A sensitive solid-phase fluoroimmunoassay for detection of opiates in urine.

An automated flow fluorometer designed for kinetic binding analysis was adapted to develop a solid-phase competitive fluoroimmunoassay for urinalysis of opiates. The solid phase consisted of polymer beads coated with commercial monoclonal antibodies (MAbs) raised against morphine. Fluorescein-conjugated morphine (FL-MOR) was used as the fluorescein-labeled hapten. The dissociation equilibrium constant (K(D)) for the binding of FL-MOR to the anti-MOR MAb was 0.23 nM. The binding of FL-MOR to the anti-MOR MAb reached steady state within minutes and was displaced effectively by morphine and other opiates. Morphine-3-glucuronide (M3G), the major urinary metabolite of heroin and morphine, competed effectively with FL-MOR in a concentration-dependent manner for binding to the antimorphine MAb and was therefore used to construct the calibration curve. The sensitivity of the assay was 0.2 ng/mL for M3G. The assay was effective at concentrations of M3G from 0.2 to 50 ng/mL, with an IC50 of 2 ng/mL. Other opiates and heroin metabolites that showed >50% crossreactivity when present at 1 microg/mL included codeine, morphine-6-glucuronide, and oxycodone. Methadone showed very low crossreactivity (<5%), which is a benefit for testing in patients being treated for opiate addictions. The high sensitivity of the assay and the relatively high cutoff value for positive opiate tests allows very small sample volumes (e.g., in saliva or sweat) to be analyzed. A double-blind comparison using 205 clinical urine samples showed good agreement between this single-step competitive assay and a commercially performed enzyme multiplied immunoassay technique for the detection of opiates and benzoylecgonine (a metabolite of cocaine).

Reusable, real-time, immuno-optical protein C biosensor.

A Protein C (PC) biosensor can be used to diagnose PC deficiency, to monitor the PC level in the blood of PC deficient patients, and to measure the PC concentration in other PC-containing samples, such as PC producing animal cell culture broth or transgenic animal milk. A fully functional biosensor requires extremely high sensitivity and specificity, and real-time measurement. To satisfy these requirements, it is proposed to develop an immuno-optical fiber biosensor that utilizes PC-specific biomolecules (PC probes) tagged with fluorophores. The method involves immobilizing monoclonal antibody against PC (anti-PC) on the surface of an optical fiber. When PC in a sample is adsorbed to the anti-PC on the fiber, it can be reached with the fluorophore tagged PC-probe. The intensity of light transported through the optical fiber, therefore, can be correlated with the concentration of PC in the sample. The sensor will be designed so it can be reused, following a simple elution step, thus reducing diagnostic expense. The preliminary study shows encouraging future for the real-time optical PC biosensor.

A fiber-optic cocaine biosensor.

A fiber-optic biosensor was developed for detection of cocaine, its metabolites, and other coca alkaloids, using a monoclonal antibody (mAb) against a derivatized benzoylecgonine (BE). The mAb was immobilized noncovalently on quartz fibers and a flow fluorometer was used to detect changes in evanescent wave fluorescence. A fluorescein (FL) conjugate of BE bound to the mAb specifically in a saturable manner and with high affinity (Kd = 7.6 nM). Cocaine or other test compounds competed with FL-BE for binding to the mAb in a concentration-dependent manner, thereby reducing the initial rate or steady-state fluorescence. Addition of cocaine to the flow buffer after reaching steady-state fluorescence enhanced the dissociation of bound FL-BE, and cocaine removal allowed fiber regeneration for multiple measurements. The detection limits for cocaine, cocaethylene, norcocaine, and BE were 5, 5, 29, and 30 ng/ml, respectively, but for ecgonine it was 4600 ng/ml and for methylecgonine it was 2000 ng/ml. Tropacocaine was detected at 10 ng/ml, but atropine was detected at 2900 ng/ml. The biosensor discriminated by 833-fold between cocaine and its stereoisomer pseudococaine. Structural features necessary for high-affinity recognition by this mAb are benzoate and 3 beta configuration, both of which are found in BE, cocaine, norcocaine, and cocaethylene.

Choline derivatives and sodium fluoride protect acetylcholinesterase against irreversible inhibition and aging by DFP and paraoxon.

