Radioimmunoassay for 3,3',5'-triiodothyronine (reverse T3, R-T3) in unextracted human serum

Highly specific antibodies against 3,3',5'-triiodothyronine (reverse T3, R-T3) have been produced in rabbits. The crossreaction with T4 is about 0.05%. A radioimmunoassay for R-T3 in unextracted serum was developed. ANS is used for blocking the binding of tracer and endogenous R-T3 to TBG. The sensitivity to the assay is 0.06 ng/ml plasma. The mean normal R-T3 concentration is 0.20 ng/ml. Thyrotoxic patients show elevated levels; in most hypothyroid patients R-T3 concentrations are below the detection limit.     

Changes in the binding capacity of thyroxine-binding globulin (TBC), of total thyroxine (T4), of the free thyroxine index (FT4-I) and of thyrotropin (TSH) during normal pregnancy and in hydatidiform mole

TBC-index and total serum thyroxine were measured in 100 healthy nonpregnant and in 163 pregnant women during the 8. and 41. weeks of gestation. The free thyroxine index was calculated. The TBC-index was found to be elevated in pregnant women and rose continously with duration of pregnancy. The amount of total serum thyroxine was greater in pregnant women (p less than 0,01) without difference between early and late pregnancy. The free thyroxine index decreased continously during pregnancy (p less than 0,01). Serum TSH level were elevated during the first two trimesters of pregnancy. At the third trimester the TSH level were found within the normal range. In patients with hydatidiform mole TSH, total thyroxine, FT4-index, ETR-index as well as TSH levels were increased.     

Immunoassay for thyroxine (T4) in serum using capillary electrophoresis and micromachined devices

As part of our ongoing effort to develop electrophoretic assay technology for clinical diagnostics, we describe a competitive immunoassay for the determination of serum thyroxine (T4) based on electrophoresis and laser induced fluorescence (LIF). Measurements of total T4 are useful for the clinical evaluation of thyroid function. A fluorescein thyroxine conjugate was utilized in conjunction with a polyclonal antibody preparation as assay reagents. Capillary electrophoresis (CE) conditions tolerant of the direct injection of serum without extraction or other sample preparation steps were developed and used for quantitation of total T4 in serum. We have been exploring the use of micromachined devices with arrays of channels for high assay throughput. Our assay protocol was carried in a microchip format. The results illustrate that gains in speed can be additionally achieved, with the electrophoretic separation of free from bound labelled T4 being performed in about 15 s for serum samples.     

Triiodothyronine (T3) and thyroxine (T4) levels in Rana curtipes during development and metamorphosis

During premetamorphosis, levels of circulating triiodothyronine (T3) and thyroxine (T4) were below the limits of detection of RIA. They became detectable in late prometamorphic stages. A gradual increase in T3 and T4 was observed during this period. A sharp rise in hormone levels was apparent at the onset of metamorphic climax. Peak levels of both hormones were found at Taylor-Kollros stage XXI. The T3 reached a peak level of 101.4 ng/dl, about 3 fold increase over the level at prometamorphosis. Thereafter the circulating hormones (particularly T4) decreased rapidly and reached levels similar to prometamorphic stages. Significant high levels of thyroid hormones (T3 and T4) in metamorphic climax suggest that like other anurans, elevation of these hormones is required for normal metamorphosis in R. curtipes tadpoles.     

A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S

Human prothrombin, factor IX, and factor X have been idolated in high yield and characterized as the their amino-terminal sequence, molecular weight, amino acid composition, and migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional human plasma protein, called protein S, has also been purified and its properties have been compared with those of prothrombin, factor IX, and factor X. Prothrombin (mol wt 72 000), factor IX (mol wt 57 000), and protein S (mol wt 69 000) are single-chain glycoproteins, while factor X (mol wt 59 000) is a glycoprotein composed of two polypeptide chains held together by a disulfide bond(s). The amino-terminal sequence of the light chain of human factor X is homologous with prothrombin, factor IX, and protein S. The heavy chain of human factor X is slightly larger than the heavy chain of bovine factor X and differs from bovine factor X in its amino-terminal sequence.     

