Hepatocellular carcinoma diagnosis using a novel electrochemiluminescence immunoassay targeting serum IgM-free AIM.

BACKGROUND: Recent increases in the number of patients with non-alcoholic steatohepatitis (NASH) warrant the identification of biomarkers for early detection of hepatocellular carcinoma (HCC) associated with NASH (NASH-HCC). IgM-free apoptosis inhibitor of macrophage (AIM), which generally associates with IgM in blood and exerts its biological function by dissociation from IgM, may serve as an effective biomarker for NASH-HCC. Here, we established a fully automatic and high-throughput electrochemiluminescence immunoassay (ECLIA) to measure IgM-free AIM and investigated its efficacy in diagnosing NASH-HCC and viral HCC. METHODS: IgM-free AIM levels were measured in 212 serum samples from patients with, or without, HCC related to NASH, hepatitis B virus, and hepatitis C virus, using ECLIA. We also developed an ECLIA for measuring both IgM-free and IgM-bound AIM and investigated the existing form of AIM in blood by size-exclusion chromatography. RESULTS: IgM-free AIM levels were significantly higher in the HCC group than in the non-HCC group, regardless of the associated pathogenesis. Moreover, the area under the receiver operating curve for IgM-free AIM was greater than that for conventional HCC biomarkers, alpha-fetoprotein or des-γ-carboxy prothrombin, regardless of the cancer stage. ECLIA counts of IgM-free AIM derived from samples fractionated by size-exclusion chromatography were significantly higher in patients with NASH-HCC than in healthy volunteers and in patients with non-alcoholic fatty liver and NASH. CONCLUSIONS: Serum IgM-free AIM may represent a universal HCC diagnostic marker superior to alpha-fetoprotein or des-γ-carboxy prothrombin. Our newly established ECLIA could contribute to further clinical studies on AIM and in vitro HCC diagnosis.

Activation of apoptosis inhibitor of macrophage is a sensitive diagnostic marker for NASH-associated hepatocellular carcinoma.

BACKGROUND: A diagnostic marker is needed enabling early and specific diagnosis of hepatocellular carcinoma (HCC) associated with non-alcoholic steatohepatitis (NASH). Our recent findings have indicated that circulating apoptosis inhibitor of macrophage (AIM), which usually associates with IgM pentamer in the blood, is activated by its dissociation from IgM. We investigated the serum levels of IgM-free AIM for AIM activation and its possible relationship with development of HCC in NASH. METHODS: Serum levels of IgM-associated and IgM-free AIM were evaluated in patients with non-alcoholic fatty liver, NASH, and NASH-HCC using enzyme-linked immunosorbent assays and immunoblots. Liver biopsy specimens were graded and staged using Brunt's classification. RESULTS: Forty-two patients with fatty liver, 141 with NASH, and 26 with NASH-HCC were evaluated. Patients with stage 4 or grade 3 NASH (with or without HCC) exhibited significantly higher levels of both IgM-free and total AIM than those with fatty liver, whereas the ratio of IgM-free-to-total AIM was equivalent in these groups. Among patients with the same fibrosis stage of NASH, those with HCC had significantly higher IgM-free but not total AIM levels, resulting in a proportional increase in the IgM-free/total AIM ratio. Analysis of the areas under the receiver operating characteristic curves indicated the high sensitivity of the IgM-free AIM for NASH-HCC. CONCLUSIONS: Our observations suggest the activation of AIM in blood in the presence of NASH-HCC, with a significant increase in IgM-free AIM levels. IgM-free AIM serum levels appear to be a sensitive diagnostic marker for NASH-HCC.

Variation in antigen-antibody affinity among serotypes of Salmonella O4 serogroup, determined using specific antisera.

Serotyping is widely used for typing Salmonella during surveillance, and depends on determining the lipopolysaccharide (LPS) O-antigen and the flagellar protein (H-antigens) components. As the O-antigen is highly variable, and structurally unique to each serotype, we investigated the binding affinities of LPS from Salmonella serotypes of O4 serogroup with specific anti-antigen serum via immunoblot and enzyme-linked immunosorbent assays. Since the serotypes from O4 serogroup also express the O-antigen factor 12, O12 antiserum was also used for the analysis. LPS from the different serotypes showed different binding affinities with the antisera. Therefore, based on the antigen-antibody affinity, a modified agglutination assay was carried out by using O4 and O12 antisera. Although serotypes from O4 serogroup have the common O-antigen factors 4 and 12, the analysis showed that the degree of agglutination reaction is different for each of the serotypes. We suggest that Salmonella serogroup O4 serotypes exhibit different binding affinities with specific antisera despite the presence of common O-antigen factors 4 and 12.

