Antibodies In Rabbit Antisera Research Articles

First Evidence for a Covalent Linkage between Enterobacterial Common Antigen and Lipopolysaccharide in Shigella sonnei Phase II ECALPS

Enterobacterial common antigen (ECA) is expressed by Gram-negative bacteria belonging to Enterobacteriaceae, including emerging drug-resistant pathogens such as Escherichia coli, Klebsiella pneumoniae, and Proteus spp. Recent studies have indicated the importance of ECA for cell envelope integrity, flagellum expression, and resistance of enteric bacteria to acetic acid and bile salts. ECA, a heteropolysaccharide built from the trisaccharide repeating unit, →3)-α-D-Fucp4NAc-(1→4)-β-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→, occurs as a cyclic form (ECA(CYC)), a phosphatidylglycerol (PG)-linked form (ECA(PG)), and an endotoxin/lipopolysaccharide (LPS)-associated form (ECA(LPS)). Since the discovery of ECA in 1962, the structures of ECA(PG) and ECA(CYC) have been completely elucidated. However, no direct evidence has been presented to support a covalent linkage between ECA and LPS; only serological indications of co-association have been reported. This is paradoxical, given that ECA was first identified based on the capacity of immunogenic ECA(LPS) to elicit antibodies cross-reactive with enterobacteria. Using a simple isolation protocol supported by serological tracking of ECA epitopes and NMR spectroscopy and mass spectrometry, we have succeeded in the first detection, isolation, and complete structural analysis of poly- and oligosaccharides of Shigella sonnei phase II ECA(LPS). ECA(LPS) consists of the core oligosaccharide substituted with one to four repeating units of ECA at the position occupied by the O-antigen in the case of smooth S. sonnei phase I. These data represent the first structural evidence for the existence of ECA(LPS) in the half-century since it was first discovered and provide insights that could prove helpful in further structural analyses and screening of ECA(LPS) among Enterobacteriaceae species.

Characterization of anti-ECA antibodies in rabbit antiserum against rough Yersinia enterocolitica O:3

Enterobacterial common antigen (ECA) is a characteristic surface component in bacteria belonging to the Enterobacteriaceae family. It is generally integrated in the outer membrane via a linkage to phosphatidylglycerol (ECA(PG)) and at the same time in some special cases via a linkage to lipopolysaccharide (ECA(LPS)); the latter form is immunogenic. Yersinia enterocolitica O:3 expresses both ECA(PG) and ECA(LPS). To study whether ECA-immunogenicity of Y. enterocolitica O:3 is temperature-regulated, rabbits were immunized with ECA-expressing Y. enterocolitica O:3 bacteria grown at 22 and 37°C. To induce minimal amount of anti-LPS antibodies, immunization was performed with YeO3-c-trs8-R, an LPS mutant missing both O-polysaccharide and the outer core hexasaccharide. However, abundant antibodies specific for LPS core were still present in the obtained antisera such that the reactivity of ECA-specific antibodies could not be detected. To obtain "monovalent" anti-ECA antisera, the sera were absorbed with ECA-negative bacteria. Absorption with live bacteria removed efficiently the anti-LPS antibodies, whereas this was not the case with boiled bacteria. Western blotting revealed that the specificity of the monovalent anti-ECA antiserum was different from that of a monoclonal anti-ECA antibody (mAb 898) as it did not react with ECA(PG), and this suggested that in Y. enterocolitica O:3 ECA(LPS) only one or two ECA repeat unit(s) is/are linked to LPS. Both ECA(PG) and ECA(LPS) expression were found to be regulated by temperature and repressed at 37°C.

