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Biomedical subjects

H La

Publications and source records attributed to H La.

7 recordsLinked to original sources
PubMed2001

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.

3C Viral Proteases↗
PubMed2001

Silicon-based biosensors for rapid detection of protein or nucleic acid targets.

BACKGROUND: We developed a silicon-based biosensor that generates visual, qualitative results or quantitative results for the detection of protein or nucleic acid targets in a multiplex format. METHODS: Capture probes were immobilized either passively or covalently on the optically coated surface of the biosensor. Intermolecular interactions of the immobilized capture probe with specific target molecules were transduced into a molecular thin film. Thin films were generated by enzyme-catalyzed deposition in the vicinity of the surface-bound target. The increased thickness on the surface changed the apparent color of the biosensor by altering the interference pattern of reflected light. RESULTS: Cytokine detection was achieved in a 40-min multiplex assay. Detection limits were 4 ng/L for interleukin (IL)-6, 31 ng/L for IL1-beta, and 437 ng/L for interferon-gamma. In multianalyte experiments, cytokines were specifically detected with signal-to-noise ratios ranging from 15 to 80. With a modified optical surface, specificity was also demonstrated in a nucleic acid array with unambiguous discrimination of single-base changes in a 15-min assay. For homozygous wild-type and homozygous mutant samples, signal-to-noise ratios of approximately 100 were observed. Heterozygous samples yielded approximately equivalent signals for wild-type and mutant capture probes. CONCLUSIONS: The thin-film biosensor allows rapid, sensitive, and specific detection of protein or nucleic acid targets in an array format with results read visually or quantified with a charge-coupled device camera. This biosensor is suited for multianalyte detection in clinical diagnostic assays.

Biosensing Techniques↗
PubMed1999

Multicolor immunofluorescence and flow cytometry utilizing cascade blue to purify murine hematopoietic stem cells from fetal liver and bone marrow.

BACKGROUND: Here we demonstrate the utility of cascade blue (CB), to purify hematopoietic stem cells by flow cytometry. Multicolor immunofluorescence and the sensitivity (signal-to-noise) of the fluorochromes are essential for the identification and isolation of rare stem cell populations. METHODS: We isolated hematopoietic stem cells utilizing a 407 nm laser line to excite CB and propidium iodide (PI) in combination with FITC, PE, and Red670 which were excited at 488 nm. RESULTS: CB is maximally excited using a 407 nm laser line, when compared to UV or 413 nm excitation. The increase in sensitivity of CB at 407 nm can be contributed to higher absorption of CB and a reduction of autofluorescence at this excitation wavelength (Ropp et al.: Cytometry 21: 309-317, 1995). CONCLUSIONS: Despite the fact that the CB antibody conjugate has a tendency to adhere specifically to a B cell subpopulation in bone marrow, we nevertheless could purify stem cells by using CB for the detection and elimination of lineage positive cells. Isolated stem cells from mouse fetal liver (Lin-CD34(+)Sca-1(+)c-Kit(high)) and adult bone marrow (Lin-CD34(-/low)Sca-1(+)c-Kit(+)) were transplanted into lethally irradiated mice, and the sorted stem cells had the ability to efficiently repopulate all mature hematopoietic lineages in recipient mice.

Animals↗
PubMed1998

Hematopoietic stem cell maintenance and differentiation are supported by embryonic aorta-gonad-mesonephros region-derived endothelium.

Hematopoietic stem cells are capable of extensive self-renewal and expansion, particularly during embryonic growth. Although the molecular mechanisms involved with stem cell maintenance remain mysterious, it is now clear that an intraembryonic location, the aorta-gonad-mesonephros (AGM) region, is a site of residence and, potentially, amplification of the definitive hematopoietic stem cells that eventually seed the fetal liver and adult bone marrow. Because several studies suggested that morphologically defined hematopoietic stem/progenitor cells in the AGM region appeared to be attached in clusters to the ventrally located endothelium of the dorsal aorta, we derived cell lines from this intraembryonic site using an anti-CD34 antibody to select endothelial cells. Analysis of two different AGM-derived CD34(+) cell lines revealed that one, DAS 104-8, efficiently induced fetal-liver hematopoietic stem cells to differentiate down erythroid, myeloid, and B-lymphoid pathways, but it did not mediate self-renewal of these pluripotent cells. In contrast, a second cell line, DAS 104-4, was relatively inefficient at the induction of hematopoietic differentiation. Instead, this line provoked the expansion of early hematopoietic progenitor cells of the lin-CD34(+)Sca-1(+)c-Kit+ phenotype and was proficient at maintaining fetal liver-derived hematopoietic stem cells able to competitively repopulate the bone marrow of lethally irradiated mice. These data bolster the hypothesis that the endothelium of the AGM region acts to mediate the support and differentiation of hematopoietic stem cells in vivo.

Animals↗