonal antibodies to sperm whale myoglobin

Monoclonal antibodies of high affinity (approximately 10(9) M-1) for sperm whale myoglobin were studied to pinpoint the antigenic determinants with which they interact. None of 6 different monoclonal antibodies tested reacted with any of the 3 CNBr cleavage fragments which encompass the whole sequence of myoglobin, an indication that they react with determinants present only on the native structure. To identify these sites, we compared the affinities of each antibody for a series of 14 mammalian myoglobins of known sequence and similar tertiary structure. Correlation of sequence differences with relative affinities allowed us, thus far, to identify critical antigenic residues recognized by 3 of the antibodies. Two of these antibodies recognize groups of residues which are far apart in primary structure but close together in the 3-dimensional structure of the native myoglobin molecule, i.e. topographic determinants. The third antibody distinguishes 140 Lys leads to Asn plus, probably, surface residues nearby. These determinants differ from previously reported antigenic sites on sperm whale myoglobin both in that they are topographic, rather than sequential, and in that almost all the critical residues recognized by these antibodies are outside the previously reported sites. Monoclonal antibodies are sensitive to subtle changes, e.g. Glu leads to Asp, in the antigenic site.     

Monoclonal antibody studies of the antigenic determinants of human plasma retinol-binding protein

A battery of monoclonal antibodies (MoAbs) against human retinol-binding protein (RBP) was produced to obtain useful probes for the study of the antigenic determinants of RBP. The 12 antibodies all reacted with human RBP by immunoblotting. Based on antibody cross-competition radioimmunoassays, four distinct and different groups of antibodies were identified: group I, 1A4 and 2F4; group II, 1G10, 5C5, 6F4, and 7G3; group III, 5H6, 6C7, 10G5, and 14E3; and group IV, 5H9 and 13A1. Information about the epitopes of RBP recognized by these MoAbs was obtained by testing the reactivity of each antibody with human, rabbit, and rat RBPs by immunoblotting. Group I and group IV antibodies reacted to a similar extent with human, rabbit, and rat RBPs. Group II antibodies reacted strongly with human and rabbit RBPs, but reacted very weakly with rat RBP. Group III antibodies reacted strongly with human RBP, but did not react with rabbit or rat RBP. Thus, the epitopes for group I and group IV antibodies appear to be regions of the RBP molecule that are conserved across the three species, whereas group III antibodies recognized only human RBP. In a preliminary study, the reactivity of each antibody with purified cyanogen bromide fragments of RBP was tested by slot immunoblotting. None of the MoAbs reacted with any of the cyanogen bromide fragments. This study shows that MoAbs specific for at least four different regions of the RBP molecule can be produced; hence, RBP contains at least four major antigenic domains.     

Immunochemical cross-reactivity between cyanogen bromide fragments of human serum albumin

The antigenic structure of human albumin was investigated in order to establish whether or not there was any similarity between its antigenic sites. Using immunoadsorbent columns prepared with cyanogen bromide fragments of human serum albumin, antibodies directed against different portions of the albumin molecules were isolated. Measurement of the amount of the antibodies isolated and study of their specificity by inhibition techniques show that these subpopulations of antibodies reacted not only with the fragment used for their isolation (homologous) but also with the other fragments (heterologous). Heterologous fragments were inhibiting only at a very high concentration with regard to the homologous ones. These results show that there is a weak cross-reactivity between different portions of the albumin molecule. This reaction is most probably due to the homology existing in the sequence of the human albumin molecule which has arisen by gene duplication. The same type of behavior can be predicted to extent to other molecules which have evolved by similar mechanisms.     

The distribution and relative immunogenicity of calf alpha-crystallin antigenic determinants on different subunits.

In the native alpha-crystallin molecule, 45.9% of all reactive antigenic determinants were found to be located on SH-containing subunits. Of these, the majority (35.3%) were reaggregation dependent, and 10.6% were reactive on monomeric subunits. By contrast, only 10.9% of all antigenic determinants were located on SH-free subunits, and the ratio of aggregation-dependent determinants (4.4%) to those of monomeric subunits (6.5%) was reversed compared to SH-containing subunits. Among all antigenic determinants reactive in native alpha-crystallin, 44.1% were dependent on the presence of both types of subunits. These data indicate that the antigenic determinants requiring subunit interaction were formed from SH-containing and SH-free subunits in a ratio of 1:1. Direct analysis showed that in the alpha-crystallin molecule, the ratio of these subunits is 2:1. The experiments indicate that some conformations of subunits in the native molecule persist in separated subunits. The relative immunogenicity of each type of antigenic determinant expressed as the ratio of the percentage of the determinant reactive in the native calf lens alpha-crystallin to the percentage of corresponding antibodies induced by native alpha-crystallin was found to be close to 1.     