Expression of HIV env gene in a human T cell line for a rapid and quantifiable cell fusion assay

Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins present at the surface of infected cells are known to mediate fusion with CD4-positive target cells. In this study we have developed a novel Env-expressing cell line for investigating the fusion process in a biologically significant system. Cell surface expression of the HIV-1 env gene, isolated from the highly fusogenic strain SF33, was obtained in the CD4-negative T cell line A2.01. To render the system versatile and efficient, HIV-1 regulatory proteins Tat and Rev were supplied in trans. The presence of Env at the cell surface was shown by cytofluorometry and immunofluorescence and precursor processing of gp160 to gp120/gp41 was demonstrated by Western blot. The fusion capacity of A2.01-Env cells was assessed by coculture with CD4-positive T lymphocytes or the fusion indicator cell line, HeLa-CD4-LTR-beta-Gal. By coincubation with CD4-positive T cells such as SupT1, A2.01-Env cells were observed to mediate rapidly numerous well-defined syncytia in a reproducible fashion. By expressing Tat, they also had the capacity to trans-activate the LTR-linked reporter beta-Gal gene following fusion with HeLa-CD4-LTR-beta-Gal cells. The fusion-inhibiting anti-CD4 monoclonal antibodies Q425 and Q428 were used to block specifically Env-mediated fusion with CD4-positive cells and to demonstrate application of this system to the search for potential fusion-blocking agents. Our system thus offers a biologically significant model for studying fusion events with the advantages of being rapid, reproducible and versatile.     

A simple and rapid microSepharose assay for GAD65 and ICA512 autoantibodies in diabetes. Diabetes Incidence Study in Sweden (DISS)

Three scales, the Fake Bad Scale, the Fake Good Scale, and the Fake Bad-Fake Good scales were developed and evaluated with respect to their capacity to detect response manipulation on the Multiple Affect Adjective Check List-Revised. Cutting scores for each scale were cross-validated in two samples consisting of three groups: (1) college students simulating either "fake good" or "fake bad," (2) college students under standard instructions, and (3) psychiatric patients. Cutting scores on the three scales were compared with cutting scores established for the MAACL-R Dysphoria and Positive Affect plus Sensation Seeking. Analysis indicated that these scales were more accurate than the Positive Affect plus Sensation Seeking and the Dysphoria scales in detecting response manipulation.     

Determination of 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in human plasma by a simple and rapid high-performance liquid chromatography assay

In order to study the pharmacodynamic effects of drugs on dopamine and serotonin metabolism, a reversed-phase HPLC assay coupled with electrochemical detection (ECD) for measuring plasma concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA) was developed. The system was operated isocratically using a mobile phase of aqueous 0.03 M KH2PO4 buffer containing 0.15 mM EDTA in methanol (8.75:1.25), with a final pH of 4.0. The flow rate was set at 1.5 mL/min, and potentials at +450 mV. Using a signal to noise ratio of > 3, the minimum detection limit assessed by direct on-column injection of a standard solution for DOPAC and 5-HIAA was < 1 pg. The assays were linear from basal concentrations (1-10 ng/mL) to 100 ng/mL. The intra- and interassay variations were < 10% and < 20%, respectively.     

Sensitive enzyme-linked immunosorbent assay for human serum amyloid A (SAA) and application to clearance study

Serum amyloid A (SAA), an apolipoprotein of high density lipoprotein (HDL), is a sensitive acute phase reactant. We established a sandwich type enzyme-linked immunosorbent assay for human SAA utilizing a monoclonal antibody and polyclonal antibodies. This assay was sensitive enough to detect SAA at 40 pg/ml. The use of nonionic detergent, Tween-20, in reaction buffer was essential to enhance specific binding of SAA to antibodies and reduce nonspecific binding to plastic. The values by this assay showed a good agreement with those by previously established latex agglutination immunoassay. Plasma clearance of human SAA was studied in mice by injection with SAA-rich human HDL and serial SAA measurement by the present assay. Half-life of injected SAA was 55 minutes, similar to mean of the reported value of murine SAA isotypes.     

Molecular modelling of (A4T4NN)n and (T4A4NN)n: sequence elements responsible for curvature

The molecular modelling program JUMNA has been used to investigate the origins of the strikingly different curvature of the two sequences, (A4T4NN)n and (T4A4NN)n. Gel electrophoresis and cyclisation studies have shown that only the former of these two sequences is significantly curved. By developing novel superhelical symmetry constraints we were able to study the energetic and structural aspects of polymeric DNA having a controlled curvature. The results obtained (which do not take into account specific hydration effects) correlate well with the experimental data and offer a molecular level explanation of curvature. Although curvature is found to be initiated by specific dinucleotide junctions, deformations spread to surrounding dinucleotide steps and, moreover, sequence effects beyond the dinucleotide level are observed.     

