RESUMO
BACKGROUND: Drug tolerance of anti-drug antibody (ADA) assays is becoming increasingly important due to the high number of newly emerging long-acting drugs. Methods to estimate and improve drug tolerance in ADA assays are needed. RESULTS: The relevance and precision of drug tolerance estimates in a bridging ELISA depended on characteristics and concentration of the surrogate control antibody together with assay cut point level. Stepwise and coincubation procedures were optimized for drug tolerance and sensitivity by adapting concentrations of reagents used for capture and detection of ADAs. In combination with acid treatment of samples, increase in drug tolerance of over 20-fold was achieved without losing sensitivity in two different assays. Acid treatment reduced drug interference observed in preclinical samples and prevented underestimation of ADA response. CONCLUSION: In a risk-based approach it may be possible to reliably predict in vivo drug tolerance using carefully chosen surrogate controls. General guidelines for development of drug-tolerant bridging ELISAs are presented.
Assuntos
Anticorpos Anti-Idiotípicos/sangue , Tolerância a Medicamentos , Ensaio de Imunoadsorção Enzimática , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Complexo Antígeno-Anticorpo/imunologia , Biotina , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Imunidade Humoral , Proteínas Recombinantes/imunologia , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Serina Proteases/imunologia , EstreptavidinaRESUMO
We identify a human mutation (E1053K) in the ankyrin-binding motif of Na(v)1.5 that is associated with Brugada syndrome, a fatal cardiac arrhythmia caused by altered function of Na(v)1.5. The E1053K mutation abolishes binding of Na(v)1.5 to ankyrin-G, and also prevents accumulation of Na(v)1.5 at cell surface sites in ventricular cardiomyocytes. Ankyrin-G and Na(v)1.5 are both localized at intercalated disc and T-tubule membranes in cardiomyocytes, and Na(v)1.5 coimmunoprecipitates with 190-kDa ankyrin-G from detergent-soluble lysates from rat heart. These data suggest that Na(v)1.5 associates with ankyrin-G and that ankyrin-G is required for Na(v)1.5 localization at excitable membranes in cardiomyocytes. Together with previous work in neurons, these results in cardiomyocytes suggest that ankyrin-G participates in a common pathway for localization of voltage-gated Na(v) channels at sites of function in multiple excitable cell types.
Assuntos
Anquirinas/metabolismo , Regulação da Expressão Gênica , Proteínas Musculares/metabolismo , Mutação de Sentido Incorreto/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Canais de Sódio/metabolismo , Sequência de Aminoácidos , Animais , Anquirinas/antagonistas & inibidores , Linhagem Celular , Membrana Celular/metabolismo , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Humanos , Cinética , Camundongos , Dados de Sequência Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Miocárdio/citologia , Miocárdio/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5 , Ligação Proteica , Canais de Sódio/química , Canais de Sódio/genética , SíndromeRESUMO
Interactions with ankyrinG are crucial to the localization of voltage-gated sodium channels (VGSCs) at the axon initial segment and for neurons to initiate action potentials. However, the molecular nature of these interactions remains unclear. Here we report that VGSC-alpha, but not -beta, subunits bind to ankyrinG using pull-down assays. Further dissection of this activity identifies a conserved 9-amino acid motif ((V/A)P(I/L)AXXE(S/D)D) required for ankyrinG binding. This motif is also required for the localization of chimeric neurofascin/sodium channel molecules to the initial segment of cultured hippocampal neurons. The conserved nature of this motif suggests that it functions to localize sodium channels to a variety of "excitable" membrane domains both inside and outside of the nervous system.