Recombinant hyaluronate associated protein as a protective immunogen against Streptococcus equi andStreptococcus zooepidemicus challenge in mice

The capsule of Streptococcus equi, the cause of strangles, and Streptococcus zooepidemicus, associated with equine lower airway disease, plays an important role in evasion of phagocytosis by polymorphonuclear leucocytes. It is composed of hyaluronate, making it non-immunogenic. A hyaluronate associated protein (HAP) fromS. equisimilis , whose gene has been sequenced [1], was investigated (a) for its presence in S. equi andS. zooepidemicus and (b) as an immunogen able to interfere with capsule structure and protect against experimental challenge of mice. The purified capsule of S. equi contained a protein of similar molecular mass to the S. equisimilis protein (approximately 53 kDa). Polymerase chain reaction (PCR) using primers derived from the published sequence of S. equisimilis HAP yielded a product from S. equi and S. zooepidemicus of the expected size and susceptibility to restriction endonucleases. Subcloning of two large in frame StuI/SspI fragments of the HAP gene from S. equi, approximately equivalent to the two halves of the molecule, into the expression vector pGEX-3X yielded only the carboxy half in the correct orientation. This latter recombinant produced a GST fusion protein (HAP-GST) of the expected size that was affinity purified. Antibodies in rabbit antiserum to the native protein in purified hyaluronate reacted strongly in immunoblots with HAP-GST. Antiserum to HAP-GST, when soaked into filter paper strips, caused a diminution of capsule production by S. equi cultured on blood agar. Antiserum added into fresh rabbit blood was not opsonic for S. equi. Immunization with HAP-GST significantly reduced rhinitis in Balb/C mice challenged nasally with S. equi and significantly increased survival time and clearance of bacteria in CBA/CA mice challenged intraperitoneally with S. zooepidemicus.

Legumin-like storage polypeptides of conifer seeds and their antigenic cross-reactivity with 11S globulins from angiosperms

The aim of the present work was to investigate the homology of seed storage proteins in a wide variety of conifers for most of which the gene sequences are not yet identified. Rabbit antiserum antibodies against the purified 57 kDa non-reduced crystalloid protein complex of white spruce seed were obtained. The antibodies were used in the immunoblot assays with seed proteins of various members of Pinaceae; Ginkgo biloba, and several representatives of angiosperms. Under reducing conditions, 35 kDa and 22 kDa range polypeptides, homologous to white spruce crystalloids, were identified in all members of the Pinaceae examined except Abies amabilis and Tsuga heterophylla

Quantitative analysis by reversed Mancini test of cross-reacting antibodies in rabbit antisera against porcine and humanEscherichia coliheat-labile enterotoxins and cholera toxin

Immunological similarities of heat-labile Escherichia coli enterotoxins pathogenic for man (LTh) and piglets (LTp) and cholera enterotoxin (CT) were examined quantitatively by the reversed Mancini test. The following results were obtained by analysis of rabbit antisera against these toxins. (1) 86% and 61% of the immunoglobulins in anti-CT antisera were antibodies cross-reacting with LTh and LTp, respectively; (2) 77% and 66% of the immunoglobulins in anti-LTh antisera were antibodies cross-reacting with LTp and CT, respectively; (3) 75% and 59% of the immunoglobulins in anti-LTp antisera were antibodies cross-reacting with LTh and CT, respectively.

Quantitative analysis by reversed Mancini test of cross-reacting antibodies in rabbit antisera against porcine and human Escherichia coli heat-labile enterotoxins and cholera toxin

Quantitative analysis by reversed Mancini test of cross-reacting antibodies in rabbit antisera against porcine and human Escherichia coli heat-labile enterotoxins and cholera toxin

A common-immunogenic Vibrio outer membrane protein

The presence of antibodies in rabbit antisera to cell envelope proteins of Vibrio cholerae has been examined using a two-dimensional system, in which the cell envelopes are electrophoresed in sodium dodecyl sulfate (SDS) in polyacrylamide gels in the first dimension, and in agarose containing antibodies in the second. The results show that a 25-kDa protein is markedly immunogenic and appears to be common to Vibrio strains; it is not present in a number of other organisms. This 25-kDa protein is an outer membrane protein as judged by sucrose gradient centrifugation and it is accessible to iodination by lactoperoxidase, suggesting that it is exposed on the cell surface.

