Structural and functional characterization of complement C8 gamma, a member of the lipocalin protein family.

Human complement component C8 exhibits an unusual structure in that it contains three chains, two of which, alpha and beta, display high sequence homology to other complement and CTL pore-forming proteins. The third chain, C8 gamma, is covalently linked to C8 alpha by a disulfide linkage; it is demonstrated that Cys40 of C8 gamma is linked to Cys164 of C8 alpha, a unique cysteine located in a loop located between the cysteine-rich LDL-receptor class A module and the membrane-inserting region of C8 alpha. C8 gamma was recently identified as a member of the lipocalin protein family, in which all proteins were either shown to, or are believed to bind small hydrophobic ligands. The present results now demonstrate that C8 gamma incorporates retinol and retinoic acid in the presence of 2 M NaCl. Molecular modeling of C8 gamma, based on the crystal structure of the homologous beta-lactoglobulin, reveals a structure of eight antiparallel beta-strands, bearing a highly hydrophobic binding pocket. The residues participating in the pocket formation are highly conserved when compared with the structures of beta-lactoglobulin and retinol-binding protein, both of which are known to interact with retinol. It is therefore proposed that C8 gamma may act as a retinol transporting protein in plasma.     

The Anaplasma marginale msp5 gene encodes a 19-kilodalton protein conserved in all recognized Anaplasma species.

Immunization with Anaplasma marginale outer membranes induced immunity against clinical disease which correlated with antibody titer to outer membrane proteins, including a 19-kDa protein (N. Tebele, T. C. McGuire, and G. H. Palmer, Infect. Immun. 59:3199-3204, 1991). This 19-kDa protein, designated major surface protein 5 (MSP-5), was encoded by a single-copy 633-bp gene. The molecular mass of MSP-5, defined in immunoblots by binding to monoclonal antibody ANAF16C1, was conserved among all recognized species of Anaplasma: A. marginale, A. centrale, and A. ovis. Recombinant MSP-5, which absorbed the antibody reactivity of bovine immune serum to native MSP-5, was recognized by anti-A. marginale and anti-A. centrale immune sera in a competitive inhibition assay with monoclonal antibody ANAF16C1. The presence of antibody to the epitope defined by monoclonal antibody ANAF16C1 in all postinfection sera tested indicates that this epitope is a potential diagnostic antigen for use in identifying persistently infected cattle.     

Epitope analysis of peanut allergen Ara h1 with human monoclonal IgM antibody clone #86

A human-mouse hybridoma clone #86 secreting IgM-class human monoclonal antibody to peanut allergen protein Ara h1 was newly established. To detect an antibody-binding sequence (epitope) on Ara h1, the monoclonal antibody #86 was reacted with multi-pin apparatus with a series of peptides synthesized from the amino acid sequence of Ara h1. The antibody #86 was found to bind to a peptide with amino acid sequence of 481EEEEDEDEEEEGSNREVRRY500. Further analysis with shorter pin-peptides with ten amino acid-long showed that the peptides reacted with the antibody #86 contained a sequence of 485DEDEEEE491. This might be an essential linear sequence of this epitope. When the 485DED487 part of the peptide was replaced by alanine, decreased binding of antibody #86 was observed.     

VH and VL Gene Complexes Encoding an Anti-Spectrin Antibody are Defined by Nucleotide Sequencing of cDNA from a Hybridoma Generated from Hu-PBL-SCID Mouse

Previously we reported that human imunocompetent cells engrafted into scid mice mount a sustained and vigorous humoral immune response to murine erythrocytes. One of the dominant and consistently observed reactivity pattern of these antibodies in immunoblot analysis is with the α and β isoforms of spectrin. In order to define the human xenoreactive response more completely, a hybridoma was generated (from a hu-PBL-scid mouse) whose antibody reacted with two high molecular weight species 225 to 250 kDa. We report here that this conserved antibody species reacts with both the murine and human erythrocyte proteins and cDNA nucleotide sequence analysis of the light and heavy chain genes encoding this antibody reveals that the light chain variable region gene has been previously observed in association with an autoreactive antibody. In addition to characterizing a conserved human B cell clonotype this is the first report of a human monoclonal antibody being generated from the hu-PBL-scid model using the standard hybridoma technology.     

