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Commercially available immunoassays generally recommend that 0. However, typical concentrations of LSD in urine are below this amount. Overall assay sensitivity is largely dependent on the affinity of the antibody 23 24 , although the conditions used in the ELISA affect the optimum assay range.
It is known that the method used for coupling hapten to the carrier protein influences the dose—response behavior. Heterologous assays in which the immunogen and immobilized antigen are different, with exception of the hapten, are known to produce the most sensitive results This is of particular importance for the detection of small haptens by ELISA, in which the antigen attached to the solid phase should use a different carrier protein, chemical linkage, and site of attachment with respect to the immunizing antigen This immunoassay, which shows improved sensitivity, uses a unique heterologous system whereby the hapten-protein conjugate used for immunization was prepared with a unique chemical attachment 21 that is distinct from that of the surface immobilized hapten-carrier conjugate.
A four-parameter logistical curve fit was calculated according to the following equation:.
ELISA | Immunalysis
Interpolated concentrations of LSD were The indirect nature of the assay, which produces increased absorbance with decreased concentration of drug in the urine, is reflected in the precision of replicate measurements. The CV values for Intraassay precision was estimated from the CV values of calibration standards that were run in the same assay.
As expected, the overall precision decreases as the concentration of analyte increases because of the indirect nature of the assay. The precision profile of the immunoassay, in which intra- and interassay CV values are plotted against the concentration of drug, are shown in Fig.
The precision profile indicates the same overall trend for between- and within-run CV values, the former of which are somewhat more exaggerated, as expected. Reagent changes such as new bovine serum albumin-LSD coating antigen, TMB stock solution, or enzyme-labeled antibody did not adversely affect precision over the range of concentrations that were tested.
Assay specificity was estimated by measuring the degree of cross-reactivity of various compounds of interest. The concentration of derivative that caused a decrease in signal that was equivalent to 0. Of the 24 compounds cited here, only 3 were found to cross-react substantially.
The first of these is the only confirmed human metabolite to date 16 , although the last compound has also been tentatively identified. As such, cross-reactivity with nor-LSD, and to a lesser extent with 2-oxohydroxy-LSD, might be considered advantageous from a drug-screening perspective. Minor cross-reactions were also observed with iso -LSD 0. A new immunoassay for LSD in urine that compares favorably with existing methods is described. This sensitivity is probably the result of the high affinity antibody to LSD that is used in the procedure.
This method offers a substantial improvement in the detection limit compared with a number of commercially available immunoassays currently available. This approach facilitates the detection of LSD in urine at concentrations that were previously only attainable by RIA but with less expense and without the need for radioisotopes. This work was supported by Grant MT to D. LSD was added to normal human urine at The degree of cross-reactivity was calculated from the concentration of substance that produced a decrease in signal that was equivalent to 0. Skip to main content.
Other Drug Monitoring and Toxicology. Sarah Kerrigan , Donald. Published May Sarah Kerrigan. Materials and Methods reagents Disposable well polystyrene plates were obtained from Corning. Results and Discussion limits of detection and quantification Typical calibration data obtained using the competitive binding assay are depicted in Fig. Figure 1. Calibration of LSD in urine. View this table: View inline View popup. Table 1. Table 2. Figure 2. Precision profile for the determination of LSD in urine.
Table 3. Determination of assay specificity.
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