Antigenic specificity of monoclonal antibodies to human myoglobin

Two monoclonal antibodies directed against different sites of the human myoglobin molecule have been tested for their cross-reactivities against several myoglobins including seven from mammalian species. The relation between their cross-reactivities and their amino acid sequences had led to a possible localization of two antigenic domains in human myoglobin. Each domain includes residues previously considered not to be directly involved in the antigenic structure of myoglobin. Unlike polyclonal serum antibodies, monoclonal hybridoma antibodies directed to a native protein often fail to bind to supposedly antigenic protein fragments. This is explicable in terms of the concept of antigenic domains. Such domains are numerous and overlapping, each comprising a number of contributory amino acid side chains which need not necessarily include continuous sequences of amino acids and which need not exhibit measurable antigenicity in isolation from the rest of the domain.     

Localization of antigenic determinants on a molecule of trophoblast-specific beta 1-glycoprotein

Spatial localization of antigenic determinants of trophoblast-specific beta I-glycoprotein (TSG) has been elucidated using chemical modifications of the sugar and protein moieties of the molecule. Various deglycosylation procedures of TSG afforded fragments slightly soluble even in the presence of powerful detergents. Treatment of TSG with boric acid and its salts, accompanied with a conformational change of the sugar moiety, failed to alter conformation of the protein portion as evidenced by CD spectral data. This modification was found to increase the antigenic activity of TSG only scarcely. Modification of tryptophane or tyrosine residues of TSG changed spatial structure of the protein portion to can be a considerable loss of the TSG antigenic activity. The data obtained led to the conclusion that antigenic determinants of TSG are localized at the protein portion of the molecule and are topographic. A tryptophane residue is an indispensable constituent of the antigenic determinants.     

Immunochemical analysis of the domain structure of CAD, the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells

CAD, is a multidomain polypeptide, with a molecular weight of over 200,000, that has glutamine-dependent carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase activity as well as regulatory sites that bind UTP and 5-phosphoribosyl 1-pyrophosphate. The protein thus catalyzes the first three steps of de novo pyrimidine biosynthesis and controls the activity of the pathway in higher eukaryotes. Controlled proteolysis of CAD isolated from Syrian hamster cells, cleaves the molecule into seven major proteolytic fragments that contain one or more of the functional domains. The two smallest fragments, which had molecular weights of 44,000 and 40,000, corresponded to the fully active dihydroorotase (DHO) and aspartate transcarbamylase (ATC) domains, respectively, but the larger fragments have not been previously characterized. In this study, enzymatic assays of partially fractionated digests and immunoblotting with antibodies specifically directed against the purified ATC domain, the purified dihydroorotase domain and an 80-kDa fragment of the putative carbamyl-phosphate synthetase domain established the precursor-product relationships among all of the major proteolytic fragments of CAD. These results indicate that 1) only the intact molecule had all of the functional domains, 2) a species with a molecular weight of 200,000 was produced in the first step of proteolysis which had glutamine-dependent carbamyl-phosphate synthetase and dihydroorotase activity, but neither aspartate transcarbamylase activity nor the antigenic determinants present on the isolated ATC domain, and 3) cleavage of the 200-kDa species produced a species, with a molecular mass of 150,000 which lacked both aspartate transcarbamylase and dihydroorotase domains. This 150-kDa species, containing the postulated carbamyl-phosphate synthetase, glutamine, and regulatory (UTP, 5-phosphoribosyl 1-pyrophosphate) domains, had two elastase-sensitive sites that divided this region of the polypeptide chain into 10-, 65-, and 80-kDa segments. The location of the functional sites on these segments has not yet been established. The immunochemical analysis also revealed the existence of possible precursors of the stable aspartate transcarbamylase and dihydroorotase domains, suggesting that the chain segments connecting the functional domains of CAD are extensive and that the overall size of the intact polypeptide chain has been underestimated. On the basis of these studies we have proposed a model of the domain structure of CAD.     

