Cross-reactivity Of Antiserum Research Articles

Immunogenic Properties and Antigenic Similarity of Virus-like Particles Derived from Human Polyomaviruses.

Polyomaviruses (PyVs) are highly prevalent in humans and animals. PyVs cause mild illness, however, they can also elicit severe diseases. Some PyVs are potentially zoonotic, such as simian virus 40 (SV40). However, data are still lacking about their biology, infectivity, and host interaction with different PyVs. We investigated the immunogenic properties of virus-like particles (VLPs) derived from viral protein 1 (VP1) of human PyVs. We immunised mice with recombinant HPyV VP1 VLPs mimicking the structure of viruses and compared their immunogenicity and cross-reactivity of antisera using a broad spectrum of VP1 VLPs derived from the PyVs of humans and animals. We demonstrated a strong immunogenicity of studied VLPs and a high degree of antigenic similarity between VP1 VLPs of different PyVs. PyV-specific monoclonal antibodies were generated and applied for investigation of VLPs phagocytosis. This study demonstrated that HPyV VLPs are highly immunogenic and interact with phagocytes. Data on the cross-reactivity of VP1 VLP-specific antisera revealed antigenic similarities among VP1 VLPs of particular human and animal PyVs and suggested possible cross-immunity. As the VP1 capsid protein is the major viral antigen involved in virus-host interaction, an approach based on the use of recombinant VLPs is relevant for studying PyV biology regarding PyV interaction with the host immune system.

A single amino acid change in hemagglutinin reduces the cross-reactivity of antiserum against an equine influenza vaccine strain.

Equine influenza virus is an important pathogen for the horse industry because of its economic impact, and vaccination is a key control measure. Our previous work suggested that a mutation at position 144 in the hemagglutinin of Florida sublineage clade 2 viruses reduces the cross-neutralizing activity of antiserum against a former vaccine strain. To confirm this suggestion, here, we generated viruses by reverse genetics. Antibody titers against the mutated viruses were one-tenth to one-sixteenth of those against the former vaccine strain. Our findings confirm that this single amino acid substitution reduces the cross-reactivity of antiserum against this former Japanese vaccine.

Corynebacterium kroppenstedtii in granulomatous mastitis: Analysis of formalin-fixed, paraffin-embedded biopsy specimens by immunostaining using low-specificity bacterial antisera and real-time polymerase chain reaction.

Granulomatous mastitis (GM) is a rare inflammatory disease of the post-lactation breast, clinically mimicking breast cancer. GM is microscopically characterized by formation of epithelioid granulomas and abscess (suppurative granulomas) with lipid droplet-centered inflammation. Corynebacterium kroppenstedtii (Ck) is known as a causative bacterium of GM, and identification of Ck infection within the lesion should thus be essential for confirming the diagnosis. In the present study, we analyzed formalin-fixed, paraffin-embedded (FFPE) biopsy specimens of a total of 18 GM lesions with immunostaining and real-time PCR for Ck genome. Widely cross-reactive rabbit antisera against Bacillus Calmette-Guerin (BCG), Bacillus cereus, Treponema pallidum and Escherichia coli were chosen. With real-time PCR, Ck genome was demonstrated in 7 of 18 GM lesions. Immunohistochemically, the low-specificity antisera reacted with the cytoplasm of phagocytes and/or granuloma-engulfed lipid droplets in 12 of 18 GM lesions. Antigenic positivity was observed in the following order: BCG > B. cereus > T. pallidum > E. coli. Real-time PCR using DNA extracted from FFPE sections was useful but not consistent for identifying the Ck genome in GM, while immunostaining using cross-reactive antisera against four kinds of bacteria was not Ck-specific but was applicable to visualizing bacterial infection within the GM lesions.

First Identification of Human Adenovirus 57 (HAdV-57) in Japan

Neutralization tests have been routinely used for the identification of human adenovirus C species (HAdV-C) in Japan until 2007. The aim of this study was to clarify the serological cross-reactivity of antiserum that has been used exclusively in Japan and to describe the first identification of HAdV type 57 (HAdV-57) in Japan. Anti-HAdV serum to HAdV-1, 2, 5, and 6 was quantitatively evaluated for cross-reactivity to the HAdV-57 isolates. Anti-HAdV-6 serum neutralized HAdV-57 with a concentration that was 32 to 64-fold higher than what was necessary to neutralize homologous HAdV-6. HAdV-1, 2, and 5 strains were not neutralized by anti HAdV-6 serum. Furthermore, 28 HAdV-6 strains isolated from 6,476 clinical samples were re-examined for HAdVs detected in the Shimane Prefecture of Japan from 2005 to 2014. These 28 strains were re-examined by PCR-sequencing techniques using the penton, hexon, and fiber regions. 3 isolates were determined to be HAdV-57. These data show that HAdV-57 had already invaded Japan as early as 2005, and that HAdV-57 strains were misidentified as HAdV-6.

