MICROFORMAT IMAGING ELISA FOR PESTICIDE DETERMINATION

A flat-well microformat competitive enzyme-linked immunosorbent chemil uminescent assay for the detection of the pesticide 2,4-dichlorophenox yacetic acid (2,4-D) is described. Thick-film technology was used to p attern a hydrophobic layer 100 mu m thick onto glass microscope slides to form an array of 2 x 2 mm(2) squares. These flat wells were able t o hold 2 mu L of reagents, corresponding to a height of similar to 500 mu m with minimal contamination risk. The hydrophobic ink used to pat tern the surfaces allowed significantly larger volumes of samples to b e applied when compared with surfaces patterned with nonhydrophobic in ks. This reduced, evaporation effects and permitted greater pipetting accuracy, thereby improving assay reproducibility, A competitive immun oassay was developed based on the ability of free 2,4-D hapten to inhi bit binding of anti-2,4-D monoclonal antibodies to 2,4-D-bovine serum albumin conjugate adsorbed onto the glass support. The support was sub sequently incubated with alkaline phosphatase (AP) labeled anti-mouse IgG. The amount of AP to conjugate bound was determined by quantitatin g 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 x 10(-11) M, or 6 pg o f 2,4-D. The linear ranges of the single-sample and multisample assays were 4.5 x 10(-8)-4.5 x 10(-11) and 4.5 x 10(-7)-1.66 x 10(-10) M, re spectively. In comparison, the detection limits of a tube-based chemil uminescent assay using standard luminometer and of a colorimetric ELIS A were 45 x 10(-11) and 9.9 x 10(-8) M, respectively. The ability to s cale the thick-film-based microformat assay makes it an ideal candidat e for the development of affinity arrays and high-throughput assay for mats. Prospects for further improvements of this imaging ELISA strateg y will be discussed.