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Biomedical subjects

M C Stuart

Publications and source records attributed to M C Stuart.

52 records · Page 3Linked to original sources
PubMed1986

Incidence and specificity of interference in two-site immunoassays.

Using a modified "two-site" immunoradiometric assay, termed an "interference assay," we have demonstrated the occurrence of non-analyte antibody-binding substances in approximately 40% of serum samples. These substances multivalently bind antibodies from any of several species at a site other than the antigen-binding site. Using a two-site immunoradiometric assay for human choriogonadotropin, we have investigated their effect on analyte quantification. In this system these antibody-binding substances mimic the presence of analyte; when analyte is actually present, they can also cause over- or underestimates. Addition of excess nonspecific immunoglobulin from an appropriate species eliminates this interference. However, the amount of nonspecific immunoglobulin added to an assay system may not always be enough to block interference in all samples.

Adult↗
PubMed1985

Covert cross reactants in a two-site immunoassay studied with monoclonal antibodies.

A one-step two-site immunoradiometric assay for the measurement of free beta subunit of human chorionic gonadotrophin (beta-hCG) was developed using monoclonal antibodies. The immobilized antibody was specific for free beta subunit and the radiolabeled antibody recognized both intact human chorionic gonadotrophin (hCG) and free beta subunit. Although the level of hCG "cross-reaction" was low when studied using conventional techniques, the apparent beta-hCG content of samples was found to be inversely proportional to the hCG level. From both experimental evidence and computer simulation studies this was found to be due to the binding of hCG to the limited amount of 125I-labeled antibody present. The term covert cross reactants has been introduced to describe substances which bind to only one of the antibodies in a two-site immunoassay. When establishing such an assay the effect of covert cross reactants on the response of an analyte should be investigated.

Antibodies, Monoclonal↗
PubMed1983

The production of high affinity monoclonal antibodies to human growth hormone.

The primary aim of this work was to produce specific monoclonal antibodies to human growth hormone (hGH) for use in a diagnostic RIA of hGH levels in serum. Three different schedules were used for immunization of BALB/c mice and the splenocytes from each mouse were fused with myeloma cells Sp 2/0 Ag 14. Each fusion resulted in the production of hundreds of hybridomas secreting hGH-directed antibodies. Six antibodies have been fully characterized and have been grouped into pairs which recognize 3 different epitopes on the hGH molecule. One pair exhibits no cross reaction with the structurally related placental hormone, human placental lactogen (hPL), a second pair has low cross reaction with hPL (1.6-3%) and a third pair reacts equally well with hGH and hPL indicating binding to a common epitope in the 2 molecules. The highest affinity antibody, 74/6, which has an affinity constant of 4.4 X 10(10) l/mol and 3% cross-reactivity with hPL, has been used to establish a RIA for serum hGH measurements. Evidence is provided that hGH levels measured in this assay correlate well with those obtained in a conventional rabbit antiserum assay.

Animals↗
PubMed1983

The production of monoclonal antibodies to human chorionic gonadotrophin and its subunits.

The difficulties encountered in producing highly specific antisera to human chorionic gonadotrophin (hCG) were overcome by the use of hybridoma technology. A panel of monoclonal antibodies directed toward hCG and its subunits was produced. Of the four antibodies which were fully characterized, one recognized the intact hCG molecule only, a second recognized only the free beta-subunit, a third recognized only the free alpha-subunit and the fourth bound to the beta-subunit of hCG both when it was in the free form and when it was associated with the alpha-subunit forming the intact hCG molecule. There was no significant cross-reaction of any of these antibodies with the pituitary glycoprotein hormones. The four antibodies had high binding affinities which should permit their use in immunoassays for measurement of circulating levels of hCG and its subunits.

Animals↗
PubMed1983

Use of immunoaffinity chromatography for purification of 125I-labeled human prolactin.

We assessed a simple method for purifying 125I-labeled human prolactin, taking advantage of the abundant supplies of monoclonal antibodies available. 125I-Labeled human prolactin purified by immunoaffinity chromatography is compared with that purified by gel filtration on Sephadex G-100. We used monoclonal antibodies to prolactin to prepare the affinity chromatography columns. Prolactin was radiolabeled by the Chloramine T method, purified by each of the above procedures, and the binding and displacement characteristics were studied in radioimmunoassays in which either monoclonal antibodies or a rabbit anti-prolactin serum was the first antibody. A nonimmune fraction of 125I-labeled prolactin that co-eluted with the immunoreactive hormone from Sephadex G-100 was removed by affinity chromatography, which increased the antibody binding of 125I-labeled prolactin in the radioimmunoassay in the absence of unlabeled antigen (B/T0, in percent) twofold or more, increased the assay sensitivity, and increased the slope of antigen displacement measured by the 50% intercept. Several advantages make this the purification method of choice.

Antibodies, Monoclonal↗
PubMed1982

A monoclonal antibody suitable for the radioimmunoassay of prolactin in human serum.

Monoclonal antibodies directed toward human PRL (hPRL) have been produced by fusion of mouse myeloma cells (Sp2/0-Ag 14) with spleen cells from mice immunized with hPRL. Total immunizing doses of 20 microgram and 64 microgram hPRL resulted in the production of three highly specific hPRL antibodies. The high affinity antibody, with a Ka value of 0.23 X 10(10) M-1, was used to establish a RIA highly suitable for the measurement of hPRL levels in human serum. The correlation of serum hPRL levels measured using the antibody and those in a conventional rabbit anti-hPRL assay was 0.99 (y = 1.16 - 7.2). These results demonstrate that using the mouse hybridoma technique, it is possible to produce high affinity monospecific monoclonal antibody suitable for the measurement of hPRL in human serum.