A novel hepatitis B virus species discovered in capuchin monkeys sheds new light on the evolution of primate hepadnaviruses

All known hepatitis B virus (HBV) genotypes occur in humans and hominoid Old World non-human primates (NHPs). The divergent woolly monkey HBV (WMHBV) forms another orthohepadnavirus species. The evolutionary origins of HBV are unclear. We analysed sera from 124 Brazilian monkeys collected during 2012-2016 for hepadnaviruses using molecular and serological tools, and conducted evolutionary analyses. We identified a novel orthohepadnavirus species in capuchin monkeys (capuchin monkey hepatitis B virus [CMHBV]). We found CMHBV-specific antibodies in five animals and high CMHBV concentrations in one animal. Non-inflammatory, probably chronic infection was consistent with an intact preCore domain, low genetic variability, core deletions in deep sequencing, and no elevated liver enzymes. Cross-reactivity of antisera against surface antigens suggested antigenic relatedness of HBV, CMHBV, and WMHBV. Infection-determining CMHBV surface peptides bound to the human HBV receptor (human sodium taurocholate co-transporting polypeptide), but preferentially interacted with the capuchin monkey receptor homologue. CMHBV and WMHBV pseudotypes infected human hepatoma cells via the human sodium taurocholate co-transporting polypeptide, and were poorly neutralised by HBV vaccine-derived antibodies, suggesting that cross-species infections may be possible. Ancestral state reconstructions and sequence distance comparisons associated HBV with humans, whereas primate hepadnaviruses as a whole were projected to NHP ancestors. Co-phylogenetic analyses yielded evidence for co-speciation of hepadnaviruses and New World NHP. Bayesian hypothesis testing yielded strong support for an association of the HBV stem lineage with hominoid ancestors. Neither CMHBV nor WMHBV was likely the ancestor of the divergent human HBV genotypes F/H found in American natives. Our data suggest ancestral co-speciation of hepadnaviruses and NHP, and an Old World origin of the divergent HBV genotypes F/H. The identification of a novel primate hepadnavirus offers new perspectives for urgently needed animal models of chronic hepatitis B. The origins of HBV are unclear. The new orthohepadnavirus species from Brazilian capuchin monkeys resembled HBV in elicited infection patterns and could infect human liver cells using the same receptor as HBV. Evolutionary analyses suggested that primate HBV-related viruses might have emerged in African ancestors of New World monkeys millions of years ago. HBV was associated with hominoid primates, including humans and apes, suggesting evolutionary origins of HBV before the formation of modern humans. HBV genotypes found in American natives were divergent from those found in American monkeys, and likely introduced along prehistoric human migration. Our results elucidate the evolutionary origins and dispersal of primate HBV, identify a new orthohepadnavirus reservoir, and enable new perspectives for animal models of hepatitis B.

Rational selection of broadly cross-reactive family 2 PspA molecules for inclusion in chimeric pneumococcal vaccines.

Pneumococcal surface protein A (PspA) is a widely studied pneumococcal protein, exposed at the surface of all strains. It is an important virulence factor, preventing complement deposition as well as inhibiting the lytic effects of lactoferrin over pneumococci. Several studies have investigated the use of PspA as a candidate in alternative pneumococcal vaccines, with great success. However, PspA presents sequence variability - there are six clades, grouped in three families - and PspAs within the same clade exhibit different levels of cross-reactivity. Therefore, the aim of this work was to select, from a panel of eight pneumococcal isolates expressing family 2 PspAs, the molecule with the broadest reactivity within this family. Antisera to these PspA fragments were initially screened by immunoblot against thirteen pneumococcal extracts; the three most cross-reactive antisera were tested for their ability to enhance the deposition of complement factor C3b on the bacterial surface and to promote their phagocytosis invitro. PspA from strain P490 was the most effective, increasing phagocytosis of all but one pneumococcal isolate. Thus, this molecule was selected for inclusion in chimeric protein-based pneumococcal vaccines. In conclusion, the rational selection of cross-reactive molecules is an important step in the development of vaccines with broad coverage.

