Improving sensitivity of cervical cytology by removal of cervical secretions before sampling: a prospective study in Mexico.

UNLABELLED: Sensitivity of cervical cytology is suboptimal, especially in developing countries such as Mexico, despite available guidelines aimed at improving this. When obtaining cervical samples, whether the samples are taken from the transformation zone and whether abnormal cells are missing must be considered. Cervical secretions (CS) are always present in variable proportions, and when cleaning the cervix, better samples may be obtained. In this study, we analyzed samples obtained with or without cleaning the cervix, and compared their contents in order to determine the sensitivity and specificity of these two methods. METHODS: Of 500 patients who underwent cytology and colposcopy, 271 (54.2%) required a second opinion due to a diagnosis of cervical intraepithelial neoplasia (CIN). CS was removed and compared with the clean, second sample (SS) using in both liquid-based cytology. The quality of samples according to the Bethesda System, the presence of CIN, and inflammatory reactions were recorded. The sensitivity and specificity were calculated using biopsy as the gold standard. RESULTS: The SS resulted in a higher proportion of adequate samples being obtained (97.6% vs. 44.8%), and in increased sensitivity (88.2% vs. 58.8%). CIN was detected in the SS 26% more often than in the CS (34 vs. 27 samples), whereas inflammatory reactions were noted more often in the CS (91.4% vs. 74%). CONCLUSION: Cervical sampling including CS results in lower sensitivity and CIN detection rates, and in more inflammatory reactions. By excluding CS from cervical samples, the sensitivity could be improved and the false negative rate could be reduced.

¿Qué profesional médico es el más adecuado para impartir cuidados en salud a niños en Atención Primaria en países desarrollados? Revisión sistemática / What medical professional is the most adequate to provide health care to children in primary care in developed countries? Systematic review

Introducción: existe controversia sobre el tipo de profesional más adecuado –pediatras (PED) o médicos de familia/generales (MF/MG)– para prestar atención sanitaria a niños y adolescentes en Atención Primaria (AP). No existen revisiones sistemáticas previas que hayan estudiado este aspecto. El objetivo de este estudio es comparar la atención sanitaria proporcionada por PED y MF/MG en los siguientes aspectos de la práctica clínica: la prescripción de antibióticos (ATB), la indicación de pruebas diagnósticas, el manejo de la otitis media (OMA), del asma, del síndrome febril y de diversas alteraciones psicopatológicas, así como la realización de actividades preventivas. Material y métodos: diseño de estudio: revisión sistemática. Fuente de los datos: hasta diciembre de 2008 se revisaron las bases de datos MEDLINE y CENTRAL, el metabuscador TRIP Database y el buscador Google Académico para recuperar artículos originales y revisiones sistemáticas que compararan la práctica clínica de ambos tipos de profesionales. No se efectuó restricción por idioma. Selección de estudios: se incluyeron estudios de cualquier tipo de diseño (transversal, cohortes, casos y controles, experimentales) que compararan la práctica clínica del PED y el MF/MG. Se excluyeron todas las referencias que no contuvieran investigación original (cartas al director o editoriales). Asimismo, se evaluó la calidad metodológica de cada estudio con el instrumento “OSTEBA; Fichas de lectura crítica”. Dicha calidad era valorada de forma independiente por dos revisores, que llegaban a un consenso en caso de discrepancia. La extracción de datos fue realizada por siete parejas de revisores de forma independiente. Las discrepancias se resolvieron mediante consenso. Resultados: como promedio, los MF/MG prescribieron más ATB que los PED en infecciones del tracto respiratorio superior de probable etiología vírica –odds ratio (OR): 1,4; intervalo de confianza del 95% (IC 95%): 1,1-1,8–. Los PED tuvieron más probabilidades de adherirse a las recomendaciones de guías de práctica clínica sobre el manejo del síndrome febril (OR: 9; IC 95%: 3-25) y del trastorno por déficit de atención con/sin hiperactividad (OR: 5; IC 95%: 3-11), y una mayor capacidad de resolución para otras enfermedades de elevada prevalencia durante la infancia y la adolescencia (como asma y OMA). Los PED presentaban porcentajes de vacunación superiores a los de los MF/MG en todos los estudios que evaluaron este resultado. Conclusión: en vista de los resultados expuestos, parece recomendable mantener la figura del PED en los equipos de AP y reforzar su función específica como primer punto de contacto del niño con el sistema sanitario (AU)

