Comparison of enzyme-linked immunosorbent assay, electron microscopy, and reversed passive haemagglutination for detection of human rotavirus in stool specimens

An enzyme-linked immunosorbent assay (ELISA) using microplates as solid phase, rabbit antiserum against human rotavirus Wa strain as catching antibody, and the same reagent labeled with beta-D-galactosidase as conjugate, has been developed for detection of human rotavirus antigen(s) in stool specimens from patients with acute gastroenteritis. The limit of detection of purified human rotavirus by ELISA was 15.6 ng/ml (1.56 ng/well) of viral protein. The sensitivities of ELISA, electron microscopy, and the reversed passive haemagglutination method (ROTA-CELL) were compared. ELISA was more sensitive than electron microscopy and the reversed passive haemagglutination method. The ELISA blocking assay was useful for detection of an antibody response to human rotavirus in paired sera from children in two institutions during outbreaks of rotavirus gastroenteritis.     

An improved method for concentrating rotavirus from water samples

A modified adsorption-elution method for the concentration of seeded rotavirus from water samples was used to determine various factors which affected the virus recovery. An enzyme-linked immunosorbent assay was used to detect the rotavirus antigen after concentration. Of the various eluents compared, 0.05M glycine, pH 11.5 gave the highest rotavirus antigen recovery using negatively charged membrane filtration whereas 2.9% tryptose phosphate broth containing 6% glycine; pH 9.0 was found to give the greatest elution efficiency when a positively charged membrane was used. Reconcentration of water samples by a speedVac concentrator showed significantly higher rotavirus recovery than polyethylene glycol precipitation through both negatively and positively charged filters (p-value <0.001). In addition, speedVac concentration using negatively charged filtration resulted in greater rotavirus recovery than that using positively charged filtration (p-value = 0.004). Thirty eight environmental water samples were collected from river, domestic sewage, canals receiving raw sewage drains, and tap water collected in containers for domestic use, all from congested areas of Bangkok. In addition, several samples of commercial drinking water were analyzed. All samples were concentrated and examined for rotavirus antigen. Coliforms and fecal coliforms (0->1,800 MPN/100 ml) were observed but rotavirus was not detected in any sample. This study suggests that the speedVac reconcentration method gives the most efficient rotavirus recovery from water samples.     

Comparison of methods for rotavirus detection in water and results of a survey of Jerusalem wastewater

Methods for the detection of viable rotaviruses and rotavirus antigen in water were developed and compared. The methods included laboratory-developed enzyme-linked immunosorbent assays (ELISAs) with chromogenic and luminescent substrates, commercial Rotazyme and Enzygnost ELISAs, and an indirect immunofluorescent assay. Of the methods tested, the immunofluorescent assay and the Enzygnost ELISA were the most sensitive for the simian rotavirus SA-11. All of the methods were positive for human rotavirus from clinical specimens. Seeded SA-11 rotavirus was concentrated from water by absorption to and elution from Zeta Plus filters followed by organic flocculation. Interference with the assays by components of the wastewater concentrates was minimal for the ELISAs, although the undiluted organic flocs were cytotoxic for the immunofluorescent assay. A survey of Jerusalem wastewater was carried out over the course of 1 year, and samples were assayed for rotaviruses and enteroviruses. Although enteroviruses were found in almost all of the samples, all samples were negative for rotaviruses. The concentration of rotaviruses in the wastewater was thus below the detection limit of the method used.     

Comparison of methods for rotavirus detection in water and results of a survey of Jerusalem wastewater.

Methods for the detection of viable rotaviruses and rotavirus antigen in water were developed and compared. The methods included laboratory-developed enzyme-linked immunosorbent assays (ELISAs) with chromogenic and luminescent substrates, commercial Rotazyme and Enzygnost ELISAs, and an indirect immunofluorescent assay. Of the methods tested, the immunofluorescent assay and the Enzygnost ELISA were the most sensitive for the simian rotavirus SA-11. All of the methods were positive for human rotavirus from clinical specimens. Seeded SA-11 rotavirus was concentrated from water by absorption to and elution from Zeta Plus filters followed by organic flocculation. Interference with the assays by components of the wastewater concentrates was minimal for the ELISAs, although the undiluted organic flocs were cytotoxic for the immunofluorescent assay. A survey of Jerusalem wastewater was carried out over the course of 1 year, and samples were assayed for rotaviruses and enteroviruses. Although enteroviruses were found in almost all of the samples, all samples were negative for rotaviruses. The concentration of rotaviruses in the wastewater was thus below the detection limit of the method used.     

