By DNA microarray and protein 2-DE screens for
Caenorhabditis elegans genes up-regulated by acrylamide, we selected the
gst-4 gene and constructed a
gst::gfp fusion gene, which was used to transform
...C. elegans into a biosensor for acrylamide. This biosensor detects acrylamide as a GFP-expression signal in a dose- and time-dependent manner. When the biosensor was exposed to acrylamide together with commercially available powdered green tea, GFP levels decreased to the control level, suggestive of acrylamide detoxification or prevention of GST induction. The present methodology should be applicable for screening of not only harmful substances but also substances that reduce or counteract their harmfulness or action, with appropriately constructed visible biosensors.
Enzyme-linked immunosorbent assay (ELISA) for human granulocytic ehrlichiosis (HGE) using two different recombinant P44 proteins (rP44 and rP44-2hv) of the HGE agent as antigens was evaluated. Sera ...from a total of 72 healthy humans both from regions where HGE is nonendemic and regions where HGE is endemic were used as negative controls to determine the cutoff value for ELISA. Sera from a total of 14 patients (nine from whom the HGE agent was isolated and five who were HGE-PCR positive) were used as positive controls. One hundred nine sera from 72 patients in an area where HGE is endemic who were suspected of having HGE were examined by ELISA and indirect immunofluorescence assay (IFA). All IFA-negative sera were negative by both ELISAs. Of 39 sera that were IFA positive, 35 and 27 were positive by ELISA using rP44 and rP44-2hv, respectively, indicating that the use of rP44 is more sensitive. Western blot analysis of the four rP44-ELISA-negative IFA-positive sera using whole HGE agent as antigen suggests that these four sera were false IFA positive. There was no difference in results with or without the preabsorption of sera with Escherichia coli or with or without the cleavage of the fused protein derived from the vector. There was a significant positive correlation between IFA titers and optical densities of ELISAs. Four Ehrlichia chaffeensis-positive and 10 Borrelia burgdorferi-positive sera were negative by ELISA. However, two Babesia microti-positive sera showed strong cross-reactivity to the fused vector protein, which was eliminated after cleavage of the protein. Thus, ELISA using rP44 nonfusion protein would provide a simple, specific, and objective HGE serologic test which can be easily automated.
Four types of commercially available feline calicivirus (FCV) vaccine were compared in terms of their efficacy on the basis of the ability of the sera of specific-pathogen-free cats immunized by two ...injections of each type of vaccine to neutralize FCV field isolates. Each vaccine immune serum neutralized relatively well strains F4, F9, and 255, which were FCV laboratory strains. As to 36 strains of field isolates, however, vaccines A, B, C, and D immune sera did not neutralize 18-20 of the strains (50.0%-55.6%), 19-22 of the strains (52.8%-61.1%), 22-25 of the strains (61.1%-69.4%), and 8-16 of the strains (22.2%-44.49%), respectively. These results indicate that there is much difference in neutralizing antigenicity between the existing vaccine strains and the FCV strains that are prevalent in Japan, suggesting the need for improvement of FCV vaccines
The major dog allergens, Can f 1 and Can f 2, are members of the lipocalin protein family. The characterization of both dog allergens is still not complete. Their deduced amino acid sequences ...indicate the presence of three cysteine residues, probably connected with a disulfide bridge. We compared the biochemical and immunological properties of Can f 1 with those of Can f 2 using gel filtration, electrophoresis, and immunological assays.
The rCan f 1, rCan f 2 and dog salivary proteins containing natural Can f 1 and Can f 2 were analyzed by HPLC gel filtration. The recombinant Can f 1 (rCan f 1) and rCan f 2 were analyzed by native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) with or without reduction. The binding ability of rabbit IgG purified by protein G affinity chromatography from the antiserum against rCan f 1 and rCan f 2 was examined after a reduction in the recombinant allergens. The immunological cross-reaction between rCan f 1 and rCan f 2 was examined by an enzyme-linked immunosorbent assay (ELISA) using the rabbit IgG against rCan f 1 and rCan f 2. The cross-reaction of human IgE in the serum of a patient with dog allergy between rCan f 1 and rCan f 2 was also analyzed by competitive ELISA.
The molecular weights of rCan f 1 and of rCan f 2 were 18 and 21 kDa, respectively, using SDS-PAGE under reducing conditions, but the natural Can f 1 and Can f 2 were separated by HPLC gel filtration into fractions containing proteins of 31 and 34 kDa, respectively. rCan f 1 and rCan f 2 migrated as multiple bands (30-100 kDa) in native PAGE in the presence or absence of a reductant. The molecular weights of natural Can f 1 and of Can f 2 were 20 and 23 kDa, respectively, in SDS-PAGE under reducing conditions. The ability of rabbit IgG to bind to rCan f 1 and rCan f 2 increased after the reduction of the recombinant allergens. The rabbit IgG against rCan f 1 bound to rCan f 2. Cross-reaction of human IgE was observed between rCan f 1 and rCan f 2.
