Mouse Polyclonal Antiserum Research Articles

Characterization of a Sulfhydryl Oxidase From Plasmodium berghei as a Target for Blocking Parasite Transmission.

Quiescin sulfhydryl oxidase (QSOX), present in a wide variety of eukaryotic species, catalyzes the insertion of disulfide bonds into unfolded, reduced proteins. Here we characterized the QSOX protein from the rodent malaria parasite Plasmodium berghei (PbQSOX), which is conserved in all sequenced malaria parasite species. The PbQSOX protein was not expressed in asexual erythrocytic stages, but was most abundantly expressed in ookinetes. Indirect immunofluorescence assays revealed PbQSOX was not only localized in cytoplasm of gametocytes, gametes and ookinetes, but also expressed on the surface of gametes and ookinetes. Western blot identified extracellular presence of PbQSOX in the culture medium of ookinetes suggestive of secretion. Pbqsox deletion (Δpbqsox) did not affect asexual intraerythrocytic development, but reduced exflagellation of male gametocytes as well as formation and maturation of ookinetes. Pbqsox deletion also led to a significant increase in the reduced thiol groups of ookinete surface proteins, suggesting that it may play a role in maintaining the integrity of disulfide bonds of surface proteins, which might be needed for ookinete development. Mosquitoes that fed on Δpbqsox-infected mice showed a significant reduction in ookinete and oocyst numbers compared to those fed on wild-type parasite-infected mice. Further, both polyclonal mouse antisera and a monoclonal antibody against the recombinant PbQSOX exhibited substantial transmission-blocking activities in in vitro and mosquito feeding assays, suggesting QSOX is a potential target for blocking parasite transmission.

Isolation of Probiotic Piliated Lactobacillus rhamnosus Strains from Human Fecal Microbiota Using SpaA Antiserum-Based Colony Immunoblotting.

Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect 2.5 × 103 CFU/ml of pLR colonies spiked in 106 CFU/ml of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.

Expression, purification of metallothionein genes from freshwater crab (Sinopotamon yangtsekiense) and development of an anti-metallothionein ELISA.

Using the phoA-fusion technology, the recombinant metallothionein (MT) from freshwater crab (Sinopotamon yangtsekiense) has been successfully produced in Escherichia coli. MT purified from the bacterial suspension showed one polypeptide with a molecular weight of 7 kDa by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). Western-blotting confirmed the polypeptides had a specific reactivity with mouse polyclonal MT anti-serum. Based on the purified MT and MT anti-serum, the reaction parameters for an enzyme-linked immunosorbent assay (ELISA) were developed. The direct coating ELISA showed a higher linear relationship compared to antibody sandwich coating ELISA. The optimal dilution rates of purified MT anti-serum and coating period were shown to be 1:160,000 and 12 hours at 4°C. At 37°C, the appropriate reaction duration of the first antibody and the second antibody were 2 hours and 1 hour, respectively. According to these optimal parameters, the standard linear equation, y = 0.0032x + 0.1769 (R2 = 0.9779, x, y representing MT concentration and OD450 value), was established for the determination of MT concentration with a valid range of 3.9–500 ng/ml. In verification experiments, the mean coefficients of variation of the intra-assay and inter-assay were 3.260% and 3.736%, respectively. According to the result of MT recovery, ELISA with an approaching 100% MT recovery was more reliable and sensitive than the Cd saturation assay. In conclusion, the newly developed ELISA of this study was precise, stable and repeatable, and could be used as a biomarker tool to monitor pollution by heavy metals.

