Flow-through Enzyme Immunoassay Research Articles

Rapid flow-through enzyme immunoassay of progesterone in whole cows' milk

A rapid flow-through immunoassay using an enzyme (horseradish peroxidase) as a label for quantitative and semi-quantitative determination of progesterone in whole cows' milk was developed. The flow-through test device consisted of a porous nitrocellulose membrane coated with antibodies and an absorbent membrane. The substrate solution containing 3,3′,5,5′ -tetramethylbenzidine was used for colour visualization. The detection limit of 0.4 ng/mL P4 was obtained by this method; analysis time did not exceed 15 min. To eliminate matrix interference a simple sample preparation procedure was used. Results of analysis of whole cows' milk samples with flow-through method were in good correlation with ELISA results (R = 0.96, n = 34). The developed rapid flow-through test system showed high efficiency for the determination of progesterone level in whole cow's milk and can be used on-site for quick identification of milk samples with low and high progesterone concentration.

Development of rapid flow-through-based dot-immunoassay for serodiagnosis of leptospirosis in dogs

An IgG-ELISA used recombinant antigen and a rapid flow-through enzyme immunoassay were developed for rapid screening of leptospiral antibodies in dogs using recombinant LipL41, which is one of the conserved outer membrane proteins in pathogenic leptospires as the coating antigen. Results from this study were compared with the standard microscopic agglutination test and found that the sensitivity and specificity of the enzyme-linked immunosorbent assay were 75.46% and 93.29% and whereas that of flow-through-based dot-immunobinding assay were 87.73% and 89.63%, respectively. Relative merits of these tests were also assessed. The flow-through-based dot-immunobinding assay was thus proved to be a valid screening test for canine leptospirosis.

Novel developments in rapid mycotoxin detection

Rapid antibody-based mycotoxin screening techniques are designed to be used outside a laboratory environment, at the place of sampling. Results are expected immediately, so that commodities can be further processed without delay. Because they are used for mycotoxin analysis, very low levels (ppb and ppt range) should be detected. A further requirement is that the obtained results are accurate with a false negative rate of <5% at the level of interest.At first, plastic microtiter plates were used as solid phase materials for immobilizing antibodies (enzyme-linked immunosorbent assays). However, to increase speed and user-friendliness, plastics were replaced by microporous membranes. As an example a flow-through enzyme immunoassay for the detection of fumonisins in cornflakes with a cut-off value of 275 μg/kg is described. No false negative results were observed and the false positive rate was 18%. However, enzyme labels, used to enable visual evaluation of results, did not seem to be completely satisfactory in terms of stability and repeatability of the generated signal. Therefore microparticle labels such as colloidal gold particles are used more and more,e.g. in a lateral flow dipstick immunoassay. When applied to the detection of aflatoxin B1 in pig feed a cut-off value of 5 μg/kg could be reached with no false negative results and a false positive rate of only 10%. Sample pretreatment for screening techniques should be rapid and simple. Preferably a simple solvent extraction is used, followed by a filtration and dilution step. However, for strongly coloured or complex food matrices, this did not seem to work. The combination of clean-up and detection in one single test device is a new approach. When using this clean-up tandem assay column for the detection of ochratoxin A in roasted coffee, a cut-off value of 6 μg/kg was reached. No false positive results were obtained, however, the false negative rate was 8%.

Evaluation of Fumonisin Contamination in Cornflakes on the Belgian Market by “Flow-Through” Assay Screening and LC−MS/MS Analyses

A total of 205 cornflake samples collected in Belgian retail stores during 2003-2004 were surveyed for the natural occurrence of fumonisin B1 (FB1), B2 (FB2), and B3 (FB3). These cornflake samples, originating from conventional as well as from organic production, were analyzed using an intralaboratory-validated LC-MS/MS method. Additionally, 90 cornflake samples were subjected to rapid screening using a flow-through enzyme immunoassay method to demonstrate the practicability of a screening test coupled to a validated confirmatory LC-MS/MS method for the management of food safety risks. FB(1) concentrations ranged from not detected (nd) [LOD (FB1) = 20 microg/kg] to 464 microg/kg with mean and median concentrations of respectively 104 +/- 113 and 54 microg/kg. For FB2 and FB3, the concentration ranges varied respectively from nd [LOD (FB2) = 7.5 microg/kg] to 43 microg/kg and from nd [LOD (FB3) = 12.5 microg/kg] to 90 microg/kg. Mean concentrations for FB2 and FB3 were respectively 12 +/- 8 and 21 +/- 15 microg/kg, while the median concentration was 11 microg/kg for FB2 and 19 microg/kg for FB3. From the statistical tests (chi2 and ANOVA model III), it could be concluded that the agricultural practice did not have any significant effect on the fumonisin concentrations but that the variation between different batches was significant (p < 0.0001).

A flow-through enzyme immunoassay for the screening of fumonisins in maize

The format of an existing flow-through enzyme immunoassay has been optimised for the detection of fumonisin B 1 (FB 1) and B 2 (FB 2) in maize. The visual detection limit i.e. the smallest mycotoxin concentration resulting in no colour development, is 1000 μg/kg. Assay validation was performed using samples spiked with respectively only FB 1 and a FB 1 + FB 2 mixture (ratio 1:1). Results showed that within-day and between-day coefficients of variation ranged from respectively 0.4–10.2% and 1.1–9.0%. Naturally contaminated samples were screened with the developed flow-through method and results were compared with fumonisin contamination values obtained by a validated HPLC method. The assay was demonstrated to be accurate and reliable giving no false compliant and only a low percentage of false non-compliant results. The described method offers a simple, rapid and cost-effective screening tool, thus contributing to a better consumers’ health protection.

