Microformat imaging ELISA for pesticide determination

A flat-well microformat competitive enzyme-linked immunosorbent chemiluminescent assay for the detection of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) is described. Thick-film technology was used to pattern a hydrophobic layer 100 μm thick onto glass microscope slides to form an array of 2 × 2 mm(2) squares. These flat wells were able to hold 2 μL of reagents, corresponding to a height of ∼500 μm, with minimal contamination risk. The hydrophobic ink used to pattern the surfaces allowed significantly larger volumes of samples to be applied when compared with surfaces patterned with nonhydrophobic inks. This reduced evaporation effects and permitted greater pipetting accuracy, thereby improving assay reproducibility. A competitive immunoassay was developed based on the ability of free 2,4-D hapten to inhibit binding of anti-2,4-D monoclonal antibodies to 2,4-D-bovine serum albumin conjugate adsorbed onto the glass support. The support was subsequently incubated with alkaline phosphatase (AP) labeled anti-mouse IgG. The amount of AP conjugate bound was determined by quantitating the chemiluminescent emission produced from the enzymatic breakdown of CSPD substrate by AP using a cooled CCD camera. The detection limit of the single-sample microformat assay was 2.7 × 10(-)(11) M, or 6 pg of 2,4-D. The linear ranges of the single-sample and multisample assays were 4.5 × 10(-)(8)-4.5 × 10(-)(11) and 4.5 × 10(-)(7)-1.66 × 10(-)(10) M, respectively. In comparison, the detection limits of a tube-based chemiluminescent assay using standard luminometer and of a colorimetric ELISA were 45 × 10(-)(11) and 9.9 × 10(-)(8) M, respectively. The ability to scale the thick-film-based microformat assay makes it an ideal candidate for the development of affinity arrays and high-throughput assay formats. Prospects for further improvements of this imaging ELISA strategy will be discussed.