Sensitive chemiluminescent immunoassay of triclopyr by digital image analysis

An image based detection of chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) for the quantification of triclopyr has been developed. The immunoassay was an indirect competitive immunoassay with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP). Chemiluminescence was produced by the luminol/H2O2/HRP reaction, detected by a monochrome video CCD camera and digitized with an Imagraph IC-PCI frame grabber using a custom program developed in C++ (Microsoft Visual C++ 6.0). Two main improvements are reported in the data processing software: the implementation of a circular mesh covering the perimeter of each well, eliminating diffuse light from the neighboring wells, and the use of volume (the integration of light intensity of all pixels that define a well) as an analytical signal instead of CL intensity or area (as usual in commercial plate readers) to improve precision for normalization of the total light output. The standard curve was produced for 0.01–10 ng/L triclopyr. The limit of detection was 0.8 ng/L and the variation coefficient was 3.07% (n=10, P=0.05).