Introduction: There is controversy about which health professional is the most adequate –pediatricians (PED) or family practitioners/general physicians (FP/GP)– to provide health care services to children and adolescents in Primary Care (PC). There are not previous systematic reviews approaching this subject in the previously published literature. The objective of this study is to compare health care provided between PED and FP/GP in the following aspects of the clinical practice: antibiotic (ATB) prescription; diagnostic test indication; acute otitis media (AOM), asthma, febrile syndrome and several psychopathological conditions’ management; and preventive measures accomplishment. Material and methods: study design: systematic review. Data sources: MEDLINE and CENTRAL databases, TRIP Database and Google Scholar, were searched until December 2008 to retrieve original papers and systematic reviews comparing the clinical practice of both kinds of health professionals. No language restriction was made. Studies’ selection: studies of any kind of design were included (cross-sectional, cohorts, case-controls and experimental) comparing the clinical practice of PED and FP/GP. The references without original research were excluded (letters to the editor, editorials). The methodological quality of each study was assessed with the tool “OSTEBA; Critical Appraisal Cards”. Two reviewers assessed the quality of the studies independently, achieving consensus in case of discrepancy. Seven pairs of reviewers made the data extraction independently. Discrepancies were achieved by consensus. Results: On average, FP/GP prescribed more ATB than PED in upper respiratory tract infections of probable viral etiology –odds ratio (OR): 1.4; 95% confidence interval (95% CI): 1.1-1.8–; PED were more likely to adhere to clinical guidelines recommendations on febrile syndrome management (OR: 9; 95% CI: 3-25) and on attention deficit disorder with/without hyperactivity (OR: 5; 95% CI: 3-11), and showed more resolution capacity on other highly prevalent conditions in children and adolescents (such as asthma and AOM). PED showed higher vaccination coverage than FP/GP in all the studies assessing this result. Conclusion: based on the presented results, it seems reasonable to recommend maintaining the PED figure in PC health centers and reinforcing its specific task as the first point of contact of the child with the health care system (AU)

Carbon nanotube reinforced polylactide-caprolactone copolymer: mechanical strengthening and interaction with human osteoblasts in vitro.

This study proposes the use of carbon nanotubes (CNTs) as reinforcement to enhance the mechanical properties of a polylactide-caprolactone copolymer (PLC) matrix. Biological interaction of PLC-CNT composites with human osteoblast cells is also investigated. Addition of 2 wt % CNT shows very uniform dispersion in the copolymer matrix, whereas 5 wt % CNT shows severe agglomeration and high porosity. PLC-2 wt % CNT composite shows an improvement in the mechanical properties with an increase in the elastic modulus by 100% and tensile strength by 160%, without any adverse effect on the ductility up to 240% elongation. An in vitro biocompatibility study on the composites shows an increase in the viability of human osteoblast cells compared to the PLC matrix, which is attributed to the combined effect of CNT content and surface roughness of the composite films.

Massive exophthalmos after traumatic carotid-cavernous fistula embolization.

An 18-year-old man without previous clinical problems developed a left traumatic carotid-cavernous fistula after a traffic accident. An endovascular embolization with coils was performed without success. The drainage was derived to the superior ophthalmic vein solely and clinical worsening occurred. Left eye proptosis, chemosis and intraocular pressure increased. Complete ophthalmoplegia developed and visual acuity decreased due to a central retinal vein obstruction. After a second embolization attempt the fistula was closed successfully but proptosis, chemosis and intraocular pressure remained uncontrolled despite medical treatment. Therefore an orbital decompression surgery was performed. Ophthalmoplegia, proptosis and chemosis improved and intraocular pressure was controlled. Although retinal hemorrhages persist, no neovascularization has developed. Central retinal vein occlusion in young patients seems to have a different etiology than in the elderly. In young patients, local factors are more frequently identified than systemic vascular diseases. Early detection of central retinal vein obstruction may prevent deterioration of visual acuity.

En niños con laringitis aguda una dosis de dexametasona oral es más eficaz que una de prednisolona / In children with croup one oral dexamethasone doses is more effective than one of prednisolone

No disponible

Quorum signaling via AI-2 communicates the "Metabolic Burden" associated with heterologous protein production in Escherichia coli.

