RESUMEN
Anaesthetists are thought to be at increased risk of suicide amongst the medical profession. The aims of the following guidelines are: increase awareness of suicide and associated vulnerabilities, risk factors and precipitants; to emphasise safe ways to respond to individuals in distress, both for them and for colleagues working alongside them; and to support individuals, departments and organisations in coping with a suicide.
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Anestesistas/psicología , Anestesistas/estadística & datos numéricos , Trastornos Mentales/diagnóstico , Estrés Psicológico/diagnóstico , Prevención del Suicidio , Suicidio/psicología , Guías como Asunto , Humanos , Trastornos Mentales/complicaciones , Trastornos Mentales/psicología , Factores de Riesgo , Estrés Psicológico/complicaciones , Estrés Psicológico/psicología , Suicidio/estadística & datos numéricos , Reino UnidoRESUMEN
AIMS: The control of the wine spoilage yeast Brettanomyces bruxellensis using biological methods such as killer toxins (instead of the traditional chemical methods, e.g. SO2 ) has been the focus of several studies within the last decade. Our previous research demonstrated that the killer toxins CpKT1 and CpKT2 isolated from the wine yeast Candida pyralidae were active and stable under winemaking conditions. In this study, we report the possible mode of action of CpKT1 on B. bruxellensis cells in red grape juice. METHODS AND RESULTS: Brettanomyces bruxellensis cells were exposed to CpKT1 either directly or through co-inoculation with C. pyralidae. This exposure yielded a temporary or permanent decline of the spoilage yeast population depending on the initial cell concentration. Scanning electron microscopy revealed cell surface abrasion while propidium iodide viability staining showed that CpKT1 caused plasma membrane damage on B. bruxellensis cells. Our data show that the exposure to CpKT1 resulted in increased levels of ß-glucan, suggesting a compensatory response of the sensitive cells. CONCLUSIONS: The toxin CpKT1 causes cell membrane and cell wall damage in B. bruxellensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Candida pyralidae shows potential to be used as a biocontrol agent against B. bruxellensis in grape juice/wine.
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Brettanomyces/efectos de los fármacos , Candida/metabolismo , Pared Celular/efectos de los fármacos , Micotoxinas/farmacología , Brettanomyces/ultraestructura , Pared Celular/ultraestructura , Microbiología de Alimentos , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Micotoxinas/aislamiento & purificación , Propidio , Vitis/microbiología , Vino/microbiología , Levadura Seca , beta-Glucanos/metabolismoRESUMEN
Sequence-based typing (SBT) for Legionella pneumophila (Lp) has dramatically improved Legionnaires' disease (LD) cluster investigation. Microbial whole genome sequencing (WGS) is a promising modality for investigation but sequence analysis methods are neither standardised, nor agreed. We sought to develop a WGS-based typing scheme for Lp using de novo assembly and a genome-wide gene-by-gene approach (core genome multilocus sequence typing, cgMLST). We analysed 17 publicly available Lp genomes covering the whole species variation to define a core genome (1,521 gene targets) which was validated using 21 additional published genomes. The genomes of 12 Lp strains implicated in three independent cases of paediatric humidifier-associated LD were subject to cgMLST together with three 'outgroup' strains. cgMLST was able to resolve clustered strains and clearly identify related and unrelated strains. Thus, a cgMLST scheme was readily achievable and provided high-resolution analysis of Lp strains. cgMLST appears to have satisfactory discriminatory power for LD cluster analysis and is advantageous over mapping followed by single nucleotide polymorphism (SNP) calling as it is portable and easier to standardise. cgMLST thus has the potential for becoming a gold standard tool for LD investigation. Humidifiers pose an ongoing risk as vehicles for LD and should be considered in cluster investigation and control efforts.