The cat lipocalin Fel d 7 and its cross‐reactivity with the dog lipocalin Can f 1

We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat-sensitized Swedish patients and elucidated its allergenic activity and cross-reactivity with the dog lipocalin Can f 1. Sixty-five of 140 patients had IgE to rFel d 7 whereof 60 also had IgE to rCan f 1. A moderate correlation between IgE levels to rFel d 7 and rCan f 1 was found. rFel d 7 activated basophils in vitro and inhibited IgE binding to rCan f 1 in 4 of 13 patients, whereas rCan f 1 inhibited IgE binding to rFel d 7 in 7 of 13 patients. Fel d 7 and Can f 1 showed high similarities in protein structure and epitopes in common were found using cross-reactive antisera. Fel d 7 is a common allergen in a Swedish cat-sensitized population that cross-reacts with Can f 1, and may contribute to symptoms in cat- but also in dog-allergic patients.

Micrurus snake species: Venom immunogenicity, antiserum cross-reactivity and neutralization potential

Micrurus snakebites can cause death by muscle paralysis and respiratory arrest a few hours after envenomation. The specific treatment for these snake envenomations is the intravenous application of heterologous antivenom. In Brazil, this antivenom is produced from horses that are immunized with a mixture of Micrurus corallinus and Micrurus frontalis venoms, which are snakes that inhabit the south and southeastern regions of the country. Previously, we demonstrated that the coral antivenom, which is used in human therapy, was not able to neutralize several of the toxic venom effects from some Micrurus species that inhabit the country, as measured by in vitro and in vivo assays. The present study aimed to investigate the immunogenic properties of Micrurus spp. venoms, as well as the cross-reactivity and neutralization potential of experimental monovalent and polyvalent sera that were produced in different animal species. The present data showed that Micrurus venoms exhibited the same immunogenicity pattern in the three utilized animal species and that the specific antisera presented a large cross-reactivity when analyzed with ELISA and Western blot assays. Nonetheless, these positive results were not well correlated with the neutralizing potential of the antisera. Thus, the establishment of a new antigenic mixture to produce novel more efficient therapeutic Micrurus antivenom is not a simple task. Further studies, particularly with the Micrurus lemniscatus, Micrurus altirostris and Micrurus surinamensis venoms, are necessary to establish new strategies for the production of antivenoms with broad neutralizing activity for the treatment of accidents involving coral snakes throughout the country.

Expression of <I>OmpK</I> gene of <I>Vibrio alginolyticus</I> and development of a detection method of indirect ELISA

Vibrio alginolyticus is the main Vibrio pathogen in aquaculture in the south of China,the number of which is the largest in marine Vibrio class.In the present study,the outer membrane protein K gene(OmpK)of V.alginolyticus strain ATCC17749 was amplified by PCR and cloned into high efficient expression vector pET28a.The results of sequencing and restriction enzyme analysis combined with agarose gel electrophoresis showed that the open reading frame sequence was correct and fully consistent with what had reported.The recombinant plasmid of pET-28a-OmpK was successfully constructed and transformed into E.coli BL21 pLys E.The fusion protein was expressed under the IPTG inducing condition,and the expression product was purified by an affinity chromatographic method.Mouse anti-OmpK poly-clonal antibodies(PAbs)were obtained via applying protein OmpK as antigen to immune mice.Western-blotting analysis proved the recombinant protein has a good reactive ability against OmpK positive serum.Then,an indirect Enzyme-Linked Immunosorbent Assay(ELISA)for the rapid diagnosis of V.alginolyticus has been developed using the PAbs.The lowest V.alginolyticus suspension was 104 CFU/mL.Cross reactions of antisera with other bacteria were detected,and all results were negative.The results indicated the suitability and simplicity of the test as a rapid,field diagnostic tool for V.alginolyticus and it can be used for the rapid detection of V.alginolyticus.Further investigation will be focused on the development of an indirect ELISA Kit for the detection of V.alginolyticus.

Evidence for a new avian paramyxovirus serotype 10 detected in rockhopper penguins from the Falkland Islands.

