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Biomedical subjects

T T Ngo

Publications and source records attributed to T T Ngo.

At least 37 records · Page 2Linked to original sources
PubMed1990

Chemistry and preparation of affinity ligands useful in immunoglobulin isolation and serum protein separation.

A number of synthetic affinity gels having high affinity for immunoglobulins and albumin have been prepared by first reacting hydroxyl groups of a polymer with pentafluoropyridine and 4-dimethylaminopyridine in an anhydrous polar organic solvent and then reacting the gel further with nucleophiles such as ethyleneglycol or glycine in basic aqueous solutions. Immunoglobulins can be adsorbed to the gel in either high-salt or low-salt buffers, while albumin can only be adsorbed under low-salt conditions. The identity of the eluted proteins was analyzed by gradient polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay techniques. Human, goat, mouse and rabbit serum proteins were fractionated on these gels by using different adsorption and desorption conditions. The possible structures of the ligand are discussed. The results showed that the chromatographic behavior of these new gels with synthetic, low-molecular-weight ligands was remarkably similar to that of the more complex immunoglobulin binding gel such as immobilized Protein A or Protein G.

Animals↗
PubMed1990

Simple, quick and efficient site-directed antibody immobilization in a cartridge.

A method is described for the simple, quick and efficient attachment of antibody within a cartridge for use as an immunoaffinity chromatography column. Antibodies are immobilized via their Fc regions through the use of periodate-oxidized carbohydrate functionalities of the immunoglobulin G. The method allows for the in situ coupling of the immunoglobulin G without prior removal of the oxidizing periodate solution. The entire procedure can be completed in 50 minutes. This method is especially useful for quick determinations of a particular monoclonal antibody's functionality or avidity towards a specific antigen. It may also be used in place of a conventional immunoaffinity column for the rapid isolation of small amounts of an antigen. This method will reduce the lengthy process of preparing an immunoaffinity column from several days to less than an hour.

Antibodies↗
PubMed1987

Spectrophotometric assay for ornithine decarboxylase.

A rapid and sensitive spectrophotometric assay for ornithine decarboxylase is described. It is based on the observation that the product of ornithine decarboxylase, putrescine, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a colored product soluble in 1-pentanol whereas ornithine does not. The amount of putrescine produced by the enzyme was determined by measuring the absorbance of the 1-pentanol extract of the reaction mixture at 420 nm, and by comparing the results to those obtained by the trapping of 14CO2 and by HPLC assays. The three assays were found to be equivalent in sensitivity, with the spectrophotometric assay having the advantages of being relatively rapid, requiring only common laboratory equipment, and not requiring the use of radioactive isotopes.

Ornithine Decarboxylase↗
PubMed1986

Single-enzyme-based amperometric assay for phosphate ion.

An amperometric assay for phosphate ion, using a single enzymatic process, has been developed. The enzyme used was pyruvate oxidase, which catalyzes the oxidation of pyruvate in the presence of oxygen and phosphate ion. The products of the enzymatic reaction are acetyl phosphate, carbon dioxide, and hydrogen peroxide. The latter was monitored by means of a hydrogen peroxide electrode and an oxidase meter. Phosphate ion in the concentration range of 50-500 microM can be measured within 4 min. Anions, such as pyrophosphate, sulfate, nitrate, nitrite, and acetate, gave only marginal responses.

Electrochemistry↗
PubMed1986

A simple spectrophotometric determination of solid supported amino groups.

A simple spectrophotometric method for the quantitative determination of solid phase supported amino groups is described. The method involves reacting the solid support with an excess of activated acylating agent, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and an efficient acylation catalyst, 4-dimethylaminopyridine, and after thoroughly removing the unreacted SPDP, the solid support is reacted with an excess of dithiothreitol to quantitatively release pyridine-2-thione from the solid support to the solution. After an appropriate dilution, the released pyridine-2-thione which has a strong absorbance at 343 nm, is quantified by reading its absorbance in a spectrophotometer at 343 nm.

4-Aminopyridine↗
PubMed1985

Identification of potential GABA-mimetics by their actions on brain GABA recognition sites.

In an attempt to identify potential anticonvulsant compounds, 18 structural analogues of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) were tested for their ability to inhibit GABA receptor binding, sodium-dependent GABA binding and GABA aminotransferase activity in synaptic membranes from mouse brain. Nine inhibitors of receptor binding were found. The most potent was N-(thiocarbamoyl)glycine (Ki = 18 microM). However, this compound had no real effect on Na+ -dependent GABA binding nor on the activity of GABA aminotransferase. In addition, it was unable to enhance the binding of [3H]flunitrazepam as GABA agonists usually do. This could indicate that this inhibitor is, rather, a GABA receptor antagonist. Even though no particularly potent inhibitors of any of the GABA recognition sites were found, this technique nevertheless demonstrates how simple in vitro assays can be used to find drugs exhibiting potential GABA-mimetic activity.

4-Aminobutyrate Transaminase↗
PubMed1983

Antibody-induced conformational restriction as basis for new separation-free enzyme immunoassay.

