Use of a thin film biosensor for rapid visual detection of PCR products in a multiplex format.

Rapid, sensitive assays for nucleic acid amplification products have utility for the identification of bacterial or viral infections. We have developed a nucleic acid hybridization assay utilizing thin film technology that permits visual detection of hybrids. The silicon-based biosensor detects the presence of target sequences by enzymatically transducing the formation of nucleic acid hybrids into molecular thin films. These films alter the interference pattern of light on the biosensor surface, producing a perceived color change. We have applied this technology to the development of a chip containing capture probes specific for human respiratory virus sequences including respiratory syncytial virus, influenza virus A and B, parainfluenza virus types 1 and 3, and rhinovirus. In a ten-minute assay, the biosensor permits unambiguous identification of viral-specific RT/PCR products from infected cell lysates.

Rapid multiserotype detection of human rhinoviruses on optically coated silicon surfaces.

BACKGROUND: More than 100 immunologically distinct serotypes of human rhinoviruses (HRV) have been discovered, making detection of surface exposed capsid antigens impractical. However, the non-structural protein 3C protease (3Cpro) is essential for viral replication and is relatively highly conserved among serotypes, making it a potential target for diagnostic testing. The thin film biosensor is an assay platform that can be formatted into a sensitive immunoassay for viral proteins in clinical specimens. The technology utilizes an optically coated silicon surface to convert specific molecular binding events into visual color changes by altering the reflective properties of light through molecular thin films. OBJECTIVE: To develop a rapid test for detection of HRV by developing broadly serotype reactive antibodies to 3Cpro and utilizing them in the thin film biosensor format. STUDY DESIGN: Polyclonal antibodies to 3Cpro were purified and incorporated into the thin film assay. The in vitro sensitivity, specificity and multiserotype cross-reactivity of the 3Cpro assay were tested. Nasal washes from naturally infected individuals were also tested to verify that 3Cpro was detectable in clinical specimens. RESULTS: The 3Cpro assay is a 28-min, non-instrumented room temperature test with a visual limit of detection of 12 pM (picomolar) 3Cpro. In terms of viral titer, as few as 1000 TCID(50) equivalents of HRV2 were detectable. The assay detected 45/52 (87%) of the HRV serotypes tested but showed no cross-reactivity to common respiratory viruses or bacteria. The thin film assay detected 3Cpro in HRV-infected cell culture supernatants coincident with first appearance of cytopathic effect. Data are also presented demonstrating 3Cpro detection from clinical samples collected from HRV-infected individuals. The assay detected 3Cpro in expelled nasal secretions from a symptomatic individual on the first day of illness. In addition, 9/11 (82%) concentrated nasal wash specimens from HRV infected children were positive in the 3Cpro test. CONCLUSION: We have described a novel, sensitive thin film biosensor for rapid detection of HRV 3Cpro. This test may be suitable for the point of care setting, where rapid HRV diagnostic test results could contribute to clinical decisions regarding appropriate antibiotic or antiviral therapy.

Differential expression and activation of p38 mitogen-activated protein kinase alpha, beta, gamma, and delta in inflammatory cell lineages.

Four p38 mitogen-activated protein kinases (p38alpha, beta, gamma, delta) have been described. To understand the role of p38 family members in inflammation, we determined their relative expression in cells that participate in the inflammatory process. Expression was measured at the level of mRNA by reverse-transcriptase PCR and protein by Western blot analysis. p38alpha was the dominant form of p38 in monocytes; expression of p38delta was low and p38beta was undetected. In macrophages, p38alpha and p38delta were abundant, but p38beta was undetected. p38alpha and p38delta were also expressed by neutrophils, CD4+ T cells, and endothelial cells. Again, p38beta was not detected in neutrophils, although low amounts were present in CD4+ T cells. In contrast, p38beta was abundant in endothelial cells. p38gamma protein was not detected in any cell type, although p38gamma mRNA was present in endothelial cells. Immunokinase assays showed a strong activation of p38alpha and a lesser activation of p38delta in LPS-stimulated macrophages. Abs specific for mono- and dual-phophorylated forms of p38 suggested that LPS induces dual phosphorylation of p38alpha, but primarily mono-phosphorylation of p38delta. IL-1beta activated p38alpha and p38beta in endothelial cells. However, p38alpha was the more activated form based on kinase assays and phosphorylation analysis. Expression and activation patterns of p38alpha in macrophages and endothelial cells suggest that p38alpha plays a major role in the inflammatory response. Additional studies will be needed to define the contribution of p38delta to macrophage, neutrophil, and T cell functions, and of p38beta to signaling in endothelial cells and T cells.

STCP-1 (MDC) CC chemokine acts specifically on chronically activated Th2 lymphocytes and is produced by monocytes on stimulation with Th2 cytokines IL-4 and IL-13.

STCP-1 stimulated T cell chemoattractant protein-1 (STCP-1) (macrophage-derived chemokine; MDC), a recently described CC chemokine for chronically activated T lymphocytes, was found to act specifically on a subset of memory CD4 lymphocytes that displayed a Th2 cytokine profile. Also, STCP-1, thymus and activation regulated chemokine (TARC), eotaxin, and eotaxin-2 acted specifically on in vitro derived Th2 lymphocytes, while IP-10 (IFN-gamma-inducible 10-kDa protein) showed some preference for Th1 lymphocytes. The corresponding receptors for eotaxin, TARC, and IP-10 are also differentially expressed on Th1 and Th2 lymphocytes. In desensitization Ca flux experiments, TARC and STCP-1 bound to a common receptor and therefore at least one chemokine receptor for STCP-1 is CCR4. STCP-1 expression is restricted to immune cells. Dendritic cells, B cells, and macrophages produce STCP-1 constitutively, while NK cells, monocytes, and CD4 lymphocytes produce STCP-1 upon appropriate stimulation. Production of STCP-1 is positively modulated by Th2 cytokines IL-4 and IL-13 but inhibited by IL-10.