The biological, serological, and genomic characterization of a paramyxovirus recently isolated from rockhopper penguins (Eudyptes chrysocome) suggested that this virus represented a new avian paramyxovirus (APMV) group, APMV10. This penguin virus resembled other APMVs by electron microscopy; however, its viral hemagglutination (HA) activity was not inhibited by antisera against any of the nine defined APMV serotypes. In addition, antiserum generated against this penguin virus did not inhibit the HA of representative viruses of the other APMV serotypes. Sequence data produced using random priming methods revealed a genomic structure typical of APMV. Phylogenetic evaluation of coding regions revealed that amino acid sequences of all six proteins were most closely related to APMV2 and APMV8. The calculation of evolutionary distances among proteins and distances at the nucleotide level confirmed that APMV2, APMV8, and the penguin virus all were sufficiently divergent from each other to be considered different serotypes. We propose that this isolate, named APMV10/penguin/Falkland Islands/324/2007, be the prototype virus for APMV10. Because of the known problems associated with serology, such as antiserum cross-reactivity and one-way immunogenicity, in addition to the reliance on the immune response to a single protein, the hemagglutinin-neuraminidase, as the sole base for viral classification, we suggest the need for new classification guidelines that incorporate genome sequence comparisons.

Lack of evidence for the μ‐opioid receptor splice variant MOR1C in rats

We previously reported the existence of MOR1(C) mRNA and MOR1(C)-immunoreactivity (-ir) in rats. However, the sequence that we reported for rat MOR1(C) appears not to be present in the rat genome. We have therefore reexamined whether MOR1(C) mRNA or MOR1(C)-ir exist in rats. We used reverse-transcription polymerase chain reaction (RT-PCR) to attempt to amplify MOR1, MOR1(A), MOR1(B), the rat MOR1(C) sequence we previously reported, and MOR1(C1) and MOR1(C2) (which have recently been reported to exist in rats). In RNA extracted from rats, we were able to demonstrate PCR products representing MOR1, MOR1(A), and MOR1(B) splice variants. All three products were confirmed as related to MOR1 by Southern blot. However, we were unable to detect either the MOR1(C) product reported previously by us or the MOR1(C)-like products reported to exist in rats by others. In RNA extracted from mice we were able to detect MOR1, MOR1(A), MOR1(B), and MOR1(D)-like products. To test the specificity of our MOR1(C) antiserum, we examined MOR1(C)-ir in control and knockout mice. MOR1(C)-ir had a distribution in control mice similar to that previously reported in rats, including coexisting with vGLUT2. However, although MOR1-ir was absent in MOR1 knockout mice, the density and distribution of MOR1(C)-ir were unchanged, suggesting that the antiserum crossreacts with another molecule in tissue. We find no evidence for MOR1(C) mRNA in rats. Furthermore, we conclude that MOR1(C)-ir represents crossreactivity.

Studies on lipolytic isoenzymes from a thermophilic Bacillus sp.: Production, purification and biochemical characterization

An isolated thermophilic Bacillus sp. produced two extracellular lipases. The expression of the two lipases was governed by the age of the culture. Hydrophobic interaction chromatography separated the lipolytic activity into two peaks. The second lipase got eluted with 80% ethylene glycol. The apparent molecular mass of Lip2 was approximately 60,000 Da from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 62,000 ± 3000 Da from gel filtration. The enzyme showed optimal activity at 60–65 °C and retained 100% activity after incubation at 60 °C and pH 8.0 for 1 h. The enzyme showed maximum activity at pH 8.0 and was very stable at pH 7.0–8.5 with optimum stability at pH 8.0. At this pH and 60 °C temperature, the half-life of enzyme was 170 h. It exhibited 50% of its original activity after 45 min incubation at 70 °C. It was demonstrated that SDS, DTT (100 mM each) and eserine (5 mM) did not affect enzyme activity while activity was inhibited in the presence of PMSF, DEPC and EDTA (100 mM each). Enzyme activity was stimulated in the presence of benzene or hexane (each 30%, v/v). With p-nitrophenyl laurate as a substrate, the enzyme exhibited a K m and V max of 0.19 mM and 0.032 μM/min/ml. The enzyme showed preference for long chain triacylglycerol and hydrolyzed triolein at all positions. The enzyme had very high hydrophobic amino acid content (60.2%) with about 50% of total amino acids as alanine and 21% as glycine. Western blot analysis confirmed the cross-reactivity of antiserum against Lip1 with Lip2. Dot blot analysis suggested that antibodies are specific for these isoforms as it did not react with a purified lipase from a mesophilic Bacillus sp.