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Variación Genética , Legionella pneumophila/clasificación , Legionella pneumophila/genética , Tipificación Molecular/métodos , Tipificación de Secuencias Multilocus/métodos , Análisis de Secuencia de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Genoma Bacteriano , Humanos , Legionella pneumophila/aislamiento & purificación , Epidemiología Molecular/métodos , Datos de Secuencia Molecular , Polimorfismo de Nucleótido SimpleRESUMEN
Operation GRITROCK saw the first operational deployment of the Maritime In Transit Care team from the Role 2 (Enhanced) (R2(E)) Medical Treatment Facility, which is able to provide Damage Control Surgery and the limited holding of patients, situated on board RFA ARGUS. Whilst the Medical Emergency Response Team demonstrated the capability of advanced military Pre-Hospital Emergency Care (PHEC) on Op HERRICK, the need to provide a similar high level of care on contingency operations was recognised. Op GRITROCK allowed for the continued exploration of a maritime capability from an established R2(E) platform whilst providing medical evacuation capability for a significant population at risk distributed over a large Joint Operation Area. Although the patient load during the operation was low, key lessons were learnt and opportunities identified to further develop the newly recognised sub-speciality of PHEC, both medically and logistically, and these will be discussed in this article.
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Campaña Afgana 2001- , Servicios Médicos de Urgencia/organización & administración , Medicina Militar , Medicina Naval , Humanos , Reino UnidoRESUMEN
The aim of this study was to assess the immune response and the protective efficacy elicited by the vaccination with the recombinant Fasciola hepatica myosin regulatory light chain (FhrMRLC) in Adjuplex® adjuvant against the infection with F. hepatica in rats. Four groups of 15 animals each were used for the study, one group was immunized with the recombinant F. hepatica MRLC in Adjuplex® adjuvant and the other groups remained as adjuvant, positive and negative control groups. The parasitological study showed that a statistically significant reduction of 65.1% and 82.1% in fluke burden and fecal egg count, respectively, was detected in vaccinated animals. In addition, vaccination with FhrMRLC induced a well-defined humoral and cellular immune response characterized by a significant production of specific IgG and IL-2, IL-12, TNF-α and IFN-γ; which confirms the immunogenic capacity of the FhrMRLC.
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Fasciola hepatica/fisiología , Fascioliasis/inmunología , Inmunización , Cadenas Ligeras de Miosina/uso terapéutico , Células TH1/inmunología , Animales , Inmunidad Celular , Inmunidad Humoral , Masculino , Cadenas Ligeras de Miosina/inmunología , Ratas , Ratas Wistar , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéuticoRESUMEN
BACKGROUND: Advanced life support of patients contaminated with chemical, biological, radiological or nuclear (CBRN) substances requires adequate respiratory protection for medical first responders. Conventional and powered air-purifying respirators may exert a different impact during resuscitation and therefore require evaluation. This will help to improve major incident planning and measures for protecting medical staff. METHODS: A randomised crossover study was undertaken to investigate the influence of conventional negative pressure and powered air-purifying respirators on the simulated resuscitation of casualties contaminated with hazardous substances. Fourteen UK paramedics carried out a standardised resuscitation algorithm inside an ambulance vehicle, either unprotected or wearing a conventional or a powered respirator. Treatment times, wearer mobility, ease of communication and ease of breathing were determined and compared. RESULTS: In the questionnaire, volunteers stated that communication and mobility were similar in both respirator groups while breathing resistance was significantly lower in the powered respirator group. There was no difference in mean (SD) treatment times between the groups wearing respiratory protection and the controls (245 (19) s for controls, 247 (17) s for conventional respirators and 250 (12) s for powered respirators). CONCLUSIONS: Powered air-purifying respirators improve the ease of breathing and do not appear to reduce mobility or delay treatment during a simulated resuscitation scenario inside an ambulance vehicle with a single CBRN casualty.