Time-resolved immunofluorometric assay for the quantification of lipoprotein(a) in serum

Although two recent studies have failed to reveal lipoprotein(a) (LP(a)) serum concentrations > 300 mg/l to be an independent risk factor for early onset of atherosclerosis, Lp(a) serum concentrations are frequently measured to evaluate the additional risk of coronary heart disease. We describe a time-resolved immunofluorometric assay (TRIFMA) for quantifying Lp(a) levels in humans serum using commercially available reagents, which is rapid, robust and simple to perform. The two-site immunometric assay was based on microtitre plates as solid phase coated with a polycloncal anti Lp(a) antibody. The liquid-phase antibody was labelled with biotin and detected by europium labelled streptavidin in the DELFIA 1232 fluorometer. The measuring range was 2-1600 mg/l. The intra-assay imprecision was < 7% (CV), the inter-assay imprecision < 12% (CV). No interference was detected with plasminogen concentration up to 2.2 g/l. There was an acceptable correlation with a commercially available enzyme immunoassay (r = 0.95) and with electroimmunodiffusion (r = 0.85) on 100 routine serum samples measured. The assay appeared to detect different Lp(a) isoforms as dilution curves were parallel for B/F, S2 and S4 isoforms.     

In vitro displacement of phenytoin from protein binding by nonsteroidal antiinflammatory drugs tolmetin, ibuprofen, and naproxen in normal and uremic sera

Displacement of phenytoin (90% bound to albumin) by other highly albumin-bound drugs like salicylate has been well documented. Other widely used nonsteroidal antiinflammatory drugs like tolmetin, ibuprofen, and naproxen are also strongly bound to albumin and can potentially displace phenytoin. However, phenytoin-ibuprofen interaction has been poorly studied in the past, and interaction of phenytoin with tolmetin or naproxen has not been studied before. For normal serum pool (albumin 3.7 g/dl), we observed significant increases in free phenytoin concentrations only with antiinflammatory drug concentrations at the upper end of therapeutic or above therapeutic concentrations. However, for the uremic pool (albumin 2.9 g/dl), displacement of phenytoin was significant even at the lower end of therapeutic concentrations of those antiinflammatory drugs. Of the three antiinflammatory drugs we studied, ibuprofen caused the highest displacement of phenytoin.     

Photochemical labeling of human erythrocyte membrane proteins with radioiodinated 4-azidosalicylic acid derivatives of G(M3), G(D3), G(M1), and FucG(M1) gangliosides

Photoreactive gangliosides of high specific radioactivity may prove useful for studies on glycosphingolipid functions. We prepared 4-azidosalicylic acid (ASA) acylated derivatives of GM3, GD3, GM1, and FucGM1 gangliosides (gangliosides-ASA). Gangliosides-ASA were characterized by their TLC mobility, UV spectra, carbohydrate composition, and digestion with leech endoceramidase. After radioiodination to about 200 Ci/mmole gangliosides-ASA were used for photochemical labeling of human erythrocytes. Radioiodinated gangliosides-ASA were incorporated into erythrocytes in a time and concentration dependent manner, the kinetics and extent of incorporation being similar for all the gangliosides-ASA used. Radioiodinated gangliosides-ASA incorporated into erythrocytes were resistant to trypsin digestion while treatment with 1% BSA removed about 90% of the label. Incubation with cholera toxin protected radioiodinated GM1-ASA and, to a lesser extent, FucGM1-ASA but not GM3-ASA and GD3-ASA, against removal with BSA. After photolysis about 40-50% of radioactivity was firmly bound to erythrocyte lipids and proteins. The ratio of lipid- to protein-bound radioactivity ranged from 2.2:1 to 3.2:1. Photolabeled proteins were analyzed by SDS/PAGE followed by autoradiography. Band 3 was the most extensively photolabeled protein with all the radioiodinated gangliosides-ASA used. DIDS, an inhibitor of band 3 protein activity, caused reduction in photolabeling of this protein by about 20%.     

Evaluation of two commercial kits for serum gastrin assay, and comparison with a conventional radioimmunoassay procedure

We compared two gastrin radioimmunoassay kits ("Immutope" kit, Squibb & Co.; "Gastrin R.I.A." kit, Schwarz/Mann) to the conventional gastrin radioimmunoassay of Yalow and Berson [Gastroenterology 58, 1 (1970)] as run by us and by a second reference laboratory. Although both kits were found to effectively discriminate above-normal and normal values for serum gastrin, they significantly underestimated very high values (greater than 1500 ng/liter). The Schwarz/Mann kit clearly had a superior quality label (lower nonspecific binding and higher specific activity) and a shorter incubation time. However, the 90-min incubation period cited for their kit caused overestimation of gastrin values in the lower range (5-300 ng/liter), which could be corrected by prolonging the incubation to 24 h. The Squibb antibody had fairly good cross reactivity to all gastrin species tested; the Schwarz/Mann antibody had poor affinity for natural human gastrin G34-II. Good correspondence was found for sera run by both reference laboratories (y = 0.96x + 10, r = 0.997), and values obtained with the Schwarz/Mann kit correlated best (+ 0.815) with those from the conventional radioimmunoassay procedure.     