Under acid denaturing conditions, hologlucose oxidase labeled with 2,4-dinitrophenyl (DNP) was dissociated into flavin adenine dinucleotide (FAD) and DNP-labeled apoglucose oxidase (DNP-AG). Both lacked catalytic activity. The activity was restored by combining FAD and DNP-AG at about pH 7. If, on the other hand, anti-DNP serum was preincubated with the DNP-AG prior to the addition of FAD, activity was not restored. Furthermore, added DNP-aminocaproic acid counteracted the effects of the antibody in inhibiting the recombining of DNP-AG and FAD to form active enzyme. The anti-DNP serum probably prevented the DNP-AG from combining with FAD to form an active holoenzyme by restricting the mobility of the polypeptide chain of DNP-AG from folding into a catalytically active conformation. Based on such an antibody-induced conformational restriction of the DNP-AG, we developed a separation-free (homogeneous) enzyme immunoassay called AICREIA.

Apoenzymes↗
PubMed1983

Tyrosine decarboxylase. Spectrophotometric assay and application in determining pyridoxal-5'-phosphate.

We have developed a highly sensitive and rapid spectrophotometric assay for tyrosine decarboxylase that can be applied to determining pyridoxal-5'-phosphate. In the assay, tyramine, a product of tyrosine decarboxylation, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a product soluble in toluene whereas tyrosine does not. We determined the amount of tyramine produced enzymatically by reading the absorbance at 340 nm of a toluene extract of the reaction mixture. This method is capable of detecting as low as 2.9 micrograms/mL of the enzyme. Using this method, we find the Km for tyrosine decarboxylase from Streptococcus faecalis to be 3.55 X 10(-4)M. We have also developed a specific and extremely sensitive method for determining pyridoxal-5'-phosphate, a cofactor of the enzyme, by using this spectrophotometric assay with apotyrosine decarboxylase.

Enterococcus faecalis↗
PubMed1983

Amperometric assay for collagenase. Amplification by the use of glucose oxidase conjugated to insoluble collagen.

A sensitive and rapid amperometric assay for collagenase has been developed. The substrate for the assay is glucose oxidase covalently linked to insoluble collagen with dimethylsuberimidate. The collagenase cleaves the insoluble collagen--glucose oxidase conjugate into smaller, soluble fragments that have glucose oxidase activity. That activity is proportional to the collagenase activity hydrolyzing the insoluble conjugate. In the absence of collagenase, no glucose oxidase activity is found in the soluble phase. Glucose oxidase activity was assayed by measuring amperometrically the rate at which hydrogen peroxide is produced. The kinetics follow that proposed for a soluble enzyme acting on an insoluble substrate.

Animals↗
PubMed1983

Enzyme modulator mediated immunoassay (EMMIA).

1. Enzyme modulator mediated immunoassay (EMMIA) is a separation-free (homogeneous) enzyme amplified immunoassay. 2. The assay is based on the ability of an analyte labeled enzyme modulator to modify the activity of an indicator enzyme and on ability of an anti-analyte antibody to abrogate the modifying action of the analyte-labeled enzyme modulator upon its binding to anti-analyte antibody. 3. The principle of EMMIA is elaborated in detail. The stages in the development of an EMMIA are described. 4. Criteria in selecting enzymes and modulators suitable for EMMIA are discussed. 5. Examples of EMMIA's using different kinds of modulators for different classes of analytes are presented.

Amino Acid Sequence↗
PubMed1983

Kinetics of rat brain soluble catechol-O-methyltransferase and its inhibition by substrate analogues.

1. The initial rates and inhibition of rat brain catechol-O-methyltransferase were studied. Double reciprocal plots of initial rates versus either S-adenosyl-L-methionine or 3,4-dihydroxybenzoic acid, in the absence of product, gave a series of lines intersecting to the left of the ordinate. 2. Inhibition in the presence of S-adenosyl-L-homocysteine was competitive but in the presence of vanillic acid was non-competitive if S-adenosyl-L-methionine was the varied substrate. 3. When 3,4-dihydroxybenzoic acid was the varied substrate, both S-adenosyl-L-homocysteine and vanillic acid gave rise to a non-competitive inhibition. 4. The initial rate and product inhibition patterns were consistent with an ordered BiBi mechanism with S-adenosyl-L-methionine being the first substrate and 3,4-dihydroxybenzoic acid the second substrate to combine with the enzyme. 5. In addition, these results suggest that vanillic acid is the first product and S-adenosyl-L-homocysteine the second product to dissociate from the enzyme. 6. The substrate analogues salsolinol and 3-carboxysalsolinol were competitive inhibitors with respect to 3,4-dihydroxybenzoic acid but were non-competitive with respect to S-adenosyl-L-methionine. For enzymes with an ordered mechanism an uncompetitive inhibition would be expected. 7. A possible explanation is that both substrate analogues can combine with either free enzyme with lower affinity or with an intermediary enzyme form with much greater affinity. 8. A scheme which is consistent with the data is presented.

