Biomedical subjects

Hung Lee

Publications and source records attributed to Hung Lee.

26 records · Page 2Linked to original sources

PubMed2003

Emerging trends in the synthesis and improvement of hapten-specific recombinant antibodies.

A key requirement for successful immunotherapeutic and immunodiagnostic applications is the availability of antibodies with high affinity and specificity. In the past, polyclonal antibodies from hyperimmunized animals or monoclonal antibodies from hybridoma cell lines were used extensively and profitably in medicine and immunotechnology. Antibody-based diagnostics, such as immunoassays, are also widely accepted because of their high sensitivity and ease of use as compared to conventional chromatographic techniques. While immunoassays have been used to monitor organic chemical contaminants such as pesticides, food preservatives, antibiotics in agricultural and food industries, hapten-specific antibodies with the desired affinity and specificity are generally difficult to obtain. With the advent of recombinant DNA technology, antibody genes can be amplified and selected through phage display, cell surface display, or cell-free display systems. A particularly useful feature common to all these display systems is the linking of the phenotype and genotype of antibodies during selection. This allows easy co-selection of the desired antibodies and their encoding genes based on the binding characteristics of the displayed antibodies. The selected antibody DNA can be further manipulated for high-level expression, post-translation modification, and/or affinity and specificity improvement to suit their particular applications. Several hapten-specific antibodies, which were successfully selected and engineered to high specificity and affinity using display technologies, have been found to be amenable to conventional immunoassay development. In this review, we will examine different formats of immunoassays designed for hapten identification and various display technologies available for antibody selection and improvement.

Antibodies↗

PubMed2002

Identification of lysine-78 as an essential residue in the Saccharomyces cerevisiae xylose reductase.

Yeast xylose reductases are hypothesized as hybrid enzymes as their primary sequences contain elements of both the aldo-keto reductases (AKR) and short chain dehydrogenase/reductase (SDR) enzyme families. During catalysis by members of both enzyme families, an essential Lys residue H-bonds to a Tyr residue that donates proton to the aldehyde substrate. In the Saccharomyces cerevisiae xylose reductase, Tyr49 has been identified as the proton donor. However, the primary sequence of the enzyme contains two Lys residues, Lys53 and Lys78, corresponding to the conserved motifs for SDR and AKR enzyme families, respectively, that may H-bond to Tyr49. We used site-directed mutagenesis to substitute each of these Lys residues with Met. The activity of the K53M variant was slightly decreased as compared to the wild-type, while that of the K78M variant was negligible. The results suggest that Lys78 is the essential residue that H-bonds to Tyr49 during catalysis and indicate that the active site residues of yeast xylose reductases match those of the AKR, rather than SDR, enzymes. Intrinsic enzyme fluorescence spectroscopic analysis suggests that Lys78 may also contribute to the efficient binding of NADPH to the enzyme.

Aldehyde Reductase↗

PubMed2002

Is human immunodeficiency virus RNA load composed of neutralized immune complexes?

During acute human immunodeficiency virus (HIV) infection, both virus load (HIV RNA) and infectivity are high (10(3)-10(7) RNA copies/mL or TCID(50)/mL) until antibody is produced, which may reduce the HIV infectivity. In HIV carriers, the HIV RNA load is elevated (10(3)-10(5) copies/mL), but infectivity is low (10(0)-10(2) TCID(50)/mL). The low infectivity in carriers could be due to neutralization by antibody in serum, resulting in immune complexes (ICs). We demonstrated that ICs in plasma, prepared with protein A beads, contained HIV RNA (80%-100%) in association with immunoglobulin G (IgG). In comparison, ICs from patients with acute HIV infection and little or no antibody contained virtually no HIV RNA. Moreover, ICs prepared by ultrafiltration contained IgG and specifically and irreversibly neutralized HIV, which indicates that the ICs contained neutralizing antibody. These findings indicate that the HIV RNA in the plasma of carriers is frequently composed of antibody-neutralized HIV as ICs.

Antigen-Antibody Complex↗

PubMed2002

Green fluorescent protein-based biosensor for detecting SOS-inducing activity of genotoxic compounds.