Antibodies, Monoclonal↗
PubMed1981

Lectins used to prepare serum free of glycoprotein hormones for use as a matrix in radioimmunoassay.

Hormone-free serum is required for use as a matrix for standards in the radioimmunoassay of hormones. The glycoprotein hormones are difficult to remove from serum by conventional techniques. We exploited the specific binding of carbohydrates by lectins to extract glycoprotein hormones from human serum. Passing serum over Concanavalin A Sepharose efficiently removed lutropin and the beta subunit of choriogonadotropin. Wheat-Germ Lectin Sepharose completely removed these, and also follitropin and thyrotropin. The latter treatment is shown to provide a suitable matrix for standards in radioimmunoassays of the four hormones.

Blood Chemical Analysis↗
PubMed1976

Somatomedin production in the neonatal rat.

The hormonal stimulus to rat fetal and neonatal somatic and skeletal growth has been investigated by a study of ciruclating somatomedin (SM), growth hormone (GH) and insulin levels in rats from 21 days in utero to 40 days of post natal life. Somatomedin activity could not be detected in the serum of fetal rats in which circulating GH and insulin levels were high. In early post natal life GH and insulin levels remained high but gradually declined reaching normal adult levels at 19 days and 40 days respectively. Somatomedin activity was measurable only at low levels until 11 days after birth and thereafter remained steady throughout the time period studied. These studies suggest that somatomedin alone is not responsible for the rapid growth of the rat in early neonatal life and it is suggested that insulin may also be active as a growth factor in this period.

Age Factors↗
PubMed1976

Hormonal regulation of fetal brain cell proliferation: presence in serum of a trophin responsive to pituitary growth hormone stimulation.

Previous studies led us to hypothesize that brain growth was regulated by a growth hormone-dependent brain trophin. An in vitro bioassay system to assess this proposal was developed. Serum was shown to stimulate the uptake of tritiated thymidine into fetal brain cell DNA. This action could not be attributed to any nutrient contribution but was due to a non-dialyzable, heat-stable serum growth factor. The levels of the growth factor in serum were reduced after pituitary removal. When added in physiological concentration, growth hormone, prolactin, placental lactogen, insulin, nerve growth factor, thyroid and steroid hormones failed to stimulate the action of serum or to act synergistically with a serum component to stimulate DNA synthesis. Thyroxine, estradiol-17beta, and corticosterone inhibited serum activity. Administration of growth hormone to hypophysectomized rats restored serum levels to normal demonstrating that the serum growth factors was a mediating trophin responsive to pituitary growth hormone stimulation. The relationship of the brain trophin to other serum growth factors and its specificity of action remain to be defined. The present findings were in accordance with in vivo studies of hormonal influence on brain growth and support the proposal that fetal brain cell proliferation is stimulated by a serum trophin responsive to pituitary growth hormone.

Animals↗
PubMed1975

Somatomedins.

An intact pituitary gland capable of secreting growth hormone has long been considered the prime requirement for the achievement of skeletal growth potential in man. Recent studies have revealed that the growth-promoting action of growth hormone is an in-vivo phenomenon which cannot be mimicked by the addition of the hormone to skeletal tissue in vitro. The humoral agent responsible for skeletal growth has now been identified as somatomedin, a peptide produced in the liver under the stimulus of pituitary growth hormone. Serum levels of somatomedin are measured in a bioassay system by monitoring the stimulation of uptake of labelled sulphate by cartilage. Low levels of somatomedin activity are detected in the serum of children with growth hormone deficiency and short stature; the levels are high in acromegalics and low in patients with cirrhosis of the liver or chronic renal failure. Undernourished children also have low levels despite reaised serum levels of growth hormone; this suggests the presence of an inhibitor which lowers the growth-promoting activity of the somatomedin molecule. Adequate nutrition in these children results in the restoration of serum somatomedin levels to normal. Attempts to isolate and purify somatomedin have led to the identification of a group of substances sharing similar actions on skeletal tissue. Insulin has also been demonstrated to share some of these growth-promoting activities but varies in its organ specificity. Nerve growth factor, epidermal growth factor and proinsulin are other molecules which form a large group of growth promoting peptides which may all be related to the somatomedins.

Acromegaly↗
PubMed1974

Fetal brain growth: selective action by growth hormone.

Growth hormone was administered to pregnant rats maintained under dietary control, and fetal and placental growth and nutrition were examined. Growth hormone had a selective action on brain growth that could not be attributed to nutrient mobilization but suggested a trophic factor which is unique to the brain.

Animals↗
PubMedDate not supplied

Dual effect of tannic acid on the preservation and ultrastructure of phosphatidyl choline vesicles.

The use of tannic acid has been proposed to improve the preservation of phospholipids in tissues. We investigated the effects of tannic acid on the preservation of small unilamellar vesicles, prepared from sonicated aqueous suspensions of phospholipids. With cryo-electron microscopy it is demonstrated that small unilamellar vesicles are formed after sonication of the phospholipid suspensions. Fixation of vesicles without tannic acid results in extraction of the phospholipids during dehydration and embedding. Fixation of vesicles containing phosphatidyl choline with tannic acid, with or without glutaraldehyde, results in a fast (within a second) aggregation of the vesicles and the resulting sediment can be dehydrated and embedded when a postfixation in osmium tetroxide is carried out. Small unilamellar vesicles fixed in this way are retrieved in thin sections as multilamellar vesicles with a periodicity of about 5 nm for dimyristoylphosphatidyl choline and about 6 nm for dioleoylphosphatidyl choline. By using 14C-phosphatidyl choline it was also demonstrated that tannic acid prevents to a large extent the extraction of phosphatidyl choline during fixation, dehydration and embedding. This dual effect of tannic acid on phosphatidyl choline, aggregation and fixation, should be considered when using tannic acid in tissue preparation.

Freezing↗