A common-immunogenicVibrioouter membrane protein

The presence of antibodies in rabbit antisera to cell envelope proteins of Vibrio cholerae has been examined using a two-dimensional system, in which the cell envelopes are electrophoresed in sodium dodecyl sulfate (SDS) in polyacrylamide gels in the first dimension, and in agarose containing antibodies in the second. The results show that a 25-kDa protein is markedly immunogenic and appears to be common to Vibrio strains; it is not present in a number of other organisms. This 25-kDa protein is an outer membrane protein as judged by sucrose gradient centrifugation and it is accessible to iodination by lactoperoxidase, suggesting that it is exposed on the cell surface.

Antigenic Specificity of Opsonophagocytic Antibodies in Rabbit Anti-Sera to Group B Streptococci

An opsonophagocytic assay has been developed which requires human polymorphonuclear leukocytes, immune serum, and complement for optimal killing of Group B streptococci. Only with all three of these components was killing of greater than 1.0 log10 of the initial inoculum achieved, using rabbit antisera directed to homologous strains of each of the five known serotypes of Group B streptococci. Titers of specific antisera which opsonized the strains and resulted in greater than 1 log 10 reduction of colony-forming units, ranged from 1:100 (serotype Ib) to 1:3200 (serotype Ia). Cross-reactions between serotype-specific sera and heterologous strains were seen in certain instances. Type Ic strain and serotype Ic antiserum demonstrated cross-reactions with types Ia and Ib which were explainable by known shared antigens among these types. The only other cross-reaction which resulted in greater than 1 log 10 reduction in colony-forming units was when unabsorbed antiserum to strain Ia was used to opsonize a strain of serotype III. Opsonization of 10 serotype III strains was demonstrated with a single type III antiserum. Killing of nine of these strains required polymorphonuclear leukocytes, complement, and antiserum, but one strain, D136C, the reference strain, could be killed (greater than 1 log 10 reduction in colony-forming units) without either complement or specific antiserum. Inhibition studies were performed utilizing large m.w. polysaccharide antigens extracted from each serotype. These antigens inhibited opsonization of homologous strains by homologous antisera with 50% inhibition points ranging between 0.5 and 4 mug.

An affinity column for ecdysone binding proteins

Under basic conditions β-ecdysone can be covalently linked to the oxirane residue of epoxy-activated Sepharose 6B. Such an ecdysone-modified column matrix retards antibodies to β-ecdysone while permitting, however, the free passage of other antibodies in rabbit antiserum. The bound anti-ecdysone antibodies can subsequently be eluted by a low pH (3.8), high salt (0.5 m) buffer. The utility of such an affinity column for the isolation of ecdysone receptors is discussed.

Use of Radioimmunoassays to Determine the Concentration of Streptococcal Group-Specific Antibodies in Rabbit Antisera

A radioimmunoassay was developed for the quantitation of antibodies to the Group A, A-variant, and C carbohydrates in rabbit streptococcal antisera. The assay employs radiolabeled purified Group carbohydrate which has been tyrosylated to allow for incorporation of 125-I. This assay has an advantage over the intitative precipitin test because it measures non-precipitating antibody in addition to precipitating antibody. Furthermore, 7S anti-IgG in rabbit antisera give a falsely elevated value for the antibody concentration in the quantitative precipitin test. This did not occur with the radioimmunoassay. The assay described is reproducible, sensitive, and uses little antisera.

The immunochemistry of Staphylococcus aureus mucopeptide. I. Antigenic specificity of the peptide subunits.