Identification and characterization of a squamate reptilian amelogenin gene:Iguana iguana

As the principal components of the developing tooth enamel matrix, amelogenins play a significant role in tooth enamel formation and organization. In order to elucidate the structure and function of amelogenins in the evolution of enamel, we have selected the Iguana iguana as a squamate model organism. Here we report the first complete squamate amelogenin sequence available as of yet and document unique features of Iguana amelogenins and enamel. Transmission electron microscopy documented randomly oriented Iguana enamel crystals during the elongation phase compared with organized enamel crystal patterns at comparable stages in mammals. Sequencing of PCR amplified products revealed a full-length I. iguana amelogenin cDNA containing 877 nucleotides with a 564 nucleotide coding sequence encoding 187 amino acids. The homologies of the newly discovered I. iguana amelogenin amino acid sequence with the published mouse, caiman (Palaeosuchus), and snake (Elaphe) amelogenin were 41.3%, 53.5%, and 55.5%, respectively. On Western blots one major protein with a molecular weight of 24 kDa, and two minor proteins with molecular weights of 28 and 13.5 kDa, respectively, were detected based on the cross-reactivity of antisera against recombinant Rana pipiens amelogenin proteins. Sequence analysis revealed a moderate sequence homology between mammalian and reptilian amelogenin genes. A significant alteration was the deletion of the hydrophilic GSP sequence from exon 3 in the mouse sequence resulting in a conversion to a hydrophobic region in Iguana. Together, these findings identified a novel amelogenin cDNA sequence in the squamate reptilian I. iguana and functional implications for the evolution of amelogenins and enamel in squamates.

Measurement of serum and peritoneal fluid LH concentrations as a diagnostic tool for human endometriosis

A rapid, sensitive enzymeimmunoassay for the measurement of LH concentrations in serum and peritoneal fluid samples of healthy women and women with endometriosis is reported. The ligand (LH) was captured by a readily available, widely used and well-characterized monoclonal antibody (mAb, 518B7) generated against the beta subunit of bovine LH. This mAb, although specific for LH, shows very little species specificity and detects LH by radioimmunoassay in humans. A polyclonal antiserum raised in rabbits against hCG was conjugated to horseradish peroxidase and was used as the second antibody signal. This anti-hCG antiserum crossreacts with LH. The enzymeimmunoassay uses the standard human LH (hLH) preparations (NIADDK-hLH-I-3, AFP-827OB) and results are based on the relative concentrations of LH in serum and peritoneal fluid. Total assay time was < 3 h. The range of the standard curve was 0.002-0.500 ng LH per well and the lowest concentration of hLH that could be distinguished from zero concentration was 0.15 +/- 0.02 ng ml(-1) serum and 0.058 +/- 0.021 ng ml(-1) peritoneal fluid. Clinical diagnostic parameters for the LH enzymeimmunoassay showed a sensitivity of 85.71%, specificity 92.50%, efficiency 88.54%, positive predictive value 94.11% and negative predictive value 82.22%. The study was retrospective. Serum LH concentrations of women with endometriosis were 13.67 +/- 7.21 ng ml(-1), whereas serum LH concentrations of women in the control group were 4.52 +/- 2.03 ng ml(-1). One-way ANOVA showed significant differences (P < 0.001) between women with endometriosis and control groups. Women in the control group had peritoneal fluid LH values of 5.65 +/- 2.43 ng ml(-1), whereas peritoneal fluid LH values of 64.06 +/- 16.44 ng ml(-1) were obtained in women with endometriosis (P < 0.001). A cycle-dependent pattern of serum and peritoneal fluid LH concentration was observed in women in the control group, which was not observed in the peritoneal fluid of the group with endometriosis. The application of this assay to serum or peritoneal fluid samples provides the attractive possibility that it could be included in the panel of markers used for diagnosis of endometriosis.

Characterization of chicken antisera raised against Aspergillus spp. by enzyme linked immunosorbent assay.

