A novel broad-specific noncompetitive immunoassay and its application in the determination of total aflatoxins.

Broad-specific immunoassays are an emerging technology that allows simultaneous determination of a class of closely related small molecular weight analytes. The current approaches are mainly based on the competitive format and involve complicated or expensive processes hindering their wide application. Herein we describe a simple approach for performing broad-specific noncompetitive immunoassays for the determination of total aflatoxins (AFB(1)+AFB(2)+AFG(1)+AFG(2)) using a highly specific polyclonal antibody against AFB(1). The method is based on blocking the free sites of the capture antibodies by an AFB(1)-protein conjugate followed by the replacement of antibody-bound AFs by an enzyme-labeled AFB(1). The rates of displacement of weakly bound AFB(1) congeners by the AFB(1)-HRP conjugate are faster than that of AFB(1). Consequently, the measured signals from cross-reactants are higher and almost linearly correlated to the AFB(1) concentration. Different proteins such as casein, ovalbumin and BSA were utilized for the preparation of AFB(1) conjugates and their binding efficiencies were investigated. The limit of detection of AFs by the present assay was 5 pg/well (0.1 microgL(-1)). Spiked and contaminated corn samples were analyzed without sample cleanup. The matrix interferences were eliminated by diluting the sample 10-fold with the assay buffer. The values obtained for the infected samples correlated well (R(2)=0.99) with estimates by the ELISA kit. This approach can be applied to any analyte for the development of noncompetitive immunoassay in broad-specific or specific format provided the corresponding anti-hapten antibody has negligible cross-reactivity with structurally related compounds.

Filtration-based staining of proteins on membranes.

A filtration-based staining method has been developed for sensitive estimation of proteins in a batch of samples. It is based on focused absorption of an applied protein standard (or sample) and dye solution on nitrocellulose membrane through an aqueous network of capillary channels formed between the membrane and a wetted filter paper. The method does not require any equipment for creating vacuum. Compared with the conventional pouring methods, the new approach reduces the consumption of staining solution and lowers the staining and destaining times (from 1.5 h to approximately 10 min) without compromising the sensitivity.

Simultaneous enzyme immunoassay for the screening of aflatoxin B1 and ochratoxin A in chili samples.

Membrane-based immunoassay has been developed for simultaneous estimation of aflatoxin B(1) (AFB(1)) and ochratoxin A (OA) in chili samples. The combined estimation of both the mycotoxins is more economical in respect of time, work and materials than two separate assays. The method uses a low cost test device consisting of a membrane with immobilized anti-AFB(1) and anti-OA antibodies and a filter paper attached to a polyethylene card below the membrane. It allows direct analysis of sample extracts containing substantial amount (40%) of methanol. This permits the use of two-fold diluted sample extracts resulting in minimum dilution error. The limit of quantitation obtained was 2 and 10 microg kg(-1) for AFB(1) and OA, respectively. The tolerance of 40% methanol was found to be due to the application of small size (0.8 mm diameter) spots on membranes, as the tolerance decreases to 20% with gradual increase in spot size. The combined method is capable of producing acceptable results to analyze AFB(1) and OA in chili with accuracy and precision. The AFB(1) and OA values obtained for spiked and naturally contaminated chili samples by the simultaneous method were in good correlation with those measured by individual ELISA. The method offers a simple, rapid and cost-effective screening tool to meet the requirements of the rapidly evolving EU legislation.

Filtration-based tyramide amplification technique--a new simple approach for rapid detection of aflatoxin B1.

The catalyzed reporter deposition (CARD) method of signal amplification, also called "tyramide signal amplification", has been used in immunoassays not only to increase sensitivity but also to reduce assay time. The current approach to tyramide amplification in immunoassays involves slow incubation with agitation. In this paper we describe new filtration-based tyramide amplification and substrate visualization techniques. Compared with the standard method, this new approach greatly enhances spot intensities in membrane immunoassay and reduces biotinylated tyramide (B-T) and substrate consumption approximately fiftyfold, without loss of specificity. An improved test device and a cost-effective method for preparation of membranes for Super-CARD amplification have also been developed. The techniques have been used for rapid detection of aflatoxin B(1) (AFB(1)) in a variety of foodstuffs with a detection limit of 12.5 microg kg(-1). The assay procedure involves sequential addition of standards or sample, AFB(1)-horseradish peroxidase (HRP) conjugate, B-T, avidin-HRP, and substrate solution over anti-AFB(1) antibody-spotted zones of the membrane surface. The method saves time, improves reproducibility, eliminates many washing steps and avoids manipulation of the membranes between the different steps, while maintaining the sensitivity of the standard method. Average recoveries from different non-infected food samples spiked with AFB(1) at concentrations from 25 to 100 mg kg(-1) were between 95 and 105%. AFB(1) results obtained on different days for Aspergillus parasiticus infection of corn and groundnut samples correlated well with estimates obtained by HPLC.

