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This article describes the conjugation between aflatoxin B1 (AFB1), one of the major mycotoxins, and alkaline phosphatase (AP), one of the most used enzymes for immunoassays. In addition, an application of the ELISA method for aflatoxin B1 determination in corn is presented. Three AFB1-AP conjugates in different toxin-enzyme ratios were prepared and tested. The ELISA results, developed with the most effective conjugate obtained, showed a satisfactory working range between 2.4 and 4000 ng of toxin/g of corn. The detection limit was 2 ng/g in corn samples, and recoveries ranged from 105 to 120%. [ABSTRACT FROM AUTHOR]

In this work are presented methods for detection of aflatoxin B1 and type-A trichothecenes, based on the use of indirect competitive ELISA format coupled with a 96-well screen-printed microplate. Electrochemical immunoassays for AFB1, T-2, and HT-2 were performed and the activity of the alkaline phosphatase or horseradish peroxidase labeled enzymes were measured using intermittent pulse amperometry (IPA) as electrochemical technique. Using standard solutions of the target analyte the LOD of the assays were 0.3 and 0.2 ng ml-1 for T-2 and AFB1 respectively, while the sensitivity was 1.2 ng ml-1 for both. For Aflatoxin B1, a stability study of electrochemical plate was also performed. Moreover, the matrix effect was evaluated using two different extraction treatments from corn. [ABSTRACT FROM AUTHOR]

The objective of the present work was to develop and evaluate an SYBR Green real time polymerase chain reaction (PCR) method for the specific detection of Salmonella spp in broth cultures and meat samples experimentally contaminated. Also, a simple and rapid multichannel electrochemical immunosensor (MEI) for this pathogen is under study. The PCR was carried out using primers ttr6 and ttr4 for the amplification of a highly conserved DNA region ( ttr sequence) specific for all Salmonella serovars. A boiling step, for the extraction of DNA, was combined with a real time PCR method based on the double‐stranded DNA (dsDNA) binding dye SYBR Green. The standard curve constructed using the mean threshold cycle and various concentrations of S. enteritidis (ranging fron 10 2 to 10 8 CFU mL -1 ) showed good linearity (R 2 =0.999) with the minimum level of detection of 10 2 CFU mL -1 . The experiments were conducted analyzing 30 Salmonella strains and 20 non‐ Salmonella strains. All Salmonella serotypes tested were ttr ‐positive and all other bacteria yielded no amplification products. The specificity of the reaction was confirmed by the melting temperature (T m ) of the amplicon obtained (T m =80.1±0.1). To verify the effectiveness of the assay, experiments were conducted on experimentally contaminated samples, which were also analyzed for comparison by the standard cultural method. A multichannel electrochemical immunosensor for detection of Salmonella also was developed. It consists of a disposable screen‐printed sensor array, coupled with a multichannel pulse monitor, which was assembled as an immunosensor through the use of specific monoclonal (MAb) and polyclonal (PAb) antibodies in a sandwich format. The limit of detection was calculated to be 2×10 6 UFC mL -1 with a working range between 5×10 6 to 5×10 8 UFC mL -1 and a total analysis time of about 3 h. This immunoelectrochemical system is economical, rapid, and easy to use but it is still under development in order to improve its analytical performance. [ABSTRACT FROM AUTHOR]

In the present work, an indirect competitive electrochemical enzyme linked immunosorbent assay (ELISA) has been used for determination of aflatoxin B 1 (AFB 1 ) in barley. The method involves the use of disposable screen‐printed carbon electrodes (SPCEs) and anti‐aflatoxin B 1 monoclonal antibodies (MAb) for immunosensor development. The specificity of the assay was assessed by studying the cross‐reactivity of the MAb relative to AFB 1 . The results indicated that the MAb could readily distinguish AFB 1 from other toxins, with the exception of AFG 1 . The stability of the coating reagents was evaluated using SPCEs coated with AFB 1 ‐bovine serum albumin (BSA) conjugate. The results showed that the coated electrodes could be used for up to one month after their preparation and storage at 4°C. Prior to evaluating the performance of the electrochemical immunosensor for AFB 1 with spiked samples, the effect of barley extract on assay performance was tested. Using this calibration method, the limit of detection (LOD) was found to be 90 pg mL -1 . The linear range was 0.1–10 ng mL -1 , and recoveries ranged from 100%–125%. The results obtained were confirmed by high performance liquid chromatography (HPLC) coupled with fluorescence detection. These results demonstrated the suitability of the proposed method for routine screening of AFB 1 in barley. [ABSTRACT FROM AUTHOR]

Different ELISA tests to detect and quantify levels of S. aureus in broth cultures were developed and compared. In all cases the assays were a modification of a "sandwich" format based on the use of common IgG as well as specific antibodies to bind protein A, an antigen localized in the cellular wall of S. aureus and partially extracted by boiling. Initially, human IgG was immobilized on the surface of microtitre plate wells in order to bind, by means of the Fc region, protein A that was present either in standard solutions or broth cultures of S. aureus treated by a boiling step. The sandwich format was completed using monoclonal (MAb) antibodies specific for protein A. The amount of bound antibody was evaluated using antiglobulins labelled with alkaline phosphatase (Ab2-AP). Reading the absorbance at 405 nm a detection limit (LOD) of 0.6ng/mL and 2 × 106 CFU/mL was found for protein A and for S. aureus, respectively. In order to improve the performance of the immunoassay, different approaches were pursued: an enzymatic amplification system (Ampli Q); the use of immunomagnetic beads employed both in a colorimetric (ELIMC Enzyme-Linked Immunomagnetic Colorimetric) and in an electrochemical (ELIME = Enzyme-Linked Immunomagnetic Electrochemistry) assay. Using these systems the detection limit decreased by a factor of about 30-fold for Ampli Q and ELIMC, and about 2000-fold for ELIME formats. In addition, a qualitative polymerase chain reaction (PCR) method, using nuc gene primers, was set up and performed in parallel and its various parameters were optimized. This method was able to detect 102 CFU/mL. In terms of minimum detectable concentration of S. aureus and total analysis time, the performance of the PCR assay and ELIME, turned out to be comparable. [ABSTRACT FROM AUTHOR]