Antigenic determinants of Staphylococcus aureus mucopeptide and corresponding antibodies in rabbit antiserum have been studied, using synthetic peptides in inhibition of indirect haemagglutination and immunoadsorbent techniques. Results are presented to show that at least 3 determinants are present in the peptide subunits. The pentapeptide L‐Ala‐γ‐D‐Glu‐L‐Lys‐D‐Ala‐D‐Ala exhibits 2 determinants, one of which is located to the C‐terminal D‐Ala‐D‐Ala, and the penta‐glycine bridge one determinant.

Quantitative studies of the specificity of anti-pneumococcal polysaccharide antibodies, Types III and VIII-V. Cross-reacting antibodies in rabbit antisera

Quantitative studies of the specificity of anti-pneumococcal polysaccharide antibodies, Types III and VIII-V. Cross-reacting antibodies in rabbit antisera

Quantitative studies of the specificity of anti-pneumococcal polysaccharide antibodies, Types III and VIII-V. Cross-reacting antibodies in rabbit antisera

The specificity of the antigen-binding sites of high-titer cross-reacting antibodies of restricted heterogeneity present in the serum of two rabbits immunized respectively with Type III and Type VIII pneumococcal vaccine have been studied and compared quantitatively. Despite the fact that the two antibodies, anti-S3,8 and anti-S8,3 could be precipitated almost completely by either S3 or S8 alone, they could be distinguished easily from one another by the following procedures: (1) Selective precipitation of the homologous polysaccharide from mixtures of the two antigen excess using 14C-internally labeled S3 as one of the reactants, (2) inhibition of homologous and heterologous precipitation by cellobiuronate, the disaccharide unit responsible for the cross-reaction and (3) equilibrium dialysis against labeled homologous and heterologous oligosaccharide ligands.

Immunological investigations of penicillium—II. Primary binding of glycopeptides and glycopeptide derivatives to specific antibodies

An exocellular glycopeptide from Penicillium charlesii has been shown previously to contain a high mol. wt polysaccharide and several oligosaccharides attached to the peptide through seryl and threonyl residues. Guinea pigs immunized with the glycopeptide conjugated to bovine gamma globulin, produced nonprecipitating antibodies to the polysaccharide portion of the glycopeptide. However, a technique was developed for measuring the primary binding of the glycopeptide by the guinea pig antibodies. It was found that the antigen-antibody complex could be precipitated by 40% ammonium sulfate, whereas negligible quantities of the glycopeptide alone or glycopeptide incubated with normal guinea pig sera were precipitated under these conditions. Equations predicting the influence of the ratio of a weighted dissociation constant representing the various antigen-antibody complexes/antibody concentrations, β/[ Ab t] , on antigen binding and the influence of this ratio on inhibition of antigen binding by hapten were derived. The data obtained with the fungal glycopeptide as an antigen were interpreted in terms of this model. The experiments showed that antibodies in the guinea pig sera were directed toward the polysaccharide portion of the glycopeptide, and not toward the peptide portion or the oligosaccharides. Furthermore, a glycopeptide deficient in galactofuranosyl and phosphorus moieties from a uridine diphosphate galactose 4-epimerase negative mutant of P. charlesii competed with wild type glycopeptide for antibody. It appeared that mannopyranosyl residues associated with an, as yet unidentified, acid labile material were the major immunodeterminant groups of the glycopeptide which bound to guinea pig sera. In contrast to its behavior in guinea pig antisera, the polysaccharide portion of the glycopeptide did not compete with the glycopeptide for antibodies in rabbit antisera. In addition the peptide portion of the glycopeptide did not compete with the glycopeptide in the rabbit antisera. Further, dilute acid treatment of the glycopeptide did not destroy its ability to bind to rabbit antibodies. These data suggest that the oligosaccharides attached to the peptide serve as the antigenic determinats which bind to rabbit antibodies.