Characterization of the teneurin C-terminal associated peptide (TCAP) in the vase tunicate, Ciona intestinalis: A novel peptide system associated with energy metabolism and reproduction

The vase tunicate, Ciona intestinalis, is a protochordate and is considered a sister lineage to the chordates. The recent sequencing of its genome has made this species a particularly important model to understand the genetic basis of vertebrate evolution. However, C. intestinalis is also a highly invasive species along the Atlantic coast of North America and other regions of the world which have caused considerable economic stress due to its biofouling actions and, in particular, negative impacts on the mussel- and oyster-based aquaculture industry. Despite this background, little is known about C. intestinalis physiology. The teneurin C-terminal associated peptides (TCAP) are a family of highly conserved peptide hormones found in most metazoans. Moreover, these peptides have been implicated in the inhibition of stress and stimulation of feeding-based metabolism. We have, therefore, identified this peptide using an in silico approach and characterized its immunological expression in tissues using a mouse polyclonal antiserum. These data indicate that its primary structure is more similar to invertebrate TCAPs relative to vertebrate TCAPs. Immunological expression indicates that it is highly expressed in the digestive tract and gonads consistent with findings in vertebrates. Synthetic mouse TCAP-1 administered into the brachial basket significantly increases the incidence of non-stress contractile behaviors. These findings support the hypothesis that TCAP is a bioactive peptide in C. intestinalis. Thus, C. intestinalis and tunicates in general may offer a simple model to investigate peptide interaction while providing information on how to control this invasive species.

Transcriptional profiling of serogroup B Neisseria meningitidis growing in human blood: an approach to vaccine antigen discovery.

Neisseria meningitidis is a nasopharyngeal commensal of humans which occasionally invades the blood to cause septicaemia. The transcriptome of N. meningitidis strain MC58 grown in human blood for up to 4 hours was determined and around 10% of the genome was found to be differentially regulated. The nuo, pet and atp operons, involved in energy metabolism, were up-regulated, while many house-keeping genes were down-regulated. Genes encoding protein chaperones and proteases, involved in the stress response; complement resistant genes encoding enzymes for LOS sialylation and biosynthesis; and fHbp (NMB1870) and nspA (NMB0663), encoding vaccine candidates, were all up-regulated. Genes for glutamate uptake and metabolism, and biosynthesis of purine and pyrimidine were also up-regulated. Blood grown meningococci are under stress and undergo a metabolic adaptation and energy conservation strategy. The localisation of four putative outer membrane proteins encoded by genes found to be up-regulated in blood was assessed by FACS using polyclonal mouse antisera, and one (NMB0390) showed evidence of surface expression, supporting its vaccine candidacy.

Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydro-lase isoform (SmATPDase 2) and immunolocalisation of this protein in the Schistosoma mansoni egg

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.

Production of recombinant protein and polyclonal mouse antiserum for ferritin from Sipuncula Phascolosoma esculenta

The iron storage protein, ferritin, plays a key role in iron metabolism, but its regulation and functions in many invertebrate species are still largely unknown. In our previous work, an inducible ferritin cDNA from Phascolosoma esculenta with a full-length of 1017 bp has been cloned. In this follow-up study, the deducted ferritin protein sequence was predicted to be a polypeptide of 175 amino acids with a molecular mass of 20.1955 kDa and an isoelectric point of 5.08. The cDNA sequence of P. esculenta ferritin was constructed into pET system expression system and efficiently expressed in E. coli BL21 under IPTG induction. The recombinant ferritin was detected as a 24 kDa protein by SDS-PAGE. After purification directly from the gel, the recombinant ferritin was used to immunize mice and the anti-serum was prepared. The antibody displayed a strong immunological reactivity and specificity when used in Western-blot analysis. For the first time, our work provided a set of molecular tools essential for the further studies of ferritin protein functions in P. esculenta.

Sexual Stage Adhesion Proteins Form Multi-protein Complexes in the Malaria Parasite Plasmodium falciparum

