OPTIMIZATION OF A CHARGE-COUPLED-DEVICE IMAGING ENZYME-LINKED IMMUNE SORBENT ASSAY AND SUPPORTS FOR THE SIMULTANEOUS DETERMINATION OF MULTIPLE 2,4-D SAMPLES

A chemiluminescent microformat enzyme linked immune sorbent assay (ELI SA) has been optimized for the simultaneous determination of multiple 2,4-dichlorophenoxyacetic acid (2,4-D) samples. The competitive immuno assay employed a 2,4-DBSA conjugate, anti-2,4-D monoclonal antibodies and alkaline phosphatase (AP) labelled anti-mouse IgG. The bound AP co njugate was determined by quantitating the chemiluminescence emission from the enzymatic decomposition of the luminogenic substrate, CSPD, b y AP using a cooled charge coupled device (CCD) camera. The detection limit for the simultaneous determination of multiple samples was 4.3x1 0(-10) M corresponding to 96 pg ml(-1) or 192 fg well with a coefficie nt of variation (CV, %) of 12.5%, The linear range of the assay was 4. 5 x 10(-7)-4.5 x 10(-10) M. The ability of gold coated silicon wafers and glass capillaries to serve as solid phase supports in the imaging ELISA was investigated. The highly reflective gold surfaces improved b oth the linear range and the sensitivity of the assay, as compared to thick-film patterned surfaces. The capillary supports, on the other ha nd, lead to a reduction in the linear range and the sensitivity of the assay, as compared to the thick-film patterned surfaces. Initial stud ies indicate that the capillaries guide the light and may provide a bu ilt-in mechanism for collecting the emitted light. Strategies for furt her development of support materials for imaging-based detectors will be discussed.