A light addressable potentiometric sensor was used to measure acetylcholinesterase (AChE) activity in order to evaluate the protective effects of quaternary compounds and NaF against enzyme phosphorylation and aging by two organophosphates. The use of the immobilized AChE made possible the quick removal of reagents (i.e., organophosphate, 2-pralidoxime, and protectant), thereby permitting accurate determination of AChE activity before and after phosphorylation and aging. Paraoxon was 15-fold more potent in inhibiting AChE than DFP, while the percent aging following phosphorylation by diisopropylfluorophosphate (DFP) was much higher. Sodium fluoride (NaF), the most effective protectant against phosphorylation and aging, and the quaternary ammonium compounds reduced significantly AChE inhibition by DFP and paraoxon, to similar degrees. Even though the percent AChE activity that was lost to aging was reduced by these agents, aging as a percent of phosphorylated AChE was not reduced. Thus, their major effect was in reducing the percent AChE phosphorylation, which consequently resulted in reduction of total aged AChE. The finding that quaternary ammonium compounds protect against phosphorylation is consonant with the proposed presence of the active site of AChE in an aromatic gorge.

Glutamate receptor inhibitors as potential insecticides.

Philanthotoxin (PhTX) is a neurotoxic constituent of the paralytic venom of the digger wasp, Philanthus triangulum. PhTX inhibits glutamate receptors of insect muscles mostly as a channel blocker, thereby producing muscle paralysis. Since glutamate receptor blockers may be of value as selective insect control agents, numerous derivatives of PhTX were synthesized and tested for their potencies as inhibitors of insect skeletal muscle glutamate receptors. Structure-activity relationship studies revealed that shortening the polyamine chain length reduced potency, and quaternarization of the nitrogen destroyed it. The potency was increased by a bulky anchoring group with moderate hydrophobicity at the end of the polyamine chain. The conversion of the tryosyl moiety to 3,5-diiodo-tyrosyl also increased potency and so did lengthening the butyryl chain from 4 to 10 carbons. Not only did PhTXs inhibit different subtypes of glutamate receptors, including the mammalian N-methyl-D-aspartate receptor, but also nicotinic receptors of insects and vertebrates. Because of this low selectively, and the hydrophilicity of the derivatives tested, which interferes with their penetration to the target receptor, these compounds cannot be used as insecticides. Nevertheless, the insect skeletal muscle glutamate receptor is a viable target for selective insecticides and major changes in PhTX structure may possibly produce derivatives that can be potential insecticides.

Synthesis and binding of [125I2]philanthotoxin-343, [125I2]philanthotoxin-343-lysine, and [125I2]philanthotoxin-343-arginine to rat brain membranes.

125I2-iodinated philanthotoxin-343 (PhTX-343), [125I2]PhTX-343-arginine, and [125I2]PhTX-343-lysine were synthesized and evaluated as probes for glutamate receptors in rat brain synaptic membranes. It was found that these probes were not specific for the glutamate receptors but may be useful for investigating the polyamine binding site. Filtration assays with Whatman GF/B fiber glass filters were unsuitable because the iodinated PhTX-343 analogues exhibited high nonspecific binding to the filters, thus hindering detection of specific binding to membranes. When binding was measured by a centrifugal assay, [125I2]PhTX-343-lysine bound with low affinity (KD = 11.4 +/- 2 microM) to a large number of sites (37.2 +/- 9.1 nmol/mg of protein). The binding of [125I2]PhTX-343-lysine was sensitive only to the polyamines spermine and spermidine, which displaced [125I2]PhTX-343-lysine with Ki values of (3.77 +/- 1.4) x 10(-5) M and (7.51 +/- 0.77) x 10(-5) M, respectively. The binding was insensitive to glutamate receptor agonists and antagonists. Binding results with [125I2]PhTX-343-arginine were similar to those of [125I2]-PhTX-343-lysine. Considering the high number of toxin binding sites (10000-fold more than glutamate) in these membranes and the insensitivity of the binding to almost all drugs that bind to glutamate receptors, it is evident that most of the binding observed is not to glutamate receptors. On the other hand, PhTX analogues with photoaffinity labels may be useful in the isolation/purification of various glutamate and nicotinic acetylcholine receptors; they could also be useful in structural studies of receptors and their binding sites.

Philanthotoxin blocks quisqualate-, AMPA- and kainate-, but not NMDA-, induced excitation of rat brainstem neurones in vivo.

1. The effect of electrophoretic ejection of philanthotoxin (the polyamine toxin, from the Egyptian digger wasp) was tested on responses of brainstem and spinal neurones in the pentobarbitone-anaesthetized rat to excitatory amino acids. 2. Philanthotoxin caused a dose-dependent reduction of responses to quisqualate, alpha-amino-3-hydroxy-5-phenyl-4-isoxazolepropionate (AMPA) and kainate with little effect on those to N-methyl-D-aspartate (NMDA). 3. The time-course of this antagonist action was slow. In particular the rate of recovery was dependent on frequency of ejection of the agonist. This agonist-dependent recovery suggests that philanthotoxin has a channel blocking mode of action on mammalian central neurones.