Extrathyroidal conversion of thyroxine to 3,3',5'-triiodothyronine (reverse-T3) and to 3,5,3'-triiodothyronine (T3) in humans

In order to estimate the relative magnitude of the two alternative pathways of monodeiodination of thyroxine (T4) in adult humans, the metabolic clearance rates (MCR) and production rates (PR) of 3,3',5'-triiodothyronine (reverse-T3,rT3) and of 3,5,3'-triiodothyronine (T3) were determined in six euthyroid control subjects (C) and in five hypothyroid patients (H) receiving L-T4 as replacement therapy (0.15-0.3 mg/day). MCR was computed by a non-compartmental method of analysis from the plasma disappearance of 125I rT3 and 131I T3 during 72 h following simultaneous injection of tracers. PR was calculated from MCR and the serum concentration of rT3 and T3, respectively, determined by radioimmunoassay. In the H subjects, rT3 MCR averaged 97.1 +/- 12.8 (SD) 1/day and rT3 PR, 34.3 +/- 12.8 microng/day; T3 MCR was 28.7 +/- 6.1 1/day and T3 PR, 20.3 +/- 6.6 microng/day (all corrected to 70 kg body weight). These results were not significantly different from those in the control group; rT3 MCR 104 +/- 24 1/day, rT3 PR 33.0 +/- 9.2 microng/day; T3 MCR 24.0 +/- 5.9, T3 PR 24.2 +/- 4.1. The proportionof total triiodothyronine (rT3 averaged 62% in H patients and was similar (57%) in the C group. The results obtained in the H subjects indicate that the production of rT3 is a major route of T4 metabolism, equal to or exceeding that of T3. From the close agreement between the mean values for rT3 PR in the C and H groups it is concluded that most, if not all of the rT3 produced in normal humans is derived by extrathyroidal conversion from T4.     

Detection of gluten in human sera by an enzyme immunoassay: comparison of dermatitis herpetiformis and celiac disease patients with normal controls

We have developed a triple sandwich enzyme immunoassay to detect circulating gluten in human sera. With human sera containing known amounts of added gluten as controls, the assay was sensitive in the range of 0.75 to 75 micrograms of gluten per ml of serum. Forty-one control subjects were compared to 21 patients with dermatitis herpetiformis and 11 patients with celiac disease. The dermatitis herpetiformis and celiac disease patients had significant elevation of serum gluten values over the control subjects. Circulating gluten antigenemia is a previously unrecognized feature which may be important in understanding the pathogenesis of dermatitis herpetiformis and celiac disease.     

Lack of germ-line mutations of CDK4, p16(INK4A), and p15(INK4B) in families with glioma

Gliomas are tumors of the central nervous system that may be inherited in some patients. The gene(s) responsible for the clustering of gliomas in families have not yet been identified. Molecular studies of sporadic high-grade gliomas have revealed mutations or deletions of the genes encoding the protein kinase inhibitors p16(INK4A) and p15(INK4B) in a large proportion of tumors. Moreover, those tumors without deletions frequently display gene amplification and/or over-expression of mRNA encoding the protein kinase cdk4. We hypothesized that germ-line mutations in the p16(INK4A), p15(INK4B), or CDK4 genes might contribute to some cases of familial gliomas. To address this issue, we analyzed 36 kindreds with a predisposition to glial tumors. Genomic DNA from index members of these families was screened by PCR-single-strand conformational polymorphism analysis. We did not detect any functional mutations in the p16(INK4A), p15(INK4B), or CDK4 genes, although two individuals did have a previously described A140T polymorphism in p16(INK4A). Thus, despite the association between the sporadic forms of high-grade glioma and abnormalities of p16(INK4A), p15(INK4B), or CDK4, we found no evidence that germ-line mutations in the coding region of these three genes predispose to inherited glial tumors.     

Anti-CD4 activity of normal human immunoglobulin G for therapeutic use. (Intravenous immunoglobulin, IVIg)

The effects of intravenously administered normal immunoglobulin G (IVIg) in autoimmune diseases are dependent on the ability of IVIg to interact with surface molecules of lymphocytes. In the present study, we demonstrate the presence of anti-CD4 activity in IVIg by showing the ability of IVIg to bind to CD4 and to inhibit CD4-dependent cellular functions. Binding of IVIg to recombinant soluble human CD4 was assessed by ELISA, immunoblotting and real time analysis of complex formation. Anti-CD4 antibodies isolated from IVIg by affinity-chromatography bound to human CD4+ T cells. These anti-CD4 antibodies inhibited proliferative responses in MLR and infection of CD4+ human T cells with HIV. These results indicate that IVIg contains antibodies reactive with human CD4 and that these anti-CD4 antibodies exhibit biological functions. The presence of anti-CD4 antibodies in IVIg may be relevant to the immunoregulatory effects of normal polyspecific immunoglobulin G.