Monoclonal antibody-based competitive enzyme-linked immunosorbent assay to detect antibodies to O:4 Salmonella in the sera of livestock and poultry.

Serotyping is an important element for surveillance of Salmonella. In this study, an anti-O:4 Salmonella monoclonal antibody-based competitive enzyme-linked immunosorbent assay that could identify Salmonella infection in cow, pig, horse, and chicken was developed. This detection system can therefore be useful for a wide range of animals and for humans.

A rapid differentiation method for enteroinvasive Escherichia coli.

Enteroinvasive Escherichia coli (EIEC) comprise 21 major serotypes defined by the presence of O and H antigens, and diagnosis depends on determining its invasive potential. Using HEp-2 cells infected with an EIEC strain, we developed a simple growth-dependent assay that differentiated EIEC strain from non-invasive strains 6 h after infection.

Characterization and identification of a novel candidate vaccine protein through systematic analysis of extracellular proteins of Erysipelothrix rhusiopathiae.

Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, is a facultative intracellular Gram-positive bacterium. It has been shown that animals immunized with a filtrate from E. rhusiopathiae cultures are protected against lethal challenge. In this study, we identified and characterized the extracellular proteins of E. rhusiopathiae to search for novel vaccine antigens. A concentrated culture supernatant from the E. rhusiopathiae Fujisawa strain, which has been found to induce protection in mice, was analyzed using two-dimensional electrophoresis. From more than 40 confirmed protein spots, 16 major protein spots were selected and subjected to N-terminal amino acid sequence determination, and 14 protein spots were successfully identified. The identified proteins included housekeeping proteins and other metabolic enzymes. We searched for surface-localized proteins by analyzing the genomes of two E. rhusiopathiae strains: Fujisawa and ATCC 19414. Genome analysis revealed that the ATCC 19414 strain has three putative surface-exposed choline-binding proteins (CBPs): CbpA, CbpB, and CbpC. Each CBP contains a putative choline-binding domain. The CbpC gene is mutated in Fujisawa, becoming a nonfunctional pseudogene. Immunogold electron microscopy confirmed that CbpA and CbpB, as well as the majority of the metabolic enzymes examined, are associated with the cell surface of E. rhusiopathiae Fujisawa. Immunization with recombinant CbpB, but not with other recombinant CBPs or metabolic enzymes, protected mice against lethal challenge. A phagocytosis assay revealed that antiserum against CbpB promoted opsonin-mediated phagocytosis by murine macrophages in vitro. The protective capabilities of CbpB were confirmed in pigs, suggesting that CbpB could be used as a vaccine antigen.

Application of West Nile virus diagnostic techniques.

West Nile virus (WNV) is an enveloped RNA virus in the family Flaviviridae and belongs to Japanese encephalitis virus serocomplex group. The WNV has a wide geographic distribution that includes Africa, Europe, Asia, America and Australia. Recently, it has re-emerged as an important pathogenic organism, illustrated by the series of WNV outbreaks in North America and in Europe. Several hundred people are sacrificed by WNV infection every year. WNV can infect many mammals, birds, reptiles and amphibians. A variety of diagnoses for WNV infection have been developed, such as virus isolation, nucleotide amplification, antigen detection and serology. Flaviviruses, including WNV, share common nucleotide sequences and antigenic epitopes. Understanding these properties that can influence cross-reactivity is important for accurate diagnosis, especially because areas with multiple flaviviruses are currently expanding. Herein, the authors outline the different diagnostic methods for detecting WNV infection as well as important considerations in using these methods.

A new competitive ELISA detects West Nile virus infection using monoclonal antibodies against the precursor-membrane protein of West Nile virus.

Precursor membrane protein (prM) is found in the envelope of immature West Nile virus (WNV) particles. Anti-prM antibodies are found in flavivirus-infected animal sera, and they are known as relatively virus species specific antibodies. However, there are no known reports of WNV-specific epitope blocking or competitive enzyme-linked immunosorbent assay (c-ELISA) that detect anti-prM antibodies. Two anti-WNV-prM monoclonal antibodies (SHW-29C2 and SHW-31B2) were generated, and c-ELISAs were developed using these monoclonal antibodies. The c-ELISAs were evaluated using WNV-infected chicken sera. Both c-ELISAs detected anti-prM antibodies in WNV-infected chicken sera and showed little cross-reactivity to antisera against Japanese encephalitis virus, St. Louis encephalitis virus, and Murray valley encephalitis virus. The average inhibition of chicken sera at 3 weeks post WNV infection was 61.6% in SHW-29C2-based c-ELISA and 71.8% in SHW-31B2-based c-ELISA. High correlation was seen between percent inhibition in the c-ELISAs and optical density values of an IgG indirect ELISA. Additionally, SHW-31B2-based c-ELISA detected antibodies against a wide variety of WNV strains. Detecting anti-PrM antibodies using c-ELISA could be useful for WNV serodiagnosis.

