Survey of total aflatoxins in camels sera by enzyme linked immunosorbent assay (ELISA)

A survey of total aflatoxins was carried out on samples of camels blood sera in order to assess aflatoxin hazards in camels following some deaths among animals during July through September, 1990 in Al Ain area, about 16 km from Abu Dhabi City. Epidemiological and laboratory based studies indicated that the outbreak was associated with the consumption of mould damaged feed.A flavus andA parasiticus and varying amount of aflatoxin were detected in samples tested. 53 samples of camel sera were collected for the analysis: 16 out of 53 samples were collected from apparently clinically healthy racing camels. All these samples were examined for total aflatoxins. All of the 16 samples were found to contain aflatoxins ranging from 5 to 50ng/mL total aflatoxin and 28 out of the 37 samples were found to contain aflatoxin at the range of 2 to 12 pg/mL total aflatoxins.     

An enzyme linked immunosorbent assay (ELISA) for plasma medroxyprogesterone acetate (MPA).

A single extraction fixed antigen enzyme-linked immunosorbent assay (ELISA) that can be completed in less than 24 h is described for the measurement of medroxyprogesterone acetate (MPA) in plasma. MPA is covalently coupled to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to a standard 96-well microtitre plate where it competes with MPA in the extracted plasma sample for goat anti-MPA. Antibody binding to the solid phase is determined via binding of a horse-radish peroxidase second antibody which reacts colorimetrically with its substrate. The reaction is stopped by addition of 1.25 M H2SO4 and absorbance read at 492 nm. All steps except for sample addition and extraction can be performed on an automatic ELISA processing machine. The assay is sensitive, specific and precise, with intra- and inter-assay coefficients of variation of less than 10 and 15%, respectively. Assay sensitivity is 0.08 ng/ml. The assay follows established methodology for other assays in this laboratory which assists standardization, cost structure and sample throughput and thus is a useful alternative to radioimmunoassays for the determination of MPA in plasma.     

Enzyme linked immunosorbent assay (ELISA) for the determination of protein-A

A competitive ELISA for the determination of protein-A concentration in culture supernatants is described. The sensitivity of the assay is 50 ng/ml and it may be used to determine concentrations of protein-A up to 1000 ng/ml. The assay is specific, rapid and suitable for routine screening of protein-A producing microorganisms.     

Detection of venom by enzyme linked immunosorbent assay (ELISA) in patients bitten by snakes in Thailand.

The ability of an enzyme linked immunosorbent assay (ELISA) to detect venom was evaluated in 251 patients bitten by four of the commonest poisonous snakes in Thailand. Serum was tested only from patients who brought the snakes that had bitten them. About one third of all bitten patients had detectable venom antigenaemia, though a smaller proportion were symptomatic. Serum venom concentrations on admission correlated with the severity of clinical manifestations. The test was sensitive and specific even for specimens that had been collected and stored under suboptimal conditions. The technique is suitable for forensic use in cases of suspected snakebite. The combination of snake identification and venom antigen detection should be a more reliable means of studying the epidemiology of snakebite than the measurement of venom antibodies in a population.     

Enzyme-linked immunosorbent assay (ELISA) using a glycolipid antigen for the serodiagnosis of melioidosis

Abstract The serodiagnosis of melioidosis is commonly performed with tests using protein or polysaccharide as antigen. However, due to the low sensitivity, specificity and difficulty in the preparation of the antigens, more simple, precise and reproducible diagnostic tests were required. A purified glycolipid antigen (GL) which is a specific lipid component of Burkholderia pseudomallei has been used in an ELISA. With this antigen, specific immunoglobulin G (IgG) was detected in 49 out of 50 melioidosis sera. IgG was also detected in 2 out of 185 (Japanese) and 16 out of 181 (Vietnamese) control sera. Thus, the sensitivity was 98.0%, and specificity was 98.9% and 91.1% in the Japanese and Vietnamese sera, respectively. When the ELISA and indirect haemagglutination (IHA) tests were combined, a sensitivity of 100% and specificity of 97.8% were achieved. The advantages of the glycolipid antigen are ease of preparation, stability, high sensitivity and specificity.     

Obtaining immunologically active bovine viral diarrhea virus (BVDV) proteins for enzyme linked immunosorbent assay (ELISA)

We report here the preparation of BVDV antigen (Ag) to obtain adequate quantities of pure active viral proteins from Madin Darby Bovine kidney (MDBK) cell culture infected with BVDV cytophatic Oregon C24V strain. SDS-PAGE, Immunodot, ECL-Western blot and ELISA showed the best specificity and activity of BVDV Ag obtained from infected cells only by mild experimental conditions. BVDV Ag preparations showed the peculiar BVDV sensitivity to proteases, nonionic surfactants and tend for protein degradation and irreversible loss of conformational antigenic determinants with subsequent inability to detect antibodies against viral Ag in hyperimmune sera. The widest panel of immunologically active and specific polypeptides, that elicit Ab production in hyperimmune sera, was obtained by ultrasonication and subsequent purification on 20%-50% sucrose cushion. We observed that BVDV tend to remain in the infected cells, to associate with components of serum and cellular origin--this is of crucial importance towards the specificity of ELISA. BVDV antigenic properties are determined by the labile conformational antigenic epitopes.     