Animals↗
PubMed1982

Enzymes as versatile labels and signal amplifiers for monitoring immunochemical reactions.

Enzymes have proven to be sensitive and versatile labels for immunochemical assays. The sensitivity of an enzyme label stems from its extraordinary catalytic power which in turn provides a great amplification of signals. Its versatility, however, stems from the fact that enzyme activity can be modulated by a number of substances. Enzyme labeled immunoassays can be divided into two categories: (a) heterogeneous and (b) homogeneous (non-separation). In the heterogeneous systems, the quantitation of the antibody bound and unbound fractions requires a physical separation of these two fractions, whereas the homogeneous or non-separation systems do not require such a separation. In the homogeneous systems, the unbound and antibody bound fractions can be distinguished functionally. A total of 11 unique principles used in the development of enzyme labeled immunoassays are described. The advantages, disadvantages and limitations of them are considered, as well as the future paths for research and developments.

Antigens↗
PubMed1982

Competitive inhibition of gamma-aminobutyric acid synaptosomal uptake by 4-(4'-azidobenzoimidylamino)butanoic acid.

4-(4'-Azidobenzoimidylamino)butanoic acid (ABBA) is a potent inhibitor of rat brain synaptosomal [3H]gamma-aminobutyric acid uptake. Ki values were calculated to be 8 microM and 16 microM with respect to the high-affinity and the low-affinity uptake processes. These values are of the same order as those reported for nipecotic acid and guvacine, which until now have been the most potent uptake inhibitors available. Since ABBA contains a phenyl group, it might be capable of penetrating the blood-brain barrier, thus becoming a useful GABA mimetic.

Animals↗
PubMed1982

Optimizing the o-phenylenediamine assay for horseradish peroxidase: effects of phosphate and pH, substrate and enzyme concentrations, and stopping reagents.

Horseradish peroxidase, assayed with o-phenylenediamine, is irreversibly inactivated when incubated in phosphate buffer, 100 mmol/L, at pH 5. The inactivation depends on both duration and incubation and phosphate concentration. Phosphate was the most potent inactivator and citrate the least potent of a series of buffers tested. The inactivation is not attributable to ionic strength per se or to Na+ or K+. The observed inactivation did not occur at high concentrations (2500 nmol/L, 0.1 g/L) of enzyme; however, this "protective" effect could not be reproduced by adding bovine serum albumin or a surfactant (Tween 20) to lower concentrations of enzyme. The inactivation was independent of commercial source of the enzyme or the kind of chromogenic assay used. On the basis of this information, we optimized the assay so that it gave eightfold greater absorbance values than those reported by others. The improved assay was sensitive to as little as 0.4 pmol/L (16 ng/L) of peroxidase, and was linear over the range of 0.4 to 5 pmol/L (16-200 ng/L).

Buffers↗
PubMed1981

p-Azidophenylglyoxal. A heterobifunctional photoactivable cross-linking reagent selective for arginyl residues.

1. The synthesis described is of p-azidophenylglyoxal (p-APG) by diazotization of p-aminoacetophenone to an intermediate which when reacted with sodium azide gives p-azidoacetophenone; oxidation of the latter with selenium dioxide gives rise to p-APG (corrected melting point, 103-105 degrees C). The phenylglyoxal moiety was designed to react with arginine residues, whereas the p-azidoaryl function generates a reactive nitrene when activated with UV light; p-APG reacts most selectively with arginine and to a lesser extent with cystine and histidine. 2. p-APG has absorption peaks at 205 and 280 nm which decrease on photolysis. 3. Bovine heart lactic dehydrogenase, egg white lysozyme, horse liver alcohol dehydrogenase, and yeast alcohol dehydrogenase, all enzymes having arginyl residues at their active sites, are inhibited by p-APG in the dark. 4. Gel electrophoresis of oligomeric enzymes having arginyl residues at their active sites and exposed to p-APG and to UV irradiation gave varying proportions of monomers and photocross-linked dimers, trimers, tetramers, and larger molecular weight aggregates.

Alcohol Oxidoreductases↗
PubMed1981

Homogeneous substrate-labeled fluorescent immunoassay for IgG in human serum.

A homogeneous substrate-labeled fluorescent immunoassay for IgG has been developed. Purified IgG was covalently labeled with 6-(7-beta-galactosylcoumarin-3-carboxamido)-hexylamine to form a stable conjugate, GU-IgG. The galactosyl residue was hydrolyzed from GU-IgG by beta-galactosidase and the progress of the hydrolysis was monitored by the increase in fluorescence emission at 450 nm with excitation at 400 nm. Antibody to IgG diminished the activity of GU-IgG as a substrate for beta-galactosidase. Competitive binding immunoassays were conducted by allowing added IgG and GU-IgG to compete for a limited number of antibody binding sites. Hence, the fluorescence produced by enzymic hydrolysis increased with the level of added IgG. This method provides a simple and reliable immunoassay for IgG and is applicable to other proteins.

Antibodies↗