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Sustancias Peligrosas/toxicidad , Dispositivos de Protección Respiratoria , Resucitación/instrumentación , Técnicos Medios en Salud , Estudios Cruzados , Diseño de Equipo , Humanos , Simulación de Paciente , Ropa de Protección , Factores de TiempoRESUMEN
Evidence is limited regarding whether periodontal treatment improves hemoglobin A1c (HbA1c) among people with prediabetes and periodontal disease, and it is unknown whether improvement of metabolic status persists >3 mo. In an exploratory post hoc analysis of the multicenter randomized controlled trial "Antibiotika und Parodontitis" (Antibiotics and Periodontitis)-a prospective, stratified, double-blind study-we assessed whether nonsurgical periodontal treatment with or without an adjunctive systemic antibiotic treatment affects HbA1c and high-sensitivity C-reactive protein (hsCRP) levels among periodontitis patients with normal HbA1c (≤5.7%, n = 218), prediabetes (5.7% < HbA1c < 6.5%, n = 101), or unknown diabetes (HbA1c ≥ 6.5%, n = 8) over a period of 27.5 mo. Nonsurgical periodontal treatment reduced mean pocket probing depth by >1 mm in both groups. In the normal HbA1c group, HbA1c values remained unchanged at 5.0% (95% CI, 4.9% to 6.1%) during the observation period. Among periodontitis patients with prediabetes, HbA1c decreased from 5.9% (95% CI, 5.9% to 6.0%) to 5.4% (95% CI, 5.3% to 5.5%) at 15.5 mo and increased to 5.6% (95% CI, 5.4% to 5.7%) after 27.5 mo. At 27.5 mo, 46% of periodontitis patients with prediabetes had normal HbA1c levels, whereas 47.9% remained unchanged and 6.3% progressed to diabetes. Median hsCRP values were reduced in the normal HbA1c and prediabetes groups from 1.2 and 1.4 mg/L to 0.7 and 0.7 mg/L, respectively. Nonsurgical periodontal treatment may improve blood glucose values among periodontitis patients with prediabetes (ClinicalTrials.gov NCT00707369).
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Diabetes Mellitus Tipo 2/sangre , Hemoglobina Glucada/análisis , Periodontitis/terapia , Estado Prediabético/epidemiología , Adulto , Anciano , Antibacterianos/uso terapéutico , Glucemia , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/epidemiología , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Periodontitis/sangre , Periodontitis/complicaciones , Estado Prediabético/sangre , Estudios Prospectivos , Resultado del TratamientoRESUMEN
Emergency room personnel are threatened by secondary poisoning when treating victims affected by chemical warfare agents. Therefore, resuscitation skills practised with respiratory protection equipment in place require evaluation. We investigated the influence of wearing air-purifying respirators on the simulated resuscitation of chemical warfare agent casualties. We studied 22 anaesthetic trainees in a simulated resuscitation scenario requiring five set tasks, either unprotected, wearing a binocular visor respirator or a panoramic visor respirator in a randomised, crossover study. Treatment times did not differ between the three groups, with mean (SD) times to complete the tasks being 122 (8) s without a mask, 126 (7) s when wearing the panoramic visor mask and 129 (8) s when wearing the binocular respirator mask. All anaesthetists preferred the panoramic visor in terms of visual orientation but 88% of them rated the binocular mask as being more comfortable. Modern respirators have a negligible effect on simulated resuscitation scenarios for victims affected by chemical warfare agents. Panoramic visor respirators allow better visual orientation for anaesthetists during simulated resuscitation.
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Sustancias para la Guerra Química/envenenamiento , Servicio de Urgencia en Hospital/normas , Exposición Profesional/prevención & control , Dispositivos de Protección Respiratoria , Resucitación/normas , Anestesiología/instrumentación , Anestesiología/normas , Actitud del Personal de Salud , Competencia Clínica , Estudios Cruzados , Diseño de Equipo , Alemania , Humanos , ManiquíesRESUMEN
AIM: Platelet-activating factor acetyl hydrolase 1B1 (PAFAH1B1, also known as Lis1) is a protein essentially involved in neurogenesis and mostly studied in the nervous system. As we observed a significant expression of PAFAH1B1 in the vascular system, we hypothesized that PAFAH1B1 is important during angiogenesis of endothelial cells as well as in human vascular diseases. METHOD: The functional relevance of the protein in endothelial cell angiogenic function, its downstream targets and the influence of NONHSAT073641, a long non-coding RNA (lncRNA) with 92% similarity to PAFAH1B1, were studied by knockdown and overexpression in human umbilical vein endothelial cells (HUVEC). RESULTS: Knockdown of PAFAH1B1 led to impaired tube formation of HUVEC and decreased sprouting in the spheroid assay. Accordingly, the overexpression of PAFAH1B1 increased tube number, sprout length and sprout number. LncRNA NONHSAT073641 behaved similarly. Microarray analysis after PAFAH1B1 knockdown and its overexpression indicated that the protein maintains Matrix Gla Protein (MGP) expression. Chromatin immunoprecipitation experiments revealed that PAFAH1B1 is required for active histone marks and proper binding of RNA Polymerase II to the transcriptional start site of MGP. MGP itself was required for endothelial angiogenic capacity and knockdown of both, PAFAH1B1 and MGP, reduced migration. In vascular samples of patients with chronic thromboembolic pulmonary hypertension (CTEPH), PAFAH1B1 and MGP were upregulated. The function of PAFAH1B1 required the presence of the intact protein as overexpression of NONHSAT073641, which was highly upregulated during CTEPH, did not affect PAFAH1B1 target genes. CONCLUSION: PAFAH1B1 and NONHSAT073641 are important for endothelial angiogenic function.
