Comparison of ELISAs for opiates, methamphetamine, cocaine metabolite, benzodiazepines, phencyclidine, and cannabinoids in whole blood and urine.

BACKGROUND: ELISAs are widely utilized in forensic drug analysis. A comparative assessment of microtiter plate assays for the detection of six common classes of drug in blood and urine is described. METHODS: ELISAs for opiates, methamphetamine, benzodiazepines, cocaine metabolite, phencyclidine (PCP), and tetrahydrocannabinol (THC) metabolite were evaluated in a side-by-side study. The analytical performance of 12 commercially available ELISAs was determined in terms of binding characteristics, dose-response curves, limits of detection, sensitivity, intra- and interassay imprecision, and lot-to-lot reproducibility. Assay performance was also compared using 855 forensic casework samples. RESULTS: Detection limits in whole blood for morphine, D-methamphetamine, nordiazepam, benzoylecgonine, nordiazepam, PCP, and L-11-nor-9-carboxy-delta9-THC were 3, 2, <4, 5, 25, and 3 microg/L, respectively, for the STC ELISAs. Corresponding detection limits for Immunalysis ELISAs were <1, <2, <4, 5, <1, and 1 microg/L, respectively. Intraassay CVs (n = 8) at the immunoassay cutoff concentrations were 4.1-5.6% and 3.5-11% for STC and Immunalysis ELISAs, respectively. Corresponding interassay CVs were 3.1-10% and 6.5-20%. Of the 855 casework samples, there were a total of 92 discordant results (44 cannabinoid, 15 opiate, 15 methamphetamine, 11 benzodiazepine, and 7 cocaine metabolite). Gas chromatography-mass spectrometry analysis indicated a total of three unconfirmed positive results for Immunalysis assays and one unconfirmed positive for STC assays. CONCLUSIONS: A comparative assessment of drugs-of-abuse assays from two manufacturers indicated some key differences in analytical performance. Overall, Immunalysis assays offered superior binding characteristics and detection limits, whereas STC assays offered improved overall precision and lot-to-lot reproducibility.

A comparison of the morbidity of midforceps and cesarean delivery.

Neonatal and maternal outcome in 358 midforceps and 486 cesarean deliveries was compared by retrospective analysis. Neonatal outcome was evaluated on the basis of Apgar score, cord blood gas values, admission to the neonatal intensive care unit, and birth trauma. Maternal outcome was based on intraoperative and postoperative complications, blood loss, and hospital stay. There was no increase in significant short-term neonatal morbidity in the midforceps group, while maternal morbidity was higher in the cesarean delivery group. It is concluded that, in selected cases, midforceps delivery is safe for the neonate and mother.

Tandem mass spectrometry (MS/MS) utilizing electron impact ionization and multiple reaction monitoring for the rapid, sensitive, and specific identification and quantitation of morphine in whole blood.

A single step liquid-liquid extraction procedure followed by pentafluoropropionic anhydride derivatization was developed for the analysis of free morphine in blood. The specificity of the method was enhanced with the use of tandem mass spectrometry (MS/MS) using multiple reaction monitoring. Two molecular transitions were monitored. The first transition was where the first quadrupole (Q1) transmits the molecular ion of morphine-3,6-dipentafluoropropionate, 577. The 577 ion undergoes subsequent collision induced dissociation with argon and the last quadrupole (Q3) then transmits the 414 daughter ion. The second transition monitored was where Q1 transmits the 414 base peak and Q3 transmits the resultant 266 daughter ion. Deuterated morphine was included as an internal standard and analyzed similarly. Morphine was confirmed only in instances where both the m/z 577 to 414 and 414 to 266 transitions occurred simultaneously. Linear and reproducible calibration curves have been obtained for morphine at concentrations from 1.0 to 500 ng/mL achieving correlation coefficients of greater than 0.994. A signal-to-noise ratio of approximately 5:1 was observed for the 1.0-ng/mL samples.

Region-specific deposition of dermal proteins between dermis and epidermis during induction of chick feather and scale rudiments.