Monoclonal anti-la antibody derived from a mouse with experimental SLE is similar to human anti-la antibodies

The La antigen is a highly conserved protein, originally defined by sera of patients with Sj?gren's syndrome or systemic lupus erythematosus (SLE). In the present study, we have produced and characterized a monoclonal anti-la antibody derived from mice with experimental SLE. The induction of SLE in these mice was achieved by their immunization with a murine monoclonal anti-idiotypic antibody against a common idiotype (16/6 Id) found in SLE patients. The monoclonal anti-La antibody derived from these mice was found to be virtually identical to the anti-La antibodies found in human autoimmune sera. First, its binding to different nuclear extracts, as well as to protease-digested HeLa nuclear protein extracts, was found to be identical to that of human anti-La antibodies. Second, an inhibition study on blotted proteins demonstrated a very close relationship between the epitopes recognized by the murine monoclonal anti-La antibody and the human anti-La serum. Third, the monoclonal anti-La antibody was found by immunofluorescence to be directed against a nuclear antigen that gave a speckled pattern. Finally, the monoclonal anti-La antibody immunoprecipitated the La-associated small RNAs. This report provides evidence for the similarity of murine anti-La antibody produced in experimental SLE and human anti-La antibodies formed in autoimmunity.     

Characterization of immunodominant surface antigens of Haemophilus somnus.

An immunodominant Haemophilus somnus outer membrane protein with an apparent molecular mass of 40 kDa on Western blots (immunoblots) of gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels was characterized because a monospecific antibody against this antigen was protective. This monospecific antibody was used for immunoaffinity purification of the antigen. The immunoaffinity-purified antigen reacted with a polyclonal antibody to the 40-kDa antigen but not with a monoclonal antibody (3G9) which reacted with the 40-kDa antigen in gradient gels. On 8 or 10% gels, the approximately 40-kDa antigen was resolved as two bands, a 40-kDa band which reacted with the protective monospecific polyclonal antibody (p40) and a band of lower molecular mass which reacted with monoclonal antibody 3G9. The latter antigen was designated p39. Both antigens were conserved in all H. somnus isolates tested. The specific antibodies were also used to detect cross-reacting antigens in other gram-negative bacteria. Antibody to p40 reacted with proteins of 55 to 28 kDa, with the greatest intensity shown among proteins from other members of the family Pasteurellaceae. Antibody to p40 was reduced by absorption with live H. somnus or other members of the family Pasteurellaceae, so the antigen appears to be surface exposed. Antibody to p39 only cross-reacted with a broad band (38 to 40 kDa) in Haemophilus agni. Since H. agni is not a bovine pathogen and since convalescent-phase serum from H. somnus-infected animals did recognize p39, the latter may be a good immunodiagnostic antigen, if the lack of cross-reactivity with antigens in other gram-negative bacteria is confirmed with a polyclonal antibody to p39. The cross-reactivity of antiserum to p40 with antigens of members of the family Pasteurellaceae and the ability of this antiserum to protect against H. somnus pneumonia indicate that p40 may be a useful vaccine antigen for H. somnus disease and perhaps even diseases caused by other members of the family Pasteurellaceae.     

Avidity progression of dietary antibodies in healthy and coeliac children

In most individuals minute amounts of food proteins pass undegraded across the intestinal mucosa and trigger antibody formation. Children with coeliac disease have enhanced antibody production against gliadin as well as other dietary antigens, e.g. beta-lactoglobulin, in cow's milk. Antibody avidity, i.e. the binding strength between antibody and antigen, often increases during antibody responses and may be related to the biological effectiveness of antibodies. The aim of the present study was to determine the avidity of serum IgG antibodies against beta-lactoglobulin and gliadin in healthy children during early childhood and compare these avidities to those found in children with coeliac disease. The average antibody avidity was analysed using a thiocyanate elution assay, whereas the antibody activity of the corresponding sera was assayed by ELISA. The avidity of serum IgG antibodies against beta-lactoglobulin as well as gliadin increased with age in healthy children, even in the face of falling antibody titres to the same antigens. Children with untreated coeliac disease had IgG anti-beta-lactoglobulin antibodies of significantly higher avidity than healthy children of the same age, and the same trend was observed for IgG antigliadin antibodies. The present data suggest that the avidities of antibodies against dietary antigens increase progressively during early childhood, and that this process seems to be accelerated during active coeliac disease.     