Recent reports have shown that bacterial cell-cell communication or quorum sensing is quite prevalent in pathogenic Escherichia coli, especially at high cell density; however, the role of quorum sensing in nonpathogenic E. coli is less clear and, in particular, there is no information regarding the role of quorum sensing in overexpression of plasmid-encoded genes. In this work, it was found that the activity of a quorum signaling molecule, autoinducer-2 (AI-2), decreased significantly following induction of several plasmid-encoded genes in both low and high-cell-density cultures of E. coli. Furthermore, we show that AI-2 signaling level was linearly related to the accumulation level of each protein product and that, in general, the highest rates of recombinant protein accumulation resulted in the greatest attenuation of AI-2 signaling. Importantly, our findings demonstrate for the first time that recombinant E. coli communicate the stress or burden of overexpressing heterologous genes through the quorum-based AI-2 signaling pathway.

Enhancement of organophosphorus hydrolase yield in Escherichia coli using multiple gene fusions.

It was previously shown that organophosphorus hydrolase (OPH) expression and purification could be tracked by fluorescence of green fluorescent protein (GFP) when synthesized as an N-terminal fusion with GFP (Cha et al., 2000; Wu et al., 2000). In order to enhance OPH productivity while utilizing the advantage of the reporter protein (GFP), two copies of OPH were cloned in tandem following the gfp(uv) gene (e.g., GFP-OPH(n=2)). Both anti-GFP and anti-OPH Western blots demonstrated that a higher yield was achieved in comparison to the one copy fusion (GFP-OPH). Importantly, the fusion protein was still fluorescent as determined via microscopy. In contrast, a fusion containing two copies of OPH without GFP, and an operon fusion of two OPHs with two independent ribosomal binding sites, did not result in a higher yield than one OPH expressed alone.

DNA microarray-based identification of genes controlled by autoinducer 2-stimulated quorum sensing in Escherichia coli.

Bacterial cell-to-cell communication facilitates coordinated expression of specific genes in a growth rate-II and cell density-dependent manner, a process known as quorum sensing. While the discovery of a diffusible Escherichia coli signaling pheromone, termed autoinducer 2 (AI-2), has been made along with several quorum sensing genes, the overall number and coordination of genes controlled by quorum sensing through the AI-2 signal has not been studied systematically. We investigated global changes in mRNA abundance elicited by the AI-2 signaling molecule through the use of a luxS mutant that was unable to synthesize AI-2. Remarkably, 242 genes, comprising ca. 5.6% of the E. coli genome, exhibited significant transcriptional changes (either induction or repression) in response to a 300-fold AI-2 signaling differential, with many of the identified genes displaying high induction levels (more than fivefold). Significant induction of ygeV, a putative sigma(54)-dependent transcriptional activator, and yhbH, a sigma(54) modulating protein, suggests sigma(54) may be involved in E. coli quorum sensing.

Effects of in situ cobalt ion addition on the activity of a gfp-oph fusion protein: the fermentation kinetics.

The effects of cobalt ion addition and inducer concentration were studied in the fermentation of E. coli BL21 expressing a GFP (green fluorescent protein)-OPH (organophosphorus hydrolase) fusion protein. It was found that cobalt ion addition improved the OPH activity significantly. When 2 mM of CoCl(2) was supplied during the IPTG-induction phase, OPH activity was enhanced approximately 10-fold compared to the case without cobalt or by the addition of cobalt to the cell extracts. Results indicate, therefore, that incorporation of the cobalt during synthesis is needed for enhanced activity. Also, the maximum OPH activity was not linearly related to inducer concentration. A mathematical model was then constructed to simulate these phenomena. Model parameters were determined by constrained least-squares and optimal IPTG and cobalt addition concentrations were obtained, pinpointing the conditions for the maximum productivity. Finally, the GFP fluorescence intensity was found linear to the OPH activity in each fermentation, demonstrating the function of GFP for monitoring its fusion partner's quantity in the bioreactor.

Mapping stress-induced changes in autoinducer AI-2 production in chemostat-cultivated Escherichia coli K-12.

