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G Barnard

Publications and source records attributed to G Barnard.

At least 37 records · Page 2Linked to original sources
PubMed1991

Idiometric assay, the third way: a noncompetitive immunoassay for small molecules.

A novel, noncompetitive immunoassay applicable to the measurement of small molecules including ovarian steroids is described. Using monoclonal antibodies with the ability to recognize both beta-typic and alpha-typic binding sites, a new simplified, sensitive, and specific immunoassay system has been developed. The initial work of this development is presented, along with preliminary results for a novel immunoassay for estradiol.

Antibodies, Anti-Idiotypic↗
PubMed1990

Idiometric assay: noncompetitive immunoassay for small molecules typified by the measurement of estradiol in serum.

We report here a novel noncompetitive immunoassay applicable to the measurement of small-molecular-mass compounds and typified by the direct measurement of estradiol in serum. Two types of anti-idiotypic antibody recognize different epitopes within the hypervariable region of the specific primary antibody (e.g., anti-estradiol). The first anti-idiotype (betatype) recognizes an epitope at the unoccupied binding site, which is masked in the presence of the analyte (e.g., estradiol). The second (alphatype) recognizes an epitope close to the binding site and is unaffected by the presence or absence of the analyte, but is sterically hindered from binding to the primary antibody in the presence of the betatype. The use of these matched antibodies (primary antibody, alphatype, and betatype) has enabled the development of a method for determining antibody occupancy that is not based on a conventional two-site assay. An excess amount of purified monoclonal antibody against estradiol is immobilized onto the walls of microtiter wells. After the capture of analyte, the unoccupied antibody sites are blocked by the addition of an excess amount of betatype. Subsequently, analyte occupancy is determined by the addition of excess europium-labeled alphatype, incubation, washing, and time-resolved fluorometry. The method demonstrates good sensitivity, precision, and comparability with alternative competitive immunoassays.

Antibodies, Monoclonal↗
PubMed1989

The development of non-separation time-resolved fluoroimmunoassays for the measurement of urinary metabolites.

We describe the principles of a new generation of sequential or simultaneous time-resolved fluoroimmunoassays, namely, simple, rapid, liquid-phase non-separation procedures which may be applied to the measurement of urinary steroid and drug metabolites. As an example, a method for the measurement of estrone-3-glucuronide in undiluted urine is reported. This method has a similar sensitivity, specificity and accuracy to a conventional separation fluoroimmunoassay or radioimmunoassay but in terms of speed, convenience, precision, reliability and clinical utility the new method has many advantages. The labelled antigen is a novel fluorescent europium chelate covalently linked to estrone-3-glucuronide. The antibody-binding reaction involves the incubation of the labelled antigen (2ng) with a limited concentration of polyclonal or monoclonal antibodies to estrone-3-glucuronyl-6-BSA and an aliquot of standard or sample (undiluted urine; 10 microliters) in microtitre wells. After a 10 min incubation, the fluorescence which emanates from the antibody-free label is measured in a time-resolved fluorometer and is proportional to the concentration of estrone-3-glucuronide in the standard or sample. The method may be applied for the monitoring of ovarian function in women.

Estrone↗
PubMed1989

A nonseparation, time-resolved fluoroimmunoassay to monitor ovarian function and predict potential fertility in women.

The authors describe a nonseparation, time-resolved fluoroimmunoassay involving the use of monoclonal antibodies for the measurement of estrone-3-glucuronide in urine. The method has appropriate sensitivity (12 nmol/l), better specificity than a conventional radioimmunoassay with polyclonal antibodies, and the advantages of speed, simplicity, less imprecision, and improved clinical effectiveness. The labeled antigen is a novel fluorescent europium chelate covalently linked to estrone glucuronide at carbon 6 of the glucuronide moiety. The antibodies were raised against estrone-3-glucuronyl-6-bovine serum albumin. The antibody binding reaction is performed in microtiter wells (or tubes) and involves the addition of labeled antigen in buffer (2 ng/100 microliters), a limited concentration of antibodies in buffer (100 microliters), and standard or urine sample (10 microliters). The mixture is incubated for 15 minutes at room temperature. Time-resolved fluorescence from the unbound label is proportional to the concentration of estrone glucuronide. The method may be used to monitor ovarian function and potential fertility in women.

Estrone↗
PubMed1989

Somatotropin as measured by a two-site time-resolved immunofluorometric assay.