The sexual phase of the malaria parasite Plasmodium falciparum is accompanied by the coordinated expression of stage-specific adhesive proteins. Among these are six secreted proteins with multiple adhesion domains, termed P. falciparum LCCL domain-containing protein (PfCCp) proteins, which are expressed in the parasitophorous vacuole of the differentiating gametocytes and which are later associated with macrogametes. Although the majority of the PfCCp proteins are implicated in parasite development in the mosquito vector, their functions remain unknown. In the present study we investigated the molecular interactions between the PfCCp proteins during gametocyte development and emergence. Using five different gene-disruptant parasite lines, we show that the lack of one PfCCp protein leads to the loss of other PfCCp family members. Co-immunoprecipitation assays on gametocyte lysates revealed formation of complexes involving all PfCCp proteins, and affinity chromatography co-elution binding assays with recombinant PfCCp domains further indicated direct binding between distinct adhesion domains. PfCCp-coated latex beads bind to newly formed macrogametes but not to gametocytes or older macrogametes 6 or 24 h post-activation. In view of these data, we propose that the PfCCp proteins form multi-protein complexes that are exposed during gametogenesis, thereby mediating cell contacts of macrogametes.

Oligomeric Amyloid β -Protein as a Therapeutic Target in Alzheimers Disease: Its Significance Based on its Distinct Localization and the Occurrence of a Familial Variant Form

Oligomer Abeta is the term utilized for multimeric but non-fibrillar forms of amyloid beta-protein (Abeta). The most prominent property of oligomer Abeta is considered to be its solubility and structure. Here, we examined the histochemical localization of oligomer Abeta in AD brains. At present, little information is available on the structure and function of cerebral oligomer Abeta. We therefore studied the molecular localization of oligomer Abeta using a newly generated polyclonal mouse antisera against a variant Abeta with a deletion mutation of the 22(nd) glutamate that we found recently in a patient with familial Alzheimer's disease. Intracellular as well as extracellular oligomer Abeta are herein discussed to define their structure and pathological roles in disease.

Localization of Per a 3 allergen in the gut and faecal pellets of the American cockroach (Periplaneta americana)

Cockroach-derived materials are known to be a major source of potent aeroallergens, causing allergic respiratory diseases such as asthma and allergic rhinitis. The aim of this study was to determine the localization of the major allergen, Per a 3 (Cr-PI), within the American cockroach (Periplaneta americana), which might reveal the relative importance of excreted materials and nonexcreted cockroach components as allergen sources. American cockroaches (P. americana) and their faecal pellets were embedded in paraffin, and serial sections were cut and collected on glass slides. After being stained with mouse polyclonal antiserum against Per a 3, the sections were incubated sequentially with biotin-labelled sheep antimouse immunoglobulin G (IgG) and a preformed fluorescent isothiocyanate (FITC)-avidin complex. Finally, the sections were mounted and examined under a fluorescent microscope. Examination of Per a 3 immunoreactivity on the sections of the American cockroaches (P. americana) revealed that the midgut mucosa, gut contents and faecal pellets were all strongly labelled. Per a 3 immunoreactive products were not detected in any other internal organs of the American cockroaches. These results suggest that Per a 3 allergen might be synthesized in and secreted from the epithelia of the midgut mucosa and excreted from the body in the faecal pellets.

Serum IgM levels against select marine bacteria in the Atlantic sharpnose shark ( Rhizoprionodon terraenovae) from three estuaries

The Atlantic sharpnose shark ( Rhizoprionodon terraenovae) is abundant and easily captured throughout the southeastern United States. Therefore this species serves as an ideal model for generating basic immunological reagents to establish baseline information regarding the immunophysiology of sharks in the wild, and for attempting to correlate shark immune responses to potential pathogens with the quality of the habitat in which they reside. Sharpnose shark serum IgM was purified over a protein-A column and used to generate mouse polyclonal anti-sera to develop indirect ELISAs for quantifying bacteria-specific IgM antibody titers against Vibrio anguillarium, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio charchariae, Escherichia coli, Mycobacterium fortuitum, and Mycobacterium marinum. Serum samples from Atlantic sharpnose sharks were collected in Charleston, SC, Beaufort, SC, and New Brunswick, GA estuaries during the early summer, and again from Charleston, SC in the fall of the same year. Relative antibody titers against E. coli, V. anguillarium, and V. parahaemolyticus differed among the three sampling locations, suggesting differences in microbial abundance or immunological responses in sharks from three locations. Overall, antibody titers in Charleston, SC sharks increased between summer and fall. A combination of chronic exposure to specific bacteria and increased antibody responses due to elevated water temperatures are likely responsible for elevated specific IgM in these sharks sampled in the fall. To our knowledge, this is the first study to examine IgM responses in elasmobranchs collected directly from their habitat.