Method for homogeneous spotting of antibodies on membranes: application to the sensitive detection of ochratoxin A.

Membrane-based dot immunoassays are now widely used in almost every branch of biology and medicine. However, the quality of the immobilized antigen or antibody spots on the membranes was found to be highly operator-dependent and spotting by conventional methods often leads to heterogeneous spot morphologies and deposition inconsistencies. To circumvent these problems, a spotting method has been developed which is based on focussed absorption of an applied antibody solution through an aqueous network of capillary channels formed between the membrane and a wetted absorbent body. The method does not require any equipment for creating vacuum and according to assay requirements highly homogeneous spots of uniform size, in the range of 0.8- to 9-mm diameter, can be obtained by varying the volume of the applied antibody solution. Spot intensities were sufficiently high even at high antibody dilutions. Immobilization of anti-ochratoxin A (anti-OA) antibody by this method gave 2-fold increased sensitivity in a competitive assay of the toxin compared to conventional spotting methods. The calculated CV of the colour intensity for spots of different sizes (0.8 to 9 mm) was between 4.5 and 1%. Application of this spotting technique has been demonstrated for detection of OA in wine and coffee samples with the elimination of matrix interferences in the same immunoassay system. This was achieved by selective removal of nonspecific interfering substances from the sample extract during the assay. The detection limit of OA in wine (1 microg L(-1)) and coffee (2.5 microg kg(-1)) obtained by the present new method is superior to values reported recently. Thus, the present new method will be highly useful for improved performance of membrane-based immunoassays in almost every branch of biology and medicine.

In situ sample cleanup during immunoassay: a simple method for rapid detection of aflatoxin B, in food samples.

A strategy for rapid in situ elimination of interfering substances that are present in extracts of food samples during assay is described in this article. The novel feature of this method is that the sample purification is carried out as a part of the assay, and a separate sample cleanup step is not required. The assay procedure involves the sequential addition of standard or sample, cleaning solutions, and aflatoxin B1-horseradish peroxidase conjugate (AFB1-HRP) over antibody-spotted zones of a membrane, and 3,3'-diaminobenzidine was used as the substrate for visualization. We have determined that trifluoroacetic acid and propionic acids at concentrations of 100 mM are highly effective for cleaning groundnut, wheat, corn, and poultry feed samples and that NaHCO3 (100 mM) is successful in cleaning processed soybean. In all cases, subsequent washing was performed with phosphate-buffered saline solution to facilitate the removal of traces of adhering interfering substances. A batch of 12 samples can be analyzed within 8 min either by visual comparison of the color intensity (inversely related to the analyte concentration) of a sample spot with those of reference standards or, more precisely, by densitometry. The method was tested for the analysis of AFB1 in groundnut, wheat, corn, processed soybean, chili, and poultry feed. The detection limit obtained was 5 microg/kg, except for chili, where it was 10 microg/kg. The average recoveries from different noninfected food samples spiked with AFB1 at concentrations of 5 to 100 microg/kg were between 99 and 105%. The values obtained for infected corn and groundnut samples correlated well with the estimates obtained by high-pressure liquid chromatography. The absence of a sample extraction step reduces the cost and labor involved in the assay. The method may be potentially applicable to the assay of other mycotoxins and environmental pollutants.

Development of a membrane-based immunofiltration assay for the detection of T-2 toxin.

An improved analytical device capable of performing simultaneous immunofiltration-based immunoassay on 30 samples in the presence of reference standards has been developed. The device consists of a rectangular membrane with 36 antibody spotted zones, one end of which was attached to a semirigid polyethylene card. A piece of wetted filter paper between the membrane and the polyethylene card absorbs the added reagent. The assay is a competitive one using T-2 toxin-horseradish peroxidase (T-2 toxin-HRP) as the labeled analyte and 4-chloro-1 naphthol (4CN) as the substrate. Signal amplification was done by the Super-CARD signal amplification method. Semiquantitative results were obtained by visual comparison of the color intensity of a sample spot with those of reference standards. Densitometric analysis was used for quantitation. The method allows rapid and easy determination of T-2 toxin in wheat and poultry feed with detection limits of 12.5 and 25 microg x kg(-)(1), respectively, with accuracy and precision. Matrix interference was eliminated by appropriate dilution of sample extracts with assay buffer. The detection sensitivity in ELISA was 10-fold higher than that in the membrane-based method. Noninfected samples were spiked with T-2 toxin at several concentrations and analyzed by the present method and rapid ELISA. Mean recoveries by both methods were between 80 and 108%. The correlation between the two methods was excellent (R(2) = 0.99).