Enzyme linked immunosorbent assays of three Aspergillus species have been developed. Laying hens were immunized with the exoantigens from Asp. flavus, Asp. ochreaus and Asp. versicolor. All test chickens except for one produced antisera raised against the exoantigens. The antisera production process and ELISA titer were analysed. Fourteen days after the first injection, the antisera began to produce largely, on the 35th day reached to the peak, and maintained a stable level until the 42nd day. The maximum ELISA titer of the antisera to the exoantigens from Asp. flavus, Asp. ochreaus and Asp. versicolor was 1:8,000, 1:10,000 and 1:10,000, respectively. The cross-reactivities of antisera were determined with seventeen species of Aspergillus, ten species of fungi from other genera and the buffer-extracts of grain. The antisera did not cross-react with the exoantigens from other genera and the buffer-extracts of grain. The antiserum to exoantigen from Asp. ochreaus was species-specific, whereas the antisera against Asp. flavus and Asp. versicolor tended to cross-react with other Aspergillus species to varying degrees. The results suggest that exoantigens immunoassays can be developed to indentify and detect Aspergillus genus in grains.

Subunit specificity of polyclonal antisera to the carboxy terminal regions of P2X receptors, P2X1 through P2X7

Polyclonal antisera generated to peptides corresponding to the carboxy termini of the seven cloned rat P2X receptors were compared using membranes from CHO-K1 or 1321N1 cells expressing recombinant rat or human P2X receptors (rat P2X3, P2X4, and P2X5 and human P2X1, P2X2, P2X6, and P2X7). Preimmune sera failed to recognize any bands in immunoblots against the membrane fractions. Antiserum to the P2X1 receptor recognized a band of approximately 58 kDa while those to the rP2X2 and the rP2X3 receptors recognized doublets of approximately 60 and 64 kDa. The antiserum to the rP2X4 receptor recognized a similarly sized doublet as well as a higher molecular weight species at the expected size for a receptor dimer. Antiserum to the rP2X5 receptor revealed a single band at 64 kDa while antiserum to rP2X6 and rP2X7 gave single bands at 50 kDa and 95 kDa, respectively. In all cases, immunoreactivity was eliminated by preincubation of the antisera with the cognate peptide and there was no cross-reactivity of antisera with heterologous receptors. These antisera are useful reagents for the analysis of the P2X receptor subtypes in native tissues as well as for measuring receptor expression levels in transfected cell lines. Drug Dev. Res. 47:189–195, 1999. © 1999 Wiley-Liss, Inc.

Subunit specificity of polyclonal antisera to the carboxy terminal regions of P2X receptors, P2X1 through P2X7

Polyclonal antisera generated to peptides corresponding to the carboxy termi of the seven cloned rat P2X receptors were compared using membranes from CHO-K1 or 1321N1 cells expressing recombinant rat or human P2X receptors (rat P2X 3 , P2X 4 , and P2X 5 and human P2X 1 , P2X 2 , P2X 6 , and P2X 7 ). Preimmune sera failed to recognize any bands in immunoblots against the membrane fractions. Antiserum to the P2X 1 receptor recognized a band of approximately 58 kDa while those to the rP2X 2 and the rP2X 3 receptors recognized doublets of approximately 60 and 64 kDa. The antiserum to the rP2X 4 receptor recognized a similarly sized doublet as well as a higher molecular weight species at the expected size for a receptor dimer. Antiserum to the rP2X 5 receptor revealed a single band at 64 kDa while antiserum to rP2X 6 and rP2X 7 gave single bands at 50 kDa and 95 kDa, respectively. In all cases, immunoreactivity was eliminated by preincubation of the antisera with the cognate peptide and there was no cross-reactivity of antisera with heterologous receptors. These antisera are useful reagents for the analysis of the P2X receptor subtypes in native tissues as well as for measuring receptor expression levels in transfected cell lines.

Time-Resolved Fluoroimmunoassay for Plasma Enterolactone

We present a method for the determination of the lignan enterolactone in plasma (serum). This compound, produced by intestinal bacteria from matairesinol and secoisolariciresinol in fiber-rich food, is a biomarker related to the intake of a healthy diet. The method is based on time-resolved fluoroimmunoassay using a europium chelate as a label. After synthesis of 5′-O-carboxymethoxyenterolactone the compound is coupled to bovine serum albumin and then used as antigen in immunization of rabbits. The tracer with the europium chelate is synthesized using the same 5′-derivative of enterolactone. After enzymatic hydrolysis and ether extraction the immunoassay is carried out using the VICTOR 1420 multilabel counter (Wallac Oy, Turku, Finland). No antiserum cross-reactivity with available lignans, isoflavonoids, or flavonoids could be detected. The intraassay and interassay coefficients of variation at different concentrations vary 4.6–6.0 and 5.5–9.9, respectively. The working range of the assay is 1.5–540 nmol/liter. We measured enterolactone in serum/plasma of 224 Finnish subjects: 98.8% of the subjects had values <100 nmol/liter, 38.0% had 20–39.9 nmol/liter, and 34.4% had <20 nmol/liter.