Clearing of globotriaosylceramide from endothelial vessels of ocular conjunctiva in patients with Fabry disease treated with agalsidase alfa

Abstract Introduction: Fabry disease is an X‐linked inborn metabolic error caused by a deficiency of lysosomal α‐galactosidase A. Globotriaosylceramide (Gb3) accumulates in lysosomes of various cells. At the moment, enzyme replacement therapy (ERT) is the only available specific treatment for patients with Fabry disease. Enzyme infusions reduce the Gb3 content of plasma, urine sediment and tissue. In a previous study, we reported the development of a method to specifically detect Gb3 deposits by immunofluorescence in conjunctival biopsies. Aim: The aim of this work is to evaluate the clearance of Gb3 in cells from the conjunctiva in patients with Fabry disease treated with agalsidase alfa. Methods: Conjunctival biopsies were obtained from three hemizygous and two heterozygous patients, before and after 6 months of treatment with agalsidase alfa. The specimens were processed for direct immunofluorescence using an anti‐Gb3 monoclonal antibody and light microscopy using a mouse polyclonal antiserum specific for α‐galactosidase A. Results: Positive immunofluorescence was not observed in cells from the blood vessels of patients with Fabry disease after 6 months of treatment, as compared with the specimens before the treatment, which were positive. We detected Gb3 in stromal/macrophage cells that were distal from the blood vessels where the enzyme was circulating. Specific staining for α‐galactosidase A revealed the presence of this protein in the tissue of patients after ERT, but not before. Conclusion: Agalsidase alfa eliminated Gb3 deposits from the cells of blood vessel walls in patients with Fabry disease after 6 months of treatment. The deposits in stromal cells were not removed, probably because the enzyme must diffuse into the tissue to reach these cells and/or because of the short length of therapy. This method may be useful to evaluate the effectiveness of ERT.

116. The Proposed AAV Serotypes 10 and 11 Serologically Cross-React with AAV8 and AAV4

Introduction: Vectors based on a variety of AAV isolates are currently being explored for application in gene therapy. This pursuit is driven by the fact that relatively subtle structural differences can dramatically affect vector biology in several ways relevant to gene therapy such as efficiency of gene transfer, tropism and interactions with the immune system of the host. Particularly, circulating neutralizing antibodies can blunt a successful gene therapy. Serological cross-reactivity between AAV isolates is not only used as a reflective measure of neutralization in vivo but also as a way of classification of primate AAVs. Recently AAV10 and AAV11 were proposed by Mori et al. (2004) as novel serotypes. AAV10 and AAV11 were found to be structurally closest to the previously described serotypes AAV8 and AAV4, respectively. Their study showed that the isolates from cynomolgus macaque were not cross-neutralized by polyclonal mouse antiserum generated against purified VP2 protein from AAV2, AAV4 or AAV8.

Lutzomyia longipalpisPeritrophic Matrix: Formation, Structure, and Chemical Composition

Sandflies are vectors of several pathogens, constituting serious health problems. Lutzomyia longipalpis (Lutz & Neiva, 1912) is the main vector of Leishmania chagasi, agent of visceral leishmaniasis. They synthesize a thick bag-like structure that surrounds the bloodmeal, named peritrophic matrix (PM). One of the major roles of PM in blood-fed insects includes protection against ingested pathogens by providing a defensive barrier to their development. We used traditional and modern morphological methods as well as biochemical and immunolabeling tools to define details of the PM structure of the Lu. longipalpis sandfly, including composition, synthesis, and degradation. The kinetics of PM formation and degradation was found to be related to the ingestion and time of digestion of the bloodmeal. The midgut changes its size and morphology after the blood ingestion and during the course of digestion. A striking morphological modification takes place in the midgut epithelium after the stretching caused by the bloodmeal, revealing a population of cells that was not observed in the unfed midgut. The transmission and scanning electron microscopies were used to reveal several morphological aspects of PM formation. The PM looks thicker and well formed 24 h after the bloodmeal. Presence of chitin in the PM was demonstrated by immunolabeling with an alpha-chitin monoclonal antibody. SDS-polyacrylamide gel electrophoresis showed at least five protein bands with molecular masses of 38.7-135 kDa, induced by the protein-free diet. Mouse polyclonal antiserum was produced against PMs induced by protein-free meal and used in Western blotting, which revealed at least three associated proteins.