Prevalence of antibodies to Sendai virus and rotavirus in laboratory rabbits

One hundred and sixty rabbit sera from 10 breeding colonies and 13 laboratory colonies were tested for antibodies to Sendai virus and rotavirus by enzyme-linked immunosorbent assay (ELISA). Antibodies were detected to Sendai virus in 53% and to rotavirus in 81%, indicating the prevalence of these viral infections in laboratory rabbit colonies.     

A survey of rotavirus infection in the topics

The age distribution of rotavirus antibody in the serum of inhabitants of Laos and Indonesia was studied by the neutralization test using antigenically related calf rotavirus (NCDV) as a substitute for non-cultivable human rotavirus. The results revealed that both the rate of antibody-positives and the modal titers of antibody distribution by each age group in these countries were higher than those in respective are groups of Japanese, which suggest higher endemicity of rotavirus infection in these countries. A survey of rotavirus in diarrheic patients' stools by enzyme-linked immunosorbent assay indicated that the virus infection occurred most frequently among infants between 4 and 12 months of age.     

Landscape phage probes for Salmonella typhimurium

We selected from landscape phage library probes that bind preferentially Salmonella typhimurium cells compared with other Enterobacteriaceae. The specificity of the phage probes for S. typhimurium was analyzed by the phage-capture test, the enzyme-linked immunosorbent assay (ELISA), and the precipitation test. Interaction of representative probes with S. typhimurium was characterized by fluorescence-activated cell sorting (FACS), and fluorescent, optical and electron microscopy. The results show that the landscape phage library is a rich source of specific and robust probes for S. typhimurium suitable for long-term use in continuous monitoring devices and biosorbents.     

Recovery from chronic rotavirus infection in mice with severe combined immunodeficiency: virus clearance mediated by adoptive transfer of immune CD8+ T lymphocytes.

Severe combined immunodeficient (SCID) mice lack both functional T and B cells. These mice develop chronic rotavirus infection following an oral inoculation with the epizootic diarrhea of infant mice (EDIM) rotavirus. Reconstitution of rotavirus-infected SCID mice with T lymphocytes from immunocompetent mice allows an evaluation of a role of T-cell-mediated immunity in clearing chronic rotavirus infection. Complete rotavirus clearance was demonstrated in C.B-17/scid mice 7 to 9 days after the transfer of immune CD8+ splenic T lymphocytes from histocompatible BALB/c mice previously immunized intraperitoneally with the EDIM-w strain of murine rotavirus. The virus clearance mediated by T-cell transfer was restricted to H-2d-bearing T cells and occurred in the absence of rotavirus-specific antibody as determined by enzyme-linked immunosorbent assay, neutralization, immunohistochemistry, and radioimmunoprecipitation. Temporary clearance of rotavirus was observed after the transfer of immune CD8+ T cells isolated from the intestinal mucosa (intraepithelial lymphocytes [IELs]) or the spleens of BALB/c mice previously infected with EDIM by the oral route. Chronic virus shedding was transiently eliminated 7 to 11 days after spleen cell transfer and 11 to 12 days after IEL transfer. However, recurrence of rotavirus infection was detected 1 to 8 days later in all but one SCID recipient receiving cells from orally immunized donors. The viral clearance was mediated by IELs that were both Thy1+ and CD8+. These data demonstrated that the clearance of chronic rotavirus infection in SCID mice can be mediated by immune CD8+ T lymphocytes and that this clearance can occur in the absence of virus-specific antibodies.     

Development and evaluation of various enzyme-linked immunosorbent assays for the detection of Clostridium perfringensβ anti-toxins

The aim of our work was to develop an enzyme-linked immunosorbent assay for the detection of antibodies against the Clostridium perfringens beta toxin. For this purpose, five different ways of performing an enzyme-linked immunosorbent assay were investigated. Positive and negative sera of different animals and partially purified beta toxin were used. In all enzyme-linked immunosorbent assay tests, microplates were first coated with monoclonal antibodies against the C. perfringens beta toxin. Actually, the first three ways of performing enzyme-linked immunosorbent assay proved to be an inhibition or a blocking enzyme-linked immunosorbent assay. In the first of these modifications, the examined serum was added on a microplate after the toxin. In the second two tests, they were added simultaneously after they were incubated together (60 min at room temperature or overnight at 4 degrees C, respectively). An anti-toxin conjugate was used for the detection. It was also used in a competitive enzyme-linked immunosorbent assay, where it was added together with the examined serum on the microplate, to which the toxin was already bound. The fifth way of performing an enzyme-linked immunosorbent assay differed from others by the use of conjugated anti-species immunoglobulin for the detection. The biggest differences in absorbances between positive and negative sera were found in the blocking enzyme-linked immunosorbent assay, where the mixture of the toxin and the examined serum were previously incubated overnight at 4 degrees C. The smallest differences in absorbance were found when anti-species conjugates were used.     

Purification of rough-type lipopolysaccharides of Neisseria meningitidis from cells and outer membrane vesicles in spent media.

A procedure for the purification of Neisseria meningitidis lipopolysaccharide (LPS) from outer membrane vesicles (OMV) in spent growth media was developed. Five different LPS strains of group A N. meningitidis were grown in tryptic soy broth with vigorous aeration for 36-48 h, and centrifuged to collect both cells and supernatants. The amount of LPS in the OMV in the supernatants was higher or at least equal to that in the cells. The OMV in each supernatant were concentrated, pelleted by ultracentrifugation, and treated with 2% sodium deoxycholate to dissociate LPS from OMV. The LPS was then separated from capsular polysaccharides, proteins and phospholipids by gel filtration on Sephacryl S-300 column in 1% sodium deoxycholate, and precipitated from the column fractions in 70% ethanol. In addition, LPS was also extracted from cells with hot phenol-water, ultracentrifuged once after treatment with ribonuclease, and purified on Sephacryl S-300. When compared with an improved phenol-water extraction method, the LPS obtained from either OMV or cells by the above methods gave a 40-180% increase in yield. The LPS also had much higher activities in limulus amebocyte lysate assay, rabbit pyrogenic test, and enzyme-linked immunosorbent assay. The LPS purified from cells and from OMV were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.     

Identification of nonspecific reactions in laboratory rodent specimens tested by Rotazyme rotavirus ELISA

Fecal specimens from several laboratory animal species were tested for rotavirus antigen by Rotazyme II, a commercially available enzyme-linked immunosorbent assay (ELISA) that is widely used in human diagnostic studies. Fecal samples from rabbits, hamsters, guinea pigs, dogs, and cats tested negative; whereas those from rats and mice yielded a high proportion of positive results. Rats had the highest rate with 82% of the samples being positive. However, the presence of rotavirus in positive rodent samples could not be confirmed by virus isolation, electron microscopy or blocking ELISA using anti-EDIM mouse rotavirus serum. Several lines of evidence indicated that these positive reactions were false positives, apparently due to a non-specifically reacting substance in the diet of rats and mice. All the positive fecal samples were from rats and mice that had been fed nonautoclaved diet. Samples from rodents fed autoclaved diet were consistently negative in the Rotazyme test. When rats fed autoclaved diet were subsequently fed nonautoclaved diet, their stool converted from negative to positive within 6 hours. Conversely, rats with positive stool samples converted to negative within 15 hours when fed autoclaved diet. Similar results were found with mice. Positive fecal specimens and nonautoclaved rodent diet both contained a substance that apparently attached nonspecifically to the antibody coated beads used in the ELISA and reacted directly with the substrate in the absence of the conjugate. This substance was heat labile and trypsin sensitive, suggesting that it was a protein.     