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1-Alquil-2-acetilglicerofosfocolina Esterasa/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , 1-Alquil-2-acetilglicerofosfocolina Esterasa/genética , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Células Cultivadas , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Femenino , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , ARN Polimerasa II/metabolismo , ARN Largo no Codificante/fisiología , Tromboembolia/complicaciones , Tromboembolia/metabolismo , Cicatrización de Heridas , Proteína Gla de la MatrizRESUMEN
In this review, we discuss the kps cluster of Escherichia coli as the paradigm for the ABC capsular polysaccharide exporter (CPSE) family. Components of the cluster form a multimeric protein complex consisting of both biosynthetic and export machinery. We compare the Kps exporter with capsule export systems from other members of the CPSE family.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Bacterias Gramnegativas/metabolismo , Proteínas Periplasmáticas , Polisacáridos Bacterianos/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/patogenicidad , Bacterias Gramnegativas/genética , Proteínas de Transporte de Membrana , Polisacáridos Bacterianos/genéticaRESUMEN
Bacterial pathogens must overcome a range of challenges during the process of infecting their host. The ability of a pathogen to sense and respond appropriately to changes in host environment is vital if the pathogen is to succeed. Mammalian defense strategies include the use of barriers like skin and epithelial surfaces, the production of a chemical arsenal, such as stomach acid and reactive oxygen and nitrogen species, and a highly coordinated cellular and humoral immune response. Salmonella serovars are significant human and animal pathogens which have evolved several mechanisms to overcome mammalian host defense. Here we focus on the interplay which occurs between Salmonella and the host during the infection process, with particular emphasis on the complex bacterial response to reactive nitrogen species produced by the host. We discuss recent advances in our understanding of the key mechanisms which confer bacterial resistance to nitrogen species, which in response to nitric oxide include the flavohemoglobin, HmpA, the flavorubredoxin, NorV, and the cytochrome c nitrite reductase, NrfA, whilst in response to nitrate include a repertoire of nitrate reductases. Elucidating the precise role of different aspects of microbial physiology, nitrogen metabolism, and detoxification during infection will provide valuable insight into novel opportunities and potential targets for the development of therapeutic approaches.
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Especies de Nitrógeno Reactivo/inmunología , Infecciones por Salmonella/inmunología , Salmonella typhimurium/patogenicidad , Animales , Humanos , Inmunidad Humoral , Óxido Nítrico/metabolismo , Nitrito Reductasas/metabolismo , Salmonella typhimurium/enzimologíaRESUMEN
OBJECTIVE: The aim of the present study was to determine sequence variations in the active centre of the Arg-X-specific protease encoding genes rgpA and rgpB of clinical Porphyromonas gingivalis isolates and to analyse their prevalence in periodontitis patients before and 3 months after mechanical periodontal therapy. BACKGROUND: Genetic diversity at nucleotides 281, 283, 286 and 331 has been shown to result in amino acid substitutions in the catalytic domain of RgpA and RgpB that affect the substrate specificity and thus may influence the efficacy of Arg-X-protease specific inhibitors. METHODS: Sequence analysis of rgpA and rgpB genes in clinical P. gingivalis strains isolated from subgingival plaque samples of 82 periodontitis patients before and 3 months after mechanical supra- and subgingival debridement was performed. RESULTS: No specific variation within the rgpA sequence was observed. However, the rgpB sequence in the region of the active centre showed five different rgpB genotypes, which were named NYPN, NSSN, NSSK, NYPK and DYPN according to the derived amino acid substitution. Porphyromonas gingivalis genotype NYPN was detected in 27 patients (32.9%) before and in 8 patients (9.8%) after therapy, NSSN in 26 (31.7%) and 10 (12.2%), NSSK in 22 (26.8%) and 2 (2.4%), NYPK in 5 (6.2%) and 1 (1.2%), and DYPN in 1 patient (1.2%) and 0 patients (0%), respectively. Only one patient (1.2%) harboured two P. gingivalis rgpB genotypes (NSSK/NYPN) before treatment; these were no longer detected after therapy. CONCLUSION: The results indicate that five rgpB genotypes are maintained in natural populations of P. gingivalis. These data may be of importance with regard to the development of specific rgpB inhibitors.