Evaluation of the use of chromogenic and fluorogenic substrates in solid-phase enzyme linked immunosorbent assays (ELISA)

Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus (FMDV). The detection limits for both antigens were compared using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain maximum sensitivity for both systems. Similar sensitivities were found using fluorogenic and chromogenic substrates. Tetramethyl benzidine substrate for horse-radish peroxidase enzyme conjugates was found to attain the highest sensitivity levels for chromogenic assays (0.12 ng IgG/ml and 1.0 ng/ml FMDV respectively), after 10 min incubation. Of the two fluorogenic enzyme/substrates studied, B-galactosidase was the most sensitive but required extended incubation times (2-3 h) as compared with chromogenic systems. Special microplates for fluoro-immunoassay (FIA) were compared with conventional microplates and no advantage was found to justify their use. An alkaline phosphatase anti-guinea-pig conjugate was used to confirm the equivalence of fluorogenic and chromogenic substrates in terms of sensitivity. A comparison of the amount of signal generated using various concentrations of enzyme in the absence of antigen was made for two different alkaline phosphatase conjugates to obtain theoretical sensitivity limits. One possible advantage of fluorogenic substrates is that high binding ratio can improve the confidence in discrimination of positive results.     

Enzyme linked immunosorbent assay (ELISA) for beta-endorphin and its antibodies

An Enzyme Linked Immunosorbent Assay (ELISA) is described for use in the determination of beta-endorphin antibody titers, as well as for the quantitation of naturally occurring levels of beta-endorphin in plasma and other bodily fluids. The ability of the assay to accommodate unpurified samples containing small concentrations of beta-endorphin was improved through the use of affinity purified antibodies in conjunction with a competitive inhibition ELISA. The problem of non-specific binding of beta-endorphin during competitive inhibition assays was circumvented through a two-step process in which the plate was first coated with BSA, followed by a second plate coating with poly-lysine (MW4000). The second coating with poly-lysine was found necessary in order to eliminate intermolecular void spaces following initial plate treatment with BSA. Following these procedures enabled quantitation of beta-EP at a level as low as 10 pmoles per microtitre plate well.     

Thermometric enzyme linked immunosorbent assay: TELISA.

A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.     

Use of monoclonal antibodies in enzyme linked immunosorbent assay (ELISA) for detection of botulinum type B toxins

Use of polyclonal antibodies failed to correlate mouse assay with enzyme linked immunosorbent assay (ELISA) in titration of culture fluid of different strains of Clostridium botulinum type B. If ELISA is performed with such a monoclonal antibody that is capable of neutralizing the toxin, however, the lethal toxicity can be determined quantitatively.     

Using an enzyme linked immunosorbent assay (ELISA) and a protein phosphatase inhibition assay (PPIA) for the detection of microcystins and nodularins

Cyanotoxins produced by cyanobacteria (blue-green algae) include potent neurotoxins and hepatotoxins. The hepatotoxins include cyclic peptide microcystins and nodularins plus the alkaloid cylindrospermopsins. Among the cyanotoxins the microcystins have proven to be the most widespread, and are most often implicated in animal and human poisonings. This paper presents a practical guide to two widely used methods for detecting and quantifying microcystins and nodularins in environmental samples-the enzyme linked immunosorbant assay (ELISA) and the protein phosphatase inhibition assay (PPIA).     

Enzyme linked immunosorbent assay (ELISA) for detection of Clostridium botulinum type B toxin.

The enzyme-linked immunosorbent assay using different techniques has been applied to determine botulinum type B toxin. With the so-called "sandwich" technique, about 5,000 mouse ip LD50 of type B toxin can be detected. With the "double-sandwich" technique, about 400 mouse ip LD50 of toxin is detected and different commerical antisera are useful. For accurate quantification of botulinum toxins in culture filtrates, addition of EDTA to samples seems to be necessary. Cross-reactivity of the assay depends on the specificity of the antisera against botulinum type B toxin used and is almost eliminated with antiserum prepared against the toxic component of type B toxin.     

Detection of tobacco mosaic virus and tomato mosaic virus in pepper seeds by enzyme linked immunosorbent assay (ELISA)

Pepper seed samples were tested for the infection of tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) by enzyme linked immunosorbent assay (ELISA). Out of 26 pepper seed samples tested, 17 were infected with TMV and ToMV in ELISA. About 34.7% of pepper seed samples were found to be healthy. Infections of TMV or ToMV were recorded to be 61.53% and 11.5%, respectively of the total tested seed samples.     