Derivation of Neutralizing Monoclonal Antibodies Against Rotavirus

Monoclonal antibodies were derived against the SA11 simian, NIC bovine, and Wa human rotavirus strains and characterized by enzyme-linked immunosorbent assay, plaque neutralization, and hemagglutination inhibition. Several strain SA11-specific antibodies were found to have neutralizing and hemagglutination-inhibiting capacity.     

Structural and Compositional Changes Attending the Ultracentrifugation of Very Low Density Lipoproteins

The effects of repetitive ultracentrifugation on the physical and chemical properties of very low density lipoproteins (VLDL) were investigated. VLDL recentrifuged one to seven times were characterized by chemical analyses, analytical ultracentrifugation and electron microscopy. The VLDL content of triglyceride was increased and the proportion of phospholipid decreased by ultracentrifugation. Recentrifugation of VLDL decreased the number of S_f ^o 20–100 particles and generated particles of S_f ^o > 400. The bulk of the material removed from VLDL by ultracentrifugation was lipoprotein having pre-g mobility on paper electrophoresis, flotation rates of S_f ^o 10–100 and a particle size of 300–400 Å. Two ultracentrifugations separated an average of 14% of the starting VLDL protein. Characterization of the apoproteins in this material by polyacrylamide gel electrophoresis, gel chromatography, immuno precipitation and amino acid analysis demonstrated a relatively high proportion of B-apoprotein and relatively little C-apoproteins.     

The Role of Maize in the Epidemiology of Barley Yellow Dwarf Virus in Northeast Spain

Barley yellow dwarf virus has been detected in maize by indirect enzyme-linked immunosorbent assay (ELISA) and by immunospecific electron microscopy (ISEM). Samples of maize collected in September 1988, 1989 and 1990 showed that this crop is an important reservoir of BYDV; MAV-like isolates were the most common although PAV-like and RPV-like isolates were also present. Earlier research in Spain had shown that PAV-like isolates were predominant. Thus the evidence from this work that MAV was the main isolate, and very widely spread, is important for future research on BYDV epidemiology in Spain.     

人脐血血浆外泌体提取方法的比较北大核心CSCD

为探寻高效且稳定的提取人脐血血浆外泌体的方法,利用超高速离心法、蔗糖垫密度梯度离心法、改良超速离心法和聚乙二醇(polyethylene glycol, PEG)沉淀法提取人脐血血浆外泌体,并比较4种方法的优劣。利用透射电镜、动态光散射技术观察外泌体的形态、结构及大小;聚氰基丙烯酸正丁酯(bicinchoninic acid, BCA)法测定外泌体蛋白总量;Western blotting检测外泌体表面标志蛋白CD63、HSP70以及外泌体阴性蛋白GM130 (高尔基标志蛋白)的表达。结果表明,与提取外泌体的“金标准”,即超高速离心法相比,蔗糖垫密度梯度离心法稳定性好,获取的外泌体粒径较均一,但操作较复杂,耗时长;改良超速离心法操作较简单,纯度较高;PEG沉淀法提取的外泌体蛋白量最高,操作时间最短,但杂质较多。结果表明,4种方法均能从人脐血血浆中获取外泌体,但在操作时间、纯度、提取量等方面存在一定差异。因此,应根据实验目的和具体要求选择合适的提取人脐血血浆外泌体的方法。     

Detection of cross-reactive antigens shared byFusarium oxysporum andGlycine max by indirect ELISA and their cellular location in root tissues