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Infecciones por Bacteroidaceae , Cisteína Endopeptidasas/genética , Hemaglutininas/genética , Periodontitis/microbiología , Porphyromonas gingivalis/genética , Adhesinas Bacterianas , Adolescente , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Femenino , Genotipo , Cisteína-Endopeptidasas Gingipaínas , Humanos , Masculino , Persona de Mediana Edad , Periodontitis/terapiaRESUMEN
1. The calcium antagonist, Ro 40-5967, is metabolized to a multitude of products by the rat and drug-related material is excreted predominantly via the bile. 2. Diode-array u.v. spectroscopy, following reverse phase h.p.l.c. separation of the partially purified metabolites, has been used to classify these compounds into six spectral classes which have been correlated with different metabolic reactions. 3. Connection of a mass spectrometer directly to the h.p.l.c. equipment by a thermospray interface, produced useful mass spectra. These, together with the u.v. spectra, enabled the structures of many metabolites to be elucidated. 4. Confirmation of structural assignments was provided by n.m.r. spectra of the major metabolites. 5. Major metabolic pathways included N-demethylation (16% of the biliary metabolites), hydrolysis of the ester side-chain (32%), hydroxylation at 4- (19%) and 5- (29%) positions of the benzimidazole ring, aromatization of the tetrahydronaphthyl system (26%), loss of the benzimidazole (15%) and glucuronidation of hydroxyl groups (81%).
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Bencimidazoles/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Tetrahidronaftalenos/metabolismo , Animales , Cromatografía Líquida de Alta Presión/métodos , Cromatografía en Capa Delgada/métodos , Espectroscopía de Resonancia Magnética/métodos , Masculino , Espectrometría de Masas/métodos , Mibefradil , Ratas , Espectrofotometría Ultravioleta/métodos , Análisis Espectral/métodosRESUMEN
We compared genetic differentiation among populations of the threatened massasauga rattlesnake (Sistrurus c. catenatus) using two types of nuclear molecular markers: randomly amplified polymorphic DNA (RAPD) markers and microsatellites. Analyses of molecular variance (AMOVA) and G(ST) and F(ST) analyses indicated that levels of among-population differentiation between regional populations (>100 km) were comparable for both markers. However, microsatellites were superior in population assignment tests and at discerning fine-scale genetic differentiation between subpopulations separated by tens of kilometers. These results argue that both types of markers are suitable for defining broad-scale genetic structures in snake populations and can provide important inputs into conservation initiatives of focal taxa. However, our analyses suggest that microsatellites 3re better for detecting structure at limited spatial scales.
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Crotalus/genética , Cartilla de ADN , ADN Satélite , Animales , Crotalus/clasificación , Marcadores Genéticos , Variación Genética , Técnica del ADN Polimorfo Amplificado AleatorioRESUMEN
Throughout its distribution in North America, the threatened eastern massasauga rattlesnake (Sistrurus c. catenatus) persists in a series of habitat-isolated disjunct populations of varying size. Here, we use six microsatellite DNA loci to generate information on the degree of genetic differentiation between, and the levels of inbreeding within populations to understand how evolutionary processes operate in these populations and aid the development of conservation plans for this species. Samples were collected from 199 individuals from five populations in Ontario, New York and Ohio. Our results show that all sampled populations: (i) differ significantly in allele frequencies even though some populations are < 50 km apart, and may contain genetically distinct subpopulations < 2 km apart; (ii) have an average of 23% of alleles that are population specific; and (iii) have significant FIS values (mean overall FIS = 0.194) probably due to a combination of Wahlund effects resulting from fine-scale genetic differentiation within populations and the presence of null alleles. Our results imply that massasauga populations may be genetically structured on an extremely fine scale even within continuous populations, possibly due to limited dispersal. Additional information is needed to determine if dispersal and mating behaviour within populations can account for this structure and whether the observed differentiation is due to random processes such as drift or to local adaptation. From a conservation perspective, our results imply that these massasauga populations should be managed as demographically independent units and that each has high conservation value in terms of containing unique genetic variation.
