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

Abstract

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 μg L⁻¹) and coffee (2.5 μg 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.