A surface-sensitive fluorescence measurement platform is utilised in the detection of morphine. The platform is based on a polystyrene parabolic lens that enables the simultaneous application of total internal reflection excitation and supercritical angle fluorescence detection in the measurements. The molecular recognition of morphine is based on two antibodies, one against morphine and the other against the immune complex formed between the anti-morphine antibody and a morphine molecule. The antibodies are applied in a sandwich-like format in a one-step test, where the molecular binding onto a liquid-solid-interface is monitored in real time. Morphine concentrations between 0.6 and 18.2 ng/mL were reliably determined in 60 s, while concentrations down to 2.7 ng/mL were detected already in 20 s. With appropriate recognition molecules the technique is applicable also to other drugs and small analytes.

(-)-Delta(9)-Tetrahydrocannabinol (THC) is the main psychoactive compound found in cannabis. In this study, an anti-THC Fab fragment, designed T3, was isolated from a display library cloned from the spleen cells of a mouse immunized with a THC bovine serum albumin conjugate, and the crystal structures of the T3 Fab in its free form and in complex with THC were determined at 1.9 angstrom and 2.0 angstrom resolution, respectively. The THC binding site of the T3 Fab is a narrow cavity: the n-pentyl group of THC protrudes deep into the interface area between the variable domains and the C-10 monoterpene moiety of the hapten is partially exposed to solvent. The metabolites of THC, with modifications in the C-10 monoterpene moiety, 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol and 11-hydroxy-Delta(9)-tetrahydrocannabinol, are bound by the T3 Fab with a higher affinity than THC. The crystal structures suggest that Ser52H and Arg53H of the T3 Fab are able to make hydrogen bonds with the metabolites, which leads to an increased binding against these metabolites. By developing a T3 Fab-Delta(9)-THC immunocomplex binding antibody from a nave antibody phage display library, the specificity of the Delta(9)-THC binding is highly increased, which allows a one-step, homogeneous, fluorescence resonance energy transfer-based sensitive immunoassay, with a detection limit of 20 ng/ml from saliva samples. (C) 2010 Elsevier Ltd. All rights reserved.

Antibody phage display technology is well established and widely used for selecting specific antibodies against desired targets. Using conventional manual methods, it is laborious to perform multiple selections with different antigens simultaneously. Furthermore, manual screening of the positive clones requires much effort. The authors describe optimized and automated procedures of these processes using a magnetic bead processor for the selection and a robotic station for the screening step. Both steps are performed in a 96-well microplate format. In addition, adopting the antibody phage display technology to automated platform polyethylene glycol precipitation of the enriched phage pool was unnecessary. For screening, an enzyme-linked immunosorbent assay protocol suitable for a robotic station was developed. This system was set up using human gamma-globulin as a model antigen to select antibodies from a VTT naive human single-chain antibody (scFv) library. In total, 161 gamma-globulin-selected clones were screened, and according to fingerprinting analysis, 9 of the 13 analyzed clones were different. The system was further tested using testosterone bovine serum albumin (BSA) and beta-estradiol-BSA as antigens with the same library. In total, 1536 clones were screened from 4 rounds of selection with both antigens, and 29 different testosterone-BSA and 23 beta-estradiol-BSA binding clones were found and verified by sequencing. This automated antibody phage display procedure increases the throughput of generating wide panels of target-binding antibody candidates and allows the selection and screening of antibodies against several different targets in parallel with high efficiency.

Recombinant anti-morphine Fab' fragments have been immobilised on gold by covalent attachment through the free thiol groups of the fragment. The antibody fragments were intercalated with a non-ionic hydrophilic polymer in order to suppress non-specific binding of interfering substances. The antibodies are oriented on the surface due to the thiol groups of the antibody and the layer shows a high response to antigen. Non-specific binding of bovine serum albumin is moreover very low because of the repellent polymer. Synthetic receptors composed of an imprinted self-assembled monolayer made from lipoates and the template, morphine, exhibit the same binding response to the antigen, morphine as the site-specific oriented antibody monolayer. A similar binding curve could be obtained as that for binding of morphine to an antibody Fab' fragment/polymer layer - indicating that synthetic receptors produced are comparable to those of antibody layers. Concentrations down to 0.1ng/ml have been measured with surface plasmon resonance.

VTT Technical Research Centre of Finland has developed two reader prototypes for immunodiagnostic tests. VTT has also developed a one-step, homogeneous noncompetitive immunoassay for small analytes using recombinant antibodies and morphine as the model analyte.VTT developed reader for lateral flow test. Lateral flow test is a strip, which has a sample area and a detection area. In the sample area there are antibodies attached to gold or fluorescence particles, which are captured into the detection area, if a sample has a desired analyte. The concentration of the measured sample is then calculated from the fluorescence detection or color change.The second developed prototype reader is based on Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET). In this reader samples are put on microwell array. There are two fluorophores in each of the wells and emission of both fluorophores is measured. The sample concentration is calculated from these emission signals. The optimization of homogenous FRET assays for morphine was included to this project. The first results obtained with the TR-FRET reader prototype show that the sensitivity of the current morphine test is clearly adequate.

We have developed a one-step, homogeneous noncompetitive immunoassay for small analytes using recombinant antibodies and morphine as the model analyte. A highly specific antibody against the immune complex (IC) formed between an anti-morphine antibody and morphine was selected from a naive scFv phage display library. The in vitro phage library selection procedure avoids the difficulties associated with the production of anti-IC antibodies by animal immunization. The anti-morphine and the anti-IC antibodies were labeled with a pair of fluorescence resonance energy transfer (FRET) fluorophores. In the FRET assay the labeled antibodies were incubated with saliva samples spiked with morphine, codeine, or heroin. Within 2 min, 5 ng/mL morphine, which is clearly under the recommended cutoff level, was detected without cross-reactivity to codeine or heroin. This assay principle is also widely applicable to other small analytes.

Microarray immunoassays of clinical specimens are very promising proteomics approaches for research and diagnostic purposes. The current status of this approach, including a wide spectrum of various fabrication and detection systems, is still in an early stage of development. However, the development of protein microarrays, which occurs mostly based on conventional DNA microarray platforms, is currently associated with a number of problems arisen from common biophysical and chemical properties of proteins. Further advances in surface chemistry, printing techniques, microarray processing devices, and detection methods will contribute to the establishment of the microarray technology as a reliable tool for proteomics in the future. Many of the basic problems have been identified, and ongoing research tries to solve them. Then the bottleneck will proceed from mere production issues toward the ability and capacity of identifying and isolating suitable receptor molecules.