Development of monoclonal antibodies to West Nile virus and their application in immunohistochemistry.

West Nile virus (WNV) is endemic throughout Africa, Eurasia, America, and Australia and has important implications for avian, horse, and human health. In these regions, dead birds are monitored for the presence of WNV through immunohistochemistry (IHC) and PCR. However, a number of the tools for IHC are inadequate owing to their cross-reactivity to other Japanese encephalitis serogroup viruses. Here we have established eight monoclonal antibodies (MAbs) to WNV. Four of them bound to the envelope protein, three of them bound to nonstructural protein 1 (NS1), and one bound to precursor membrane protein (prM), as shown by Western blot analysis. The anti-NS1 MAbs and the anti-prM MAb did not cross-react with Japanese encephalitis virus (JEV), Murray valley encephalitis virus, or St. Louis encephalitis virus in an indirect enzyme-linked immunosorbent assay. One NS1-specific MAb, SHW-32B1, and the previously reported NS1-specific MAb, SHW-7A11, were shown by IHC to specifically detect the cytoplasm of degenerated cells in the heart and brain of a WNV-infected goose. Neither of these MAbs were shown by IHC to cross-react with degenerated cells in the brain of a JEV-infected pig. These MAbs are the first reported anti-NS1 MAbs that can be used for WNV-specific IHC using formalin-fixed, paraffin-embedded sections. They may be useful for WNV research and surveillance.

Single intramammary infusion of recombinant bovine interleukin-8 at dry-off induces the prolonged secretion of leukocyte elastase, inflammatory lactoferrin-derived peptides, and interleukin-8 in dairy cows.

A single intramammary infusion of recombinant bovine interleukin-8 (IL-8) at 50 µg/quarter/head, but not 10 µg/quarter/head, induced clinical mastitis in three of four cows during the dry-off period, resulting in an elevated rectal temperature, redness and swelling of the mammary gland, extensive polymorphonuclear leukocyte (PMNL) infiltration, and milk clot formation from 1 to 28 days post infusion (PI). In the mammary secretions of the mastitic glands, high levels of IL-8 were sustained from 8 hours to 28 days PI, peaking at 1-3 days PI. The levels of leukocyte-derived elastase and inflammatory 22 and 23 kDa lactoferrin derived peptides (LDP) were also increased in the mammary secretions from the mastitic glands. In addition to the experimentally induced mastitis, the mammary secretions from the glands of cattle with spontaneous Staphylococcus aureus dry-period mastitis displayed milk clot formations and significant increases in their levels of PMNL counts, elastase, LDP, and IL-8, compared with those of the mammary secretions from the uninfected glands. These results suggest that after an intramammary infusion of IL-8 has elicited inflammatory responses, it induces the prolonged secretion of elastase, inflammatory LDP, and IL-8, and that long-lasting IL-8-induced inflammatory reactions are involved in the pathogenesis of S. aureus dry-period mastitis.

Cross-reactivity of chicken anti-Japanese encephalitis virus serum and anti-West Nile virus serum in serological diagnosis.

The cross-reactivity of Japanese encephalitis virus (JEV)-immunized chicken sera and West Nile virus (WNV)-immunized chicken sera in serological tests, such as the IgG indirect ELISA (IgG-ELISA), hemagglutination inhibition test (HI) and plaque reduction neutralization test (PRNT), for JEV and WNV were examined. The mean JEV/WNV ELISA ratio in IgG-ELISA of JEV-immunized sera was significantly higher than that of WNV-immunized sera (P<0.01). JEV-immunized chicken sera did not cross-react in the WNV HI. However, all the WNV-immunized chicken sera cross-reacted in the JEV HI. JEV-immunized chicken sera did not show the WNV neutralization titer at 90% plaque reduction, and WNV-immunized chicken sera did not showed the JEV neutralization titer at 90% plaque reduction. Therefore, it is possible that chicken JEV serum can be distinguished from WNV serum by comparing the titers of IgG-ELISA, HI or PRNT respectively.

New sensitive competitive enzyme-linked immunosorbent assay using a monoclonal antibody against nonstructural protein 1 of West Nile virus NY99.