Numerous gram-negative bacteria employ a cell-to-cell signaling mechanism, termed quorum sensing, for controlling gene expression in response to population density. Recently, this phenomenon has been discovered in Escherichia coli, and while pathogenic E. coli utilize quorum sensing to regulate pathogenesis (i.e., expression of virulence genes), the role of quorum sensing in nonpathogenic E. coli is less clear, and in particular, there is no information regarding the role of quorum sensing during the overexpression of recombinant proteins. The production of autoinducer AI-2, a signaling molecule employed by E. coli for intercellular communication, was studied in E. coli W3110 chemostat cultures using a Vibrio harveyi AI-2 reporter assay (M. G. Surrette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046-7050, 1998). Chemostat cultures enabled a study of AI-2 regulation through steady-state and transient responses to a variety of environmental stimuli. Results demonstrated that AI-2 levels increased with the steady-state culture growth rate. In addition, AI-2 increased following pulsed addition of glucose, Fe(III), NaCl, and dithiothreitol and decreased following aerobiosis, amino acid starvation, and isopropyl-beta-D-thiogalactopyranoside-induced expression of human interleukin-2 (hIL-2). In general, the AI-2 responses to several perturbations were indicative of a shift in metabolic activity or state of the cells induced by the individual stress. Because of our interest in the expression of heterologous proteins in E. coli, the transcription of four quorum-regulated genes and 20 stress genes was mapped during the transient response to induced expression of hIL-2. Significant regulatory overlap was revealed among several stress and starvation genes and known quorum-sensing genes.

Genomic analysis of high-cell-density recombinant Escherichia coli fermentation and "cell conditioning" for improved recombinant protein yield.

The Escherichia coli stress gene transcription profile and response to recombinant protein overexpression were substantially altered at high cell density when compared with low cell density. Reverse trascription-polymerase chain reaction RT-PCR-amplified mRNA from low (4 g[DCW]/L) and high-cell-density (43.5 g [DCW]/L) conditions were hybridized with a DNA microarray of Kohara clones encompassing 16% of the E. coli genome, and differentially displayed genes were identified. Transcript-specific RNA dot blots indicated that molecular chaperones (groEL, ibpA, degP), proteases (degP, ftsH), the lysis gene mltB, and DNA damage/bacteriophage-associated gene transcript levels (ftsH, recA, alpA, uvrB) increased 10- to 43-fold at high cell density. In addition, overexpression of recombinant green fluorescent protein (GFP(uv))/chloramphenicol acetyltransferase (CAT) fusion protein did not change the rates of cell growth or cell lysis. The stress gene transcription profile at high cell density was used to evaluate "cell conditioning" strategies to alter the levels of chaperones, proteases, and other intracellular proteins prior to recombinant protein overexpression. Interestingly, the addition of 1 g/L dithiothreitol (DTT) 20 min prior to induction of a GFP(uv)/CAT fusion protein resulted in a twofold increase in CAT activity when compared with the unconditioned controls. In addition, RNA dot blots of five stress genes confirmed that cell conditioning strategies significantly altered the dynamic stress gene response to foreign protein overexpression.

A comparative study of global stress gene regulation in response to overexpression of recombinant proteins in Escherichia coli.

Global gene regulation throughout the Escherichia coli stress response to overexpression of each of five recombinant proteins was evaluated. Reverse-transcriptase polymerase chain reaction-amplified mRNA from induced and control cells were hybridized with a DNA array of Kohara clones representing 16% (700 genes) of the E. coli genome. Subsequently, Northern analysis was performed for quantification of specific gene dynamics and statistically significant overlap in the regulation of 11 stress-related genes was found using correlation analysis. The results reported here establish that there are dramatic changes in the transcription rates of a broad range of stress genes (representing multiple regulons) after induction of recombinant protein. Specifically, the responses included significantly increased upregulation of heat shock (ftsH, clpP, lon, ompT, degP, groEL, aceA, ibpA), SOS/DNA damage (recA, lon, IS5 transposase), stationary phase (rpoS, aceA), and bacteriophage life cycle (ftsH, recA) genes. Importantly, similarities at the microscopic (gene) level were not clearly reflected at the macroscopic (growth rate, lysis) level. The use of such dynamic data is critical to the design of gene-based sensors, the engineering of metabolic pathways, and the determination of parameters (harvest and induction times) needed for successful recombinant E. coli fermentations.

A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase.

Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of the gene for green fluorescent protein (GFP) to the 5' end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP. Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations with inexpensive instrumentation based on detecting green fluorescence.

Recombinant antibodies: a new reagent for biological agent detection.