Specific agglutination of polyacrylamide derivatives of protein antigens

Proteins such as β-lactoglobulin, bovine serum albumin, α-lactalbumin, and bovine γ-globulin were coupled by covalent bonds to crosslinked copolymers of acrylamide and methyl acrylate through reaction with azide derivatives of the methyl acrylate residues. Substantial amounts of proteins (1–5 mg/100 mg polymer) were bound to the surface of the copolymer particles. The use of these derivatives to assay for the specific antibodies in rabbit antisera was tested. Specific agglutination of the protein derivatives was obseved with antisera containing as little as 0·05 μg antibody per ml. Antibody titers against the protein derivatives as high as 1:10,000 were retained on storage of the polymer-protein derivatives at 4°C in saline solutions for 3 months. The protein derivatives removed specific antibodies from immune sera.

Chemical and immunological properties of reduced and alkylated polypeptide chains of bovine fibrinogen.

The α, β and γ chains were isolated from reduced and carboxymethylated bovine fibrinogen by chromatography on CM‐cellulose. Electrophoretically pure polypeptide chains could be obtained as judged by three different methods. The chains were soluble in buffers at or above pH 8 but exhibited non‐covalent aggregation. The molecular weights of the α, β and γ chains were estimated in dodecylsulfate‐polyacrylamide gel electrophoresis as 62000, 58000 and 48000, respectively. Each chain differed considerably from the other in amino acid composition as well as in the fragment pattern obtained after CNBr treatment. The occurrence of unresolved α/β material suggested microheterogeneity of the individual chains. Sulfitolysis yielded, at least for the α chain, less pure products.Between 20% and 54% of the antibodies in rabbit antisera to native fibrinogen precipitated with the α and/or β chain. Absorption studies, gel precipitation and hemagglutination‐inhibition suggested a considerable antigenic homology of both chains. They could be clearly distinguished from the γ chain which reacted poorly in precipitation test but considerably better in passive hemagglutination. Cyanogen bromide cleavage did not destroy the serologic activity of the chains. These findings indicated that a considerable number of the antigenic determinants on fibrinogen are not dependent on intact conformation. However, antibodies specifically reacting with the native molecule occur as well.

Detection of anti-HeLa antibodies in rabbit antiserum by indirect hemagglutination.

SummaryMaterial obtained from sonically disrupted HeLa cells by extraction of trichloracetic-acid precipitate with buffered saline was used to sensitize tanned sheep erythrocytes. Sensitized erythrocytes were agglutinated to high titer by anti-HeLa rabbit serum, but minimally by normal rabbit serum. Reactivity of the sensitizing extract was considerably reduced by treatment of HeLa cells with tannic, acid prior to disruption and extraction.

Studies on Organ Specificity

Summary Beef thyroid antisera and hog thyroid antisera produced by the rabbit contain, in addition to potent antibodies against their homologous antigen, antibodies against rabbit thyroid extract. Beef thyroid and hog thyroid antisera when treated with the homologous antigen are completely deprived of their antibody content. Complete absorption with rabbit thyroid extract, on the other hand, removed only that part of the antibody spectrum which is directed against the rabbit thyroid extract but always left antibodies against the homologous thyroid antigen. Rabbit thyroid antibodies in rabbit antisera produced by immunization with thyroid extracts of foreign species are demonstrable mainly by the tanned cell hemagglutination test and occasionally by the gel diffusion precipitation test. In addition, complement-fixing antibodies against rabbit thyroid extracts could be demonstrated in certain antisera against hog thyroglobulin following an intensive and prolonged course of immunization. Histologic changes characteristic of immunologic thyroiditis were also observed in the thyroid glands of the rabbits intensively treated with hog thyroglobulin.

Hemagglutination test for detection of Candida albicans antibodies in rabbit antiserum.

SummarySaline washings from Candida albicans yeast cells have been found to yield an active hemagglutinating substance which does not appear to be protein in nature. A Candida yeast cell antigen cross reacted with Saccharomyces rabbit antiserum. On the other hand, a Candida hemagglutinating antigen, equally effective in the homologous test, eliminated the cross reaction with Saccharomyces rabbit antiserum.