RESUMEN
Most protease-substrate assays rely on short, synthetic peptide substrates consisting of native or modified cleavage sequences. These assays are inadequate for interrogating the contribution of native substrate structure distal to a cleavage site that influences enzymatic cleavage or for inhibitor screening of native substrates. Recent evidence from HIV-1 isolates obtained from individuals resistant to protease inhibitors has demonstrated that mutations distal to or surrounding the protease cleavage sites in the Gag substrate contribute to inhibitor resistance. We have developed a protease-substrate cleavage assay, termed the cleavage enzyme- cytometric bead array (CE-CBA), which relies on native domains of the Gag substrate containing embedded cleavage sites. The Gag substrate is expressed as a fluorescent reporter fusion protein, and substrate cleavage can be followed through the loss of fluorescence utilizing flow cytometry. The CE-CBA allows precise determination of alterations in protease catalytic efficiency (k(cat)/K(M)) imparted by protease inhibitor resistance mutations in protease and/or gag in cleavage or noncleavage site locations in the Gag substrate. We show that the CE-CBA platform can identify HIV-1 protease present in cellular extractions and facilitates the identification of small molecule inhibitors of protease or its substrate Gag. Moreover, the CE-CBA can be readily adapted to any enzyme-substrate pair and can be utilized to rapidly provide assessment of catalytic efficiency as well as systematically screen for inhibitors of enzymatic processing of substrate.
Asunto(s)
Farmacorresistencia Viral/genética , Pruebas de Enzimas/instrumentación , Citometría de Flujo/instrumentación , Proteasa del VIH/metabolismo , VIH-1/enzimología , Mutación , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Sitios de Unión , Proteasa del VIH/genética , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/genética , Ensayos Analíticos de Alto Rendimiento , Cinética , Microesferas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Subvisible particles in formulations intended for parenteral administration are of concern in the biopharmaceutical industry. However, monitoring and control of subvisible particulates can be complicated by formulation components, such as the silicone oil used for the lubrication of prefilled syringes, and it is difficult to differentiate microdroplets of silicone oil from particles formed by aggregated protein. In this study, we demonstrate the ability of flow cytometry to resolve mixtures comprising subvisible bovine serum albumin (BSA) aggregate particles and silicone oil emulsion droplets with adsorbed BSA. Flow cytometry was also used to investigate the effects of silicone oil emulsions on the stability of BSA, lysozyme, abatacept, and trastuzumab formulations containing surfactant, sodium chloride, or sucrose. To aid in particle characterization, the fluorescence detection capabilities of flow cytometry were exploited by staining silicone oil with BODIPY 493/503 and model proteins with Alexa Fluor 647. Flow cytometric analyses revealed that silicone oil emulsions induced the loss of soluble protein via protein adsorption onto the silicone oil droplet surface. The addition of surfactant prevented protein from adsorbing onto the surface of silicone oil droplets. There was minimal formation of homogeneous protein aggregates due to exposure to silicone oil droplets, although oil droplets with surface-adsorbed trastuzumab exhibited flocculation. The results of this study demonstrate the utility of flow cytometry as an analytical tool for monitoring the effects of subvisible silicone oil droplets on the stability of protein formulations.
Asunto(s)
Anticuerpos Monoclonales/química , Citometría de Flujo/métodos , Inmunoconjugados/química , Muramidasa/química , Albúmina Sérica Bovina/química , Aceites de Silicona/química , Abatacept , Adsorción , Anticuerpos Monoclonales Humanizados , Química Farmacéutica , Emulsiones , Tamaño de la Partícula , Espectrometría de Fluorescencia/métodos , Tensoactivos/química , TrastuzumabRESUMEN
Traditional log-scale data display of multiparameter immunofluorescence cytometry data has several perplexing intrinsic problems that are largely mitigated by recent transformation alternatives that are log-like at the high end of the scale, near linear at the low end of the scale, and symmetrical about zero. These alternative log-like display transformations provide a means for better interpretation and analysis of compensated data.
Asunto(s)
Citometría de Flujo/métodos , Fluorescencia , Técnica del Anticuerpo Fluorescente , Cómputos Matemáticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Intensificación de Imagen Radiográfica , Programas InformáticosRESUMEN
A frequent goal of flow cytometric analysis is to classify cells as positive or negative for a given marker, or to determine the precise ratio of positive to negative cells. This requires good and reproducible instrument setup, and careful use of controls for analyzing and interpreting the data. The type of controls to include in various kinds of flow cytometry experiments is a matter of some debate and discussion. In this tutorial, we classify controls in various categories, describe the options within each category, and discuss the merits of each option.