Improved bioluminescent enzyme immunoassay for the rapid detection of Salmonella in chicken meat samples

AIMS: To evaluate an improved bioluminescent enzyme immunoassay (BEIA) using biotinylated firefly luciferase for the rapid detection of Salmonella in naturally contaminated chicken meat samples. METHODS AND RESULTS: Capture agents and lipopolysaccharide (LPS) extraction reagents for Salmonella were investigated to improve the sensitivity of the BEIA. Also, the use of Oxoid SPRINT (Simple Pre-enrichment and Rapid Isolation New Technology) as a pre-enrichment and selective medium for 26-h BEIA detection of Salmonella in chicken meat samples was examined. The use of polymyxin B as a capture agent on solid support and 3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonic acid (CHAPS) for extraction of the LPS facilitated sensitive detection of Salmonella. Of 120 chicken meat samples, 25 samples were positive using the improved BEIA with the SPRINT and 25 samples were positive using the SPRINT followed by the standard isolation methods. CONCLUSIONS: The improved BEIA, in which polymxin B was used as a capture agent and CHAPS was used for extraction of the antigen, had a sensitivity of 96.0% and a specificity of 98.9% for the detection of Salmonella in chicken meat. SIGNIFICANCE AND IMPACT OF THE STUDY: The improved BEIA combined with the SPRINT medium for the detection of Salmonella in chicken meat samples produced comparable results to the culture methods in 26 h.     

Comparison of culture and immunoassay for detection of Escherichia coli O157 in raw minced meat and hamburger

Verocytotoxin-producing Escherichia coli O157 (VTEC) is an important food-borne pathogen of humans. The serious complications of VTEC infection and the established reservoir of VTEC in cattle used for mass food production are a public health concern. In this study 500 samples of hamburger and minced meat were examined for presence of E. coli O157. For E. coli detection, Tryptic Soy Broth supplemented (with novobiocin and bile salts) and Sorbitol Mc Conkey agar were used; an automated rapid enzyme linked fluorescent immunoassay (VIDAS E. coli O157) was also evaluated. E. coli O157 was found in 5 samples of hamburger, 2 strains were found to be positive for verocytotoxin production on Vero cells.     

Evaluation of lysine-iron-cystine-neutral red for the detection of salmonellas in the Bactometer

Ninety-five salmonellas and 40 non-salmonellas were screened in the Bactometer using lysine-iron-cystine-neutral red (LICNR) in order to evaluate the selectivity of the medium for the detection of salmonellas. Results for blackening of the medium in the well (indicating hydrogen sulphide, H₂S, production) and step size are presented. Five out of 95 salmonellas tested failed to produce blackening in the well, four of these five are known to be non-H₂S producers. Although salmonellas generally gave a larger step value than non-salmonellas, this criterion could not be used to distinguish reliably the two groups of organisms.     

Evaluation of an enzyme immunoassay for the detection of the mycotoxin tenuazonic acid in sorghum grains and sorghum-based infant food

An enzyme-linked immunosorbent assay (ELISA) for the Alternaria mycotoxin tenuazonic acid (TeA) was evaluated by comparative analysis of naturally contaminated sorghum grains and sorghum-based infant food, using a stable isotope dilution LC-MS assay (SIDA; limit of detection (LOD) 1.0 μg/kg) as the reference method. LODs of the ELISA were 30 μg/kg in sorghum grains and 220 μg/kg in sorghum-based infant cereals. With SIDA, 100% of the samples (n = 28) had been positive for TeA in a concentration range of 6–584 μg/kg (mean 113 μg/kg). The ELISA consistently detected TeA in all naturally contaminated samples at cut-off levels of 30–60 μg/kg (sorghum) and 200–300 μg/kg (infant cereals), as based on corresponding to SIDA values. Although the ELISA was much less sensitive than the SIDA method, it may be useful as a screening method for sorghum and sorghum-based infant foods and can be employed to identify samples containing elevated concentrations of TeA in food, well below the proposed level of concern (500 μg/kg).     

A convenient homogeneous enzyme immunoassay for estradiol detection

A convenient homogeneous enzyme immunoassay for estradiol is described. Unlike heterogeneous immunoassays, which require time-consuming separation steps or expensive automated systems, homogeneous immunoassays, wherein all reagents are freely suspended in bulk solution, can be simple and fast without costly instrumentation. The key component of this assay system, an estradiol-reporter enzyme conjugate, was prepared by covalently binding β-estradiol-6-(O-carboxymethyl)oxime to glucose-6-phosphate dehydrogenase (G6PDH) by an N-hydroxysuccinimide-enhanced, carbodiimide-mediated coupling reaction. The estradiol-G6PDH activity can be repressed up to 46% upon anti-estradiol antibody binding. The lower detection limit of the assay is 1 nM estradiol in aqueous solution, and the standard curve is linear on logit-log scale-up to 6.7 μM estradiol. A detection limit of 11.5 nM in estradiol-spiked human serum samples suggests the feasibility of applying this assay to monitor estradiol levels for the prediction and prevention of ovarian hyperstimulation syndrome.     