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Crotalus/genética , Genética de Población , Animales , Variación Genética/genética , Endogamia , Repeticiones de Microsatélite , New York , Ohio , OntarioRESUMEN
Three 5-HT receptors have been implicated in retinal processing but positive identification of the receptors and the localization of receptor subtypes in the retina have not been achieved. In this study, molecular techniques were used to identify one class of 5-HT receptor--5-HT2a--in the retina, and immunohistochemical techniques were used to localize the receptor in the retinal network. Reverse transcription polymerase chain reaction (RT-PCR) techniques were used to identify a segment of the rabbit 5-HT2a gene; a 422 base fragment was identified, cloned, and sequenced. The fragment shows a high degree (ca. 90%) of nucleotide sequence identity with the 5-HT2a receptor gene from other mammals. 5-HT2a immunoreactivity was seen in both the inner and outer plexiform (synaptic) layers of the retina. Using cell-type-specific markers, the 5-HT2a immunoreactivity was shown to be on the terminals of photoreceptor and rod bipolar cells. This association of 5-HT2a receptors with these two synapses suggests that serotonin may be a modulator of synaptic function in the retina.
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Terminales Presinápticos/química , Receptores de Serotonina/análisis , Retina/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Interneuronas/química , Datos de Secuencia Molecular , Células Fotorreceptoras de Vertebrados/química , Pliegue de Proteína , Conejos , Receptor de Serotonina 5-HT2A , Receptores Presinapticos/análisis , Receptores de Serotonina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
The development of the HIV protease inhibitor saquinavir (Ro 31-8959) required a range of analytical methods for its measurement in biological fluids. This paper describes the development of isocratic, reverse-phase HPLC/UV methods for the routine measurement of plasma levels of the drug together with a more sensitive radioimmunoassay. The performance of the two assays is compared with that of an HPLC/MS/MS method previously published and has been shown to be satisfactory, with coefficients of variation of calibration standards and quality control samples within the usual outside limits of +/- 15%. The HPLC/UV method can be routinely applied for concentrations down to 10-20 ng/ml and a lower limit of quantification of 1 ng/ml from 1 ml of human plasma is possible. The radioimmunoassay was developed for the specific measurement of saquinavir concentrations in human, HIV-positive plasma samples and has a lower limit of quantification of 0.5-1.0 ng/ml. Some preliminary findings suggested that it might not be specific in rat plasma and no attempts have been made to quantify any nonclinical samples with this technique. If still greater sensitivity is required, recourse can be made to the HPLC/MS/MS assay.
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Cromatografía Líquida de Alta Presión/métodos , Inhibidores de la Proteasa del VIH/sangre , Radioinmunoensayo/métodos , Saquinavir/sangre , Anticuerpos , Estabilidad de Medicamentos , Humanos , Control de Calidad , Trazadores Radiactivos , Reproducibilidad de los Resultados , Espectrofotometría Ultravioleta , Vacunas Sintéticas/químicaRESUMEN
A procedure for covalent immobilization of functional proteins on silica substrates was developed using thiol-terminal silanes and heterobifunctional cross-linkers. Using this procedure, a high density of functional antibodies was bound to glass cover slips and silica fibers. The amount of anti-IgG antibody immobilized was determined to be in the range of 0.66 to 0.96 ng/mm2 using radiolabeled antibody. The relative amount of IgG antigen bound by the immobilized antibody (0.37 to 0.55 mol antigen/mol antibody) was three to five times greater than other investigators have reported. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was further reduced by the addition of blocking agents so that nonspecific adsorption of protein antigens represented only 2-6% of the total antigen binding. With this low background, IgG antigen binding could be measured at levels as low as 150 fmol when an antigen concentration of 3 pmol/ml was applied. The process for antibody immobilization is straightforward, easy to perform, and adaptable for modifying mass quantities of biosensor components.