Fluorescent oligonucleotide ligation technology for identification of ras oncogene mutations

A mutation detection strategy based on multiplex PCR followed by multiplex allele-specific oligonucleotide probe ligation was developed to detect single nucleotide substitutions in ras oncogenes, a common genetic abnormality in many human cancers. Mutation-specific probes are synthesized for each possible single-base, nonsilent mutation in codons 12, 13, and 61 of H-, K-, and N-ras oncogenes. Mutations are identified by competitive oligonucleotide probe ligation to detect normal and /or mutant genotypes in one reaction. Three probes (one common and two allelic probes) are needed for analysis of each mutation. Probes hybridized to target ras oncogene DNA are joined by a thermostable ligase if there are no mismatches at their junctions; temperature cycling results in a linear increase in product. Common probes are labeled with fluorochromes, and allelic probes each have different lengths. Ligation products are analyzed by denaturing polyacrylamide gel electrophoresis on a fluorescent DNA sequencer. We have applied this technology to identify ras mutations in pancreatic cancers and lung cancers and in patients with myelodysplastic syndromes and leukemias.     

Detection of extracellular proteinase of Pseudomonas fragi by enzyme-linked immunosorbent assay

A double-antibody-sandwich, enzyme-linked immunosorbent assay was developed to detect an extracellular proteinase produced by Pseudomonas fragi. The method was capable of detecting 4 g/ml of the proteinase in spiked samples of buffer and broth and 4.2 g/ml in a broth culture of the organism. The assay detected the presence of proteinase at bacterial densities of approximately 10⁴ cfu/ml, which develop after incubation for 15 h at 25°C in a broth medium. All assays could be completed within 7 h. This assay is of value in plotting proteolytic expression in relation to the growth cycle of Ps. fragi in broth culture and may be of value, with development, in other more complex milieux.     

Detection of Aeromonas hydrophila in food with an enzyme-linked immunosorbent assay

A microtitration plate, antibody capture, enzyme-linked immunosorbent assay was developed for the detection of Aeromonas hydrophila serotype O : 11 (highly virulent strains). The assay utilizes a detector antibody which shows no cross-reactions with Aeromonas strains other than serotype O : 11 or non- Aeromonas competing organisms. The detector antibody is mixed with the sample and incubated for 1 h, microcentrifuged and the supernatant fluid (unadsorbed antibody) titred in a microtitre plate coated with A. hydrophila cells from serotype O : 11. All the A. hydrophila strains from serotype O : 11 tested reacted strongly with the detector antibody. Also by culturing and performing the immunoassay with the detector antibody we established and quantified the presence of A. hydrophila O : 11 in different foods.     

An optimized enzyme-linked immunosorbent assay for quantitative diagnosis of bovine fascioliasis

An optimized immunoassay for detection of antibody to Fasciola hepatica antigen in cattle was developed through the adaptation of a kinetics-dependent, enzyme-linked immunosorbent assay (k-ELISA) to a microplate format. Enhanced sensitivity and a strict quantitative nature were achieved with the utilization of enzyme kinetics. With this k-ELISA, significant (P less than 0.01) elevations in anti-F. hepatica antibody could be detected as early as 2 wk post-infection in experimentally infected calves. Furthermore, fluke-burden related differences in anti-F. hepatica antibody levels between 3 different levels of fluke infection were evident.     

Detection of Clostridium botulinum type G toxin by enzyme-linked immunosorbent assay.

Clostridium botulinum type G toxin was detected and quantified readily with the enzyme-linked immunosorbent assay. With the double-sandwich technique and alkaline phosphatase as the enzyme indicator, C. botulinum toxin type G was detected in quantities equaling those required for one mouse intraperitoneal median lethal dose. The time required for the procedure was approximately 6.5 h, but this requirement could have been reduced to 5.5 h or less with the use of precoated plates stored at -70 degrees C. Cross-reactions did not occur with culture extracts of C. sporogenes of C. botulinum types B, C, D, E, and F. Acidic preparations of C. botulinum type A exhibited nonspecific reactivity. Likewise, 50% of the C. subterminale isolates tested were cross-reactive in the assay. These latter isolates express similar metabolic and physiological characteristics with C. botulinum type G.     

Detection of Renibacterium salmoninarum by the enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection and identification of Renibacterium salmoninarum. The immune γ-globulin used in the assay was absorbed with two species of cross-reacting bacteria to make a specific test system. R. salmoninarum could be detected in clinically-diseased fish within 30 minutes of preparing a kidney sample, and thus because of its ease of use, the ELISA could be employed as a rapid field test for bacterial kidney disease (BKD), although isolation of R. salmoninarum was more sensitive than the ELISA for detecting individual carrier fish.     