Inhibition of rat brain glutamate receptors by philanthotoxin.

The actions of philanthotoxin (PhTX) were studied on the function of glutamate receptors expressed in Xenopus oocytes injected with rat brain mRNA and on binding of radioligands to rat brain glutamate receptors. PhTX reversibly inhibited the oocyte responses to quisqualate, N-methyl-D-aspartate (NMDA) and kainate in a dose-dependent manner. The NMDA receptor was the most sensitive to PhTX action (10-fold more than the kainate receptor) and the least sensitive was the smooth current component of the quisqualate response. Recovery from PhTX block differed among the three amino acids. NMDA responses recovered completely within a few minutes whereas responses to kainate and quisqualate recovered more slowly. PhTX had no effect on equilibrium binding of [3H]glutamate to rat brain cortical membranes studied in buffer treated to eliminate microorganisms. Based on the drug specificity of this [3H]glutamate binding, it is suggested to be mostly to the NMDA receptor. Low concentrations of PhTX (1-10 microM) potentiated binding of [3H] MK-801, a specific noncompetitive inhibitor of the NMDA receptor. However, higher PhTX concentrations inhibited this binding with an IC50 of 20 microM, similar to its inhibition of the oocyte-expressed NMDA receptor. Inhibition of [3H]MK-801 binding by PhTX was noncompetitive. It is suggested that PhTX, like the more potent MK-801, binds to an allosteric site on the NMDA receptor and inhibits its function but its binding site is not identical with the MK-801 binding site.

Acetylcholine receptors at mature and aged mouse neuromuscular junctions.

The density and distribution of junctional and perijunctional ACh receptors (AChR) were studied in young (8-12 months) and old (24-25 months) C57 mice to determine: (1) if increased amplitude of spontaneous postsynaptic potentials previously reported in old C57 muscle was due to increased junctional AChR; (2) if increased extrajunctional AChR would be found in association with previously reported nerve terminal complexity; and (3) if extrajunctional AChR was present as in disused or denervated muscle. Microdissection of individual muscle fibers combined with I125-alpha-bungarotoxin labeling, gamma counting, measurement of surface area, cholinesterase stains, and autoradiography were used to obtain the results. In both young and old mice there was a sharp gradient in AChR between the end-plate and the perijunctional region. End-plate AChR densities and total AChR per end-plate were the same at old and young end-plates, as were perijunctional values. Thus, neither end-plate nor extrajunctional AChR density changes with age. An increased mepp amplitude reported previously in old CB57 animals must be due to other factors. The perijunctional AChR in old mice show no changes characteristic of disuse or denervation, or those which might give rise to the observed nerve terminal complexity.

General and strain-specific age changes at mouse limb neuromuscular junctions.

Age changes at limb neuromuscular junctions (NMJs) of CB57 and BALB/C mice were investigated and compared with previous results in a hybrid strain, in order to identify common characteristics of neuromuscular transmission and to assess the prevalence of signs of denervation and disuse. Resting membrane potential decreased with age in soleus muscles with no change in passive membrane properties. Miniature end-plate potential, amplitude and quantal content were greatly increased, with no change in muscle fiber diameter or nerve terminal morphometry. The various observed patterns of age changes were not those of disuse or denervation. Although there were diverse age changes in spontaneous transmitter release in different mouse strains, increased transmitter release per unit length of nerve terminal in limb muscle was a notably consistent finding across strains. It is apparently less subject to epigenetic variation during aging than most other reported neuromuscular physiological parameters.

Effects of ketamine and three other anaesthetics on spinal reflexes and inhibitions in the cat.

The effects of ketamine, alphaxalone/alphadolone, methohexitone and di-isopropylphenol have been compared on synaptic excitations and inhibitions in the spinal cord of decerebrate or pentobarbitone-anaesthetized cats. Ketamine selectively and reversibly decreased polysynaptic reflexes over a wide dose range. With the other three anaesthetic drugs decreases in reflex activity were accompanied by increases in the prolonged inhibition of reflexes, and in the amplitude and time course of dorsal root potentials. It was concluded that ketamine decreases synaptic transmission at terminals of excitatory interneurones, whereas the other three anaesthetics enhance synaptic inhibitions mediated by gamma-aminobutyric acid. Such specific effects of anaesthetics on particular synaptic processes do not support a unitary hypotheses of anaesthesia.

The effect of the dioxolanes on amino acid induced excitation in the mammalian spinal cord.