Pathogenicity test ofFusarium oxysporum on ten cultivars of soybean revealed Soymax and Punjab-1 to be most resistant while JS-2 and UPSM-19 were most susceptible. Antigens were prepared from the roots of all the ten varieties of soybean and the mycelium ofF. oxysporum. Polyclonal antisera were raised against the mycelial suspension ofF. oxysporum and the root antigen of the susceptible cultivar UPSM-19. Cross reactive antigens shared by the host and the pathogen were detected first by immunodiffusion. The immunoglobulin fraction of the antiserum was purified by ammonium sulfate precipitation and DEAE-Sephadex column chromatography. The immunoglobulin fractions were used for detection of cross-reactive antigens by enzyme-linked immunosorbent assay. In enzyme-linked immunosorbent assay, antigens of susceptible cultivars showed higher absorbance values when tested against the purified anti-F. oxysporum antiserum. Antiserum produced against UPSM-19 showed cross-reactivity with the antigens of other cultivars. Indirect staining of antibodies using fluorescein isothiocyanate indicated that in cross-sections of roots of susceptible cultivar (UPSM-19) cross-reactive antigens were concentrated around xylem elements, endodermis and epidermal cells, while in the resistant variety, fluorescence was concentrated mainly around epidermal cells and distributed in the cortical tissues. CRAs were also present in microconidia, macroconidia and chlamydospores of the fungus.     

Immunochemical detection of ochratoxin A in black Aspergillus strains

One hundred and fifty-seven strains belonging to Aspergillus section Nigri were tested for ochratoxin A production using three different methods: a relatively new immunochemical method based on an enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The monoclonal antibody-based ELISA technique was successfully used to screen for low levels of ochratoxin A in the black Aspergilli without concentrating the culture filtrates. The results were confirmed by TLC and HPLC analysis and chemical derivatization. These latter methods required concentrated filtrates. Ochratoxin A was detected in the culture filtrates of five of the 12 A. carbonarius strains, none of the 45 A.japonicus strains and three of the 100 isolates in the A. niger aggregate (A. foetidus, A. awamori and A. niger).Abbreviations ELISA enzyme-linked immunosorbent assay - HPLC high-performance liquid chromatography - OA ochratoxin A - TLC thin-layer chromatography     

Cholesterol can stimulate secretion of apolipoprotein B by cultured human hepatocytes

During a 5 day cultivation of human hepatocytes in a primary culture the secretion of apolipoprotein B was measured by enzyme-linked immunosorbent assay. Density-gradient ultracentrifugation demonstrated that the majority of the secreted apolipoprotein B was associated with the very-low-density lipoprotein fraction. Exposure of the cells to cholesterol (5-100 micrograms/ml) resulted in a dose-dependent increase in apolipoprotein B secretion rate.     

Rabbit model of rotavirus infection.

A new small animal model was developed to study parameters of rotavirus infections, including the active immune response. Seronegative New Zealand White rabbits (neonatal to 4 months old) were inoculated orally with cultivatable rabbit rotavirus strains Ala, C11, and R2 and with the heterologous simian strain SA11. The course of infection was evaluated by clinical findings, virus isolation (plaque assay and enzyme-linked immunosorbent assay), and serologic response. All four strains of virus were capable of infecting rabbits as determined by isolation of infectious virus from intestinal contents or fecal samples, by seroconversion, or by a combination of these methods. The responses differed depending on the virus strain used for inoculation. Rabbits remained susceptible to primary infection to at least 16 weeks of age (upper limit examined). Virus excretion in intestinal contents was detected from 6 h to 7 days postinoculation. RNA electropherotypes of inocula and viruses isolated from rabbits were the same in all samples tested. Transmission of Ala virus and R2 virus but not SA11 virus from inoculated animals to uninoculated controls also occurred. In a challenge experiment with Ala virus, 74- and 90-day-old rabbits were rechallenged with Ala 5 weeks after a primary infection with Ala. Virus was excreted in feces from 2 to 8 days after the primary infection but was not excreted after challenge. These results indicate that the rabbit provides an ideal model to investigate both the primary and secondary active immune responses to rotavirus infections and to evaluate candidate vaccines.     

现用梅毒血清试验临床应用价值评估

通过检测梅毒患者正规治疗前、后血清抗体变化,评价梅毒实验室各种血清学试验方法对梅毒的诊断和随访的临床意义.本文采用梅毒螺旋体(TP)暗视野显微镜检查、快速血浆反应素(RPR)试验、TP明胶凝集试验(TPPA)和TP-IgM酶联免疫吸附试验(TP-IgM ELISA)等方法对135例梅毒患者正规治疗前、后的血清进行检测....