Strongyloides venezuelensis alkaline extract for the diagnosis of human strongyloidiasis by enzyme-linked immunosorbent assay

The present study was conducted to detected IgG antibodies using Strongyloides venezuelensis alkaline extract for the diagnosis of human strongyloidiasis by the enzyme-linked immunosorbent assay (ELISA). Sera from 90 subjects were analyzed (30 with strongyloidiasis, 30 with other parasites and 30 healthy individuals). Results were expressed in antibody titers, which were considered as positive when titer was >80. Sensibility and specificity of the assay were 100% and 96.7%, respectively. It can be concluded that the heterologous alkaline extract could be employed in ELISA as a diagnostic aid in human strongyloidiasis, due to its advantages as easiness of obtaining, practicability in preparing, and high indexes of sensitivity and specificity.     

Detection of Colletotrichum falcatum causing red rot of sugarcane by enzyme-linked immunosorbent assay

Red rot disease of sugarcane caused by Colletotrichum falcatum Went is one of the most destructive diseases of sugarcane (Saccharum officinarum L.) worldwide. The pathogen spreads primarily through infected sugarcane setts and hence the use of disease-free setts is essential to prevent the disease. In order to develop immunological method for detection of C. falcatum, two proteins with molecular weights of 27 kDa and 45 kDa were purified from the mycelium of C. falcatum race Cf 05 and used as antigen source to raise polyclonal antibodies in NewZealand white rabbit. The developed polyclonal antibodies were tested for detection of C. falcatum by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. The polyclonal antibodies specifically detected C. falcatum in extracts from infected plants, both in immunoblot and ELISA. The ELISA results showed that the developed polyclonal antibodies were highly specific to C.falcatum. The developed antibodies were very sensitive and could detect C.falcatum proteins even at a dilution of 1:50,000. Higher ELISA absorbance values were recorded even at an antigen dilution of 1:500. In western blot analysis, protein bands with molecular weights of 27 kDa and 45 kDa reacting to antisera raised against 27 kDa and 45 kDa mycelial proteins of C. falcatum, respectively, were detected in protein samples from red rot infected canes. The high specific reactivity and sensitivity of the antisera indicate its potential suitability for ELISA-based detection of C. falcatum.     

北京地区设施草莓8种病毒的酶联免疫吸附测定检测

[背景]病毒可以随同草莓无性繁殖材料传播扩散,导致产量和品质下降.选育无病毒种苗是草莓病毒病防治的主要措施,高效、灵敏的检测技术可为草莓病毒病防治提供技术保障.[目的]为明确8种能够侵染草莓的病毒在北京地区设施草莓上的发生情况,应用酶联免疫吸附测定(Enzyme-Linked Immunosorbent Assay,E...     

Detection of Bacillus cereus flagellar antigen by enzyme-linked immunosorbent assay (ELISA)

A serological typing scheme of Bacillus cereus has been developed by immunochemical analyses of flagellar antigen using an agglutination method. Enzyme-linked immunosorbent assay (ELISA) for the classification of flagellar serotype of Bacillus cereus had greater sensitivity. 10-500 times, than that of agglutination method. The specificity of flagellar antigen and antibody was determined by immunogold electron microscopy and ELISA inhibition assay. Application of ELISA is useful for the detection of the small amounts and many kinds of antigen-antibody reactions.     

A dot-immunobinding assay for the laboratory diagnosis of tuberculous meningitis and its comparison with enzyme-linked immunosorbent assay

V.V. RADHAKRISHNAN AND A. MATHAI. 1991. In an attempt to establish an alternative to standard bacteriological methods in the laboratory diagnosis of tuberculous meningitis (TBM), a simple dot-immunobinding assay (Dot-Iba) was standardized to detect Mycobacterium tuberculosis antigen 5 and antimycobacterial antibody in cerebrospinal fluid (CSF) specimens of patients with TBM. Sensitivity and specificity of Dot-Iba was compared with conventional enzyme-linked immunosorbent assay (ELISA) and standard bacteriological techniques. The Dot-Iba showed excellent correlation with indirect ELISA for the detection of antimycobacterial antibody in CSF and showed 60% sensitivity and 100% specificity in culture-negative patients with TBM. However Dot-Iba was less sensitive for the detection of antigen 5 in CSFs and showed false negative results (60%) in culture-positive patients with TBM.     

A dot-immunobinding assay for the laboratory diagnosis of tuberculous meningitis and its comparison with enzyme-linked immunosorbent assay.