A collaborative study to validate novel field immunoassay kits for rapid mycotoxin detection

Kits designed to detect ochratoxin A (OA) and T-2 toxin by a membrane-based flow-through enzyme immunoassay were studied collaboratively by screening cereals (wheat, rye, maize and barley) for the presence of these mycotoxins. Sample preparation and test procedure were clearly described in the instruction leaflets included in the kits. A simple methanol-based extraction followed by filtration and dilution steps was prescribed. Reagents were successively pipetted to the membrane of the device, then colour development was evaluated visually. Limits of detection for the ochratoxin A and T-2 toxin tests were 4 and 50 μg kg −1, respectively. Five laboratories took part in the first stage of this study, and five more joined the second stage. Cereal samples (blank, spiked or inoculated) were shipped with the kits to the participating laboratories, while results obtained were confirmed by high-performance liquid chromatography with fluorescence detection and by gas chromatography-mass spectrometry for ochratoxin A and T-2 toxin, respectively. Some initial difficulties were encountered. In the second stage, four ochratoxin A and four T-2 toxin kits were used by 10 collaborators to analyse 21 cereal samples. For the ochratoxin A kits, the percentage of false positive and false negative results were 2% and 4%, respectively. The results of one T-2 toxin kit were outliers and when excluded, the overall percentage false positive and false negative results were 6% and 3%, respectively.

Development of a Flow-Through Enzyme Immunoassay and Application in Screening Green Coffee Samples for Ochratoxin A with Confirmation by High-Performance Liquid Chromatography

A flow-through enzyme immunoassay has been developed for the screening of green coffee bean samples for ochratoxin A (OA) and was later used in a survey on OA in green coffee from different countries. The test has a sensitivity of 8 ng/g, and calculated recoveries ranged from 70 to 89% and from 86 to 95% for spiked and naturally contaminated samples, respectively. There were no significant differences in within-day and between-day assay performance (P > 0.05). Green coffee samples (15 Arabica and 7 Robusta) received from an international coffee trader were analyzed for intrinsic fungal contamination, screened for OA, and subsequently confirmed by high-performance liquid chromatography (HPLC). All 22 samples were contaminated by fungal species of the genus Aspergillus, while Penicillium species were isolated from a mere 13.6% of the total number of samples. Isolates were tested for their ability to produce OA, and only 3.9% were positive. There was no correlation between occurrence of OA-producing isolates and levels of OA in contaminated samples. Results of the screening procedure showed that 4 of the 22 samples were contaminated with 8 ng/g or higher. The HPLC method confirmed that the OA levels ranged from 27 to 168 ng/g. A fifth sample, which was shown to be negative during screening, had an OA concentration of 4 ng/g. There were no false negatives or positives recorded, and the flow-through enzyme immunoassay results correlated with those obtained by HPLC.

Detection of T-2 toxin in different cereals by flow-through enzyme immunoassay with a simultaneous internal reference.

In most previously described membrane-based immunoassays a separate negative control assay is always carried out to evaluate the performance of the assay. To overcome this problem, a membrane-based flow-through enzyme immunoassay with an internal control has been developed for the detection of T-2 toxin in cereals (patent pending). An Immunodyne ABC membrane was coated with 2 microL of goat anti-horseradish peroxidase (HRP) (internal control spot) (1:1000) and 2 microL of rabbit anti-mouse (test spot) (undiluted) immunoglobulins, and the free binding sites were blocked. In addition to the antibody-coated Immunodyne ABC membrane, the assay also comprises a plastic snap-fit device, absorbent cotton wool, mouse anti-T-2 monoclonal antibodies (Mab), and T-2-HRP conjugate. The color intensity (Delta) of the internal control and that of the negative sample showed no difference (P > 0.05), whereas there was a significant difference between the internal control and positive samples (P < 0.05). The minimum detectable limit that could be visually detected with confidence was 50 ng of T-2 per gram of cereal sample.

Development of a portable field immunoassay for the detection of aflatoxin M 1 in milk

Many methods for AFM 1 detection exist, but most are time consuming, employ expensive equipment and require experienced personnel. To overcome these problems a membrane-based flow-through enzyme immunoassay has been developed (patent pending). The assay comprised a nylon Immunodyne ABC membrane spotted with anti-mouse antibodies, a plastic snap-fit device, absorbent cotton wool, mouse anti-AFM 1 monoclonal antibodies (Mab), and AFB 1–horseradish peroxidase (HRP) conjugate. This assay was coupled to an immunoaffinity column (IAC). The visual detection limit was 0.05 ng/g AFM 1 in milk. Assay time for IAC clean-up was 12 min, and that for the flow-through assay was 18 min, hence the total assay time was 30 min. This method allows for a rapid screening of milk consignments which do not conform to the maximum permissible limits of 0.05 ng/g AFM 1, hence enabling the rejection of such at the farm level. Laboratory validation was done using certified reference materials (CRM) with AFM 1 concentrations of <0.05, 0.09 and 0.76 ng/g. Precision of the assay was high as shown by the high repeatability of the assay results. There were no significant differences in recoveries between standard in buffer and CRM ( P>0.05), and assay responses for these two were highly correlated (99.63%).