To date, many of the current criteria for diagnosis of somatotropin (growth hormone, GH) deficiency have been based upon measurement of this hormone by competitive radioimmunoassay (RIA) with use of polyclonal antibodies. In recent years, however, the development of hybridoma technology has led to the generation of various monoclonal antibodies (Mabs) to GH with different affinities and epitope specificities. Subsequently, these reagents have been used in the development of noncompetitive two-site immunometric assays (e.g., immunoradiometric assay; IRMA). In general, the values obtained for serum GH by IRMA have been lower than those obtained by RIA, because of the epitope-specificity profile of the Mabs in the IRMA. Attempting to obtain GH values numerically similar to those by RIA, we used a combination of Mabs to GH in developing and evaluating a two-site time-resolved immunofluorometric assay (IFMA) based on the streptavidin-biotin interaction. Fluorescence is proportional to concentration of analyte and is linearly related to concentration over the range 0.3 to 40 micrograms/L. The assay was satisfactory with respect to sensitivity, accuracy, and precision (CV less than 10% over the entire working range). In addition, the concentration of GH was determined by the IFMA and a competitive RIA in serum obtained from GH deficient and acromegalic patients. The pairing of antibodies in the IFMA gave numerical values that agreed well with those by RIA (r = 0.97; n = 100).

Acromegaly↗
PubMed1989

Measurement of estrone-3-glucuronide in urine by rapid, homogeneous time-resolved fluoroimmunoassay.

We describe a liquid-phase nonseparation time-resolved fluorescence immunoassay for measuring estrone-3-glucuronide in undiluted urine. The sensitivity, specificity, and accuracy are similar to those for a conventional separation fluoroimmunoassay or radioimmunoassay, but the speed, convenience, precision, reliability, and clinical utility of the new method are more advantageous. The labeled antigen, a fluorescent europium chelate covalently linked to estrone-3-glucuronide, is incubated for 10 min with a limited concentration of polyclonal or monoclonal antibodies to estrone-3-glucuronyl-6-bovine serum albumin and 10 microL of standard or sample (undiluted urine) in microtiter wells. The fluorescence emanating from the antibody-free label, which is proportional to the concentration of estrone-3-glucuronide in the standard or sample, is then measured in a time-resolved fluorometer. The method is useful for monitoring ovarian function in women.

Antibodies, Monoclonal↗
PubMed1988

Sexual maturity as a criterion for classification of phallometric stimulus slides.

With the acceptance of behavior modification techniques in the 1970's as the standard modality of treatment of sex offenders, the ability to diagnose the paraphilic arousal patterns became of primary importance. The use of the penile plethysmograph in the assessment of sex offenders is regarded as the most accurate measure of these sexual arousal patterns. This method measures the vasocongestive engorgement of the penile corporea while the offender is exposed to deviant and nondeviant sexual stimuli. The stimuli may be slides of nude males and females of varying ages, audiotaped descriptions of erotic scenarios or videotaped scenes of sexual interaction. Researchers who have used slides to determine the age and gender preference of the sex offenders have typically categorized the slides in arbitrarily established age categories that have varied from one research study to another. The use of age as a criterion does not take into account the variation in growth and maturation among children and adolescents. To increase reliability and standardization of research techniques, the authors propose that the stages of sexual maturity as set forth by Tanner (1962, 1978) be used in lieu of age.

Adolescent↗
PubMed1988

The development of fluorescence immunoassays.

Radioactive markers have been of inestimable value in the establishment of immunoassays for compounds of analytical interest. It is anticipated, however, that the trend towards the development of alternative fluorescence immunoassays will continue. For example, where extreme assay sensitivity is not required, the development of simple nonseparation FIAs (e.g., fluorescence polarization FIA) will continue. However, the lanthanide chelates can be measured with high sensitivity and the specific activity of the labelled conjugates may exceed that of conjugates prepared with radioisotopes. Consequently, this approach has great potential for achieving higher sensitivity than can be obtained by conventional IRMA procedures. The components of an assay are harmless and easily disposed of. The labelled materials are very stable, showing no significant deterioration after storage for more than a year. The approach has been successfully exploited in the development of two-site immunofluorometric assays of proteins and direct, competitive binding assays of haptens. In the future, it is anticipated that the introduction of new fluorescent probes and appropriate instrumentation will facilitate the introduction of tests which may be performed in the ward, in the outpatient clinic, or in the home.

Antibodies↗
PubMed1981

Use of monoclonal antibodies to pregnanediol-3 alpha-glucuronide for the development of a solid phase chemiluminescence immunoassay.