Antibodies are critical reagents used in several biodetection platforms for the identification of biological agents. Recent advances in phage display technology allow isolation of high affinity recombinant antibody fragments (Fabs) that may bind unique epitopes of biological threat agents. The versatility of the selection process lends itself to efficient screening methodologies and can increase the number of antigen binding clones that can be isolated. Pilot scale biomanufacturing can then be used for the economical production of these immunoglobulin reagents in bacterial fermentation systems, and expression vectors with hexahistidine tags can be used to simplify downstream purification. One such Fab reagent directed against botulinum neurotoxin A/B has been shown to be sensitive in a variety of assay formats including surface plasmon resonance (SPR), flow cytometry, enzyme linked immunosorbent assay (ELISA), and hand-held immunochromatographic assay. Recombinant antibodies can provide another source of high quality detection reagents in our arsenal to identify or detect pathogens in environmental samples.

Detection of Yersinia pestis by pesticin fluorogenic probe-coupled PCR.

The <> assay (PE Applied Biosystems) combines PCR with concomitant release of fluorogenic nucleotides for immediate product detection by fluorometry. Yersinia pestis, the etiological agent of bubonic plague, expresses species-specific genes known to be located on two unique plasmids (9.6-kb pPCP and 100.9-kb pMT). Pesticin (pst) is a unique gene located on pPCP which encodes for a bacteriocin. Using fluorogenic probe coupled PCR as few as three copies of pst targets were detected from total Y. pestis genomic DNA. The pst probe used in this report was positive only for pesticinogenic isolates and did not show complementarity with Yersiniae nor with other bacteria targeted in this study suggesting, that the pst probe is very specific for Y. pestis. Under optimal conditions of Mg(2+)concentration and thermal cycle number, addition of extraneous DNA to respective assay mixtures had no effect on detection.

Observations of green fluorescent protein as a fusion partner in genetically engineered Escherichia coli: monitoring protein expression and solubility.

We have constructed three plasmid vectors for the expression of green fluorescent protein (GFP) fusion proteins using the following motif: (His)(6)-GFP-EK-X, where X represents chloramphenicol acetyl-transferase (CAT), human interleukin-2 (hIL-2), and organophosphorous hydrolase (OPH), respectively, (His)(6) represents a histidine affinity ligand for purification, and EK represents an enterokinase cleavage site for recovering the protein-of-interest from the fusion. The CAT and OPH fusion products ( approximately 63 kDa GFP/CAT and approximately 70 kDa GFP/OPH) were expressed at 4.85 microg/mL (19.9 microg/mg-total protein) and 1.42 microg/mL (4.2 microg/mg-total protein) in the cell lysis supernatant, and, in both cases, enzymatic activity was retained while coupled to GFP. In the case of hIL-2 fusion ( approximately 52 kDa), however, the GFP fluorescence was significantly reduced and most of the fusion was retained in the cell pellet. Linear relationships between GFP fluorescence and CAT or OPH concentration, and with enzymatic activity of CAT or OPH, indicated, for the first time, that in vivo noninvasive quantification of proteins-of-interest, was made possible by simple measurement of GFP fluorescence intensity. The utility of GFP as a reporter was not realized without disadvantages however, in particular, an incremental metabolic cost of GFP was found. This could be offset by many benefits foreseen in expression and purification efficiencies.

Reverse transcription-PCR differential display analysis of Escherichia coli global gene regulation in response to heat shock.

A reverse transcription (RT)-PCR technique was developed to analyze global gene regulation in Escherichia coli. A novel combination of primers designed specifically for the start and stop regions of E. coli genes (based on the findings of Fislage et al. [R. Fislage, M. Berceanu, Y. Humboldt, M. Wendt, and H. Oberender, Nucleic Acids Res. 25:1830-1835, 1997]) was used as an alternative to the poly(T) primers often used in eukaryotic RT-PCR. The validity of the technique was demonstrated by applying it to heat shock analysis. Specifically, RT-PCR-amplified total RNA from heat-shocked and non-heat-shocked cells were hybridized with slot blots of the Kohara set (U. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987; S. Chuang, D. Daniels, and F. Blattner, J. Bacteriol. 175:2026-2036, 1993). The signals obtained for heat-shocked and control cultures of each clone were compared, and differences in intensity were evaluated by calculating induction ratios. Clones that were considered significantly induced were subsequently mapped by the Southern blot technique in order to determine specific gene upregulation. Also, for several genes, Northern blotting and total RNA dot blotting were performed to confirm that the transcript levels in the original RNA samples were different. This technique extended previously described methods for studying global gene regulation in E. coli by incorporating a PCR amplification step in which global, mRNA-specific primers were used. In addition, the method employed here can be easily extended to study E. coli global gene regulation in response to additional environmental stimuli.