Detection of Klebsiella pneumoniae antibodies in Aotus l. lemurinus (Panamanian owl monkey) using an enzyme linked immunosorbent assay (ELISA) test

An enzyme linked immunosorbent assay (ELISA), was adapted to detect antibodies against Klebsiella pneumoniae in Aotus l. lemurinus monkeys. It was used to define the prevalence of infection and the immunogenicity of an Al(OH)3 bacterin in a population of laboratory born A. l. lemurinus monkeys. This represents a preliminary step to reduce K. pneumoniae produced mortality. A striking finding during a cross-sectional prevalence study was that none of the babies of less than 2 months old had detectable levels of antibody. The antibody prevalence gradually increased in all other age groups reaching 87.5% in the 8-10-month-old group. These results indicate that infection with K. pneumoniae occurred sometime between 2 and 6 months of age, probably as a result of oral-faecal contamination and a change in the feeding and grooming behaviour. To determine whether infants had maternal antibodies or if they were asymptomatic carriers of the bacterium, a cross-sectional study was done in 15 infants less than 4 months old and their mothers. K. pneumoniae antibodies were detected in 11/15 mothers with serum titers ranging from 1:4 to greater than 1:256 and the bacterium was isolated from 3 babies and one mother and her baby. Results showed that no maternal antibodies remained in babies older than 3 weeks old. A prospective study indicated a reduction in mortality from 20% for the previous 3 years to 3.7% (3/79) in AL(OH)3 K. pneumoniae bacterin vaccinated infants born during 1988-89.     

Enzyme-linked immunosorbent assay (ELISA) in the paracoccidioidomycosis

An enzyme-linked immunosorbent assay (ELISA) for detection and quantification of antibodies antiParacoccidioides brasiliensis is described. Polystyrene plates have been used as solid phase to absorb P. brasiliensis metabolic yeast phase antigen. Twenty sera of proven paracoccidioidomycosis, 11 of histoplasmosis due Histoplasma capsulatum, 20 of aspergillosis and 20 human normal sera were tested. Ninety-five percent of the paracoccidioidomycosis sera had O.D. superior to 0.150 (from 0.163 to 2.650) at 1/400 serum dilution. ELISA assay was compared with counterimmunoelectrophoresis and erythro-immunoassay tests; a correlation was observed only with erythro-immunoassay. ELISA test should give new perspectives for the serodiagnosis of paracoccidioidomycosis.     

Quantitative detection of meat spoilage bacteria by using the polymerase chain reaction (PCR) and an enzyme linked immunosorbent assay (ELISA)

A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw meat has been developed using primers designed from conserved regions in the 16S ribosomal RNA gene (rRNA). Amplified PCR products generated using a digoxigenin-labelled primer were automatically hybridized to a biotinylated probe included in the PCR reaction. The hybridization was performed as part of the PCR programme. The biotin-digoxigenin hybrids were quantified by an enzyme-linked immunosorbent assay (ELISA). Streptavidin bound to the wells of a microtitre plate was used to capture the biotin-digoxigenin-labelled fragments that were detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying meat samples containing bacteria in the range 10²–10⁷ cfu cm⁻². The detection threshold for the PCR-ELISA assay developed in this work is 10² cfu cm⁻².     

Enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to human cytomegalovirus

A solid-phase enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to cytomegalovirus (CMV) is described. The assay used purified CMV and extracts of CMV infected cells as antigen. Antigens were desiccated onto the bottom surface of polystyrene microcuvettes. The antibodies bound to the antigens were assayed by anti-IgG-alkaline phosphate conjugate followed by addition of the enzyme substrate. Titration curves have been obtained from the sera of 35 blood donors and of 23 patients. Comparison of results obtained by ELISA with those obtained by complement fixation (CF) shows that there is agreement between the tests. Both purified CMV and extracts of CMV infected cells were found to be suitable antigens. Purified CMV was of value particularly in those sera which show high reactivity against control antigen. The ELISA technique described is approximately 412 to 548 times more sensitive than the CF test when purified CMV or extracts of CMV infected cells, respectively, are used as antigens. No significant heterotypic rise to CMV was observed by ELISA in three sets of sera with seroconversion to herpes simplex virus. The ELISA technique gives objective results, is easily performed, and may be adaptable as a routine test both for serological diagnosis of CMV infection and for screening of the general population.     

The serodiagnosis of human hydatid disease: 2. Additional studies on selected sera using indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS).

Sixty-one serum samples selected on the basis of reactivity in the complement fixation (CF) and latex agglutination (LA) test, were further examined for sensitivity and specificity by indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS). Twenty sera from healthy Europeans and 48 samples from patients with either schistosomiasis or trichinosis were also tested. Comparable levels of sensitivity were found between the CF and LA positive sera and IHA, ELISA and DASS. Of the CF positive LA negative group of sera, many were positive by DASS but only a few reacted in IHA and ELISA. Some cross reactivity was also observed in the schistosomiasis sera tested by IHA and ELISA.     

Optimality assessment in the enzyme-linked immunosorbent assay (ELISA)

An optimality criterion is proposed for evaluating the precision of alternative designs in the enzyme-linked immunosorbent assay. Assay profiles are represented as four-parameter logistic functions with parameter estimation based on either a weighted nonlinear regression or a simple nonlinear regression after a logarithmic transformation. Assay design changes are characterized in terms of their effects on parameters in the four-parameter logistic model. General optimality results are derived for the variance of relative potency estimates in routine assay applications.