Serodiagnosis of infectious diseases with antigen microarrays

AIMS: To generate protein microarrays by printing microbial antigens on slides to enable the simultaneous determination in human sera of antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus and herpes simplex virus (HSV) types 1 and 2. METHODS AND RESULTS: Antigens were printed on activated glass slides using high-speed robotics. The slides were incubated with serum samples and subsequently with fluorescently labelled secondary antibodies. Human IgG and IgM bound to the printed antigens were detected using confocal scanning microscopy and quantified with internal calibration curves. The microarray assay could detect as little as 0.5 pg of both IgG and IgM bound onto the glass surface. Precision profiles ranged from 1.7 to 18.5% for all the antigens. Microarrays and commercial ELISAs were utilized to detect serum antibodies against the ToRCH antigens in a panel of characterized human sera. Overall >80% concordance was obtained between microarray and ELISA kits in the classification of sera. CONCLUSIONS: These results indicate that the microarray is a suitable assay format for the serodiagnosis of infectious diseases. SIGNIFICANCE AND IMPACT OF STUDY: Antigen microarrays can be optimized for clinical use, their performance is equivalent to ELISA but they offer significant advantages in throughput, convenience and cost.     

Serodiagnosis of infection with verocytotoxin-producing Escherichia coli

Human sera (167) were screened for antibodies to lipopolysaccharide (LPS) prepared from strains of Verocytotoxin-producing Escherichia coli (VTEC) belonging to a range of serogroups, secreted proteins expressed by attaching and effacing VTEC, enterohaemolysin and H = 7 flagellar proteins. Twelve sera (about 7%) contained antibodies to the LPS of E. coli 05 (one), 026 (two), 0115 (two), 0145 (one), 0163 (one) and 0165 (five). Sera containing antibodies to the LPS of E. coli O26 and O145 also contained antibodies to secreted proteins of 100 and 40 kDa. An additional 34 sera, known to contain antibodies to the lipopolysaccharide of E. coli O157, were examined for antibodies to enterohaemolysin, H = 7 flagellar antigens and bacterial cell surface-associated proteins of 5, 6 and 22 kDa. Three sera contained antibodies to enterohaemolysin and one serum contained antibodies to flagellar proteins. Antibodies to membrane-associated proteins were not detected. It was concluded that enterohaemolysin, H = 7 flagellar proteins and the cell surface-associated proteins were unsuitable for use in immunoassays for providing evidence of infection with VTEC.     

Effect of brucellosis protective antigen on the phagocytic activity of the cells]

The authors present the results of study of the protective function of cells of the local inflammatory focus caused by the administration of live brucella culture to guinea pigs immunized with a protective antigen in the optimal (0.6) and increased (2 mg) doses. Macrophages of guinea pigs vaccinated with the optimal immunized dose of the protective antigen phagocytized brucellae actively; an increased dose of the antigen suppressed the ingestive and the digestive functions of macrophages, this apparently being caused by the manifestation of the immunodepressive activity of the residual amount of lipopolysaccharide (endotoxin) in the protective antigen.     

Mirin-making at low alcohol concentration with Koji prepared with a new mutant of Aspergillus usamii

We examined the productivity of mirin-making and the quality if the mirin produced using koji prepared with a new mutant (mutant CA) that originated from Aspergillus usamii mut. shiro-usamii. The koji prepared with the mutant CA contained a large amount of citric acid. Therefore, the concentration of the brewing alcohol added to prevent bacterial contamination of the mash was decreased to 6.0% from the 12.5% needed when the mash was made with koji of the conventional Aspergillus oryzae. A mash containing this low concentration of alcohol was incubated with koji of the mutant CA and enzyme preparations such as α-amylase (6,000 DU/kg mash) from Bacillus subtilis and acidic protease (1,000 PU/kg mash) from Aspergillus niger. The starch and protein in this mash was sufficiently digested. The yield of mirin obtained from this mash was high (96% based on the mash weight), and the resulting mirin contained much citric acid, malic acid, succinic acid, nitrogen compounds, isomaltose, isomaltotriose, and oligosaccarides. The taste of the mirin was refreshingly sour and flavorsome.