RESUMEN
It has been reported that 90% of the anti-drug antibody (ADA) to Adalimumab in human patients bound to the TNF-binding area, resulted in the annual loss of responses to Adalimumab up to 24%. It is of urgency to develop a cost-effective and easy-to-use ADA diagnostic kit for diagnosis of potential drug-resistance in patients treated with Adalimumab in clinic hospitals to avoid the tremendous economic and human costs to patients and health-care providers. In this study, we reported the generations of mouse monoclonal and monkey polyclonal antibodies against Adalimumab as assay standards and positive quality controls respectively. A Bridging ELISA assay was successfully developed with a limit of detection (LOD) between 22-80ng/ml. The preliminary validation of assay was carried out first with 50 normal human sera, further validated by screening the ADA in 192 serum samples from monkeys treated with or without Adalimumab. Our data showed that the Bridging ELISA kit is very sensitive, highly specific and ready for study in human clinic trials.
Asunto(s)
Adalimumab/inmunología , Antiinflamatorios/inmunología , Anticuerpos Monoclonales/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Adalimumab/uso terapéutico , Animales , Antiinflamatorios/uso terapéutico , Análisis Costo-Beneficio , Resistencia a Medicamentos , Femenino , Humanos , Inmunoglobulina G/metabolismo , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C , Sensibilidad y EspecificidadRESUMEN
Surface display of the active proteins on living cells has enormous potential in the degradation of numerous toxic compounds. Here, we report the codisplay of organophosphorus hydrolase (OPH) and enhanced green fluorescent protein (GFP) on the cell surface of Escherichia coli by use of the truncated ice nucleation protein (INPNC) and Lpp-OmpA fusion systems. The surface localization of both INPNC-OPH and Lpp-OmpA-GFP was demonstrated by Western blot analysis, immunofluorescence microscopy, and a protease accessibility experiment. Anchorage of GFP and OPH on the outer membrane neither inhibits cell growth nor affects cell viability, as shown by growth kinetics of cells and stability of resting cultures. The engineered E. coli can be applied in the form of a whole-cell biocatalyst and can be tracked by fluorescence during bioremediation. This strategy of codisplay should open a new dimension for the display of multiple functional moieties on the surface of a bacterial cell. Furthermore, a coculture comprised of the engineered E. coli and a natural p-nitrophenol (PNP) degrader, Ochrobactrum sp. strain LL-1, was assembled for complete mineralization of organophosphates (OPs) with a PNP substitution. The coculture degraded OPs as well as PNP rapidly. Therefore, the coculture with autofluorescent and mineralizing activities can potentially be applied for bioremediation of OP-contaminated sites.
Asunto(s)
Arildialquilfosfatasa/metabolismo , Enzimas/metabolismo , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Organofosfatos/metabolismo , Arildialquilfosfatasa/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Western Blotting , Técnicas de Cocultivo , Enzimas/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/genética , Viabilidad Microbiana , Microscopía Fluorescente , Datos de Secuencia Molecular , Nitrofenoles/metabolismo , Ochrobactrum/genética , Ochrobactrum/metabolismo , Péptido Hidrolasas/metabolismo , ARN Ribosómico 16S/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADNRESUMEN
We report, the surface presentation of organophosphorus hydrolase (OPH) and green fluorescent protein (GFP) fusions by employing the adhesin-involved-in-diffuse-adherence (AIDA-I) translocator domain as a transporter and anchoring motif. The surface location of the OPH-GFP fusion protein was confirmed by immunofluorescence microscopy, and protease accessibility, followed by Western blotting analysis. The investigation of growth kinetics and stability of resting cultures showed that the presence of the AIDA-I translocator domain in the outer membrane neither inhibits cell growth nor affects cell viability. Furthermore, the surface-exposed OPH-GFP was shown to have enzymatic activity and a functional fluorescence moiety. These results suggest that AIDA-I autotransporter is a useful tool to present heterologous macromolecule passenger proteins on the bacterial surface. Our strategy of linking GFP to OPH and the possibility to employ various bacterial species as host has enormous potential for enhancing field use.
Asunto(s)
Adhesinas de Escherichia coli/fisiología , Arildialquilfosfatasa/metabolismo , Escherichia coli/enzimología , Organofosfatos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Arildialquilfosfatasa/genética , Biodegradación Ambiental , Escherichia coli/genética , Proteínas Fluorescentes Verdes/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
The cDNA of Alocasia macrorrhiza lectin (aml, GenBank accession number: DQ340864) was cloned by RACE-PCR and its characteristics were predicted by various bioinformatics tools. GSPs (Gene Specific Primers) were designed according to the conserved regions of the genes encoded for lectins and similar proteins from the same family Araceae. Total RNAs were extracted from the tubers of A macrorrhiza by Qiagen RNeasy mini kit. The 3'- and 5'-RACE-PCRs were performed with the isolated total RNAs by SMART(TM)RACE cDNA amplification kit from BD Biosciences Clontech Company, respectively. The purified PCR products were ligated with pMD 18-T vector, and the confirmed clones were sequenced. The full-length cDNA of aml was obtained by combination of 3'- and 5'-end sequences, and was then confirmed by full-length 3'-RACE-PCR. The aml cDNA is 1 124 bp long. The deduced amino acid length of AML lectin is 270 aa. Its relative molecular weight is 29.7 kD. The results of homologous analysis showed a high similarity between AML and other mannose-binding lectins and similar proteins from Araceae family. Two typical B-lectin domains and three mannose- binding motifs were found in the sequence of AML. With all these taken together, it can be concluded that this newly cloned aml cDNA encodes for a mannose-binding lectin.