Ed Walsh
Industrial Microbiology
Article Review #2

Indirect enzyme-linked immunosorbent assay for the quantitative estimation of lysergic acid diethylamide in urine
Clinical Chemistry 44:5, pgs. 985-990, (1998)

The use of lysergic acid diethylamide (LSD) is difficult to detect in body fluids due to the lose dose ingested and its rapid biotransformation. The past techniques that have been used to detect LSD in urine and biological matrices were thin layer chromatography, RIA, high performance liquid chromatography-fluorescence and gas chromatography-mass spectrometry. Problems with the stated techniques are that they required large sample volumes, are technical demanding and poorly suited for high sample throughput. With these problems, immunoassays are advantageous in terms of their potential sensitivity, small sample requirement, and large sample capacity. However, the positive identification of LSD on the basis of an immunoassay is not considered conclusive for legal purposes because of low assay specificity. It is common for antibodies raised against the parent drug to undergo cross-reactions with structurally similar molecules, including metabolites, some of which are as yet unidentified. The samples that screen positive must be confirmed using a more rigorous technique that is both sensitive and specific. The focus of this work was to develop ELISA (enzyme linked immunosorbent assay) for LSD in urine that was as sensitive as the emerging confirmation techniques. The objective was to develop and optimize conditions for the detection of LSD in urine and characterize immunoassay performance in terms of accuracy, precision, sensitivity, and specificity. It is to be noted that the quantitative estimates of LSD in biological fluids are typically higher with immunoassays compared with confirmatory techniques, which is from the cross reaction of the antibody with drug metabolites present in the urine. To help evaluate the results, a negative control free of metabolites or any related LSD-like substances that could be present in the urine of someone who had ingested the drug. ELISA is very important to the biotech/microbiology fields because of the ability to not only produce yes or no results but also how much is or isn’t in a sample. ELISA uses the principle of antigen-antibody interactions; the assay allows detection of proteins, those making up cellular components of bacteria and viruses, in solutions such as serum, urine, or culture media. ELISA detects pathogen and has been adapted to detect food born allergies. The immunoassay procedure used ELISA plates coated overnight at 4 degrees Celsius with 100 ul of a LSD bovine serum albumin coating antigen. Uncoated sites on the microtiters were pre-blocked with 175 ul of 50 g/L SM-PBS for 30 mins at 37 degrees Celsius. The plates were then washed five times with PBS between each of the incubation steps. The LSD was diluted in undiluted urine to give varied concentrations between 20 and .002 ug/L and the immune rabbit serum was diluted in 100 g/L SM-PBS. Both urine and antibody solutions were added to the microtiter wells and the total volume in the wells was 100 ul. A pre-immune rabbit serum was used to estimate the degree of nonspecific binding in the assay. Gentle agitation was performed and then plate was incubated for two and a half hours at 37 degrees Celsius. Goat anti-rabbit IgG horseradish peroxidase diluted 1:1000 in 50 g/L SM-PBS was added, and the plates were incubated for a half hour at 37 degrees Celsius. When the final plate wash was complete, the color reaction began with 100 ul of TMB substrate solution followed by 50 ul of 1 mol/L sulfuric acid to stop the reaction 5 minutes later. The absorbance was measured at 450-620 nm using a SLT EAR 400AT plate reader. The results were displayed as percentage of the antibody that remained bound, percentage bound relative to the zero calibrator which was drug free urine. Calibration