The effects of the dissociative anaesthetic, etoxadrol, and the stereoisomers of dioxadrol, dexoxadrol and levoxadrol, were examined on the excitation of spinal neurones by electrophoretically administered amino acids in pentobarbitone- or urethane-anaesthetized rats, or pentobarbitone-anaesthetized cats. Both etoxadrol and the (+)isomer of dioxadrol, dexoxadrol, administered locally or systemically, exhibited a selective antagonism of N-methyl-D,L-aspartate relative to quisqualate and kainate. This selective antagonism was not observed with the (-)isomer of dioxadrol, levoxadrol. Since such a stereoselective antagonism of the excitation of spinal neurones by N-methyl-D,L-aspartate is also displayed by the dissociative anaesthetics phencyclidine and ketamine, it is suggested that a reduced efficiency at excitatory synapses utilising N-methyl-D,L-aspartate receptors contributes to that part of the pharmacological spectrum common to both arylcyclohexylamines and dioxolanes.

Stereoselective effects of two phencyclidine derivatives on N-methylaspartate excitation of spinal neurones in the cat and rat.

In pentobarbitone-anaesthetised cats and rats effects of stereoisomers of two methylated congeners of phencyclidine, GK4 and GK5, and (+)- and (-)-PCMP, were examined on the excitation of spinal neurones by electrophoretically administered excitatory amino acids and acetylcholine. GK5 and (+)-PCMP were more potent and selective antagonists of N-methylaspartate (NMA) than were GK4 and (-)-PCMP. None of these phencyclidine derivatives showed stereoselectivity in their effects on excitation of neurones by kainate, quisqualate and acetylcholine. The differences in potency between each pair of isomers as NMA antagonists correlates well with the differences reported in results from phencyclidine binding assays and behavioural tests.

The dissociative anaesthetics, ketamine and phencyclidine, selectively reduce excitation of central mammalian neurones by N-methyl-aspartate.

The interaction of two dissociative anaesthetics, ketamine and phencyclidine, with the responses of spinal neurones to the electrophoretic administration of amino acids and acetylcholine was studied in decerebrate or pentobarbitone-anaesthetized cats and rats. Both ketamine and phencyclidine selectively blocked excitation by N-methyl-aspartate (NMA) with little effect on excitation by quisqualate and kainate. Ketamine reduced responses to L-aspartate somewhat more than those of L-glutamate; the sensitivity of responses to these two putative transmitters was between that to NMA on one hand and that to quisqualate or kainate on the other. On Renshaw cells, ketamine and phencyclidine reduced responses to acetylcholine less than those to NMA but more than those to quisqualate or kainate. Dorsal root-evoked synaptic excitation of Renshaw cells was reduced to a greater extent than that following ventral root excitation. Intravenous ketamine, 2.5-20 mg/kg, and phencyclidine, 0.2-0.5 mg/kg, also selectively blocked excitation of neurones by NMA. Ketamine showed no consistent or selective effect on inhibition of spinal neurones by electrophoretically administered glycine or gamma-aminobutyricacid (GABA). The results suggest that reduction of synaptic excitation mediated via NMA receptors contributes to the anaesthetic/analgesic properties of these two dissociative anaesthetics.

Effects of optical isomers of ketamine on excitation of cat and rat spinal neurones by amino acids and acetylcholine.

The (+) isomer of ketamine was approximately 3 and 1.5 times as potent as the (-) isomer in reducing excitation of rat and cat Renshaw cells by N-methylaspartate (NMA) and acetylcholine (ACh) respectively. The potency ratio of the two isomers of ketamine as NMA antagonists was similar to that obtained in anaesthetic and analgesic tests [16, 19, 25].

Influence of agonists on desensitization of glutamate receptors on locust muscle.

1. The desensitization, by ionophoretically and bath-applied L-glutamate and agonists, of excitatory post-junctional receptor populations on locust extensor tibiae muscle was investigated. 2. The kinetics of onset of and recovery from desensitization were determined ionophoretically either with trains of glutamate/agonist pulses of different frequencies ('conditioning trains') followed by a single 'test' pulse at different intervals after cessation of the conditioning train or with trains of constant frequency drug pulses superimposed upon steady 'conditioning' doses. Both methods gave quantitatively similar results. 3. For approximately equipotent doses, L-glutamate and agonists produced different rates of desensitization onset. Qualitatively similar results were obtained when changes in input conductance of single muscle fibres were measured during bath application of the amino acids. 4. The time courses of recovery from desensitization for receptor populations activated by glutamate and agonists could be described adequately by single exponentials and were independent of the concentration of amino acid except when desensitization exceeded ca. 90%. 5. The rate of recovery from desensitization was different for each agonist.