In the native and recombinant forms, Can f 1 and Can f 2 possessed a dimer structure under natural (non-reduced) condition. The dimers of Can f 1 and of Can f 2 were not built with a disulfide bridge but by non-covalent association. Cleavage of a disulfide bond of rCan f 1 and rCan f 2 increased the ability of binding of rabbit IgG to the allergens. The cross-reactivity of rabbit IgG and human IgE between rCan f 1 and rCan f 2 indicates that the same epitope(s) was present in Can f 1 and Can f 2.
Recombinant dog allergens, rCan f 1 and rCan f 2, and their antibodies are good tools for the characterization of dog allergens in order to develop modern therapeutic and preventive methods for dog ...allergy.
In this study, cDNA was synthesized from the mRNA of dog salivary glands and cloned into the pGEX4T vector. rCan f 1 and rCan f 2 containing glutathione S-transferase were prepared by an Escherichia coli expression system. The antibodies against the recombinant allergens were prepared in rabbit. The serum of patients with dog allergy was evaluated by ELISA and immunoblot, using the recombinant allergens, goat anti-human immunoglobulin (Ig) E (epsilon) labeled with biotin, and enzyme-labeled streptavidin. The binding of IgE in the serum of patients with dog allergy to dog saliva as a natural antigen was determined in the presence or absence of dog saliva, rCan f 1 and rCan f 2 as competitors. The anaphylactic potential of rCan f 1 and rCan f 2 was evaluated. The body temperature of the mice sensitized with rCan f 1 and rCan f 2 was monitored after intravenous injection of the allergens. The passive cutaneous anaphylaxis reaction was examined for rCan f 1 and rCan f 2. Dog salivary glands, dog saliva and dog hair/dander extracts were analyzed with antibodies by means of an immunoblot assay. The expression of the mRNA of Can f 1 and Can f 2 was verified in various dog tissues by reverse transcription polymerase chain reaction.
The E. coli expression system revealed the yield of rCan f 1 and rCan f 2 in 36 and 30 mg/l of culture. The molecular weights of rCan f 1 and rCan f 2 were 18 and 20 kDa in SDS-PAGE, respectively. rCan f 1 and rCan f 2 were found to bind to specific IgE in the serum of dog allergy patients. The binding of IgE in the patient serum for dog saliva was partially inhibited in the presence of rCan f 1 and rCan f 2. These recombinant allergens showed positive signals in passive cutaneous anaphylaxis reaction and induced anaphylactic shock in the mouse model, resulting in a decrease in body temperature. The polyclonal rabbit antibody for rCan f 1 bound to a protein of 20 kDa in the salivary gland, saliva and hair/dander extracts of dogs. The rabbit antibody for rCan f 2 bound to proteins in the saliva and the hair/dander extracts. The proteins possessed a molecular weight of 22/ 23 kDa. Reverse transcription polymerase chain reaction showed the presence of mRNA expression of Can f 1 and Can f 2 not only in the salivary gland but also in dog skin. A clear expression of Can f 2 mRNA was observed in dog skin.
The recombinant allergens and antibodies for Can f 1 and Can f 2 are available for immunological and biochemical characterization of dog allergens. The molecular weight of the natural Can f 1 and Can f 2 in dog saliva and hair/dander extracts showed a higher molecular weight than that of rCan f 1 and rCan f 2. The significance of dog skin as the tissue producing dog allergens, especially Can f 2, should be considered in further studies.
Reactivities of feline calicivirus (FCV) field isolates with monoclonal antibodies (MAbs) were examined by enzyme-linked immunosorbent assay (ELISA). The reactivities of the viruses in ELISA were ...different from our previous results using the neutralization tests (NT). Many isolates were positive in ELISA with MAbs which recognized neutralizing epitope 3B and/or 4. However, most were negative in NT in our previous study. After absorption of two FCV strains with host cells, the non-infectious virus fluid still reacted with MAb, which recognized epitope 3B and/or 4 in ELISA. These results indicated the possibility that neutralizing epitopes are expressed on non-infectious virus particles or exist as proteinaceous molecules in virus fluid.
We used a consensus primer PCR method to amplify a region of herpesviral DNA-directed DNA polymerase gene using degenerate primers for initial characterization of the porcine cytomegalovirus (PCMV) ...genome. The sequence of the PCR product from PCMV DNA template and its alignment with other herpesvirus DNA polymerase counterparts showed that both conserved amino acid residues and conservative amino acid substitutions are in parallel. Phylogenetic analysis revealed that PCMV should be included in the clade comprising human herpesvirus 6 and 7, rather than human and mouse cytomegaloviruses, in Betaherpesvirus subfamily.
The reactivity of native bovine conglutinin (Kg) with antibody against recombinant Kg (rKg), with deletion of the N-terminal and collagen-like regions of the native Kg molecule, was studied by ...sandwich enzyme-linked immunosorbent assay. With anti-recombinant Kg antibody as the coating antibody, rKg reacted with biotinylated homologous anti-rKg and heterologous anti-Kg antibodies as probing antibodies, while native Kg did not. With anti-native Kg antibody as coating antibody, native Kg reacted with biotinylated homologous antibody as probing antibody, while recombinant Kg reacted weakly with both biotinylated homologous and heterologous antibodies. Consequently the N-terminal and collagen-like regions of native Kg molecule are essential to express the complete immunogenicity and/or antigenicity of the native Kg molecule
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