The pigeon cytochrome c-specific T cell response of low responder mice. I. Identification of antigenic determinants on fragment 1 to 65

An examination of the proliferative response to pigeon cytochrome c fragments 1 to 65 and 1 to 80 by T cells from mice that are low responders to the native molecule revealed that some of the strains could respond to antigenic determinants on these fragments. T cell clones derived from B10.A(3R) and B10.A(4R) mice were used to characterize the antigenic determinants on fragment 1 to 65. All of the clones recognized syngeneic A beta:A alpha Ia molecules as their restriction element. Three B10.A(3R) clones and six B10.A(4R) clones recognized fragment 39 to 65. Another four B10.A(4R) clones responded to fragment 1 to 38. By stimulating with a series of cytochrome c fragments from different species, as well as a synthetic peptide, it was possible to localize the antigenic determinant(s) recognized by the B10.A(3R) clones to residues 45 to 58. Each clone showed a unique pattern of responsiveness to the various fragments, suggesting a diversity of T cell receptors specific for the same peptide. One B10.A(3R) clone could be stimulated by many of the 1 to 65 fragments in association with allogeneic B10.SM presenting cells and by tuna fragment 1 to 65 in association with B10.M presenting cells, although the rank order of potency for several of the fragments was different than that observed with syngeneic antigen-presenting cells. In addition, the clone was poorly reactive to a synthetic peptide containing a conservative substitution, serine for threonine, at position 49. The implications of these results for subsite dissection (agretope and epitope) of the antigenic determinant recognized by this clone are discussed.     

Common antigenic determinants of the tubulin binding domains of the microtubule-associated proteins MAP-2 and tau.

The structural-functional aspects of the tubulin binding domain on the microtubule-associated protein MAP-2, and its relationship with the tubulin binding domain on tau, were studied using anti-idiotypic antibodies that react specifically with the epitope(s) on MAPs involved in their interaction with tubulin in addition to other tau and MAP-2 specific antibodies. Previous studies showed that MAP-2 and tau share common binding sites on tubulin defined by the peptide sequences alpha (430-441) and beta (422-434) of tubulin subunits. Furthermore, binding experiments revealed the existence of multiple sites for the interaction of the alpha- and beta-tubulin peptides with MAP-2 and tau. Most recent studies showed that the synthetic tau peptide Val187-Gly204 (VRSKIGSTENLKHQPGGG) from the repetitive sequence on tau defines a tubulin binding site on tau. Our present immunological studies using anti-idiotypic antibodies which interact with the synthetic tau peptide and antibodies against the Val187-Gly204 tau peptide indicate that MAP-2 and tau share common antigenic determinants at the level of their respective tubulin binding domains. These antigenic determinants appear to be present in the 35 kDa tubulin binding fragment of MAP-2 and in 18-20 kDa chymotryptic fragments containing the tubulin binding site(s) on MAP-2. These findings, along with structural information on these proteins, provide strong evidence in favor of the hypothesis that tubulin binding domains on MAP-2 and tau share similar structural features.     

Structural factors modulate the activity of antigenic poliovirus sequences expressed on hybrid hepatitis B surface antigen particles.

We have studied the functional expression of antigenic poliovirus fragments carried by various hybrid hepatitis B surface antigen (HBsAg) particles. Several constructions were made by using two different insertion sites in the HBsAg molecule (amino acid positions 50 and 113) and two different sequences, one derived from poliovirus type 1 (PV-1) and the other from PV-2. The inserted fragments each encompassed residues 93 to 103 of the capsid protein VP1, a segment which includes the linear part of the neutralization antigenic site 1 of the poliovirus. The antigenicity and immunogenicity of the hybrid particles were evaluated and compared in terms of poliovirus neutralization. A high level of antigenic and immunogenic activity of the PV-1 fragment was obtained by insertion at position 113 but not at position 50 of HBsAg. However, a cooperative effect was observed when two PV-1 fragments were inserted at both positions of the same HBsAg molecule. Antibodies elicited by the PV-2 fragment inserted at amino acid position 113 did not bind or neutralize the corresponding poliovirus strain. They did, however, bind a chimeric poliovirus in which the homologous antigenic fragment of PV-1 had been replaced by that of PV-2. The only virions that were neutralized by these antibodies were certain mutants carrying amino acid substitutions within the PV-2 fragment. These results show that position, constraints from the carrier protein, and nature of the inserted sequences are critically important in favoring or limiting the expression of antigenic fragments as viral neutralization immunogens.     

Location of the antigenic determinants of bovine pancreatic ribonuclease.