Monoclonal antibodies to pregnanediol-3 alpha-glucuronide were produced by hybridomas between P3-X63-Ag8 variants and spleen cell of mice immunized with a bovine serum albumin conjugate of the homologous hapten. The ascites fluid collected from mice inoculated with the cloned hybridoma cells contained antibodies with high specificity and affinity to pregnanediol-3 alpha-glucuronide. A sensitive solid-phase chemiluminescence immunoassay for urinary pregnanediol-3 alpha-glucuronide was established utilizing these antibodies. The assay was validated in terms of specificity, accuracy, sensitivity and precision. When urine samples were assayed for pregnanediol-3 alpha-glucuronide, the results obtained by the solid-phase chemiluminescence immunoassay method and the conventional gas liquid chromatographic method agreed well (n = 30, r = 0.96). The method may be of value for monitoring luteal function since it is fast, sensitive and does not require the use of radioisotopes or purification of the biological sample. Monoclonal antibody preparations facilitate rigorous standardization of the assay.

Animals↗
PubMed1980

An assay for urinary estriol-16 alpha-glucuronide based on antibody-enhanced chemiluminescence.

An immunoassay for estriol-16 alpha-glucuronide in pregnancy urine is described that utilizes antibody-enhanced chemiluminescence. The steroid glucuronide was covalently conjugated with the chemiluminescent marker aminobutyl-ethyl-isoluminol. The light yield of this conjugate upon oxidation was augmented by specific antibody, and this effect was inhibited by addition of the homologous steroid glucuronide (10-100 pg) in a dose-dependent manner. The assay does not require separation of bound and free ligand and proved satisfactory with respect to sensitivity, precision and accuracy. Assay results obtained by radioimmunoassay and chemiluminescence immunoassay were in good agreement (r = 0.98; n = 25).

Antibody Specificity↗
PubMed1979

Effect of flurazepam, pentobarbital, and caffeine on arousal threshold.

After laboratory and procedure adaptation, 6 normal subjects were randomly administered 30 mg flurazepam (twice), 100 mg pentobarbital (twice), 400 mg caffeine (once), and placebo (twice) on nonconsecutive nights. On each night subjects were aroused from standard segments of stage 2 sleep five to eight times with an ascending series of 1,000 Hz tones produced by an audiometer. Arousal threshold and awake threshold after each arousal were measured. Both thresholds were increased by flurazepam and pentobarbital and decreased by caffeine. All of the drugs appeared to modify arousal threshold in a time course fashion such that extreme effects were found during the first half of the night. However, the modifications of waking threshold by caffeine and flurazepam continued throughout the sleep period. The method may be a means of measuring the behavioral time course of drug activity during the sleep period.

Adult↗
PubMed1976

Mechanism and stereochemistry of enzymic reactions involved in porphyrin biosynthesis.

5-Aminolaevulinate synthetase cataylses the condensation of glycine and succinyl-CoA to give 5-aminolaevulinic acid. At least two broad pathways may be considered for the initial C--C bond forming step in the reaction. In pathway A the Schiff base of glycine and enzyme bound pyridoxal phosphate (a) undergoes decarboxylation to give the carbanion (b) which then condenses with succinyl-CoA with the retention of both the original C2 hydrogen atoms of glycine. In pathway B, loss of a C2 hydrogen atom gives another type of carbanion (c) that reacts with succinyl-CoA. Evidence has been presented to show that the initial C--C bond forming event occurs via pathway B which involves the removal of the pro R hydrogen atom of glycine. Subsequent mechanistic and stereochemical events occurring at the carbon atom destined to become C5 of 5-aminolaevulinate have also been delineated.(Carticle) Several mechanistic alternatices for the formation of the two vinyl groups of haem from the propionate residues of the precursor, coproporphyrinogen III, have been examined. (see article). It is shown that during the biosynthesis both the hydrogen atoms resident at the alpha positions of the propionate side chains remain undisturbed thus eliminating mechanisms which predict the involvement of acrylic acid intermediates. Biosynthetic experiments performed with precursors containing stereospecific labels have shown that the two vinyl groups of haem are formed through the loss of pro S hydrogen atoms from the beta-positions of the propionate side chains. In the light of these results, three related mechanisms for the conversion, propionate leads to vinyl, have been considered. In order to study the mechanism of porphyrinogen carboxy-lyase reaction, stereo-specifically deuterated, tritiated-succinate was incorporated into the acetate residues of uroporphyrinogen III which on decarboxylation generated asymmetric methyl groups in coproporphyrinogen III and then in haem. Degradation of the latter yielded chiral acetate deriving from C and D rings of haem. Configurational analysis of this derivate acetate shows that the carboxy-lyase reaction proceeds with a retention of configuration.

5-Aminolevulinate Synthetase↗