Increasing levels of environmental pollution demand specific and sensitive methods for detection of genotoxic agents in water, food products and environmental samples. Tests for genotoxicity assessment are often based on biosensor strains that respond to DNA damage induced by chemicals. In the present study, fluorescent reporter Escherichia coli strains have been developed, which contain a plasmid-borne transcriptional fusion between the DNA-damage inducible recA promoter and the green fluorescent protein gene (gfp) or a gene encoding a red-shifted, higher intensity GFP variant (mutant 3). GFP-based biosensors allowed the detection of a dose-dependent response to genotoxic agents such as mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and nalidixic acid (NA). A reporter strain carrying recA'-gfp mutant 3 fusion gave more dramatic and sensitive response than a strain containing the wild-type gfp. These results indicate that recA'-gfp mutant 3-based biosensor is potentially useful for detection of genotoxins.

Biosensing Techniques↗

PubMed2002

Efficacy of common laboratory disinfectants on the infectivity of Cryptosporidium parvum oocysts in cell culture.

Nine liquid disinfectants were tested for their ability to reduce infectivity of Cryptosporidium parvum oocysts in cell culture. A 4-min exposure to 6% hydrogen peroxide and a 13-min exposure to ammonium hydroxide-amended windshield washer fluid reduced infectivity 1,000-fold. Other disinfectants tested (70% ethanol, 37% methanol, 6% sodium hypochlorite, 70% isopropanol, and three commercial disinfectants) did not reduce the infectivity after a 33-min exposure. The results indicate that hydrogen peroxide and windshield washer fluid or ammonium hydroxide disinfectant may be suitable laboratory disinfectants against C. parvum oocysts.

Ammonium Hydroxide↗

PubMed2002

Green fluorescent protein-labeled recombinant fluobody for detecting the picloram herbicide.

A green fluorescent protein-labeled fluobody was designed to develop a simple immunoassay method for detecting picloram herbicide in an environmental sample. The gfp gene was successfully inserted into the pSJF2 vector harboring the picloram-specific antibody fragment to yield pSJF2GFP. Picloram spiking in an environmental river sample could be indirectly detected by observing the fluorescence intensity value of the gfp-fluobody, exhibiting specific sensitivity to free picloram with an IC50 value of 50 ppb. Using the gfp-fluobody immunoassay avoids the enzyme-substrate reaction for calorimetric detection that is required in an enzyme-linked immunosorbent assay (ELISA).

Base Sequence↗

PubMed2002

Influence of temperature on Cryptosporidium parvum oocyst infectivity in river water samples as detected by tissue culture assay.

Cryptosporidium parvum oocysts were stored in 1-ml aliquots of filtered river water at -20, 4, 10, and 21-23 C in the dark. Oocysts were also added to filter-sterilized river water samples and stored at 21-23 C. The infectivity of oocysts stored under different conditions was assayed at weekly intervals through infection of human adenocarcinoma ileocecal (HCT-8) cell monolayers. Wells containing between 10 and 100 foci of infection were enumerated by immunofluorescent microscopy, and the number of infective oocysts was calculated. No infectious oocysts were detected after 1 wk at -20 C. The number of infective oocysts stored at 4 C decreased 5-fold, and the number of those stored at 10 C decreased 2.5-fold after 14 wk. The infectivity of oocysts stored in potassium dichromate (positive control) at 4 C decreased 2-fold over 14 wk. The number of infective oocysts in filter-sterilized and non-filter-sterilized river water stored at 21-23 C decreased by 3.3 and 2.6 log units, respectively, over 12 wk, and no foci of infection were detected at 14 wk. The results show that as temperature increased from 4 to 23 C, the duration of oocyst infectivity decreased.

Animals↗

PubMed1988

Induction of Xylose Reductase and Xylitol Dehydrogenase Activities in Pachysolen tannophilus and Pichia stipitis on Mixed Sugars.

The induction of xylose reductase and xylitol dehydrogenase activities on mixed sugars was investigated in the yeasts Pachysolen tannophilus and Pichia stipitis. Enzyme activities induced on d-xylose served as the controls. In both yeasts, d-glucose, d-mannose, and 2-deoxyglucose inhibited enzyme induction by d-xylose to various degrees. Cellobiose, l-arabinose, and d-galactose were not inhibitory. In liquid batch culture, P. tannophilus utilized d-glucose and d-mannose rapidly and preferentially over d-xylose, while d-galactose consumption was poor and lagged behind that of the pentose sugar. In P. stipitis, all three hexoses were used preferentially over d-xylose. The results showed that the repressibility of xylose reductase and xylitol dehydrogenase may limit the potential of yeast fermentation of pentose sugars in hydrolysates of lignocellulosic substrates.

Journal Article↗