To begin to study the role of particular proteins in inductive tissue interactions, we have used density labelling techniques to determine whether any dermal proteins are found between embryonic chick dermis and epidermis at a stage when the dermis plays an important inductive role in epidermal differentiation. Epidermis will form feathers or scales depending on whether it interacts with dorsal or foot dermis, respectively, and the dermis can still influence epidermal differentiation when direct cell contact between the tissues is blocked by a membrane filter during culturing (Peterson & Grainger, 1985). In transfilter experiments, we detect a subset of dermal proteins within the filter between the tissues. Several of these dermal proteins are deposited in a region-specific manner, that is, they are only found associated with filters from either dorsal or foot dermis. We have previously shown that the expression of some of these proteins is specific to particular regions of dermis and is also associated with the inductive potential of the dermis (Peterson & Grainger, 1986). We detect only 17 dermal proteins which are transferred across the filter in these cultures and found in direct association with epidermis; of these 14 are common to both dorsal and foot dermis, and 3 are deposited in a region-specific manner. Our results lead us to hypothesize a significant function for certain dermal proteins in this inductive interaction either as part of the extracellular matrix or in direct association with epidermis.

High resolution KSCN/CsSCN equilibrium gradients effectively separate a population of density labeled proteins from unlabeled proteins.

The persistence of proteins in a number of biological systems has been analyzed by density labeling techniques; however, the utility of this approach has been severely hampered by poor resolution between density-labeled and unlabeled proteins on equilibrium gradients. A high resolution equilibrium salt gradient composed of KSCN/CsSCN has been developed to effectively separate density-labeled proteins (13C-15N-2H-substituted) from unlabeled proteins. The resolution of this system is approximately twofold greater than that previously achieved with cesium formate/guanidine hydrochloride equilibrium gradients which have been used in many recent protein density labeling studies. In order to examine the extent of cross-contamination between density-labeled and unlabeled proteins in a KSCN/CsSCN gradient system, density-labeled chick epidermal proteins were mixed with unlabeled Drosophila larval proteins and then separated on these equilibrium gradients. From individual gradient fractions proteins were recovered and fractionated on a sodium dodecyl sulfate-polyacrylamide gel, demonstrating the virtually complete separation between the two populations. The general utility of this system for protein stability studies is also demonstrated.

Drosophila ribosomal RNA stability increases during slow growth conditions.

We have developed density labeling pulse-chase methods which, in contrast to a conventional radiolabeling approach, allow us to determine the effectiveness of our chase and to measure RNA stability in vivo without measuring precursor pool specific activities. We have used these methods to determine the stability of the embryonic ribosomal RNA inherited by either normally or slowly growing Drosophila melanogaster larvae. If larvae are raised in a rich growth medium, embryonic rRNA decays with a half-life of 48 h. However, if larvae are raised in a poor growth medium, which slows larval growth and prolongs development, the half-life of rRNA increases to 115 h. This is the only example, of which we are aware, directly showing that rRNA half-life increases during slow growth conditions. We propose that the increased stability of rRNA that we find may enable slowly growing larvae to maintain the ribosome levels necessary to continue growth and development under conditions of nutrient deprivation.

Selective depletion of human natural killer cells on monolayers of target cells.

The specificity of human natural killer (NK) cells recently has been evaluated by examining direct cytotoxic reactions against a variety of target cells and by cold target inhibition assays. Although these studies have provided evidence for the multiplicity of NK antigens, they did not indicate whether NK cells were heterogeneous, with subpopulations recognizing different specificities. We have examined this issue by using a tumor cell-monolayer adsorption technique that allowed the removal of NK-reactive cells by adsorption to monolayers of various NK-sensitive target cells. After depletion of cells capable of binding to a particular monolayer, the remaining cells were tested for cytotoxicity against a panel of 5 target cells. With several donors, the monolayers were effective in depleting most or all reactivity against the same target cell but only removed a portion of reactivity against some other target cells. The patterns of reactivity after adsorption, although varying among the donors, were consistent upon repeat testing of the same donor. To account for the reactivities of the various donors that remained after adsorption, it was necessary to postulate at least 7 antigenic specificities. These results indicate that human NK cells may be heterogeneous, with each subpopulation recognizing different antigenic specificities on target cells.