Cytotoxicity of organophosphate anticholinesterases.

Organophosphate (OP) anticholinesterases were found to modulate metabolic activities of human neuroblastoma cells and hepatocytes, which was detectable by the Cytosensor microphysiometer. The nerve gas ethyl-S-2-diisopropylaminoethyl methylphosphorothiolate (VX), at 10 microM, produced significant reduction in cell metabolism within 2 min, as measured by changes in the acidification rate of the medium. The reduction was dose- and time-dependent and irreversible after 4 h of exposure. Two alkaline degradation products of VX produced no cytotoxicity. Exposure for 24 h to 3 microM VX caused 36% and 94% irreversible loss of metabolism in hepatocytes and neuroblastoma cells, respectively. The insecticides parathion and chlorpyrifos stimulated hepatocyte metabolism but inhibited neuroblastoma cells. Their oxons were more active. Exposure of neuroblastoma cells for 4 h to VX, parathion, paraoxon, diisopropylfluorophosphate or chlorpyrifos gave an LC50 of 65, 775, 640, 340, or 672 microM, respectively, whereas 24 h gave an LC50 of 0.7, 3.7, 2.5, 29, and 31 microM, respectively. Preincubation of hepatocytes with phenobarbital enhanced their response to parathion and VX due to metabolic bioactivation. Atropine partially blocked the effects of VX and paraoxon on both cell types, which suggests the involvement of a muscarinic receptor as the target for cytotoxicity. There was no correlation between OP in vivo neurotoxicity and in vitro cytotoxicity. It is suggested that the former results from their cholinesterase inhibition, while the latter results from action on different targets and requires much higher concentrations.

Detection of Yersinia pestis using branched DNA.

In contrast to target amplification methods, e.g. polymerase chain reaction, the branched DNA (bDNA) signal amplification method quantitates target nucleic acid at physiological levels, involving a series of hybridization reactions without thermal cycling. In this report, we describe a modification of the bDNA assay in which a <> preamplifier oligonucleotide (206 mer) is used in concert with ELISA and light addressable potentiometric sensor (LAPS) formats to detect the plasminogen activator (pla) gene of Yersinia pestis, the etiological agent of plague. Pla is encoded by a 9.6-kb plasmid pPCP, which is essential for virulence. The detection limit of the bDNA-ELISA and LAPS assays is less than 10 000 and 1000 molecules of Y. pestis plasmid DNA, respectively.

Assessment of an automated solid phase competitive fluoroimmunoassay for benzoylecgonine in untreated urine.

A new solid phase fluoroimmunoassay using a fully automated flow fluorometer adapted for urinalysis of drug metabolites is described. Fluorescein-conjugated benzoylecgonine (FL-BE) and monoclonal antibodies (mAb) against benzoylecgonine (BE) were the reagents used for demonstration. The solid phase consisted of anti-BE mAbs immobilized on the surface of polymethyl methacrylate (PMMA) beads. Free BE in solution competed with FL-BE and reduced bead-bound fluorescence in a concentration-dependent manner. The binding of FL-BE to the anti-BE mAb reached steady-state within minutes. FL-BE was not bound by uncoated beads nor beads coated with non-specific proteins or IgG. The signal-to-noise ratio was 33, and the sensitivity of the assay was 2 ng ml(-1) for BE. The effective concentration of BE was 1 to 100 ng ml(-1), with an IC50 value of 12 ng ml(-1). The mAb showed equal affinities for BE, cocaine, and cocaethylene, but a five order-of-magnitude lower affinity for ecgonine and ecgonine methylester. In a double-blind comparison using clinical urine samples, the data from this single-step competitive assay had excellent agreement with results obtained using a fiber-optic biosensor (FOB), and the EMIT assay performed commercially. The assay provided kinetic data rapidly and can be used to detect small analytes for which antibodies and fluorescein conjugates are available. The affinity of the mAb for FL-BE, calculated from kinetic analysis of the time course of the on and off reaction, was 2.25 x 10(-9) M.