Expression and characterization of HlyX hemolysin from Leptospira interrogans serovar Copenhageni: Potentiation of hemolytic activity by LipL32

The HlyX, a putative hemolysin identified from the Leptospira genomes, was cloned, expressed in Escherichia coli, purified, and its hemolytic activity was confirmed. Mouse polyclonal antiserum against the recombinant HlyX recognized HlyX-related antigens in a panel of Leptospira species extracts and it was also able to abolish the hemolytic activity of HlyX. A mixture of HlyX and LipL32, a known hemolysin from Leptospira, induced hemolysis in a synergistic way that was fully inhibited by antiserum against either protein. Moreover, sera from patients with leptospirosis also recognized the recombinant HlyX, showing that it is presented to the host immune system during Leptospira infection.

Antiserum to the gp116 glycoprotein of yellow head virus neutralizes infectivity in primary lymphoid organ cells of Penaeus monodon

Yellow head virus (YHV) is an invertebrate nidovirus that has caused mass mortality of cultured Penaeus monodon in Asia. In this study, we investigated whether mouse polyclonal antiserum raised against the YHV gp116 or gp64 structural glycoproteins could neutralize YHV infectivity as determined using an in vitro quantal assay in primary cultures of lymphoid organ cells. Anti-gp116 antiserum showed virus-neutralizing activity whereas anti-gp64 antiserum failed to inhibit infection. The results suggest that gpl16 antiserum blocks binding of virions to cellular receptors to facilitate YHV entry into lymphoid organ cells.

Purification and antigenic characteristics of a rickettsia-like organism from the oyster Crassostrea ariakensis

A rickettsia-like organism (RLO) has been suggested to be the etiological agent responsible for heavy losses of the oyster Crassostrea ariakensis Gould in China. Because of the lack of molluscan cell lines for in vitro culture of intracellular prokaryotes, antigenic analysis of RLOs has been limited by the inherent difficulties of their purification. In this report, we describe the use of differential speed centrifugation and renografin density gradient centrifugation to purify the RLO directly from infected oyster tissues. The purity and integrity of purified prokaryotes were validated by transmission electron microscopy. Thirteen major constituent proteins, with molecular weights ranging between 17 and 99 kDa, were electrophoretically identified by silver staining, and 8 major proteins were identified with Coomassie blue R staining. Specific mouse polyclonal antiserum was prepared for serological characterization of the RLO and was used in an immunoblot assay, and 3 major antigen groups were identified. The present results advance our knowledge of RLO protein antigens, and several proteins have been identified that could potentially be useful for diagnostic assays or for production of experimental immunostimulants.

Conservation of the opcL gene encoding the peptidoglycan-associated outer-membrane lipoprotein among representatives of the Burkholderia cepacia complex.