A monoclonal antibody against the immunodominant epitope of the ribosomal P2beta protein of Trypanosoma cruzi interacts with the human beta 1-adrenergic receptor.

Monoclonal antibodies were raised against a recombinant ribosomal P2beta protein of Trypanosoma cruzi. One of these reacted with the C terminus of this protein (peptide R13, EEEDDDMGFGLFD) and epitope mapping confirmed that this epitope was the same as the one defined by the serum of immunized mice, and similar to the previously described chronic Chagas' heart disease (cChHD) anti-P epitope. Western blotting showed that the monoclonal antibody recognized the parasite ribosomal P proteins, as well as the human ribosomal P proteins. Electron microscopy showed that it stained different structures in parasite and human cells. Interestingly, surface plasmon resonance measurements indicated that the affinity for the parasite ribosomal P protein epitope (R13) was five times higher than for its human counterpart (peptide H13, EESDDDMGFGLFD). Since the human epitope contained an acidic region (EESDD) similar to the AESDE peptide recognized by cChHD patients in the second extra-cellular loop of the human beta1-adrenergic receptor, the biological activity of the antibody was assessed on neonatal rat cardiomyocytes in culture. The monoclonal antibody had an agonist-like effect. These results, together with the fact that the monoclonal reacted in Western blots with the different isoforms of the heart beta1-adrenergic receptor, confirm the possible pathogenic role of antibodies against the parasite ribosomal P protein based on their cross-reaction with the human beta1-adrenergic receptor.     

Characterization of anti-P monoclonal antibodies directed against the ribosomal protein–RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice

Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus.     

A single amino acid is critical for the expression of B-cell epitopes on the helicase domain of the pestivirus NS3 protein

Truncated NS3 proteins, expressed by recombinant baculoviruses, were used to investigate the location of conserved B-cell epitopes on this non-structural bovine viral diarrhoea virus (BVDV) protein. A goat anti-pestivirus antiserum, and a panel of anti-NS3 monoclonal antibodies, including the BVDV-1 specific antibody P1D8, were used to verify the presence or absence of the epitopes. Interestingly, the monoclonal antibodies reacted only with the truncated protein encompassing the helicase domain of NS3. Expression of the B-cell epitopes was dependent on, but not within, a 57 amino acid sequence at the carboxy-terminal end of this protein, supporting observations that these conserved epitopes are conformational in nature. A comparison of deduced amino acid sequences of the helicase domain from BVDV-1, BVDV-2, BDV and CSFV isolates highlighted a single amino acid that appeared to be unique to P1D8-reactive BVDV-1 isolates. Site-directed mutagenesis studies confirmed that this amino acid is critical for the expression of the BVDV-1 specific NS3 epitope recognised by the P1D8 monoclonal antibody. Surprisingly, the amino acid was also important for an epitope recognised by two group-specific monoclonal antibodies, P1H11 and P4A11. Protein modelling studies, based on the structure of the hepatitis C NS3 helicase domain, indicated that this amino acid occupies a prominent position on the surface of the protein.     

Immunochemical studies of polioviruses: identification of immunoreactive virus capsid polypeptides

Investigation of the immunological reactions with individual poliovirus capsid polypeptides of antisera and monoclonal antibodies raised against poliovirus type 3 antigens are described. Virus polypeptides were separated by electrophoresis, transferred electrophoretically to nitrocellulose sheets and treated with antibody preparations. Antibody binding specifically to the virus polypeptides was then detected by application of 125I-labelled anti-immunoglobulin followed by autoradiography. The technique readily enabled the identification of the polypeptides recognized by the antibody. Antibodies present in polyclonal, type-specific neutralizing sera to poliovirus type 3 bound to the two largest capsid polypeptides (VP1 and VP2) of the homotypic poliovirus, and also to the VP1 of poliovirus type 1 and type 2. There was no obvious difference between the antibody binding patterns obtained with neutralizing and non-neutralizing antisera or between C-specific and D-specific antisera. VP1 appeared to be the immunodominant virus polypeptide. Among monoclonal antibodies specific for the C antigen of poliovirus type 3, a proportion reacted homotypically with the VP1 of poliovirus type 3. Other monoclonal antibodies of C antigen or D antigen specificity, or which reacted both with D and C antigens, some of which had potent virus-neutralizing activity, failed to give demonstrable binding reactions. The non-correlation of neutralization and immunoblot reactivity suggests that sequence determinants alone do not mediate virus neutralization which may depend on antigenic determinants specified by complex conformational arrangements of the virus capsid proteins.     