Four antigenic regions of native bovine pancreatic ribonuclease have been located by using antibodies that react specifically with segments 1--13, 31--79, and 80--124. These antibodies were purified by affinity chromatography on columns to which these peptide segments were bound. Analysis of precipitin curves indicates that there are at least three antigenic determinants to which antibody molecules can bind simultaneously in the presence of excess antibodies. Analysis of binding data, however, for each purified specific antibody preparation, carried out by the method of Berzofsky et al. [Berzofsky, J. A., Curd, J. G., & Schechter, A. N. (1976) Biochemistry, 15, 2113], leads to an estimate of four for the number of antigenic determinants in ribonuclease; this estimate had also been made earlier by Stelos et al. [Stelos, P., Fothergill, J. E., & Singer, S. J. (1960) J. Am. Chem. Soc. 82, 6034]. We find that one determinant is associated with each of segments 1--13 and 80--124 and two with segment 31--79. No antigenic activity could be detected for segment 14--29 either in native ribonuclease or in the free fragment. These conclusions are based on (1) the use of specific peptides to isolate purified antibodies by affinity chromatography, (2) immunoprecipitation of an antigenic peptide from the peptic digest of ribonuclease, (3) competitive inhibition studies with various peptide and protein fragments [cyanogen bromide fragments 1--13, 31--79, and 80--124, the tryptic peptides 40--61 and 105--224, S-peptide, S-protein, and des(121--124)-RNase], and (4) comparison and evaluation of the published effects on antigenicity of chemical and enzymatic modifications and changes in sequence among homologous ribonucleases. These approaches provide evidence that the four antigenic determinants are localized around the alpha-helical portion of segment 1--10, somewhere in segment 40--61, at the beta bend in segment 63--75, and either at the beta bend or beta sheet in segment 87--104 of native ribonuclease.     

Biochemical and antigenic analysis of the Epstein Barr virus/C3d receptor (CR2)

Four monoclonal antibodies (OKB7, HB-5, AB-1, and anti-B2) that recognize a 145-kDa B cell-specific membrane structure have markedly different abilities to 1) inhibit C3d and EBV binding to B cells, 2) immunoprecipitate a 145-kDa B cell protein, and 3) stimulate B cell proliferation and differentiation into Ig-secreting cells. This study was initiated to determine whether these four monoclonal antibodies (MoAb) react with the same protein; a related goal was to determine whether the structure(s) recognized by these antibodies constitutes an antigenically related family of structurally distinct molecules. In the studies presented here, the four MoAb were found to fully immunoprecipitate the purified 145-kDa B cell molecule isolated by immunoaffinity chromatography on either OKB7, HB-5, or AB-1 columns, findings that show conclusively that the antibodies all react with the same B cell protein. The variable ability to immunoprecipitate this B cell membrane protein was found to result from differences in exposure or accessibility of the relevant antigenic epitopes in the detergent extract. The 145-kDa molecule immunoprecipitated with the four MoAb was equivalently sensitive to endoglycosidase F and yielded the same banding pattern after digestion with endoglycosidase F and after partial digestion with either S. aureus V8 protease or with trypsin. Within the limits of the sensitivity of these techniques, therefore, there is no evidence for carbohydrate or protein differences in the EBV/C3d receptor (CR2) molecule recognized by the four MoAb. Additional studies showed that the four MoAb react with distinct and nonoverlapping antigenic epitopes on the 145-kDa molecule. The variable abilities of the four MoAb to inhibit CR2 function and EBV binding and to trigger B cell activation, together with the other findings noted above, indicates that the 145-kDa EBV/C3d receptor possesses discretely localized functional domains.     

Isofocusing of antigenic small nuclear ribonucleoproteins. I. Compositional analysis

This report describes the behavior of antigenic small nuclear ribonucleoproteins (snRNPs) in native isofocusing gels. These RNA-protein complexes exhibited true isofocusing characteristics only when in the complexed form. Deproteinized snRNAs migrated to pH ranges which varied according to the pH of the application site. Immunological assays using lupus sera which recognized the La, Sm, and RNP determinants on these snRNPs established that the La and the Sm/RNP antigens segregated to pH 4.7-4.9 and 5.5-7.5, respectively. RNase digestion of these snRNPs did not alter the isofocusing migration of either the Sm or the La determinants. These antigenically active fractions contained the appropriate protein and RNA species shown by immunoprecipitation studies to associate with these antigenic determinants. The isofocusing fractions containing the uridylic acid-snRNPs were fully immunoprecipitable by anti-Sm sera, confirming their particulate integrity after isofocusing.     

Construction of a fusion protein carrying antigenic determinants of enteric clostridial toxins

Clostridium difficile and Clostridium perfringens type A are infectious agents of enteric diseases. The main virulence factors of these microorganisms include toxins A and B of C. difficile (ToxA and ToxB) and enterotoxin of C. perfringens (Cpe). In this study genetic constructions have been created for the expression of ToxA, ToxB and Cpe fragments either as individual components or as a hybrid multidomain (ToxA-ToxB-Cpe) protein. Rabbit monospecific sera raised against individual peptides reacted with the chimeric product indicating that the corresponding antigenic determinants were correctly expressed on the hybrid molecule. Furthermore, mice immunized with the fusion protein produced antibodies specific to each of the three separate components. These data suggest that the constructed three-domain molecule could be used in future studies for development of a vaccine against enteric clostridial diseases.