In an attempt to establish an alternative to standard bacteriological methods in the laboratory diagnosis of tuberculous meningitis (TBM), a simple dot-immunobinding assay (Dot-Iba) was standardized to detect Mycobacterium tuberculosis antigen 5 and antimycobacterial antibody in cerebrospinal fluid (CSF) specimens of patients with TBM. Sensitivity and specificity of Dot-Iba was compared with conventional enzyme-linked immunosorbent assay (ELISA) and standard bacteriological techniques. The Dot-Iba showed excellent correlation with indirect ELISA for the detection of antimycobacterial antibody in CSF and showed 60% sensitivity and 100% specificity in culture-negative patients with TBM. However Dot-Iba was less sensitive for the detection of antigen 5 in CSFs and showed false negative results (60%) in culture-positive patients with TBM.     

Analysis of larval antigens of Oestrus ovis for the diagnosis of oestrosis by enzyme-linked immunosorbent assay

A serodiagnostic test for the diagnosis of infestation by the sheep nasal bot fly, Oestrus ovis (Linné) was examined. The enzyme-linked immunosorbent assay (ELISA) technique was used to analyze and compare the production of immunoglobulin G (IgG) antibodies against excretory-secretory products (ESP) and crude extract (CE) antigens from all the different larval stages of O. ovis in the sera of 276 adult sheep sampled in summer (n = 135) and winter (n = 141). ESP from first stage larvae was the most sensitive, coating antigen in winter and ESP from second stage larvae during summer. The most specific values were obtained by ESP against L1 in winter and by CE against L3 in summer. These results show that the stage of larval development has a significant impact on the humoral immune response over the course of a season. A significant correlation (P < 0.001) was found between the number of O. ovis larvae and the serum antibody levels using all differents antigens, except L3 CE. In Spain, where a long favourable period exists for the evolution and development of the different stage larvae between March and November, the ELISA test using L1 ESP antigen during winter and L2 ESP antigen in summer may be used for ovine oestrosis immunodiagnosis.     

Commercial enzyme-linked immunosorbent assay versus polymerase chain reaction for the diagnosis of chronic Chagas disease: a systematic review and meta-analysis

Chronic Chagas disease diagnosis relies on laboratory tests due to its clinicalcharacteristics. The aim of this research was to review commercial enzyme-linkedimmunosorbent assay (ELISA) and polymerase chain reaction (PCR) diagnostic testperformance. Performance of commercial ELISA or PCR for the diagnosis of chronicChagas disease were systematically searched in PubMed, Scopus, Embase, ISI Web, andLILACS through the bibliography from 1980-2014 and by contact with the manufacturers.The risk of bias was assessed with QUADAS-2. Heterogeneity was estimated with theI² statistic. Accuracies provided by the manufacturers usuallyoverestimate the accuracy provided by academia. The risk of bias is high in mosttests and in most QUADAS dimensions. Heterogeneity is high in either sensitivity,specificity, or both. The evidence regarding commercial ELISA and ELISA-recsensitivity and specificity indicates that there is overestimation. The currentrecommendation to use two simultaneous serological tests can be supported by the riskof bias analysis and the amount of heterogeneity but not by the observed accuracies.The usefulness of PCR tests are debatable and health care providers should not orderthem on a routine basis. PCR may be used in selected cases due to its potential todetect seronegative subjects.     

Bluetongue virus: Detection of antiviral immunoglobulin G by means of enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay was developed to detect antiviral IgG in the sere of sheep exposed to bluetongue virus. It was found that the enzyme-linked immunosorbent assay is a rapid and sensitive method for the detection of anti-bluetongue virus antibody. Bluetongue virus antigen prepared from extracts of virus infected BHK and Vero cells were equally effective. Antigen prepared from uninfected cells when used as coating antigen did not bind IgG from either exposed or unexposed animals. Sera raised against each of the four individual BTV serotypes, 10, 11, 13, and 17, found in the United States reacted equally with all four bluetongue virus serotype antigen preparations. Thus, any of the four serotypes can be used as the bluetongue virus antigen for the detection of anti-bluetongue virus antibody in the bluetongue virus-enzymelinked immunosorbent assay system. Antiviral IgG was readily detectable 6 days postinoculation. The anti-bluetongue virus antibody concentration continued to increase through the 35-day postinoculation test period. At 35 days postexposure, antibody titers of 1:1,600 to >1:3,200 were found. The rapid and sensitive nature of the bluetongue virus enzyme-linked immunosorbent assay indicates that this system should significantly extend serological studies on bluetongue virus.     

Detection of T-2 toxin in Fusarium sporotrichioides-infected corn by enzyme-linked immunosorbent assay.

A competitive enzyme-linked immunosorbent assay was used to screen for T-2 toxin in Fusarium sporotrichioides -infected corn. The assay detected T-2 toxin in diluted methanol extracts of corn samples at concentrations of 0.05 ng/ml. In infected corn samples, enzyme-linked immunosorbent assay and gas-liquid chromatography estimations of T-2 toxin concentrations were similar.