The use of synthetic peptides in the design of a consensus sequence vaccine for Pseudomonas aeruginosa.

Pseudomonas aeruginosa employs pili to mediate adherence to epithelial cell surfaces. Research has shown that the C-terminal region of the pilin monomer contains the epithelial cell binding domain, which is semiconserved in seven different strains of this bacterium. Antibodies to this region of the pilin molecule are also able to block and prevent the infection process. As there is a degree of sequence and structural homology in the C-terminal region and all strains examined have been shown to bind to the same cell surface receptor, we reasoned that it should be possible to produce a synthetic peptide consensus sequence which would provide cross-reactive antiserum from a single peptide immunogen inhibiting the adherence of the known strains of P. aeruginosa. In this article we examine the cross-reactivity of five rabbit polyclonal antisera. One has been raised against the cell-surface receptor binding domain of native PAK strain pilin (residues 128-144) while the others have been raised to analogues of this region. Analysis of the cross-reactivity of these antisera, using competitive ELISA assay, has shown that it is possible to manipulate the amino acid sequence of a peptide immunogen to generate antiserum, which exhibits enhanced cross-reactivity to various strains of P. aeruginosa. Furthermore, when this peptide is conjugated to tetanus toxoid and used to vaccinate mice it provided cross-reactive protection against heterologous challenge with PAO strain bacteria. The results of these experiments are analyzed, and the applicability of our hypothesis and the implications of this approach to the design of a strain-independent consensus vaccine for immunization against Pseudomonas aeruginosa are discussed.

Somatostatin Inhibits (d-Arg6, Pro9-NEt) Salmon Gonadotropin-Releasing Hormone- and Dopamine D1-Stimulated Growth Hormone Release from Perifused Pituitary Cells of Chinese Grass Carp,Ctenopharyngodon idellus

In this study, a heterologous radioimmunoassay (RIA) for grass carp GH has been validated and used to monitor the kinetics of GH release from perifused grass carp pituitary cells. To establish the anatomical specificity of GH antiserum used in this RIA, immunohistochemical staining was performed in grass carp pituitary sections. Somatotrophs recognized by this GH antiserum were located mainly in the proximal pars distalis without overlapping with gonadotrophs located in the same area or with lactotrophs located in the rostral pars distalis. The immunoreactivity of somatotrophs was abolished by preabsorbing GH antiserum with purified grass carp GH, suggesting that the possibility of a cross-reactivity of antiserum with other grass carp pituitary hormones is unlikely. Using125I-labeled carp GH as the RIA tracer, parallelism was observed among the displacement curves of grass carp GH standard, grass carp serum, and culture medium conditioned by grass carp pituitary cells, suggesting that this RIA can be used to quantitate grass carp GH levels in biological samples. Using anin vitrocolumn perifusion system, a superactive gonadotropin-releasing hormone (GnRH) analog (d-Arg6, Pro9-NEt)-sGnRH(sGnRHa, 0.3–30 nM), dopamine (DA, 0.1–10 μM), and the nonselective DA agonist apomorphine (0.1–10 μM) stimulated GH release from grass carp pituitary cells in a dose-dependent manner. The GH-releasing effect of DA was mimicked by the D1agonists SKF38393 (0.1–10 μM) and SKF77434 (0.1–10 μM), but not by the D2agonist LY171555 (3 μM). In addition, the GH response to DA (1 μM) was blocked by the D1antagonist SCH23390 (5 μM) but not by the D2antagonist (±) sulpiride (5 μM), suggesting that the GH-releasing action of DA is mediated through receptors resembling mammalian D1receptors. Somatostatin-14 (SRIF14, 0.01–100 nM), unlike sGnRHa and DA, induced a dose-dependent suppression on basal GH release. At a high dose (100 nM), SRIF14also abolished the GH responses to sGnRHa (100 nM), DA (10 μM), and the D1agonist SKF38393 (3 μM). These results, as a whole, provide evidence that GH release in the grass carp is under the direct regulation of GnRH, DA, and SRIF at the pituitary cell level. The present study also suggests that DA D1receptors are present in grass carp pituitary cells mediating the GH-releasing action of DA.