An anti-West Nile virus (anti-WNV) monoclonal antibody, SHW-7A11, was developed for competitive enzyme-linked immunosorbent assays (c-ELISAs). SHW-7A11 reacted with nonstructural protein 1 in Western blot analysis. SHW-7A11 was relatively specific for the WNV strain NY99 and recognized Kunjin and Eg101 strains in indirect ELISAs. Two c-ELISAs were developed for sera diluted 10 and 100 times and named c-ELISA10 and c-ELISA100, respectively. Both c-ELISAs detected antibodies against WNV NY99 and Kunjin strains. Little cross-reactivity was observed for antibodies against Japanese encephalitis virus and St. Louis encephalitis virus in these assays. Using the cutoff point for the St. Louis encephalitis virus, all WNV-infected chickens were found to be positive on day 21 after infection in both c-ELISAs. On the other hand, all infected chickens were found to be positive on day 35 after infection in a virus neutralization test. Our newly developed SHW-7A11-based c-ELISA can detect WNV infection with sera diluted 10 to 100 times. Therefore, this c-ELISA can be used for WNV serosurveillance of chickens and wild birds.

Establishment of a quantitative bovine CXCL8 sandwich ELISA with newly developed monoclonal antibodies.

Three IgG class anti-bovine CXCL8 (bCXCL8) monoclonal antibody (mAb)-secreting hybridomas, SH8-8D7, SH8-12A5 and SH8-2A1, were developed. SH8-8D7 was IgG2a, and SH8-12A5 and SH8-2A1 were IgG1. All three mAbs detected recombinant bCXCL8 (rbCXCL8) by immunoprecipitation and Western blotting. SH8-2A1 could neutralise the chemotactic activity of rbCXCL8 towards neutrophils. The quantitative bCXCL8 ELISA was constituted by the combination of SH8-12A5 and biotin-SH8-2A1. The detection range was 20-1000  pg/mL. A sandwich ELISA was used to measure native bCXCL8 derived from the supernatant of cultured bovine peripheral blood mononuclear cells stimulated with ConA, LPS or PHA. Furthermore, SH8-2A1 could detect bCXCL8 in formalin-fixed, paraffin-embedded, pneumonic calf tissues. These findings indicate that the newly developed anti-bCXCL8 mAbs could contribute to research on bovine inflammatory responses and immunology.

Expression and characterization of bluetongue virus serotype 21 VP7 antigen: C-terminal truncated protein has significantly reduced antigenicity.

In the present study, group-specific antigen VP7 of bluetongue virus (BTV) serotype 21 isolated from cattle in Tochigi prefecture in Japan in 1994 was characterized by sequencing and expression. Gene was amplified from cDNA synthesized on viral dsRNA using reverse-transcriptase-PCR. Nucleotide sequence of this isolate showed high similarity with other published BTV VP7 sequences. Full-length and C-terminal truncated forms of VP7 were expressed in insect cells by a baculovirus gene expression system under control of the viral polyhedrin promoter. Expression of full-length recombinant VP7 was confirmed by immunoprecipitation with VP7 specific monoclonal antibody (8A3B.6, ATCC). Recombinant proteins expressed with or without 6x His-tag showed good expression levels in TN5 cells and reacted well with the monoclonal antibody in the indirect ELISA. However C-terminal truncated VP7 with His-tag failed to react with this monoclonal antibody, while poor antigenicity was evident when it was reacted with infected bovine serum. Reduced antigenicity of the latter suggested that C-terminal truncation affects 8A3B.6 epitope construction probably via inhibition of VP7 trimer structure formation.

Cross-reactivity of Japanese encephalitis virus-vaccinated horse sera in serodiagnosis of West Nile virus.

Flavivirus-infected sera are known to show cross-reactions in serodiagnoses of heterologous flavivirus infections. Japanese encephalitis virus (JEV) is endemic in Asia and, in Japan, many horses are vaccinated against JEV. However, the cross-reactivity level of JEV-vaccinated horse sera in the serodiagnosis of West Nile virus (WNV) has not been clarified. The antibody cross-reactivity of JEV-vaccinated horse sera in WNV serological tests, such as the plaque reduction neutralization test (PRNT), IgG indirect ELISA (IgG-ELISA) and hemagglutination inhibition (HI) test, was examined. All JEV-vaccinated horse sera were positive for JEV antibodies with JEV PRNT at both 90% and 50% plaque reductions. In WNV PRNT, 16.7% of the horses were positive at 90% plaque reduction, and 50% of the horses were positive at 50% plaque reduction. All the JEV-vaccinated horse sera showed positive-to-negative (P/N) ratios of over 2.0 with JEV IgG-ELISA, and half of them had P/N ratios of over 2.0 with WNV IgG-ELISA. There was little difference between the JEV HI and WNV HI titers in individual horses. These results indicate that in serosurveillance of WNV, JEV-vaccinated horses can produce false-positive results in WNV IgG-ELISA, HI and PRNT.