Enzyme channeling immunoassay: A new homogeneous enzyme immunoassay technique

A homogeneous enzyme immunoassay has been developed in which an antigen and glucose-6-phosphate dehydrogenase are coimmobilized on agarose beads. Binding of hexokinase-labeled antibody to the bead-bound antigen results in an accelerated conversion of glucose, ATP, and NAD⁺ to 6-phosphogluconolactone, ADP, and NADH. Critical parameters affecting assay response are discussed.     

Electrochemical enzyme immunoassay for detection of toxic substances.

Sensors that provide reliable, rapid measurement of toxic substances are needed to solve significant human health and safety problems. We developed a new biosensor design that combines the advantages of immunoassay with electrochemical response. We established that this enzyme-linked immunosensor measures toxic substances in biological samples. The biosensor consists of two major elements: (1) an electrical conducting layer having immobilized enzyme, polyclonal or monoclonal antibodies, and other necessary reagents, and (2) the electronic components used in the signal readout. The result is an amperometric immunoassay based on coupling the immunochemical reaction to the enzyme electrode response by using a soluble, electrochemically active mediator. The specific question addressed was: Does the system's immunochemical detection reliably respond at sufficiently low analyte concentrations? We present our results in these areas: (1) enzyme immobilization on colloidal gold; (2) colloidal gold-enzyme deposition on the electrode surface; (3) mediator-antigen conjugate synthesis; (4) antibody incorporation at the electrode surface; (5) bioelectrode characterization and optimization; and (6) immunosensor demonstration to detect antigen. Sensors that employ immunochemical detection will have broad applicability to detect/diagnose toxic substances in biological samples such as blood and urine and in environmental samples such as wastewater and drinking water.     

Evaluation of the diagnostic application of an enzyme immunoassay for Clostridium perfringens type A enterotoxin

The diagnostic application of an enzyme immunoassay for Clostridium perfringens type A enterotoxin was evaluated. Test results from 100 individuals associated with C. perfringens gastroenteritis outbreaks and 111 control individuals were included. The assay sensitivity was 93.7%, and the assay specificity was 98.7%.     

Capillary electrophoretic enzyme immunoassay with electrochemical detection for thyroxine

A capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) has been developed. In this method, antigen (Ag) competes with horseradish peroxidase (HRP)-labeled antigen (HRP-Ag) for a limited number of antibody (Ab) binding sites. The free HRP-Ag and the bound HRP-Ag-Ab complex are separated by capillary electrophoresis in a separation capillary. Then they catalyze the oxidation of their enzyme substrate 3,3',5,5'-tetramethylbenzide (TMB (reduced form)) with H(2)O(2) in a reaction capillary, which follows the separation capillary. The reaction product (TMB (oxidized form)) is amperometrically determined using a carbon fiber microdisk bundle electrode at the outlet of the reaction capillary. Due to the amplification of the enzyme, the concentration of TMB(Ox) is much higher than those of free HRP-Ag and the bound HRP-Ag-Ab complex. Therefore, the limit of detection (LOD) of CE-EIA-ED is very low. The method has been used to determine thyroxine in human serum. A concentration of LOD of 3.8 x 10(-9)mol/L, which corresponds to a mass LOD of 23.2 amol, was achieved.     

Rapid detection of salmonellas in raw meats using a fluorescent antibody-microcolony technique

A fluorescent antibody-microcolony technique was developed for the rapid detection of salmonellas in pure cultures. Examination of microcolonies made the detection of salmonellas by epifluorescence microscopy easier and more reliable than using fluorescent antibody and single cells. After a study of the most effective selective enrichment media for increasing the number of salmonellas, the technique was examined with various samples of raw meats. It was able to detect salmonellas in 24 h and appeared to be as sensitive as conventional cultural techniques. Of the 101 samples studied, complete agreement was obtained with conventional methods for 94 but six apparently false positive results and one false negative result occurred. and accepted 22 June 1989     

Rapid detection of salmonellas in raw meats using a fluorescent antibody-microcolony technique

A fluorescent antibody-microcolony technique was developed for the rapid detection of salmonellas in pure cultures. Examination of microcolonies made the detection of salmonellas by epifluorescence microscopy easier and more reliable than using fluorescent antibody and single cells. After a study of the most effective selective enrichment media for increasing the number of salmonellas, the technique was examined with various samples of raw meats. It was able to detect salmonellas in 24 h and appeared to be as sensitive as conventional cultural techniques. Of the 101 samples studied, complete agreement was obtained with conventional methods for 94 but six apparently false positive results and one false negative result occurred.     

Development of an enzyme immunoassay for cryptococcal antibody

An enzyme immunoassay (ELISA) for measurement of cryptococcal Ig G antibody in human serum is described. Clinical studies indicate that the assay is a useful addition to the currently available techniques for measuring antibodies in cryptococcosis. IgG-specific antibody (titers 4 to 1,024) was detected in the serum of 78 % of the cryptococcosis patients tested and in 61 % of the serum from healthy individuals with positive delayed skin hypersensitivity to cryptococcin. The micro-ELISA for cryptococcal antibody is of potential value in patient management, and in epidemiological studies.     