Identification of a Novel B Cell Epitope on the Nucleocapsid Protein of Porcine Reproductive and Respiratory Syndrome Virus by Phage Display

A phage display peptide library targeting the nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) strain CH-1a was generated and used for epitope mapping. After 3 rounds of biopanning with the monoclonal antibody (MAb) N3H2 directed against the N protein, 3 positive phages were screened and sequenced. These phages share a consensus sequence, IQTAFNQGA, which corresponds to the amino acid (AA) 79–87 segment of the CH-1a N protein. A small DNA fragment coding for IQTAFNQGA was expressed as a fusion product, and reacted to N3H2 in Western blots and indirect ELISA. Four truncated peptides (IQTAFNQG, IQTAFNQ, QTAFNQGA, and TAFNQGA) expressed as GST fusion products failed to react with N3H2. The sequences around the N3H2-binding site among the N proteins of 57 PRRSV strains were compared. Our results indicate that the IQTAFNQGA motif is highly conserved among North American and European isolates. We concluded that the precisely defined nona-peptide epitope is a novel conserved Linear B cell epitope on the N protein of PRRSV.     

Localization of conserved and variable antigenic domains of equine infectious anemia virus envelope glycoproteins using recombinant env-encoded protein fragments produced in Escherichia coli

Previous characterizations of equine infectious anemia virus (EIAV) glycoprotein variation by DNA sequence analysis and epitope mapping using monoclonal antibodies (MAbs) have revealed the presence of conserved and variable regions within the EIAV env gene. To extend these studies, fragments of the EIAV envelope proteins gp90 and gp45 were expressed in Escherichia coli and used in Western blot analysis with a diverse panel of equine immune sera to identify antigenic segments. All sera from EIAV-infected animals reacted with the carboxyl terminal portion of gp90 and the amino terminal portion of gp45, indicating the highly conserved and immunodominant nature of these regions. Other gp90 segments, both from conserved and variable env sequences, displayed variable reactivities with the panel of equine sera. A panel of MAbs was also used in Western blot assays with the recombinant protein fragments for physical localization of previously identified MAb epitopes. The binding sites of two neutralizing MAbs were localized to a highly variable region of gp90, while nonneutralizing epitopes were localized to conserved and variable regions of the envelope glycoproteins. These results, in addition to localizing important antigenic sites on EIAV glycoproteins, indicate that previously defined conserved and variable env nucleotide sequences indeed encode protein sequences constituting conserved and variable immunogens during persistent infection by EIAV.     

Immunoreactive epitopes on an expressed recombinant flagellar protein of Borrelia burgdorferi.

A recombinant Borrelia burgdorferi flagellin protein expressed in Escherichia coli is bound by a murine monoclonal antiflagellin antibody (H9724) and by antibodies in the sera of patients with Lyme disease. Immunoreactive epitopes on the flagellar protein were identified by immunoblot analysis of antibody binding to expressed truncated flagellar proteins. The epitope recognized by the murine monoclonal antibody is within the central heterologous region of the flagellar protein (amino acids 90 to 266). However, antiflagellin antibodies in the sera of patients with Lyme arthritis bound an epitope entirely within, or whose conformation was partly formed by, the 90 NH2-terminal amino acids of the flagellar protein. The binding of antibodies in the sera of patients with Lyme arthritis to the NH2-terminal region of the flagellar protein, a region with sequence homology to the flagellar proteins of other bacterial species, suggests the possibility that antigenic mimicry contributes to the immunopathogenesis of Lyme disease. The fact that human antibodies bind to a highly conserved and hence shared portion of the flagellin reduces the specificity of serological assays for the diagnosis of Lyme disease which use the flagellar protein as antigen.     

Antigenic and sequence diversity in gonococcal transferrin-binding protein A

Neisseria gonorrhoeae is a gram-negative pathogen that is capable of satisfying its iron requirement with human iron-binding proteins such as transferrin and lactoferrin. Transferrin-iron utilization involves specific binding of human transferrin at the cell surface to what is believed to be a complex of two iron-regulated, transferrin-binding proteins, TbpA and TbpB. The genes encoding these proteins have been cloned and sequenced from a number of pathogenic, gram-negative bacteria. In the current study, we sequenced four additional tbpA genes from other N. gonorrhoeae strains to begin to assess the sequence diversity among gonococci. We compared these sequences to those from other pathogenic bacteria to identify conserved regions that might be important for the structure and function of these receptors. We generated polyclonal mouse sera against synthetic peptides deduced from the TbpA sequence from gonococcal strain FA19. Most of these synthetic peptides were predicted to correspond to surface-exposed regions of TbpA. We found that, while most reacted with denatured TbpA in Western blots, only one antipeptide serum reacted with native TbpA in the context of intact gonococci, consistent with surface exposure of the peptide to which this serum was raised. In addition, we evaluated a panel of gonococcal strains for antigenic diversity using these antipeptide sera.     

Antigenic diversity of the circumsporozoite proteins in the Plasmodium cynomolgi complex

Antigenic diversity was observed in the circumsporozoite (CS) proteins of five of the six Plasmodium cynomolgi isolates (NIH, Mulligan, London, Gombak, Ceylon, RO) that we examined. Monoclonal antibodies were produced against salivary gland sporozoites of three of the isolates. Interaction of these monoclonal antibodies with the sporozoites was isolate specific, the exception being the anti-NIH monoclonals which also reacted with Mulligan strain sporozoites. Inhibition of binding between the different monoclonal antibodies indicated that for each of the NIH, London, and Gombak strains, the homologous monoclonals were recognizing the same or a topographically close immunodominant epitope on the respective CS protein. Also the binding of a polyvalent anti-NIH rhesus serum to the homologous antigen could only be inhibited by anti-NIH monoclonal antibody. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of sporozoite extracts demonstrated clear differences in the apparent molecular weights of the CS proteins of four of the six isolates. This is the first study which provides evidence of antigenic diversity in the CS proteins of different isolates of a primate plasmodial species.     

Amyloid protein of vessels in leptomeninges, cortices, choroid plexuses, and pituitary glands from patients with systemic amyloidosis

The cerebrum, cerebellum, and choroid plexuses from 16 patients with systemic amyloidosis, and the pituitary glands from 14 of these patients, were investigated histologically and immunohistochemically. Cerebrovascular amyloid (CVA) was found in the leptomeninges and cortices of six patients with systemic amyloidosis, including two patients with amyloid A protein (AA) amyloidosis related to serum amyloid A protein, one with AL amyloidosis related to immunoglobulin light chain (AL), two with familial type I amyloidotic polyneuropathy (FAP), and one with senile systemic amyloidosis (SSA). CVA protein from two patients with FAP reacted with anti-human prealbumin antibody similar to that of the visceral organs of these two patients. CVA in SSA reacted with anti-human prealbumin antibody and anti-beta protein antibody. Vascular amyloid was frequently noted in the pituitary glands and choroid plexuses of patients with systemic amyloidosis, and was found to be identical to that in the visceral organs (heart, kidney, and intestine) of these patients. CVA in the leptomeninges and cortices from two patients with AA amyloidosis and one with AL amyloidosis reacted with anti-beta protein monoclonal antibody but not with anti-human AA monoclonal antibody, anti-human A lambda antisera, and anti-human A kappa antisera. We suggest that amyloid proteins of AA and AL amyloidosis do not readily accumulate in the vessels in the leptomeninges and cortices even though the proteins circulate, and that beta protein is not derived from a serum precursor.     

Epitopic mapping of linear and conformation-dependent antigenic determinants on GP5 of five U.S. bluetongue viruses.

Two distinct antigenic determinants of the major outer capsid protein, GP5, of five U.S. bluetongue viruses have been identified and mapped using monoclonal and oligoclonal antibodies. One antigenic site, identified by oligoclonal antibody AK-15, was found to be common and conserved in all five U.S. BTV serotypes. This linear epitope was located between amino acid residues 175 and 189 (ALQREAAERSEDEIK). The second determinant identified by monoclonal antibody 34.7 was present in BTV-2, -10, -11, and -17 but absent in BTV-13. The binding of this monoclonal antibody to GP5 could be blocked specifically by one of three short synthetic peptides located among amino acid residues 33-42 (KAAERFAESE), 159-168 (EKILKEEDSK), and 206-215 (EIERDGMQEE), indicating that this antigenic determinant is conformation-dependent. Oligoclonal antibody (AK-15) reacted with denatured GP5 immobilized on nitrocellulose membrane after Western transfer as well as with native GP5 present on the surface of purified BTV virions. Monoclonal antibody (34.7) reacted only with denatured GP5 but not native GP5 using an ELISA assay. However, these two antigenic epitopes alone did not elicit detectable neutralizing antibodies as determined by plaque reduction assay.     

Common Peptide Epitope in Mumps Virus Nucleocapsid Protein and MHC Class II-Associated Invariant Chain

The present study describes a 7 amino acid-long sequence (YQQQGRL) which is identical in HLA-associated invariant chain and mumps virus nucleocapsid protein and is additionally followed by one conservative amino acid pair. As such a long amino acid homology is extremely rare in two evolutionarily unrelated proteins the possibility that it could induce immunological cross-reactivity was evaluated. Several antigenicity indices suggested high antigenic potential within this region. Synthetic peptides containing this sequence were reactive with 31% of monoclonal antibodies specific for mumps virus nucleocapsid protein in ELISA. High antibody levels against this epitope were found in 7% of mumps-seropositive human sera and antibody levels clearly increased after natural mumps infections and mumps vaccinations. Rabbit antibodies raised against a synthetic invariant chain peptide AYF-LYQQQGRLDKL-C reacted with corresponding nucleocapsid peptide RFAKYQQQGRLEAR-C and antibodies against the nucleocapsid peptide reacted with the invariant chain peptide. Rabbit antibodies against the invariant chain peptide also reacted with nucleocapsid molecules in formaldehyde-fixed mumps virus-infected cells, and antibodies against the nucleocapsid peptide reacted with invarianl chains expressed in methanol-fixed cells. One monoclonal antibody specific for the nucleocapsid molecule also reacted with cells expressing invariant chains. In immunoprecipitation rabbit antibodies against the invariant chain peptide bound to invariant chains while antibodies against the nucleocapsid peptide did not. The results suggest tha t there is antigenic similarity in mumps virus nucleocapsid molecule and HLA-associated invariant chain which may cause immunological cross-reactivity between these molecules.     

Immunoblot reactivity of polyclonal and monoclonal antibodies with periplasmic flagellar proteins FlaA1 and FlaB of porcine Serpulina species.

The periplasmic-flagellum (PF) proteins of Triton X-100-soluble and Triton X-100-insoluble sodium dodecyl sulfate-treated fractions from reference and field strains of Serpulina hyodysenteriae, Serpulina innocens, and Serpulina pilosicoli were characterized by Western blotting with a rabbit polyclonal antibody (PAb) specific for the 44-kDa PF sheath protein of S. hyodysenteriae (Z. Li, F. Dumas, D. Dubreuil, and M. Jacques, J. Bacteriol. 175:8000-8007, 1993) and a murine monoclonal antibody (MAb), designated 7G2, specific for the PF core FlaB proteins of S. hyodysenteriae. The MAb 7G2 reacted with a conserved epitope present in the 37-, 34-, and 32-kDa PF core FlaB proteins of all Serpulina species. This suggested that the core FlaB proteins are conserved among porcine Serpulina species. An immunoreactive band of approximately 44 kDa was present with all S. hyodysenteriae, S. innocens, and S. pilosicoli strains that were reacted with the PAb. The specificities of the PAb and the MAb for the FlaA1 and FlaB proteins of Serpulina species were confirmed by N-terminal amino acid sequencing of 44- and 37-kDa proteins, respectively, of S. hyodysenteriae and S. pilosicoli. Results from this study provide further evidence that the 44-kDa protein FlaA1 and the 37-, 34-, and 32-kDa FlaB proteins are conserved among porcine Serpulina species.