Members of the Burkholderia cepacia complex are Gram-negative beta-proteobacteria that are classified into nine genomic species or genomovars. Some representatives of this group of bacteria, such as Burkholderia multivorans (genomovar II) and Burkholderia cenocepacia (genomovar III), are considered to be dangerous pathogens for cystic fibrosis (CF) patients because of their capacity to colonize CF lungs. The opcL gene, which encodes the peptidoglycan-associated outer-membrane lipoprotein (PAL), was detected in the genome of Burkholderia sp. LB 400 by a similarity search that was based on the sequence of the Pseudomonas aeruginosa PAL, OprL. Primers that could amplify part of opcL from B. multivorans LMG 13010(T) were designed. This PCR fragment was used as a probe for screening of a B. multivorans genomic bank, allowing cloning of the complete opcL gene. The complete opcL gene could be PCR-amplified from DNA from all genomovars. The sequences of these opcL genes showed a high degree of conservation (> 95 %) among different species of the B. cepacia complex. OpcL protein that was purified from B. multivorans LMG 13010(T) was used to generate mouse polyclonal antisera against OpcL. The OpcL protein could be produced in Escherichia coli and detected in outer-membrane fractions by Western blot. Burkholderia cells were labelled by immunofluorescence staining using antibodies against OpcL, but only after treatment with EDTA and SDS. The opcL gene could be amplified directly from the sputa of 15 CF patients who were known to be colonized by B. cepacia; sequence data derived from the amplicons identified the colonizing strains as B. cenocepacia (genomovar III, n = 14) and B. multivorans (n = 1).

Localization of the Thyroid Peroxidase Autoantibody Immunodominant Region to a Junctional Region Containing Portions of the Domains Homologous to Complement Control Protein and Myeloperoxidase

Thyroid peroxidase (TPO) autoantibody epitopes are largely restricted to an immunodominant region (IDR) on the extracellular region of the native molecule. Localization of the IDR has been a longstanding and difficult goal. The TPO extracellular region comprises a large myeloperoxidase-like domain, linked to the plasma membrane by two smaller domains with homology to complement control protein (CCP) and epidermal growth factor (EGF), respectively. Recent studies have focused on the CCP- and EGF-like domains as the putative location of the TPO autoantibody IDR. To address this issue, we attempted to express on the surface of transfected cells native TPO in which the CCP- and EGF-like domains were deleted, either together or individually. We used a quartet of human monoclonal autoantibodies that define the TPO IDR, as well as polyclonal TPO autoantibodies in patients' sera, to detect these mutated TPO molecules by flow cytometry. The combined CCP/EGF-like domain deletion did not produce a signal with TPO autoantibodies but did not traffic to the cell surface. In contrast, both monoclonal and polyclonal autoantibodies recognized TPO with the juxtamembrane EGF-like domain deleted equally as well as the wild-type TPO on the cell surface. TPO with the CCP-like domain deleted expressed normally on the cell surface, as determined using the polyclonal mouse antiserum. Nevertheless, this modified TPO molecule was recognized very poorly by both the human monoclonal autoantibodies and the polyclonal autoantibodies in patients' sera. In conclusion, we have clearly excluded the juxtamembrane EGF-like domain as being part of the IDR. In contrast, a component of the CCP-like domain does contribute to the IDR. These data, together with findings from other studies, localize the TPO autoantibody IDR to the junction of the CCP-like domain and the much larger myeloperoxidase-like domain on TPO.

Epitope analysis of the FanC subunit protein of the K99 (F5) fimbriae of enterotoxigenic Escherichia coli using a recombinant fusion technique.

We have used a recombinant approach to characterise the B- and T-cell epitopes of FanC, the major subunit polypeptide of K99 (F5) fimbriae of enterotoxigenic Escherichia coli strains. This involved the fusion of FanC and its carboxy-terminal truncated derivatives to a reporter, the E. coli alkaline phosphatase (PhoA), generating stable, recombinant fusions. The B-cell epitopes of FanC were characterised by Western blotting of FanC::PhoA fusion proteins with a polyclonal mouse antiserum directed against K99 fimbrial antigen, and with a panel of monoclonal antibodies generated to the K99 antigen. An attempt to characterise the T-cell epitopes of the fimbrial subunit was made by standard in vitro T-cell proliferation assay. Our results suggest that the B-cell epitopes of FanC are likely to be continuous, with a potentially immunodominant epitope at the carboxy-terminus. However, T-cell proliferation assays with the FanC::PhoA fusion proteins did not indicate any immunodominant T-cell epitope(s). We hypothesise that fusion of FanC peptides to PhoA had resulted in altered folding of the peptides for antibody and T-cell recognition, highlighting the potential problems and drawbacks of the recombinant fusion technique in defining the epitopes of certain proteins.