Evaluation of the diagnostic application of an enzyme immunoassay for Clostridium perfringens type A enterotoxin.

The diagnostic application of an enzyme immunoassay for Clostridium perfringens type A enterotoxin was evaluated. Test results from 100 individuals associated with C. perfringens gastroenteritis outbreaks and 111 control individuals were included. The assay sensitivity was 93.7%, and the assay specificity was 98.7%.     

Development of an enzyme immunoassay for the detection of Clostridium novyi type B alpha toxin

Clostridium novyi type B alpha toxin was purified to homogeneity and shown to have a molecular weight of 200 kD by SDS polyacrylamide gel electrophoresis. The toxin was toxoided and used to produce a pair of non-interfering monoclonal antibodies. Their specificity was confirmed by immunoblotting and bioassay. The monoclonal antibodies were used to develop an enzyme immunoassay which was more sensitive than bioassay, and permitted less than 1 ng/ml toxin to be detected in a rapid 10 min assay format. Use of the assay can eliminate the requirement for in vivo testing of novyi toxin and toxoid, provided measurements of biological activity are not required. Because of its speed and sensitivity, the assay can be used to monitor toxin production during fermentation and as an alternative to bioassay to measure antigen content during toxoiding and vaccine formulation.     

Double-antibody solid-phase enzyme immunoassay for the detection of staphylococcal enterotoxin A.

A simple double-antibody enzyme immunoassay that uses a microtechnique was developed for detecting staphylococcal enterotoxin A in food products. Sample preparation can be completed in less than 15 min. Assay sensitivity ranges from 0.4 ng (20-h test time) to 3.2 ng (1- to 3-h test time) of toxin per ml of prepared sample. Separation and detection of enterotoxin from spiked food products ranged between 72 and 98% of the amount added.     

Double-antibody solid-phase enzyme immunoassay for the detection of staphylococcal enterotoxin A.

A simple double-antibody enzyme immunoassay that uses a microtechnique was developed for detecting staphylococcal enterotoxin A in food products. Sample preparation can be completed in less than 15 min. Assay sensitivity ranges from 0.4 ng (20-h test time) to 3.2 ng (1- to 3-h test time) of toxin per ml of prepared sample. Separation and detection of enterotoxin from spiked food products ranged between 72 and 98% of the amount added.     

Reusable immunomagnetic beads in an enzyme immunoassay

Multiple antigen peptides (MAPs) were synthesized according to the epitope sequence of human cytomegalovirus phosphoprotein pp150 and used as antigens to coat the surface of Dynabeads M-450 Tosylactivated covalently. The coating efficiency peaked at 79% when the concentration of MAP8 was 100 μg/ml. Based on the immunomagnetic beads, an enzyme immunoassay was established to detect anti-MAP8 immunoglobulin G in sera of Balb/c mice, which were immunized with MAP8. We showed in this study that, with optimized working conditions, the immunomagnetic beads could be regenerated after removal of the antibody complexes from their surface with 0.2 M citric acid buffer (pH 2.5) and reused at least 16 times without significantly influencing the antibody binding efficiency. The results suggest the possibility of developing reusable immuno-diagnostic kits in the near future. F. C. Han and J. Luo contributed equally. They are co-first author.     

Sensitivity of an antigen detection enzyme immunoassay for diagnosis of Trypanosoma congolense infections in goats and cattle

The sensitivity of a monoclonal antibody-based antigen-detection enzyme immunoassay (antigen-ELISA) for the diagnosis of Trypanosoma congolense was evaluated using sera from experimentally infected goats and cattle. Ten goats (Galla x East African Masai) and 7 steers (Bos indicus) were infected with different clones of T. congolense and left to run a chronic course for 46 and 24 mo, respectively. During this period, monthly blood samples were collected and analyzed for the presence of trypanosomes and antigens in peripheral blood. Of 383 caprine blood samples, 361 (94.3%) were positive for circulating antigens whereas only 42 (10.9%) had demonstrable trypanosomes as revealed by the microhematocrit centrifugation technique. In cattle, 570 (82.5%) of 691 blood samples were antigen-ELISA positive compared to 136 (19.7%) samples with detectable trypanosomes. In an analysis of serum samples from goats in an area known to be endemic for trypanosomiasis, 106 (80.9%) of 131 were positive for T. congolense antigens whereas none of the corresponding blood samples had detectable trypanosomes. Control sera from 24 goats in a trypanosomiasis-free region were all antigen-ELISA negative. Hence, the antigen-ELISA was at least 4 times more sensitive than the microhematocrit centrifugation technique in monitoring T. congolense infections in goats and cattle.     

A magnetic particles-based chemiluminescence enzyme immunoassay for rapid detection of ovalbumin

Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles–chemiluminescence enzyme immunoassay (MPs–CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol–H₂O₂ chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs–CLEIA method had a linear range from 0.31 to 100 ng/ml with a detection limit of 0.24 ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA.